©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Endopeptidase 24.16B
A NEW VARIANT OF ENDOPEPTIDASE 24.16 (*)

Donald Rodd , Louis B. Hersh (§)

From the (1) Department of Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0084

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 Kor k. Variation of peptide size with a series of dynorphin A peptides showed chain length to be significant.

The peptidase cleaved dynorphin A-8 at both Leu-Argand Arg-Arg, and neurotensin 1-13 at Pro-Tyrand Arg-Arg. In contrast, rat endopeptidase 24.16 cleaves dynorphin A-8 at Gly-Leuand 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


INTRODUCTION

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-Tyrbond (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.


MATERIALS AND METHODS

Purification of Endopeptidase from Rat Testes All purification steps were carried out at 4 °C.

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.

Protein was determined by either absorbance at 280 nm (1 A= 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 Kor Kwas 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) .

For the determination of kthe 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, AsnVal-Ang I, Ala-Ang I, Ser-Ang I, Gln-Ang I, Gly-Ang I, 25%; Ang I, Val-Ang I, ValSer-Ang I, ValAsn-Ang I, and Leu-Ang I, 26%; and, AsnValTyr-Ang I, AsnValPro-Ang I, AsnValVal-Ang I, and AsnValGlu-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.

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.


RESULTS

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 Vof 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.

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 Kfor 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.

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 Kand kvalues 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 kand k/ Kwhen 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.

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 Kvalues 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-Argand the other at ProTyr.


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.




DISCUSSION

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,() but does cleave acetylneurotensin 1-8, which is not a substrate for endopeptidase 24.15 (25) .

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-Tyrbond, while endopeptidase 24.16B cleaves this substrate at both the Pro-Tyrand Arg-Argbonds, 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-Argand 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-Argbond, with a minor cleavage at the Phe-Leubond. Endopeptidase 24.16B also cleaves this substrate primarily at the Leu-Argbond; however, a secondary cleavage site was found at the Arg-Argbond, indicating that there may be species differences as well as variants within a species.

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, 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-Argand 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.

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.

  
Table: Purification of endopeptidase activity from rat testes

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.


  
Table: Effect of inhibitors on endopeptidase activity

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.


  
Table: Kinetics of the reaction of physiologic substrates


  
Table: Kinetic constants for the reaction of angiotensin I analogs

Angiotensin I: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu. Conditions for Kand Kdeterminations were as described in Table III.


  
Table: Kinetics of the hydrolysis of enkephalin-related peptides

Conditions for Kand Kdeterminations were as described in Table III. NA, not applicable.



FOOTNOTES

*
This research was supported in part by National Institute on Drug Abuse Grant DA 02243. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, University of Kentucky, Chandler Medical Center, 800 Rose St., Lexington, KY 40536-0084. Tel.: 606-323-5549; Fax: 606-323-1037.

The abbreviations used are: Mcc, methoxycoumarin-3-carboxyl; Dnp, dinitrophenylamide; CFP, N-1-( R, S)-carboxyl-3-phenylpropyl; pAB, p-aminobenzoate; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; Ang I, angiotensin I; Dyn, dynorphin; Enk, enkephalin; NT, neurotensin.

D. Rodd and L. Hersh, unpublished results.


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