Note: Descriptions are shown in the official language in which they were submitted.
2 1L ~ 5 `
-- 1 --
ENDOTHB~IN CONVER~ING ~N~YM~
Backqround of the I~vention
Endothelin (ET) is a 21-amino acid vasoconstrictor
peptide originally isolated from porcine endothelial
cells, and having the amino acid sequence shown in Figure
la and a molecular weight of 2492 (Yanagisawa et al.,
Nature 332, 411 ~1988)). Porcine endothelin is synthe-
sized first in a preproendothelin form of 203 amino
acids, which is cleaved by known processing endopepti-
dases to a precursor form of 39 amino acids, which iscalled Big ET or BET. BET is then further cleaved to
form ET by a putative enzyme designated by Yanagisawa
et al. as "endothelin converting enzyme" (ECE), which
cleaves off the C-terminal 18 amino acids from BET to
form the active 21 amino acid peptide ET. The existence
of ECE was postulated because the cleavage which occurs
to produce ET from BET occurs at an amino acid sequence
for which there is no known protease having that speci-
ficity, e.g., to cut only between Trp-Val within such a
sequence.
The human precursor form of ET consists of 38 amino
acids, which must be cleaved by an enzyme recognizing an
essentially identical cleavage site (Y. Itoh, et al. FEBS
Lett. 231, 440, 1988). Three human isoforms of ~T have
2r, been identified, and an ECE activity was inferred for the
cleavage of all isoform precursors ~A. Inoue, et al.
Proc. Natl. Acad. Sci. USA 86, 2863, 1989).
-` 2~3~ ~
ET is one of the most potent vasoconstrictors known.
It is postulated to have an important function in the
control of the cardiovascular system. For example, ele-
vated levels have been implicated in various cardio
vascular-related diseases such as essential hypertension,
vasospastic angina, acute myocardial infarction, conges-
tive heart failure, pulmonary hypertension, renal failure
and shock. These data support the proposal that one or
more of the endothelin isoforms, e.g., endothelin-l, may
contribute to the pathogenesis of these diseases. There-
fore, the putative "endothelin converting enzyme" t which
produces active ET from its precursor BET, is also pre-
sumed to play an important role in the regulation of ET.
Thus, for example, a pharmacologically active inhibitor
of endothelin production, e.g., an ECE inhibitor, would
be clinically useful in treating such cardiovascular
diseases. For example, even in the absence of isolating
and identifying the enzyme, researchers are studying the
inhibition of the putative ECE enzyme in vivo, in whole
cells and in cell-free extracts, as a means of providing
therapies for the above diseases, based theoretically on
preventing the formation of the contractilely active ET
from the precursor BET. Other cardiovascular therapies
based upon administration of ECE itself are also contem-
plated. Furthermore, the enzyme itself would be usefulas an endopeptidase having an amino acid specificity
which is not otherwise available to protein biochemistry.
Various enzymes have been purified to varying
degrees, characterized and shown to cleave BET to ET;
however, in each case, the cleavage of ET is not spe-
cific, in that further cleavages of ET occur, resulting
in a degraded product. Some of these enzymes which non-
specifically cleave BET to ET have been identified as
already-known peptidases such as pepsin, cathepsin D and
E, and other aspartic proteases.
However, despite the interest in the field~ the
putative ECE has thus far not been isolated or charac-
~~~ 2~33a
terized. Thus very valuable information is lacking,
especially as it relates to the control of production of
ET from its precursor forms.
Summary of the Inventio~l
This invention provides a proteinaceous substance
having the biological activity of cleaving big endothelin
specifically to endothe]in at pH 7.2, with substantially
no further cleavage of endothelin, and having a specific
activity of at least about 30 U/mg of protein, and
preferably at least 500 U/mg.
In particular, the invention provides a metalloendo-
proteinase having said properties. In addition, the
invention provides a metalloendoproteinase containing a
metal ion, capable of binding strongly to an anion
exchange resin and capable of being substantially
completely inhibited by 1 ~M phosphoramidon, as well as
being less than 50% inhibited by 10 ~M 1-[(phenyl-
methoxy)carbonyl]-L-prolyl-L-leucyl N-hydroxyglycinamide
(CK4919), in particular wherein the metal ion is a
catalytically effective metal ion, e.g., zinc.
Another aspect of this invention provides a process
for isolating endothelin converting enzyme from cells
containing said enzyme, comprising
disrupting the cells;
isolating the high speed membrane-containing
fraction;
solubilizing the membrane-bound proteins;
separating the solubilized proteins by anion
exchange,
separating by size the thus-obtained fractions of
similar charge density proteins containing endothelin
converting enzyme activity; and
isolating the thus-obtained fraction having an
apparenk molecular weight as measured on a Superose 12
column o~ between 232 and 440 kilodalkons.
~ 2~3~
Still another aspect of the invention relates to
additional purification steps in the purification of ECE.
In these cases, ECE activity-containing pools are ob~
tained which generally contain protein bands correspond-
ing to molecular weights of, respectively~ 100 and 140
kD, when analyzed on SDS PAGE. These additional steps
can be performed in any order after solubilization of the
membrane fractions:
Heparin-Sepharose fractionation and/or
ConA-Sepharose fractionation.
Yet another aspect of the invention provides endo-
thelin converting enzyme, preparable by a process as
described above, in particular when prepared from human
bronchiolar smooth muscle cells or human lung fibroblast-
like cells.
Still another aspect of this invention provides a
method o~ screening compounds suspected of having endo-
thelin converting enzyme inhibitory activity, comprising
determining the amount of conversion of big endothelin to
endothelin by a protein having the hiological activity of
converting big endothelin to endothelin in the presence
of said compound and, e.g., comparing with the amount of
such conversion in the absence of such compound.
Upon further study of the specification and appended
claims, further objects and advantages of this invention
will become apparent to those skilled in the art.
Various other objects, features and attendant advan-
tages of the present invention will be more ~ully appre-
ciated as the same becomes better understood when con-
sidered in conjunction with the accompanying drawings,
and wherein:
Figure l shows the proposed proteolytic processing
pathway for the conversion of preproendothelin to endo-
thelin. The preproform of porcine endothelin-1 which
contains 203 amino acids is believed to be converted to
the 39 amino acid form referred to as big endothelin-1 by
dibasic endopeptidases (cross-hatched arrows) and car-
21~3 ~ 3 ~ ~
boxypeptidases (small curved arrows) as shown. Big
endothelin-1 is then cleaved at the Trpn-Val74 bond by a
specific endopeptidase referred to as endothelin convert-
ing enzyme (cross-hatched arrow). The final product is
the 21 amino acid peptide, endothelin, containing amino
acids Cys53 to Trp~. Figure la shows the amino acid
sequence of endothelin-l.
Figure 2 shows ECE elution from a Mono Q FPLC
column. A 170 ~1 aliquot containing 1.411 mg of deter-
gent solubilized 150,000 x g HBSM cell pellet is loaded
in 50 mM Tris, pH 8.0 (20C), 25 mM n-octyl-B~D-gluco-
pyranoside. The sample is eluted with a 0 to 1.0 M NaCl
gradient at 0.2 ml/min at 4C and 0.5 ml fractions are
collected and assayed for ECE activity in the presence of
1 mM PMSF. ECE activity is expressed as pmol of ET pro-
duced in a 200 ~1 assay.
Figure 3 shows ECE elution from a Superose 12 FPLC
column. Separate 100 ~1 aliquots of Mono Q fractions 14,
15 or 16 aré run on a Pharmacia Superose 12 column (10 mm
x 30 cm; 10 ~ particle size) and eluted with 50 mM MOPS
pH 7.2, 250 mM NaCl and 25 mM n-octyl-~-glucopyranoside
with a flow rate of 0.5 ml/min at 4C. One minute frac-
tions (0.5 ml) are collected over a 60 min period and
assayed for ECE activity in the presence of 1 mM PMSF as
described below.
Figure 4 shows ECE activity in individual Superose
12 column fractions. Fractions in the high molecular
weight range (>100 kDa) from each of the individual frac-
tions making up the pooled samples in Figure 2 are
assayed in the presence of 1 mM PMSF.
Figure 5 shows separation of Mono Q fraction 14 on
Superose 12. Top trace: absorbance at 280 nm monitored
during run, scale 1% = 0.002 OD. Middle trace: activity
profile of fractions 14 and 15 separated on Superose 12.
(Data offset o~e fraction for detector-to-collector
delay.) Bottom trace. size standards for this Superose
column.
2 1 ~ ~ 3 ~ 3
Figure 6 shows the elution profile of ECE activity
and optical density at 280 nm of a solubilized membrane
ECE preparation passed over Heparin-Sepharose as
described in Example l(g)(i);
Figure 7 shows the elution profile of ECE activity
and optical density at 280 nm of a solubilized membrane
ECE preparation passed over ConA-Sepharose as described
in Example l(g)(ii);
Figure 8 shows the elukion profile of ECE activity
and optical density at 280 nm off of a Superose 12 column
of an ECE preparation passed over Heparin-Sepharose
before the Superose 12 purification step as described in
Example l(g)(iii);
Figure 9 shows the kinetics of ET production by ECE
and NEP.
The present invention provides purified endothelin
converting enzyme, as well as a process for the isolation
and purification of an enzyme which specifically converts
human proendothelin-l (amino acids 1-38) or Big ET (B~T)
to endothelin-l (amino acids 1-21) or ET. The enzyme ob-
tained by this procedure produces only one major detect-
able product which coelutes with genuine ET from a C-18
~PLC column. The enzvme also gives only one major
detectable band when run on a 7.5% SDS-PAGE gel. These
data imply that the enzyme is homogeneous, e.g., substan-
tially free of other proteases, inter alia, and that its
major enzymatic activity towards the substrate, human
BET-1, is conversion to the product, ET-1. By these
criteria, the enzyme purified by this process is the
proteolytic activity referred to by Yanagisawa et al. as
"endothelin convertiny enzyme" or ECE.
By "proteinaceous substance" is meant a single
molecular species or a complex, comprising one or more
covalently or otherwise linked amino acid or protein
sequences, with or without covalent modifications such as
glycosylation or fatty acid acylation, with or without
.: ' '. :
,
2 .1
-- 7 ~
one or more bound metal ions, etc., which have the
indicated biological activity. Examples include dimers
or higher oligomers of a single molecular species, or
covalent or tightly bound non-covalent complexes of two
or more different protein sequences.
With respect to the several proteinaceous substances
described herein, the term "an apparent monomeric
molecular weight of greater than x kilodaltons", is meant
that the proteinaceous material has a property usually
associated with proteins of that size on, e.g.,
electrophoresis or gel filtration, but is not necessarily
actually a single protein chain of that size. For exam-
ple, a protein may have an anomalous shape which causes
it to behave similarly to a higher molecular weight spe-
cies when analyzed on, e.g., gel filtration or electro-
phoresis, which anomalous shape may be endogenous to the
protein itself or may be due to covalent modifications
such as glycosylation; it may be a single gene product
that is later processed into two or more covalently
linked protein chains, analogously to, e.g., insulin,
etc.; it may result from proteolytic fractionation of a
larger to one or more smaller species; from dimeric vs.
monomeric molecular weights, etc., or may be due to
different gene products. Whether or not the activity in
these various bands is due to different molecular spe-
cies, in each case the proteins detected are present in
fractions containing ECE activity as measured by the
various assays disclosed herein. The molecular weight of
the species as disclosed herein, therefore, is a physical
measurement which is only one property of the claimed
polypeptides. In every case, it is the enzymatic and
other functional properties as disc]osed below which
particularly define the claimed polypeptides.
Several proteinaceous substances have been purified
according to the methods disclosed herein. The apparent
molecular weights of these materials ~ary according to
the method used for its detection. Thus, the material
2 ~0~3
-- 8
prepared from HBSMC cells which is eluted from a
Superose-12 column elutes between molecular weight
standards of 232 and 440 kilodaltons, corresponding to an
apparent molecular weight of about 400 k:ilodaltons. This
matarial eluted from the Superose 12 column and analyzed
using electrophoresis on a 7.5% sodium dodecyl sulfate
polyacrylamide gel under reducing condit:ions ran as an
about 205 kilodalton molecular weight species.
Subsequent analysis of material isolated the same way,
but prepared from MRC-5 cells, and analyzed on SDS~PAGE
under reducing conditions demonstrated two bands
corresponding to proteins having molecular weights of lO0
and 140 kilodaltons. Each of the proteins which
correspond to the bands detected on SDS-PAGE, as well as
the proteinaceous material detected in the 232-440
kilodalton molecular weight eluate on Superose-12 possess
the enzymatic activities described herein. The 100 and
140 kilodalton proteins are further isolated ~rom each
other using routine protein purification techniques. The
100 kilodalton protein has the biological activity
disclosed herein. The 140 kilodalton protein has the
biological activity disclosed herein.
The phrase "cleaving big endothelin specifically to
endothelin at a pH of 7.2", of course, does not imply
that the proteinaceous substance or protein of this
invention will not cleave at other lower or higher pHs,
only that the proteinaceous substance or protein of this
invention will cleave at a pH of 7.2 and will have the
other characteristics described herein.
As a measure of purity, the term "with substantially
no further cleavage of endothelin" as used herein means
that a sample having a specific activity of at least 30
U/mg, and preferably at least 500 U/mg, and most
preferably greater than 10,000 U/mg, has an absence of
detectable amounts of proteolytic activity which cleaves
ET to pieces smaller than 21 amino acids, i.e., upon
cleavage of 3 ~M BET for one hour, essentially no peaks
2 ~
g
of material containing a (tryptophan) fluorescence-
emitting group other than BET and ET are detectable after
HPLC analysis of the reaction mixture, as disclosed below
for analysis of the ECE reaction. Similarly, purity of
ECE in this respect can be defined as the absence of
substantial ET degradation in the ECE assay conditions
described herein with ET-1 added at 0.2 ~M in lieu of
BET-1.
As another indication of purity, the protein of this
invention has a specific activity of at least about 30
U/mg of protein, and preferably at least 500 U/mg, e.g.,
values greater than 1000, 2000, 3000, 4000, 5000, 6000,
7000, 8000, 9000, etc., and most preferably greater than
10,000 U/mg, e.g., up to 50,000, 100,000, 500,000 U/mg,
i.e., the activity of a 100% purified protein, wherein a
unit of activity is defined as the amount of enzyme which
converts 1 nmol of substrate, e.g., human BET-l (hBET 1),
to product, e.g., ET-1, in one hour, at a starting
concentration of 3 ~M of substrate, at 37C, pH 7.2.
These activities can be obtained by further purification
steps, e.g., using affinity chromatography wherein an ECE
inhibitor is bound to a support resin; using preparative
gel electrophoresis; using chromatofocusing; etc., and
especially wherein the ECE is produced by recombinant
techniques, as discussed below.
As a further measure of purity, the protein of this
invention is characterized by the presence of
substantially only a single proteinaceous substance as
determined by SDS-PAGE, in that there is no more than one
defined stained band using a Pharmacia PHAST System
silver stain kit, when > 4 ng of protein is loaded in a
lane, or, if more than one band is present, every band
corresponds to a polypeptide involved in ECE activity.
As is true of other proteases, which can be
identified with families of proteins having similar
enzymatic, biochemical and structural properties, the
activity of ECE has similarities with other proteases,
21~3 '~
-- 10 --
notably ACE (angiotensin converting enzyme; EC 3.4.15.1)
and NEP (neutral endopeptidase, enkephalinase; EC
3.4.24.11). Thus, both ACE and NEP can cleave hB~T 1;
however, these proteases do not have the speci~icity nor
activity for hBET-l of ECE. The activity of ECE is
distinguished from these enzymatic activities using
various biochemical and kinetic tests. For example, ECE
is clearly distinguished from ACE by the fact that it is
not inhibited by 10 ~M of the potent ACE inhibitors
enalaprilat and benzaprilat. Distinguishing ECE from NEP
is less unambiguous, in that kinetic, biochemical and
immunological properties are used to distinguish them
comparatively. See Table VI, in which the comparative
properties of the two enzymes are summarized. In
particular:
1. The specific activity, and thus KCat/Km of ECE for
hBET-1 is very much higher than the specific activity of
NEP ~or hBET-1: see Table IV;
2. NEP will cleave BET to ET (and other products),
but unlike the result using ECE, the amount of ET
produced by NEP is limited by the greater intrinsic rate
of ET degradation by this enzyme: see Fig. 9;
3. NEP does not show the strict dependence on Cl
which ECE shows, similarly to the distinction between ACE
and NEP (J.P. Swerts et al., Eur. J. Pharm. 53, 209-210
(1979)); and
4. Immunological crossreactivity using antibodies
raised to NEP ([provided by Prof. A.J. Turner, Univ. of
Leed, UK) can be used to show that preparations of ECE
contain little if any crossreacting material as a percent
of activity present and these amounts at worst correlate
with an insignificant amount of NEP activity.
In addition, two commercially available compounds,
CK4590 (= carboxyphenylpropionyl-Leu; N-([R,S]-2-carboxy-
3-phenylpropionyl)-L-leucine, a NEP inhibitor) and CK4919
(=Cbz-Pro-Leu-Cly-hydroxamate; l-[(phenylmethoxy3-
carbonyl]-L-prolyl L-leucyl-N-hydroxyglycinamide, a
2~
collagenase inhibitor) (both compounds from Sigma, St.
Louis, M0) have been shown to inhibit NEP significantly
more than they inhibit ECE, when tested in the ECE assay
under conditions in which NEP generates ET from BET
linearly. See Table VII.
Substantially purified EOE according to this
invention has numerous advantages over the unpurified ECE
disclosed in the prior art, e.g., the material as it
occurs in nature or in partially purified form, in all
cases at substantially lower purity. This invention for
the first time provides ECE at specific activities at
which the following advantages become apparent, e~g., at
specific activities of about 20 U/mg and higher:
~1) substantially purified ECE can be used to screen
potential ECE inhibitors most effectively at purities
where the measurable results of the conversion of BET to
ET result primarily from the specific cleavage of BET by
ECE, as opposed to measuring the non-specific cleavage by
other proteases which may be present in a less purified
sample;
(~) substantially purified material can be used to
prepare antibodies to ECE, whereas the use of less
purified material would make this process much more
difficult;
t3) substantially purified protein can be sequenced,
which is extremely difficult if not impossible to do with
impure proteins, in order to provide information that can
be used to tailor nucleic acid probes for identifying and
retrieving a clone containing the ECE gene from a genomic
or cDNA library for further cloning and expression of the
ECE gene; etc.
The proteinaceous substance or protein of this
invention is isolated using a sequence of steps which are
each per se routine in the isolation of membrane bound
proteins of high molecular weight. The following
description is intetlded to be illustrative of one method
for isolating ECE.
2~3~
- 12 -
Suitable cell or tissue types for use as a source of
ECE are any cell or tissue types which contain ECE that
is expressed; for examples of cell or tissue types which
have already been shown to contain ET, and thus by
inference, ECE, see Table A:
~, 2~3
- 13 -
Table A
~I8~UE DIS~RIB~ION OF ~NDOT~BLIN
Oriqin Tissue 8pecies ~ef.
8rain Cortex Pig
Spinal Cord Pig 2
Hypothalamus Pig
Cerebellum Rat 3
Cerebrum Rat 3
Medulla Oblongata Rat 3
Pituitary Rat 3
~eart Atrium Pig
Atrium Rat 4
Ventricle Pig
Ventricle Rat 4
Blood Ve~sel Aorta Rat 3
Umbilical Artery Human 5
Umbilical Vein Human 5
Ridney Inner Medulla Pig
Outer Medulla Pig
Inner Medulla Rat 6
Cortex Rat 6
Cortex Pig
Kidney Rat 3
~astrointe~tinal Duodenum Pig
Traat Duodenum Rat 3
Stomach Rat 3
Intestine Rat 3
Colon Rat 3
Pancreas Rat 3
Other Lung Pig
Lung Rat 3
Liver Pig
Liver Rat 3
Spleen Pig
Spleen Rat 3
Adrenal Gland Rat 3
Urinary Bladder Rat 3
Testis Rat 3
Placenta Human 5
Refs.:
1. Kitamura, K., et al., Biochem. Biophys. Res.
Commun. 161, 348-352 (1989).
2. Shinmi, O., et al., Biochem. Biophys. Res.
Commun. 164, 587-593 (1989).-
4S 3. Matsumoto, H., et al , Biochem. Biophys. Res.
Commun. 164, 74-80 (1989)-.
4. Yanagisawa, M., et al., Proc. Natl. Acad. Sci.
USA 85, 6964-6968 ~1988~.
5. Haegerstrand, A., et al., Acta Physiol. Scand.
137, 541-542 (1989).
6. Xitamura, K , et al., Biochem. Biophys. Res.
- Commun. 162, 38-44 ~1989).
2~3~
In addition to HBSM cells, ~RC-5 cells, which are a human
fetal lung "fibroblast-like" cell line, publicly
available from ATCC, Rockville, MD as CCL171, have been
used as a source of ECE in the results disclosed herein.
Other cell types can be routinely screened by one of
skill in the art for the presence of ECE to determine if
they would be suitable as a source of the enzyme.
In order to screen cell types for the presence of
ECE and monitor samples for the presence of ECE during
purifications, any suitable ECE assay can be used, e.g.,
an enzymatic assay, e.g., in which the production of ET
from BET is detected, an immunoassay, an HPLC
chromatogram, an electrophoretic gel analysis, etc. A
preferred method is to perform an assay in which the
production of ET from BET is detected. This assay is
performed by placing a sample suspected to contain ECE in
an assay mixture containing a suitable buffer, e.g., any
biologically suitable buffer effective in the neutral pH
range, e.g., about pH 6.5-8.0, e~g., MOPS, TRIS,
phosphate, etc., and a predetermined amount of BET for a
certain amount of time, and then detecting the formation
of ET from BET by ECE. Suitable concentrations of NaCl
or other source of chloride ion are also required for
maximal ECB activity. As controls are used blanks
containing no ECE and samples containing known amounts of
ECE for quantifying the results. The detection can be
performed by a number of means, as noted above. In a
preferred method, the detection of ET is monitored by
passing the assay mixture, after the reaction is
terminated, e.g., by the addition of EDTA, over an
appropriate liquid chromatography column, e.g., a Vydac
C-l~ reverse phase HPLC column, and monitoring the eluted
material, e.g., by fluorescence spectroscopy at, e.g, an
excitation wavelength of 225 nm and an emission
wavelength of 340 nm, for the presence of the peptide
peak characteristic of the smaller ET polypeptide. Any
of the usual methods of eluting material from colu~ns can
,,,
3 ;.~ ~
- 15 -
be used, e.g., a decreasing gradient of a buffer, e.g.,
sodium acetate, and an increasing gradient of an organic
solvent, e.g., acetonitrile.
Since ECE is associated with the membrane ~raction
of cells which express it, cells containing ECE are
disrupted by routine methods, e.g., by high pressure, low
pressure, grinding, osmotic disruption, etc., followed by
removal of large material, such as unbroken and only
partially disrupted cells and lysosomes, from the
suspension, e.g., by low speed centrifugation, e.g., at
20,000 x g for 1 hr. The ECE-containing plasma membrane
fraction is then separated from the soluble fraction by,
e.g., high speed centrifugation for a suitable period of
time; this is called the high speed membrane fraction,
and is distinct from other membrane-containing fractions
which contain additional undesired components, e.g., the
lysosomes, or which do not contain the plasma membrane.
Thus, by high speed fraction is meant the fraction
pelleted, by centrifugation at 150,000 x g for about 1
hr, from the supernatant remaining after pelleting the
crude homogenate at 20,000 x g for about 1 hr. The
membrane-bound proteins are then solubilized from the
lipid portion of the membrane using a solubilization
buffer containing a detergent suitable for gently
solubilizing membrane proteins, e.g., n-octyl-~-D-
glucopyranoside. Other suitable detergents are those
which gently solubilize proteins from membranes without
irreversibly denaturing the protein of interest,
including, e.g., non-ionic detergents such as Triton
X-100, Nonidet P-40, digitonin, Lubrol PX, C12E8,
zwitterionic CHAPS, etc. In particular, Triton X-lOOR is
useful when purification over Con~-Sepharose is used, as
the sugar on ~-octylglucoside interferes with the enzyme
binding to this matrix. Other suitable detergents can be
determined according to methods known to one of skill in
the art, e.g., according to methods in Methods in
Enzymology 182, Guide to Protein Purification, M.P.
"
2~
- 16 -
Deutscher, ed., Academic Press, NY (1990), especially
pages 247-255. The solubilized membrane fraction is then
subjected to a preliminary fractionation on the basis of
a particular physical characteristic, e.g., charge
density, e.g., on a column, e.g., a Pha~macia MONO Q HR
5/5 column, and eluted from the column, ~e.g., with a
detergent-containing buffer in a gradient of an agent
suitable for eluting proteins from such a column, e.g.,
NaCl. Fractions are collected and tested for the
presence of ECE, and ECE-containing fractions are pooled.
Other preliminary fractionation methods may also be used,
e.g., based on other physical methods, e.g., size~ or
based on affinity, e.g., by antibodies specific for ECE
bound to a column or by binding to a non-cleavable
reversible inhibitor for ECE.
If analysis of the pooled ECE-containing fractions
from said preliminary fractionation, e.g., by HPLC or gel
electrophoresis, indicates that the ECE is not
substantially purified, e.g., to at least a specific
activity of 30 U/mg, and preferably at least 500 U/mg,
most preferably greater than 10,000 U/mg, the pooled
fractions are then subjected to a further purification
step, preferably based upon a different physical
property, e.g., a size fractionation on a column having a
suitable size differentiative ability consistent with the
unusually large apparent size (about 400 kDa) of ECE,
e.g., on a Superose 12 HR 10/30 column, and eluted with a
suitable buffer, again containing a suitable detergent to
maintain the solubility of membrane proteins, such as the
above-mentioned n-octyl-~-D-glucopyranoside. Fractions
are collected and assayed for the presence of ECE, and
EC~-containing fractions pooled. Similarly, other
purification matrices can be used, preferably based upon
different physical, biological or enzymatic properties,
e.g., Heparin-Sepharose or ConA-Sepharose, each of which
provides a differentiative ability based upon a property
of ECE. In each case, the partially purified ECE is
2 ~
- 17 -
added to a column containing the matrix and eluted with a
suitable buffer, again containing a suit:able detergent to
maintain the solubility o~ membrane proteins. Depending
on the origin of the material, the size of the batch to
be purified, and other routinely determinable factors,
these various purification steps can be used in any order
to best effectuate the purification required.
The pooled material from each step is tested for
purity, e.g., on a polyacrylamide-SDS gel, run against
molecular weight standards, and protein bands detected
by, e.g., silver stain or Coomassie Brilliant Blue dye.
It was found in this assay to be a monomeric protein of a
very high molecular weight of greater than about 205
kilodaltons, substantially free of any other protein-
stainable material, i.e., when 4-40 ng of protein was
loaded on the gel, and using a stain which is capable of
detecting bands containing > O.S ng or more of protein,
no band of protein not in the larger-than 205 kDa band
was detectable.
Further purifications of HBSM ECE as well as ECE
isolated from MRC-5 cells, when analyæed on SDS gels
under reducing conditions, have resulted in the detection
of essentially pure protein bands which migrate on the
gel in positions which indicate molecular weights of 100
and 140 kilodaltons.
0.5 ng of the purified enzyme was subjected to 1 ~M
phosphoramidon, which is known to inhibit metallo-
proteinases, and assayed for ECE as above, and was found
to be completely inhibited thereby. It is also inhibited
by other metal chelating agents, e.g., EDTA, EGTA and
o-phenanthroline. Therefore, the puxified enzyme is also
a metalloproteinase. Since ECE is a metalloenzyme,
containing one or more metal ions, eOg., Zn'~ ions, it can
also be prepared in an apoprotein form by removing (or,
in the case of synthetic ECE prepared by recombinant DNA
methods, by not adding) zinc, e.g., by chelation usin~ a
2~
- 18 -
chelator such as, e.g., EDTA. Glycosylated and
unglycosylated forms of the protein can also be prepared.
The conditions employed in each of these steps
individually are conventional and routinely optimizable
using conventional considerations, e.g., as described in
Methods in Enzymology 182, Guide to Protein Purification,
M.P. Deutscher, ed., Academic Press, NY (1990).
The purified ECE obtained by this process can be
used for various purposes. In particular, it can be used
to screen compounds which are suspected of having E OE
inhibitory activity. Such compounds are of interest as
they may be useful in treating conditions in which excess
levels of ET have been implicated, e.g., various
cardiovascular related diseases such as essential
hypertension, vasospastic angina, acute myocardial
infarction, congestive heart failure, pulmonary
hypertension, renal failure and shock. As a screening
test, various methods may be employed to determine if ECE
is being inhibited, for example, a modification of an ECE
assay as described above, whereinl instead of a sample
suspected of containing ECE being added to an assay
mixture containing BET and buffer, a sample of the
compound suspected of having ECE-inhibitory activity is
added to a sample containing either a known amount of ECE
in buffer or a known amount of BET in buffer, and the
reaction started by adding the missing ingredient (i.e.,
BET or ECE, respectively). The production (or lack
thereof) of ET is then monitored in the usual ways
described above, e.g., by comparison with blanks not
containing any inhibitor. The ECE of this invention can
also be used as a tool to characterize the optimum
conditions for preparing ET from BET.
Suitable methods for performing screening tests for
inhibitors of ECE according to this invention are
analogous to those for screening for inhibitors of other
enzymes, given the ECE of this invention, and particular
protocols can be routinely optimized-~Y one of ordinary
-- 19 --
skill in the art. For example, 0.08 U/ml of E OE are
incubated for 1 hr at 37C in a buffer c:ontaining 100 ~M
Na-MOPS, pH 7.2, 150 mM NaCl, 2.5 mM ~-octylglucoside, 30
~M CaCl2, and 3 ~M BET, in the presence or absence of the
compound to be screened. The reaction is stopped by
addition of EDTA to 3 mM, and the resulting mixture is
analyzed for the production of ET by separation o~ the
mixture on HPLC, and quantification of the resulting ET
peak area.
Another use of the purif~ed ECE includes using it as
an antigen for the production of antibodies, using
routine immunological protocols, as well as for the
production of monoclonal cell lines producing such
antibodies, for use in immunoassays for detecting the
presence of ECE in, e.g., clinical samples. All of the
methods for this use are routine to one of ordinary skill
in the art, using standard protocols, e.g., as described
in Galfre, G. and Milstein, C., Preparation of Monoclonal
Antibodies: Strategies and Procedures, in Methods in
Enzymology 73, 3-46 (1973).
Yet another use for purified ECE is for the
det~rmination of the amino acid sequence of the protein.
Either the entire sequence can be determined using
standard protocols, or, particularly in view of the large
size of the protein, partial amino acid sequences can be
determined sufficient to enable one of ordinary skill in
the art to prepare one or more probes, i.e., nucleic acid
sequences, or sequences complementary to such nucleic
acid sequences, which encode the amino acid partial
sequences of ECE; such probes can then be used to probe a
genomic or cDNA library, e.g. any of the standard
available libraries, for clones containing the DNA
sequence for the ECE gene. Due to its size, probes ~rom
more than one location in the protein, e.g.~ three, will
preferably be used to ensure that the entire correct gene
is present in a given clone; it may be necessary ~o join
together two or more clones to Qht~ir~ a full~length gene,
- 20 -
but such procedures are routine to one of ordinary skill
in the art without undue experimentation. After a full-
length cloned gene is obtained, the entire sequence of
the gene and thereby also of the protein may be
determined using routine DNA sequence analysis.
A further use for ECE, after the above protocol has
been performed and a cloned full-length gene is obtained,
is to transfer the cloned gene into an expression vector
capable of expressing such a cloned protein in a host
cell, e.g., using a baculovirus expression system ancl
insect cells or a vector with a retroviral promoter and a
mammalian cell system, in order to produce large amounts
o purified ECE using standard genetic engineering
protocols.
Methods for performing the sequencing and genetic
engineering aspects of this invention are routine for one
of ordinary skill in the art, e.g., by reference to any
one of a number of standard references, e.g., Fritsch,
E.F. and Maniatis, T., Molecular Cloning: A Laboratory
Manual (2nd. ed.), Cold Spring Harbor, Cold Spring Harbor
Laboratory Press (1990).
The ECE gene(s) so obtained can be used to prepare
transgenic animals using fully conventional methods,
e.g., according to the methods disclosed in U.S.
4,736,866. These cloning techniques can be further used,
e.g., to provide gene therapies, and to create
experimental animal models for disease states.
Still another use of ECE is for administration to
patients to treat conditions characterized by lack of ECE
or insufficient ET, or a condition for which increased
amounts of ECE or ET would be effective; for example, low
blood pressure. The corresponding pharmaceutical
preparations could be prepared and administered
analogously to other cardiovascularly active enzymes
which are administered for various purposes, e.g., tissue
plasminogen activator, which can be used to regulate
blood pres.sure, particularly for intravenous
~,,
2 1 0 ~ 3 ~ ~
administration. Typically, these compositions would be
formulated with carriers usual in galenic pharmacy, such
as, e.g., water and serum albumin for intravenous
administration, in the presence of the usual additives,
e.g., buffers, etc.
Without further elaboration, it is believed that one
skilled in the art can, using the preceding description,
utilize the present invention to its fullest extent. The
following preferred specific embodiments are, therefore,
to be construed as merely illustrative and not limitative
of the remainder of the disclosure in any way whatsoever.
In the foregoing and in the following examples, all
temperatures are set forth in degrees Celsius; and,
unless otherwise indicated, all parts and percentages are
by weight.
The entire disclosures of all applications, patents
and publications, cited above and below, if any, are
hereby incorporated by reference~
2~0~3~ja
- 22 -
E ~ A M P L E 8
Exam~le 1: PuriPicatio~ of ~n~othelin Converting
E~zyme ~EC~
a. Crude ~ell preparation
The source of ECE is cultured human bronchiolar
smooth muscle cells obtained according to the procedure
of Twort and Van sreemen (Tissue ~ Cell 20 (3), 339-344
(1988)).
Cultured bronchiolar smooth muscle (HBSM) cells at
passage 16 are grown to confluency in DMEM media
containing 10~ fetal bovine serum, 0.05 mg gentamicin/ml,
105 U penicillin/ml, and 105 ~g streptomycin/ml in eight
850 cm2 (surface area) roller bottles. At this time, the
cells are washed twice with 50 ml of physiological saline
solution (PSS) containing 140 mM NaCl, 10 mM glucose, 5.3
mM KCl, 13 ~M EDTA, 1.5 mM CaCl2 and 5 mM HEPES, pH 7.4.
The cells in each roller bottle are scraped from the
surface of the roller bottles into 25 ml of PSS, pooled,
and then pelleted in a tabletop centrifuge at 100 x g.
The supernatant is discarded and the cell pellet weighed.
The pellet is then resuspended to a single cell
suspension in PSS containing 10% sucrose and 0.1% sodium
azid~ (10 ml solution/1.5 g wet cell pellet weight.) The
cells are disrupted using a Parr Cell Disruption Bomb
under 700 psi nitrogen for 5 min at 4C. The broken
cells are centrifuged at 370 x g ~2000 rpm, Sorvall SS-34
rotor) at 4C. The supernatant is saved and the pellet
resuspended in 2 ml PSS containing 10% sucrose and 0.1%
sodium azide. The cell disruption is repeated and the
3Q broken cells centrifuged at 370 x g. The supernatants
are pooled-and centrifuged at 20,000 x g (16,000 rpm,
Sorvall T-865 rotor) for 45 min at 4C. The resulting
supernatant is centrifuged again at 150,000 x g (45,000
rpm, Sorvall T-865 rotor) for 60 min at 4C. The
resulting pellet is resuspended in 0.5 ml PSS containing
---- - I0~-s~cros~ --o ~ diu~ azide. The protein
- 23 -
concentration is dete~nined using the PIERCE Protein
Assay Reagent with BSA as the skandard protein.
b. Solubilization of the 150,000 ~ g cruaa Nembrane
~ssM Cell Pellet _ _
The 150,000 x g crude HBSM cell membrane preparation
is solubilized by adding Tris Solubilization Buffer
containing 100 mM Tris-Cl pH 8.0 ~at 4C), 1% n-octyl-~-
D-glucopyranoside and 1 mM PMSF. 170 ~1 containing 1.411
mg protein of the 150,000 x g HBSM cell pellet is added
to 330 ~1 of Tris Solubilization Buffer and vortexe~.
The solution is centrifuged at 19,500 x g for 10 min.
The pellet is saved, and purification continued with the
supernatant, which is loaded onto a Pha~nacia FPLC Mono Q
HR 5/5 (5 X 50 mm) column, pre-equilibrated with 50 mM
Tris-Cl, pH 8.0 (20C), 25 mM n-octyl-~-D-
glucopyranoside. Unbound material is washed through the
column using this initial buffer at a flow rate of 0.2
ml/min for 15 min. At the end of the 15 min period, a 25
min linear gradient from 0 to 1 M NaCl in 50 mM TRIS-Cl,
pH 8.0 (20C) and 25 mM n-octyl-~-D-ylucopyranoside is
run. The 1 M NaCl buffer is then pumped through the
column for 5 min. Eighteen fractions of 0.5 ml each are
collected throughout the run and assayed for endothelin
converting enzyme (ECE) activity. The results are shown
in Figure 2.
c. ECE A~say of Mono Q Fractio~s from the ~olubilized
150,000 x ~ ~B~M Cell Pellet
Each fraction is assayed at a 1:10 dilution in the
presence of 50 mM MO~S pH 7.4, 30 ~M CaCl2 and in the
presence or absence of 1 mM phenylmethylsulfonyl fluoride
(PMSF). The diluted column fractions and the assay
buffer are pre-incubated for 30 min at room temperature
before starting the reaction by the addition of 3 ~M
hwnan big endothelin (hBET). The reaction is terminated
after 1 h at 37C by adding EDTA to 3 ~M and placing the
samples on ice. Quantitation of product is done by
loading 200 ~1 of the assay mixture onto a Vydac c 18
2 ~
- 24 -
Reverse Phase HPLC column. The program for sample
elution is shown in Table I~
~ABLE~ IPLC GRl~DIENq! I~LUTION PROGR2~M
.. ,,,.,~ , -~.,.. _,, .. ~
5-5 75 _ lO
13 5 75 25
I . .
26 0 50 50
I .
1 26.01 90_ 10_
35.0 90 10
,
Flow rate is 1.5 ml/min, Vydac 218TP54 RP,
4.6 x 250 mm, 5 ~, 300 A pore size.
2 Time at which condition is reached.
3 Buffer A - 50 mM sodium acetate pH 6.5.
Buffer B - 100% acetonitrile.
Initially, the 90% 50 mM sodium acetate, pH 6.5, and 10%
acetonitrile is pumped ~hrough the column at a flow rate
~of 1.5 ml/min at 30C for 1 min. For the next 7.5 min, a
linear gradient of 90% to 75% 50 mM sodium acetate and
10% to 25% acetonitrile is run, followed by 5 min of 75%
50 mM sodium acetate and 25% acetonitrile. The linear
gradient is then continued from 75% 50 mM sodium acetate
and 25% acetonitrile to 50% 50 mM sodium acetate and 50%
acetonitrile over 12.5 min. Finally, over the last 9 min
the column is reequilibrated with 90% 50 mM sodium
acetate and 10% acetonitrile.
a. Application of ~v~o Q Fractions 13, 1~, 15, 1~ on
~upero~e 1~ _
Separate 100 ~l aliquots of Mono Q fractions 13, 14,
15 and 16 are loaded individually onto a Pharmacia
Superose 12 HR 10/30 (10 mm x 30 cm, 10 ~ particle size)
-" 2 ~ 3 ~ ~
- 25 -
column connected to an FP~C and eluted with 50 m~ ~OPS,
pH 7.2 containing 250 mM~NaCl and 25 mM n-octyl-~-D-
glucopyranoside at a flow rate of 0.5 ml/min at 4C. One
minute fractions of 0.5 ml are collected over a 60 min
period and assayed for ECE activity as described below.
o. ECE As~aY o~ Su~erose Column Fractio~s
To determine which fractions contain ECE activity,
50 ~1 of each succassive five or six fractions are pooled
and assayed. The void volume of the column is
approximately 9 ml; therefore, the fractions are pooled
as follows: 14-19, 20-24, 25-30, 31-34, 35-40, 46-50, 51-
55, and 56-60. These fractions are assayed undiluted in
the presence of 30 ~M CaCl2 and 1 mM PMSF. The assay
mixture is pre-incubated for 30 min at room temperature
and the reaction initiated with the addition of 3 ~M
hBET . The reaction is incubated for 6 h at 37C and
finally terminated with the addition of EDTA to 3 mM and
placed on ice. 200 ~1 of each assay mixture is loaded
onto a Vydac C-18 Reverse Phase HPLC column and analyzed
as described previously. The fractionation scheme,
yields and purification are shown in Table II:
~ABLE II - FRaCTIONATION 8C~ENE FOR ~CE
100 xg Cell 0.15 g/ml 10 ml 4
370 xg Disrupted 12 ml
Cell Supernatants2
I _ I
20,000 xg 12 ml
Supernatants
150,000 xg Pellet 8.3 mg/ml0.17 ml 0.3175 1
19,500 xg 2.38 mg/ml O.S ml
Supernatants of
the 150,000 xg
Solubillzed Pellet3
Mono Q Fraction0.088 0.5 ml 2.41 7.6
14 mg/ml
Mono Q Frac~on4-40 ng/ml 4.0 ml 588-58801855-18,550
14 (100 ~1),
Superose
Fractions 20-27
.
Cell pellet from 8 roller bottles of human bronchiolar
smooth muscle cells, total yield was 1.5 g net weight of
cells.
2 Cells were disrupted with a Parr Cell Disruption Bomb
3 One volume of the 150,000 xg pellet was solubilized with
2 volumes of 100 mM Tris-Cl pH 8.0 at 4C, 1~ n-octyl-~-
D-glucopyranoside, 1 mM PMSF.
4 --, Not determined.
5 These data represent an apparent specific activity due to
substantial ET breakdown in the impure stages of
preparation.
2:~t~3~5
ECE activity is found in the pooled fractions
containing Superose tubes 20-24 and 25-29 from Mono Q
fractions 14, and 15 and 16 (Figure 3). Each of the
individual fractions making up the above pools is assayed
individually at a 1:2.5 dilution in the presence of 30 ~M
CaCl2 and 1 mM PMSF. ~he assay mixture is pre-incubated
for 30 min at room temperature and the reaction started
~y the addition of 3 ~M hBET. The reaction is incubated
for 6 h at 37C and stopped by adding EDTA to 3mM and
placing the samples on ice. 200 ~1 is loaded onto a
Vydac C-18 column and run as stated previously. The
results are shown in Figure 4. Comparison of the
activity profile with the elution locations at standard
proteins, shown in Figure 5, indicates a molecular weight
of approximately 400 kDa.
f. SD~-PAGE of Mono O 14 8uperose Fraction~ 20-27
The Mono Q 14 Superose fractions 20 through 27 are
pooled for a total volume of 2.5 ml and placed in an
Amicon Centricon 10 centrifugal microconcentrator with a
molecular weight cut off of 10,000 Da. It is centrifuged
at 5000 xg for 90 min at 4C until a deadstop volume of
100 ~1 is reached. Two ml of 0.02M TRIS pH 8.0 is added
in order to dilute the remaining detergent. The solution
is again centrifuged at 5000 xg for 90 min at 4C until a
final volume of 100 ~1 is reached. The 100 ~1 is then
quick frozen in dry ice/ethanol and lyophilized.
The lyophilized powder is resuspended in 2.5 ~1
PHAST SDS-PAGE sample buffer which contains 10 mM TRIS-
- HCl pH 8.0, 1 mM EDTA, 2.5% SDS t 20 mM DTT and 0.01%
Bromophenol blue. The sample is loaded onto a 7.5%
homogeneous SDS gel and run on a Pharmacia PHAST System.
The gel is stained with Pharmacia PHAST System silver
stain kit. The number of proteins is noted and their
molecular weights are estimated by comparison with SDS-
PAGE standards. Only one prominent band of a molecular
weight substantially greater than 205 kDa is observed.
2 ~
- 28 -
g. ~urther affinity ~ri~ic~tion
In addition to the above-described ]purification
procedure, which results in purifications of much higher
specific activity than was previously possible, other
affinity methods have been used to even further purify
ECE.
i. Heparin-Sepharose purification:
HBSMC cells were processed as described above, and
the membrane proteins solubilized. Solubilized membrane
protein, 6 ml, was added to 120 ml of Heparin-Sepharose
CL-6B (Pharmacia), in 50 mM Tris-HCl, pH 8.0, 50 mM NaCl,
25 mM ~-octylglucoside, and incubated overnight at 4 C
with gentle rotation. The solution was then poured into
a chromatographic column and allowed to equilibrate and
settle for three days. ~Excess buf~`er was removed from
the top, and the column was attached to a gradient
elution and UV detection system. The column was washed
through with starting buffer, then eluted with a linear
gradient of 0.05 to 1.5 M NaCl in 50 mM Tris-HCl, pH 8.0,
25 mM ~-octylglucoside, over 5 column volumes (280 ml) at
0.67 ml/min. Fraction of 10 ml each were collected, then
assayed for ECE activity, with the results shown in Fig.
6.
ii. ConA-Sepharose purification:
About 400 mg of MRC-5 membrane proteins, solubilized
as described above, except that Triton X-lOOR was used in
place of ~-octylglucoside, was mixed with 54 ml of
ConA-Sepharose (Sigma) in a total volume of 210 ml o~ 50
mM Tris-HCl, pH 8.0, 0.5% Triton X-lOOR, and equilibrated
in the cold for 65 h. The slurry was poured into an FPLC
column and attached to an FPLC system (Pharmacia). The
column was washed through, then eluted with a dual linear
gradient of O to 0.~ M NaCl plus O to 0.5 M
~-methylmannoside over 9.4 column volumes (510 ml~ at O.3
ml/min. Fractions of 7 ml each were collected and
assayed for ECE activity, with the results shown in Fig.
7.
,,,
2~ 3
- 29 -
iii. Heparin-Sepharose purification followed by
Superose 12 purification:
Superose 12 (10/30, Pharmacia) was equilibrated with
50 mM Tris-HCl, pH 8 0, 50 mN NaCl, 25 ~M
~-octylglucoside. 0.5 ml of the Heparin-Sepharose pool
isolated in Example l(g)(i), above was :Loaded and eluted
in this buffer at 0.5 ml/min. Fractions of 0.5 ml each
were collected and assayed for ECE activity, with the
results shown in Fig. 8.
These exemplary additional purification steps can be
used in any order after solubilization of the membrane-
bound proteins from the crude cell homogenate. Thus,
depending upon the technical requirements of the size of
the purification, etc., one or more of these additional
steps can be conducted before or after Superose-12
purification, or before or after Mono Q purification.
A summary of a pilot purification of ECE from ~BSMC
is shown in Table III, showing a specific activity yield
of 25000 U/mg protein. (The step of using a
Blue-Sepharose column purification is omitted in
subsequent purifications due to its negligible effect on
enhancing purification.)
. : . . . . . . ... .
2 ~
- -- 30 --
T~BL~ III
Pilot Purification of ~CB fro~ 30 ~oller Bottles ~s~
Tatal Protein Total Specif1c Activity ¦ Total Act. h:tivity Puri-
Vol. (ml) Cnnc. (mg/ml)Prote1n (~9) (Units/~93 ¦ (Ihlits) Reoovery (X) Fold
_ . _ - _ _ _ -
Membrane 0.41 16.9 7.0 _4.6 _ 32.2
Solub11i~ed _ 1.36 3.0 4.1 4.2 ~ 17.2 = 100
Q-Seph Pool 20. 0.0115 0.23 43.0 9.9 57 10.2
Blue-Seph Pool 3. 0.038 0.08 66.2 5.D 29 15.8
Dialy2ed Pool 3. ND* ND ND 4.2 _ 25 _ ND
Heparin-Seph 7. 0.0000660,000471 1062. O.S 3 253.
IPool_ l
Superose 12 Pool 19 ND ND ~ S000.(est) 0.2 1 ~es~c) l
,
* ND = Not Determined. Protein concentration is too low to
detect by standard methods.
= Estimated from SDS-PAGE
BxamPle 2: Characterizatio~ Of ECE
Gel filtration studies as disclosed herein show that
~CE has an usually high apparent molecular weight of
approximately 400 kDa under these conditions. However,
SDS-PAGE under reducing conditions on various
preparations have indicated that ECE preparations of high
specific activity contain proteins migrating on such gels
which correspond to molecular weight markers of
approximately 205, 140 and/or 100 kilodaltons. The SDS-
PAGE data indicate this is a monomeric molecular weight.
The purified enzyme is completely inhibited by 1 ~M
phosphoramidon, which indicates that it is a metallo-
(Zn2-containing) endoproteinase.
Exa~ple 3: Distinguishing ~CB from N~P
Studies were conducted to distinguish ECE from NEP
(neutral endopeptidase, enkephalinase, EC 3.4.24.11).
NEP was obtained from purified recombin~nt rabbit
2.~3~
preparations. ECE was prepared from HB~C,MC cells, as
disclosed above.
a. Ri~eticq
NEP has properties which are superficially similar
to ECE; e.g. NEP will produce ET, as well as other
products, under conditions identical to those for ECE
assay; however, the kinetic rate constant for B~T --> ET
is ~uch lower than for ET --> degradation products. See
Table IV (KCat and Km are determined from least-squares fit
of concentraton-dependence of the rates, according to
methods routine in the art):
TAsLE IV
~INæTIC8 OF r~P A~D h~C~
ENZYME-- SUBSTRATE - (~) C(Amin~1) K ~ M
NEP Enkephalin l.0 24332433
NEP ET-l 2.3 131 57
NEP big ET-l 12. l.2 0.l
ECE big ET-l 12.0 83 7
______.__________________________________________________
aEstimated
bAssumptions: ~AX = 50, 000 nmol hr~1mg~
M.W. = l00,000 daltons
Another comparison which can be made is based upon
the kinetic prediction of the activity of NEP as an
endothelin converting enzyme. Thus, the question that
was posed was: using rNEP, at what % conversion of BET
- will the yield of ET-l plateau? This was answered using
the ~ollowing calculations (Table V), by finding the
concentration of ET at which the rate of BET --> ET-l
equals the rate of further hydrolysis of ET-l:
~;
:
?,~0~
- 32 ~
TABLB V
r2a~P ~ ~C~: pR~DIcTIo~æ F~O~ ~TIC8
[bET-l] ET-l Synth [ET-l] ET-l Degr.
UM nm/min/mq ~M nm~min/mq
INITIAL 3.0 26 0 0
AFTER 1% 3.0 26 0.03 19
CONVERSION
__________________ ________________~_~_ _________________
Therefore, ET-1 production from NEP should plateau
at about 1% conversion = 6 pmol ET-l produced.
ET reaches an equilibrium amount in the reaction
with NEP, but continues linear production with ECE (see
Fig. 9). Furthermore, the rate of ET production from BET
by NEP is very much lower than that for ECE (50 U/mg vs
>10,000 U/mg). Another important distinction is the
NaCl-dependence of the activity. ECE is completely
dependent on the presence of chloride ions (this has also
been observed for metalloproteinase ACE) for substantial
activity. Maximal activity is obtained at 150-200 mM
NaCl. For NEP with the same substrate and same assay
conditions, much of the activity is present in the
absence of salt, and maximal activity is not reached
until 1.5 M NaCl. Finally, immunological assays (ELISA)
for NEP in the above ECE preparations, both from HBSMC
and MRC-5 cells, were performed. Amounts reported were
converted to "units of ECE" and compared to our
measurements of the units actually observed. Less than
0.03% of the activity could be accounted for by any NEP
contamination. These results are summarized in Table VI:
2 ~ 3 ~ ~
~rABLE VI
Properties o~ B~ ~C,B Versu~
Purifie~l ~eco~ nt R~bbilt ~JEP
BET Cleavage Products ET E~ ETIS-21 ET(S.6
_ __ 1
Specific Activity (U/mg) _ 10 000 _ 50 _ ¦
Km for BET-1 12~ -10 aM
Time Course of ETIncreases linearly for Plateaus at 2h
Production at Equivalent ~6 h ~1% conversion
Initial Rates 3% conversion
Molecular Weight
Gel Filtration ~232-440 kDa 232 kDa
SDS-PA6E (reduced)100 140 205 kDa 94 kDa
Behavior on anion exchange Binds; eluted 0.2-0.3M Binds; eluted by 0.2- l
(pH 8) . salt0.3M salt ._ _ _
Behavior on heparinBinds; eluted by 0.6-0.7M Binds; eluted by 0.6-
agarose (pH B) _ ~ 0.7M salt _
Behavlor on Con A agarose Binds weakly; eluted by Binds; eluted by a-
~pH 8) low a-methylmannoside methylmannoside
plus NaCl
Snake Plasma Inhibits contaminating No inhibiton of any
proteases leaving ET as of the NEP products
the sole ECE product from from BET
_ _ '3ET _
Salt Dependence No Activity: 0 M NaCl 25-50X Max Activity: Max Activity: 150-20û mM 0 M NaCl
NaCl Max Activity:
40% Inhibition: ~ 1.5 M l.S M NaCl
NaCl _
Inmunoprecipitation by
Mouse Anti-human CALLA <20X ppt ND2
Mouse Anti-rabbit NEP 0% ppt ND
18BS 0% ppt ND
23B11
% Activ3ity accounted for ~0.03 100
by NEP _ _
1 Assayed according to ECE protocol with 3 aM BET as substrate
2 ND = Not Determined
3 NEP by ELISA (AJ Turner)
9~
~ 34 -
b. ~nhibition
ECE and the other mammalian neutral neutral
metallopeptidases NEP and ACE can also b~ distinguished
by their inhibition by various standard metalloproteinase
inhibitors. Neither NEP nor ECE is inhibited by the ACE
inhibitor enalaprilat (Merck). ECE is inhibited about
5-fold less potently, under identical assay conditions,
by the NEP inhibitor CK4590 (= carboxyphenylpropionyl-
Leu; N-([R,S]-2-carboxy-3-phenylpropionyl~-L-leucine,
purchased from Si~ma: Ref.: M.-C. Fournie-Zaluski et al.,
J. Med. Chem. 26, 60 (1983)). It is inhibited about
14-fold less potently by the collagenase inhibitor CK4919
(=Cbz-Pro-Leu-Cly-hydroxamate; 1-[(phenylmethoxy)-
carbonyl]-L-prolyl-L-leucyl-N-hydroxyglycinamide, Si~ma:
Ref. W.M. Moore et al., Biochem. 25, 5189 (1986)). In
the latter case, a clear distinction can be made between
the inhibition of ECE and NEP by use of 10 ~M CK4919:
under these conditions, ECE is less than 50% inhibited,
while NEP is more than 50% inhibited. See Table VII:
T~BLE VII
INHIBITOR POTENCY - ICso's in "ECE" SCREEN
ICso (nanomolar) Ratio
CK # ECE NEP ECEJNEP Structure
4590 3600 (4) 770 (2) 4.7 O ~ '-P'
HO2~J~` Nli C02H
i'h
4919 33~000 (1) 2300 (2) 14.4 ' O
N~
-e.
1 Value determined by assay of NEP according to ECE
protocol, with 3 ~M BET as substrate, lin2ar ran~e of ET
~oductii~ ._._ _ ---------- ---- -- ~-~
____________ ____________________________ _________ ____
2 ~
Although a variety of screening protocols can be
used to find new inhibitors of ECE (see above), a
suitable screening protocol is provided iLn Table VIII,
below:
TABLB VII
~creeni~g Protocol
Criteria which should be met by each ECE prep before
screening:
1. ET-1 production from hBET-l is linear over time
2. ET degradation rate is <10% of production rate
at final [ET] produced in (1)
3. ICso for phosphoramidon is approximately 1 nM
Assay conditions (and why):
1. 50 mM MOPS-, pH-7.2 (physiological pH, pH
optimum)
2. 2.5 mM ~-octylglucoside (detergent solubilized
membrane enzyme)
3. 30 ~m CaC12 (enzyme is calcium-activated)
4. 150 mM NaCl (enzyme is chloride-activated;
essentially no activity in absence; compare to
ACE)
5. 1 mM PMSF (inhibit serine proteinases)
6. 1 ~M amastat=i-n--~-inhibit aminopeptidases; allows ---
easier observation of breakdown products)
7. 3 ~M hBET-l (substrate by definition; time and
cost effective amount which is significantly
below Km of 12 ~M)
. 37'C, time and enzyme sufficient for 2-3%
reaction (initial rate measurement; typically
3-5 milliunits, 4-5 h; 1 unit = 1 nmol/hour ET
produced under these conditions)
9. Stop reaction with 3 mM EDTA, 4OC; quantify by
HP~C, tryptophan fluorescence (sensitivity <1
pmole; <5% of control reaction)
.
:
,. .~ 2 ~ r~ ,r~
It is also noted that screening for various
inhibitors can be based upon either apparent IC50 or upon
apparent K~: See Table IX:
AsL~ IX
CO~PARI~ON OF APPA~$~ IC50 vs APPAR~NT
~ASSUMPTION: Reversible, competitive inhibition)
IC50
FO~ULA: Kl = ---------
(1 + [S]/XM~
KM K,
ENZYME SUBSTRATE r s ] UM UM
NEP ENKEPHALIN 0. 5 1. 0 0. 67*IC50
ECE Big ET-l 3.0 12.0 o. 80*IC50
________________________________________._________
Therefore, expressing screening data as apparent Kl
will lower the reported values for both enzymes, but will
have little influence on the evaluation of the
selectivity of individual CK compounds for these two
enzymes. The calculation for BET as a substrate for NEP
can be calculated correspondingly.
The preceding examples can be repeated with similar
success by substituting the generically or specifically
described reactants and/or operating conditions of this
invention for those used in the preceding examples.
From the foregoing description, one skilled in the
art can easily ascertain the essential characteristics of
this invention, and without departing from the spirit and
scope thereof, can make various changes and modifications
of the invention to adapt it to various usages and
conditions.
