From the
A peptidase, isolated from rat testes, is inhibited by 1
mM o-phenanthroline, 1 µM N-(1-( R, S)-carboxyl-3-phenylpropyl)-Ala-Ala-Phe- p-aminobenzoate,
and 6 mM Pro-Ile, properties similar to those ascribed to
endopeptidase 24.16. The enzyme hydrolyzes dynorphin A-8, neurotensin
1-13, angiotensin I, and substance P. Kinetic analysis of a
series of angiotensin I analogs showed that substitutions at P-1, P-1`,
or P-2` had little effect on K
The
peptidase cleaved dynorphin A-8 at both Leu
Recent interest in peptidases has been stimulated by the finding
that these enzymes can regulate the physiological action of peptides by
inactivating them or by changing their mode of action. One of the best
documented cases is the regulation of blood pressure through the action
of angiotensin converting enzyme (EC 3.4.15.1) which converts
angiotensin I to angiotensin II
(1, 2) . Another example
is the peptidase neprilysin, also referred to as neutral endopeptidase
(3) , enkephalinase
(4) , or CALLA
(5, 6) (EC 3.4.24.11) which is involved in regulating enkephalin
levels in brain
(7) , substance P levels in the lung
(8) , and endothelin levels in the endometrium
(9) .
These, as well as other peptidases acting on biologically active
peptides, exhibit a rather broad tissue distribution, with their
physiological role dependent on substrate availability rather than on
substrate specificity.
Another such peptidase, endopeptidase 24.16
(EC 3.4.24.16), was first identified based on its ability to cleave
neurotensin 1-13 at the Pro
Purification of Endopeptidase from Rat Testes All purification steps were carried out at 4 °C.
Protein was determined by either absorbance at 280
nm (1 A
For the determination of k
Determination of the cleavage sites in
angiotensin I analogs was made by comparison of the product obtained,
with all but the seven substituted analogs, to authentic angiotensin
1-7 by gradient HPLC analysis and in some cases by mass
spectrometry. Cleavage of the angiotensins substituted at the 7
position was determined by identification of the tripeptide Phe-His-Leu
which corresponds to residues 8 through 10 in each of these analogs.
Determination of the cleavage sites in the various dynorphins and
met-enkephalin analogs was determined by gradient HPLC by comparison of
the elution position of the product to authentic standards.
A number
of angiotensin 1 analogs were custom-synthesized by Chiron Mimotopes
Peptide Systems, Emeryville, CA. The concentration of these peptides
was determined from their UV spectrum over the range of 220-330
nm. Each peptide had an absorbance maximum at 274 nm. The
o-phthalaldehyde assay
(17) was used to confirm the
spectral data. In both methods the concentrations of peptide was
calculated using commercial angiotensin I as a standard.
Authentic
rat endopeptidase 24.16 was kindly provided by Dr. Frederick Checler,
Institut De Pharmacologie Moleculaire et Cellulaire, Valbonne, France,
while purified rat endopeptidase 24.15 and
N-(1-( R, S)-carboxyl-3-phenylpropyl)-Ala-Ala-Phe- p-aminobenzoate
were generous gifts from Dr. Marion Orlowski, Mt. Sinai College of
Medicine, New York, NY.
It has recently been established that rat endopeptidases 24.15 and
24.16 can be distinguished by the susceptibility of the latter enzyme
to inhibition by the dipeptide Pro-Ile
(20) . As shown in
Fig. 2
, the endopeptidase isolated in this study was in fact
inhibited by Pro-Ile, whereas purified rat endopeptidase 24.15 was much
less susceptible to inhibition by this dipeptide. The
K
To gain additional insight
regarding the specificity of the enzyme, the hydrolysis of two series
of peptides, one based on angiotensin I and the other based on
dynorphin A, were studied in more detail. The K
shows the results obtained
with analogs of dynorphin A as substrate. Although Leu-enkephalin is
not a substrate, extension of this pentapeptide by one amino acid
rendered it a substrate. As the size of the peptide increased, multiple
cleavages were observed. The site of cleavage as well as the relative
cleavage at each site is given in . With these peptides as
substrates, it is difficult to see a definitive trend except perhaps
that a di- or tripeptide appears to be released as product. This is
consistent with the results obtained with the angiotensin series, where
cleavage was essentially independent of the residues occupying the P-1
and P-1` positions.
In those cases where comparisons can be made,
the K
The results of this study indicate that a soluble variant of
the enzyme endopeptidase 24.16 has been isolated. Although
endopeptidases 24.15 and 24.16 are similar in many of their properties,
they can be distinguished by several criteria. First, only
endopeptidase 24.16 is inhibited by the dipeptide Pro-Ile
(20) .
Second, the peptidase described in this study is poorly inhibited by
benzoyl-Gly-Ala-Ala-Phe-pAB, a potent inhibitor of endopeptidase 24.15
(15) , yet is effectively inhibited by CFP-Ala-Ala-Phe-pAB, a
inhibitor of both peptidases
(21) . Last, the endopeptidase
isolated in this study does not cleave neurotensin 1-8, a
substrate for endopeptidase 24.15,
The enzyme described here is
similar to rat membrane endopeptidase 24.16 in that both are
metallo-peptidases subject to inhibition by the dipeptide Pro-Ile and
both share similarities in terms of the substrates hydrolyzed and in a
general sense their substrate specificity. Both enzymes prefer
substrates containing more than 5 amino acids, and both cleave a
variety of peptide bonds with a preference for the release of
carboxyl-terminally derived di or tripeptides. On the other hand, the
enzymes differ quantitatively in their sensitivity to inhibition by
Pro-Ile; rat membrane endopeptidase 24.16 exhibiting a >60-fold
higher affinity for this dipeptide
(21) . In addition the
peptidase isolated in this study did not hydrolyze angiotensin II,
while rat membrane endopeptidase 24.16 does
(22) . Based on
these observations and the results described below, we propose that the
enzyme described in this study be referred to as endopeptidase 24.16B,
and the enzyme first described by Checler et al. (10) as endopeptidase 24.16A
Other differences between rat
endopeptidase 24.16A and endopeptidase 24.16B are the products formed
with dynorphin A 1-8 and neurotensin 1-13 as substrates.
Rat endopeptidase 24.16A cleaves neurotensin 1-13 only at the
Pro
It is of interest that dynorphin A 1-8 is a
substrate for endopeptidase 24.16B since the products of its cleavage,
leucine enkephalin and leucine enkephalin-Arg
Similarly, the ability of endopeptidase 24.16B to convert
angiotensin I to angiotensin 1-7 may be of physiological
significance. Angiotensin 1-7 is found in relatively high
concentrations in the central nervous system, where it mimics some of
the actions of angiotensin II
(26) , while peripherally this
metabolite acts as a vasodilator, being formed under conditions in
which angiotensin converting enzyme is inhibited
(27) . Although
angiotensin 1-7 can be formed either by the action of neprilysin
(28) or by prolyl endopeptidase
(29) , the contribution
of endopeptidase 24.16 to the formation of angiotensin 1-7 must
be considered.
Dynorphin A 1-13 binds to the enzyme with a
high affinity, but with a very low turnover. Thus dynorphin A
1-13, once formed, could act as a competitive inhibitor elevating
the concentration of smaller peptides more susceptible to hydrolysis by
this peptidase. Thus there can be a complex set of relationships which
regulate the concentration of these peptides in vivo.
The
results of this study imply that a family of 24.16 endopeptidases may
exist. These 24.16 endopeptidase variants may be species-specific as
evidenced by differences between the rat and porcine membrane derived
enzymes or tissue specific as seen in this study.
The enzyme was isolated from 209 g of rat testes. Activity
was determined by using the fluorescent substrate
Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp as described under
``Materials and Methods.'' One unit is defined as the amount
of enzyme that cleaves 1 µmol of substrate/min.
Enzyme activity was determined as described under
``Materials and Methods'' with inhibitors preincubated with
enzyme for 5 min prior to initiating the reaction. The uninhibited
activity corresponded to 0.08 nmol/min.
Angiotensin I:
Asp
Conditions for K
or
k
. Variation of peptide size with a series of
dynorphin A peptides showed chain length to be significant.
-Arg
and Arg
-Arg
, and neurotensin 1-13
at Pro
-Tyr
and
Arg
-Arg
. In contrast, rat endopeptidase 24.16
cleaves dynorphin A-8 at Gly
-Leu
and
Leu
-Arg
, and neurotensin 1-13 only at
Pro
-Tyr
. These findings, as well as the
observation that endopeptidase 24.16 exhibits a considerably higher
affinity for Pro-Ile, K
= 90
µM, indicates the peptidase isolated in this study is
related to, but distinct from, rat endopeptidase 24.16. We propose that
this new endopeptidase be referred to as endopeptidase 24.16B, while
the originally described enzyme be referred to as endopeptidase 24.16A
-Tyr
bond
(10) . This peptidase appears to co-localize with neurotensin
receptors in primary cultures of mouse embryonic neurons, suggesting a
physiological role in neurotensin metabolism
(11) . Subsequent
studies of the enzyme showed it exhibits a rather broad substrate
specificity including angiotensin I, bradykinin, and dynorphin-8 as
potential physiological substrates
(12) . The specificity of
this enzyme in terms of bond cleavage is complex and yet to be clearly
defined. The peptidase exhibits a rather broad tissue distribution and
exists in both a soluble and membrane-associated form
(13) .
During a study of peptidases in rat testes, we have found an enzyme
with properties similar to, yet distinct from, rat endopeptidase 24.16.
In this report we describe the purification and characterization of
this enzyme and its similarities and differences to rat endopeptidase
24.16.
Tissue Homogenization
Frozen rat testes (209 g) were
homogenized with a Tissuemizer (Tekmar, Cincinnati, OH) for 30 s at
maximal speed in 5 volumes of 100 mM Tris-HCl buffer, pH 8.5,
containing 1.0 mM dithiothreitol. The homogenate was
centrifuged for 60 min at 10,000 g and filtered
through cheesecloth.
Ammonium Sulfate Fractionation
Solid ammonium
sulfate was slowly added to the supernatant to give 35% saturation (209
g/liter). The precipitated proteins were removed by centrifugation at
10,000 g for 60 min. The supernatant was adjusted to
60% ammonium sulfate saturation (164 g/liter), and the precipitated
proteins were collected as described above. The precipitate was
redissolved in a minimal volume of 20 mM Tris-HCl buffer, pH
7.5, (buffer A) and dialyzed overnight against 4.0 liters of this
buffer.
Ion Exchange Chromatography
The ammonium sulfate
dialysate was loaded onto a 100-ml anion exchange column, prepared from
Accell Plus QMA resin (Waters), equilibrated with buffer A. After
washing the column with 5 volumes of starting buffer, the enzyme was
eluted with a 750-ml linear gradient of 0 to 0.5 M NaCl at a
flow rate of 2.0 ml/min. Gradient elution for all columns was performed
on a Waters 650 protein purification system. The active fractions were
pooled and concentrated to 15 ml using an Amicon concentrator equipped
with a YM-30 filter membrane.
First Hydrophobic Chromatography
The concentrate
from the QMA column was equilibrated to 30% ammonium sulfate by the
addition of 9.0 ml of 80% ammonium sulfate prepared in buffer A, then
loaded onto a 15-ml TosoHaas butyl 650-S column equilibrated with 30%
ammonium sulfate in buffer A. After washing the column with 60 ml of
starting buffer, the enzyme was eluted using a decreasing gradient of
ammonium sulfate (30% to 0) in a total gradient volume of 150 ml. The
active fractions were pooled yielding a volume of 10 ml.
Second Hydrophobic Chromatography
The active pool
from the butyl column was adjusted to 20% ammonium sulfate by the
addition of 3.0 ml of 80% ammonium sulfate prepared in buffer A and
loaded onto an 8.0-ml TosoHaas phenyl 650-S column equilibrated with
20% ammonium sulfate in buffer A. After washing the column with 50 ml
of starting buffer, enzyme was eluted with a decreasing gradient (20%
to 0) of ammonium sulfate over a total gradient volume of 60 ml. The
active fractions were pooled and concentrated to 2.0 ml as described
above.
Molecular Sieve Chromatography
The phenyl 650-S
column concentrate was loaded onto a Pharmacia Biotech Inc. Superdex
200 16/60 gel filtration column equilibrated with buffer A. The enzyme
was eluted with this buffer at a flow rate of 0.2 ml/min and
concentrated as described above.
Chromatofocusing
The concentrated gel filtration
pool was applied to a 4.0 ml Mono P (Pharmacia) column equilibrated
with bis-Tris buffer, pH 5.0. The enzyme was eluted with a gradient of
decreasing pH from 5.0 to 4.0 using Pharmacia Polybuffer 74. Fractions
were collected into tubes containing 20 µl of 0.5 M Tris
base, pH 11.0. The active fractions were pooled in a total volume of 10
ml and concentrated to 2.0 ml.
Molecular Sieve Chromatography
The concentrated
Mono P pool was applied to a second Superdex 200 16/60 gel filtration
column equilibrated with buffer A. The enzyme was eluted with buffer A
at a flow rate of 0.2 ml/min. The active fractions were pooled in a
volume of 6.0 ml.
= 1 mg/ml) or by the method of
Bradford
(14) using bovine serum albumin as a standard. Determination of Endopeptidase Activity Throughout the purification, enzyme activity was followed by measuring
the hydrolysis of the fluorogenic substrate
7-methoxycoumarin-3-carboxyl-Pro-Leu-Gly-Pro-D-Lys-dinitrophenylamide
(Mcc
-PLGPL-Dnp, Nova-Biochemicals, (
)La Jolla,
CA)
(15) . With this substrate, an increase in fluorescence
occurs when a peptide bond is cleaved separating the N-terminal Mcc
fluorescent group from the fluorescent quenching C-terminal Dnp group.
Reaction mixtures contained 20 mM Tris-HCl buffer, pH 7.5, 30
µM Mcc-PLGPL-Dnp, and enzyme in a final volume of 200
µl. The reaction was initiated by the addition of enzyme and
monitored for 3-5 min at an excitation wavelength of 345 nm and
an emission wavelength of 405 nm using an Aminco-Bowman
spectrofluorometer equipped with a strip chart recorder. Linear rates
were obtained in all cases. A standard curve for Mcc-PLGPL-Dnp
hydrolysis was constructed by measuring the total fluorescence change
which occurs upon complete hydrolysis of varying concentrations of the
substrate. Determination of Kinetic Constants K
or K
was
determined by the above described fluorescent assay using the peptide
in question as a competitive inhibitor. Reaction mixtures contained 20
mM Tris-HCl buffer, pH 7.5, 30 µM Mcc-PLGPL-Dnp
( K
= 33 µM), peptide
and enzyme in a final volume of 100 µl. Data were plotted as
1/velocity versus [peptide] with kinetic constants
determined by a fit of the data to the computer programs of Cleland
(16) .
the
rate of substrate hydrolysis was determined by HPLC. Reaction mixtures
contained 20 mM Tris-HCl buffer, pH 7.5, peptide and enzyme in
a final volume of 125 µl. At various time points a 25-µl
aliquot was removed and added to 75 µl of 0.1% trifluoroacetic acid
to stop the reaction. The rate of substrate disappearance, or in some
cases product appearance, was then determined by isocratic HPLC.
Samples were applied onto a VYDAC C-4 reverse phase column and eluted
isocratically with a solvent composed of acetonitrile and 0.1%
trifluoroacetic acid at a flow rate of 1.0 ml/min. The following
peptides were eluted at the indicated acetonitrile concentration:
Ala
-Ang I, Gln
-Ang I, and Ser
-Ang
I, 20%; Dyn A-6, and Dyn A-7, 23%; Leu-Enk and Dyn A-8, 24%;
NT-1-13, Dyn A-9, Asn
Val
-Ang I,
Ala
-Ang I, Ser
-Ang I, Gln
-Ang I,
Gly
-Ang I, 25%; Ang I, Val
-Ang I,
Val
Ser
-Ang I,
Val
Asn
-Ang I, and Leu
-Ang I, 26%;
and, Asn
Val
Tyr
-Ang I,
Asn
Val
Pro
-Ang I,
Asn
Val
Val
-Ang I, and
Asn
Val
Glu
-Ang I, 28%. Peptide peaks
were monitored at 214 nm using a Waters 484 variable wavelength
detector. In some cases gradient HPLC was used to identify and
quantitate products.
Purification and Characterization of Peptidase
Activity
A peptidase was purified from rat testes as described
under ``Materials and methods'' using
Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp as a substrate. A summary of the
purification is given in . Starting with 209 g of frozen
testes, 80 µg of enzyme were obtained in 1% yield and
3,000-fold purification. Comparison of the activity in the
membrane fraction to the cytosolic activity showed the latter to
contain greater than 90% of the activity. Analysis of the purified
enzyme by SDS-PAGE revealed a single protein, molecular mass of 72 kDa
(Fig. 1). This enzyme had an apparent pI of 4.3-4.5, based
on its elution from a chromatofocusing column.
Figure 1:
SDS-PAGE of purified endopeptidase
isolated from rat testes. One (1.0) µg of the purified enzyme was
analyzed by SDS-PAGE on a 10% polyacrylamide gel. Protein was stained
with Silver Stain Plus (Bio-Rad).
The reaction of this
enzyme with the synthetic substrate
Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp was initially characterized. A
Kof 33 µM with a
V
of 13.1 µmol/min/mg of protein was
obtained. In order to classify this peptidase, the effect of a number
of inhibitors on its activity were tested and are summarized in
. It can be seen that the enzyme is not inhibited by the
sulfhydryl reagents iodoacetamide or N-ethylmaleimide,
indicating it is not a thiol protease. Similarly no inhibition was
observed with phenylmethylsulfonyl fluoride indicating the enzyme is
not a serine protease. The enzyme is inhibited weakly by EDTA, but more
potently by o-phenanthroline, indicating it is a
metallopeptidase. The activity was unaffected by known inhibitors of
specific peptidases; the neprilysin inhibitor phosphoramidon, the
aminopeptidase inhibitor bestatin, or the dipeptidylcarboxypeptidase
inhibitor captopril. Enzymatic activity was inhibited by
N-(1-( R, S)-carboxyl-3-phenylpropyl)-Ala-Ala-Phe- p-aminobenzoate
(CFP-AAF-pAB) which is a known inhibitor of two peptidases,
endopeptidase 24.15 and endopeptidase 24.16
(18) . In addition,
activity slightly increased when dithiothreitol was added, another
characteristic of endopeptidase 24.15
(19) . However,
benzoyl-Gly-Ala-Ala-Phe-pAB, a potent inhibitor of endopeptidase 24.15
( K
= 0.02 µM) and a
relative weak inhibitor of endopeptidase 24.16
( K
= 0.66 µM)
(15) , inhibited the activity only 15% at 4 µM.
for Pro-Ile was calculated to be 6
mM, which is considerably higher than the value of 0.09
mM reported for rat endopeptidase 24.16
(21) .
Figure 2:
Effect of Pro-Ile on endopeptidase
activity () and endopeptidase 24.15 (
). Reaction mixtures
contained 20 mM Tris-HCl buffer, pH 7.5, 30 µM
Mcc-Pro-Leu-Gly-Pro-DLys-Dnp, Pro-Ile in increasing
concentrations and the appropriate enzyme in a final volume of 200
µl (100% activity = 0.08
nmol/min).
Substrate Specificity of Endopeptidase
The
substrate specificity of the enzyme was examined by first testing a
number of potential physiological peptide substrates for their ability
to inhibit Mcc-PLGPL-Dnp hydrolysis (I). For those
peptides which were inhibitory, their rate of cleavage was determined
by following substrate disappearance by HPLC. The results of this
analysis showed that bradykinin, dynorphin B, dynorphin A 1-8,
neurotensin 1-13, leutinizing hormone-releasing hormone,
angiotensin I, dynorphin A 1-17, and substance P are all
substrates for the enzyme (I). Although binding to the
enzyme, as determined by their ability to act as an inhibitor,
dynorphin A 1-13 and somatostatin were not cleaved at a
significant rate. Met-enkephalin and
Leu
-enkephalin neither bound to the enzyme with significant
affinity nor were they cleaved at a significant rate. Although not
shown in I, it was found that neither angiotensin II nor
neurotensin 1-8, at 0.1 mM were cleaved at a detectable
rate, but acetylneurotensin 1-8 was cleaved at approximately the
same rate as neurotensin 1-13.
and k
values for a group of angiotensin I
analogs is shown in . Cleavage of all of the peptides
occurred at the same position, between the 7 and 8 bond. Substitution
of the phenylalanine at position 8 with serine, leucine, alanine, or
glutamine was without significant effect. Similarly substitution of
histidine at position 9 with serine, glutamine, valine, tyrosine,
proline, valine, glutamate, and asparagine had no effect on the
kinetics of substrate hydrolysis. The only significant effect observed
was a slight increase in k
and
k
/ K
when the
proline, at the 7 position, was substituted with alanine, serine, or
glutamine. Thus, as shown in , the results of this
analysis indicate that substitutions of residues at P-1, P-1`, or P-2`
are rather unremarkable.
values obtained are similar to
those reported for endopeptidase 24.16
(12) . However, a
significant difference in the position at which bond cleavage occurred
was noted between the results obtained in this study and those reported
for rat endopeptidase 24.16. Analysis of the reaction of the peptidase
described in this study with dynorphin A 1-8
(Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile) as substrate revealed the presence of
two products, Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) and
Leu-enkephalin-Arg
(Tyr-Gly-Gly-Phe-Leu-Arg) (
and Fig. 3, top. The rates of product
formation were linear with Leu-enkephalin being formed 4 times faster
than Leu-enkephalin-Arg
(Fig. 3, inset).
With the addition of 20 mM Pro-Ile, the rate of dynorphin A
1-8 cleavage was inhibited more than 80%, and no products could
be detected. As shown in Fig. 3, authentic rat endopeptidase
24.16 cleaved dynorphin A 1-8 differently, yielding
Tyr-Gly-Gly-Phe and Leu-enkephalin as products, a finding in agreement
with the published results of Barelli et al. (12) . In
the case of rat endopeptidase 24.16, Leu-enkephalin was formed
4
times faster than Tyr-Gly-Gly-Phe (Fig. 3, inset).
Figure 3:
Peptidase cleavage of dynorphin A
1-8. Top, HPLC chromatogram of dynorphin A 1-8
cleavage by the endopeptidase isolated in this study. The reaction
mixtures contained 20 mM Tris-HCl buffer, pH 7.5, 2.5 nmol of
dynorphin A 1-8, and enzyme (40 ng) in a final volume of 125
µl. At the indicated times the reaction was stopped by the addition
of 0.1% trifluoroacetic acid and analyzed by HPLC as described under
``Materials and Methods.'' The arrow indicates the
elution time of the substrate dynorphin A 1-8. The inset shows the formation of the products Leu-enkephalin () and
Leu-enkephalin-Arg
(
). Bottom, HPLC
chromatogram of dynorphin A 1-8 cleavage by authentic rat
endopeptidase 24.16. The same procedure as described above was
employed. The arrow indicates the elution time of the
substrate dynorphin A 1-8. The inset shows the formation
of the products Leu-enkephalin (
) and Tyr-Gly-Gly-Phe
(
)
Similarly, we found a difference in the cleavage pattern of
neurotensin 1-13 by the peptidase isolated in this study as
compared to rat endopeptidase 24.16. As shown in Fig. 4, and in
agreement with the studies of Checler et al. (10) , rat
endopeptidase 24.16 catalyzed a single cleavage of neurotensin
1-13 at Pro-Tyr
. In contrast the enzyme
described in this study shows two distinct cleavages; one at
Arg
-Arg
and the other at
Pro
Tyr
.
Figure 4:
Peptidase cleavage of neurotensin.
Reaction mixtures and product analysis were as described in Fig. 3 with
2.5 nmol of neurotensin 1-13 replacing dynorphin 1-8.
Curve A is the HPLC chromatogram of neurotensin 1-13
cleavage by authentic rat endopeptidase 24.16 after an incubation time
of 4 h. Curve B is the same as A using the peptidase
isolated in this study (15 ng) and an incubation time of 1 h. Formation
of neurotensin 9-13 was not detected as a unique product peak,
but may co-elute with neurotensin
11-13.
(
)
but does
cleave acetylneurotensin 1-8, which is not a substrate for
endopeptidase 24.15
(25) .
-Tyr
bond, while endopeptidase 24.16B
cleaves this substrate at both the Pro
-Tyr
and Arg
-Arg
bonds, the latter being the
preferred site. A possible explanation for these differences is that
endopeptidase 24.16A is derived from membranes, while endopeptidase
24.16B is cytosolic. However, Millican et al. (18) ,
purified endopeptidase 24.16 from both porcine brain cytosol and
membranes and found that both enzymes exhibited the same substrate
specificity including cleavage of neurotensin 1-13 at both
Arg
-Arg
and Pro
-Tyr
,
a finding confirmed by Dahms and Mentlein
(22) . Furthermore,
Millican et al. (18) concluded the membrane form of
porcine endopeptidase 24.16, which represents
10% of the total
activity, is simply the cytosolic form loosely bound to the membrane.
Interestingly, endopeptidase 24.16A and porcine endopeptidase 24.16
cleave dynorphin A 1-8 primarily at the Leu
-Arg
bond, with a minor cleavage at the Phe
-Leu
bond. Endopeptidase 24.16B also cleaves this substrate primarily
at the Leu
-Arg
bond; however, a secondary
cleavage site was found at the Arg
-Arg
bond,
indicating that there may be species differences as well as variants
within a species.
, are both
physiologically active peptides
(23, 24) . It has been
shown that dynorphin A 1-8 can act as a precursor for leucine
enkephalin in the rat CNS
(25) . Thus, endopeptidase 24.16B may
act as a processing enzyme for dynorphin A 1-8. This suggestion
is further supported by examining the cleavage sites of dynorphin B.
Dynorphin B is cleaved to liberate leucine enkephalin, leucine
enkephalin-Arg
and leucine
enkephalin-Arg
-Arg
. Thus, the 24.16 family of
enzymes may play a dual role of inactivating peptides as well as
converting them to other peptides with a distinct activity.
Table:
Purification of endopeptidase activity from rat
testes
Table:
Effect of inhibitors on endopeptidase
activity
Table:
Kinetics of the reaction of physiologic
substrates
Table:
Kinetic constants for the reaction of
angiotensin I analogs
-Arg
-Val
-Tyr
-Ile
-His
-Pro
-Phe
-His
-Leu
.
Conditions for K
and K
determinations were as described in Table III.
Table:
Kinetics of the hydrolysis of enkephalin-related
peptides
and
K
determinations were as described in Table III.
NA, not applicable.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.