An alternative quenched fluorescence substrate for Pz-peptidase

7-Methoxycoumarin-3-carboxylyl-Pro-Leu-Gly-Pro-D-Lys(2,4-dinitr oph enyl) is introduced as a new quenched fluorescence substrate for assaying Pz-peptidase (also known as soluble metallo-endopeptidase and endo-oligopeptidase). The value of Km for partially purified Pz-peptidase from rat muscle was 8.6 microM. High protein concentrations did not interfere with the assay, so that for the first time continuous assays of Pz-peptidase in crude tissue extracts became possible.     

Influence of the carboxyl terminus of luteinizing hormone-releasing hormone and bradykinin on hydrolysis by brain endo-oligopeptidases

A homogeneous preparation of endo-oligopeptidase A from rabbit brain cleaves luteinizing hormone-releasing hormone (less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) at the Tyr-Gly bond only after the removal of Gly-NH2 from the COOH-terminal position of the molecule. The influence of the carboxyl terminus on hydrolysis by brain endo-oligopeptidases was studied using bradykinin as a model substrate. The substitution of the carboxyl group of bradykinin by the amide reduces by 2.5-fold the rate of Phe-Ser bond hydrolysis by endo-oligopeptidase A but has no effect on the rate of hydrolysis of the Pro-Phe bond by endo-oligopeptidase B. On the other hand, the deletion of Phe-Arg from the COOH-terminal portion of bradykinin makes the peptide resistant to hydrolysis by endo-oligopeptidase A whereas it increases by 5-fold the rate of hydrolysis of the Pro-Gly bond by endo-oligopeptidase B.     

Chicken liver Pz-peptidase, a thiol-dependent metallo-endopeptidase.

Pz-peptidase was purified from chicken liver as a protein of Mr 80,000 and pI 5.2. The purified enzyme hydrolysed phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg, 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys. 7-methoxycoumarin-3-carboxylyl-Pro-Leu-Gly-Pro-D-(2,4-dinitropheny l)Lys, benzoyl-Gly-Ala-Ala-Phe-p-aminobenzoate, Ac-Ala4 (at the Ala-1-Ala-2 bond) and bradykinin (at the Phe-5-Ser-6 bond). No hydrolysis of proteins was detected. Loss of activity in the presence of EDTA or 1,10-phenanthroline was time-dependent. Metal ions found to restore activity after treatment with EDTA were Zn2+, Mn2+, Ca2+, Co2+ and Cd2+, in decreasing order of effectiveness. Ni2+, Fe2+ and higher concentrations of Zn2+ were inhibitory. Inhibition by N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate and related compounds showed Ki values (down to 5 nM) somewhat lower than those for the rat enzyme. Pz-peptidase was activated by low concentrations of 2-mercaptoethanol and dithiothreitol, but inhibited by higher concentrations. p-Chloromercuribenzoate and some other thiol-blocking reagents were inhibitory. Inactivation by diethyl pyrocarbonate that was reversible by hydroxylamine showed the presence of essential histidine residue(s). We conclude that chicken Pz-peptidase is a metallo-endopeptidase with thiol-dependence. Moreover, the properties of chicken Pz-peptidase agree with those described for mammalian soluble metallo-endopeptidase and endo-oligopeptidase A. consistent with the view that these three types of activity are all attributable to the single enzyme for which the name thimet peptidase has been proposed.     

Brain Endo-Oligopeptidase A, a Putative Enkephalin Converting Enzyme

Endo-oligopeptidase A, highly purified from the cytosol fraction of bovine brain by immunoaffinity chromatography, has been characterized as a thiol endopeptidase. This enzyme, known to hydrolyze the Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin, has been shown to produce, by a single cleavage, Leu5-enkephalin or Met5-enkephalin from small enkephalin-containing peptides. Enkephalin formation could be inhibited in a concentration-dependent manner by the alternative substrate bradykinin. The optimal substrate size was found to be eight to 13 amino acids, with enkephalin the only product released from precursors in which this sequence is immediately followed by a pair of basic residues. However, the specificity constants (kcat/Km) obtained for endo-oligopeptidase A hydrolysis of bradykinin, neurotensin, and dynorphin B are of the same order, a result indicating that the substrate amino acid sequence is not the only factor determining the cleavage site of this enzyme.     

Identification and preliminary characterization of two distinct bovine seminal Pz-peptidases

Two peptidases hydrolyzing the Pz-peptide substrate were identified in bovine semen. Each Pz-peptidase was strongly inhibited by chelating agents, suggesting both were metallopeptidases. However, these peptidases could be distinguished by other properties and were designated Pz-peptidases A and B. Pz-peptidase A hydrolyzed the Pz-peptide at the Leu-Gly bond, was inhibited by tosylphenylethylchloromethylketone (TPCK) but not by phosphoramidon and had a pH optimum near 6, whereas Pz-peptidase B cleaved the Pro-Leu bond, was inhibited by phosphoramidon but not by TPCK and had a pH optimum near 7. Seminal plasma, light particulates and cytoplasmic droplets contained almost exclusively Pz-peptidase A, and Pz-peptidase A predominated in sperm extracts. Pz-peptidase B was found primarily in sperm extracts, but Pz-peptidase B activity was also present in ultralight particulates. Pz-peptidase A of spermatozoa required Triton X-100 for complete extraction, but Pz-peptidase B was solubilized from spermatozoa by nitrogen decompression without detergents. Pz-peptidase B was inhibited by several detergents. In particular, addition of 0.1% Hyamine 2389 to sperm extracts inhibited 99% of the Pz-peptidase B activity. Thus, Pz-peptidase B may have been overlooked in previous studies employing extraction of spermatozoa with Hyamine 2389. The properties of both seminal PZ-peptidases were different from those of purified bovine testicular PZ-peptidase, suggesting that PZ-peptidases from these sources were not identical.     

Liberation of enkephalins from enkephalin-containing peptides by brain endo-oligopeptidase A

Endo-oligopeptidase A, highly purified from the cytosol fraction of bovine brain by immunoaffinity chromatography, has been characterised as a thiol endopeptidase. This enzyme, known to hydrolyse the Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin has been shown to produce, by a single cleavage, [Leu]enkephalin or [Met]enkephalin from small enkephalin-containing peptides. Enkephalin formation could be inhibited in a concentration dependent manner by the alternative substrate bradykinin. The optimal substrate size was found to be 8-13 amino acids, with enkephalin the only product released from precursors in which this sequence is immediately followed by a pair of basic residues. However, the specificity constants (kcat/Km) obtained for endo-oligopeptidase A hydrolysis of bradykinin, neurotensin and dynorphin B are of the same order. Taken together, these results indicate that the substrate amino acid sequence is not the only factor determining the cleavage site of this enzyme. Finally, endo-oligopeptidase A and metalloendopeptidase EC 3.4.24.15 are two different enzymes. The latter is not able to liberate enkephalins from metorphamide and dynorphin.     

Isolation and characterization of Pz-peptidase from Bacillus licheniformis N22

Pz-peptidase is an endopeptidase that cleaves the synthetic substrate, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg (Pz-peptide), which was originally developed for the assay of Clostridium histolyticum collagenase (Wünsch and Heidrich, Hoppe-Seyler's Z. Physiol. Chem., 333, 149–151, 1963; Morales and Woessner, J. Biol. Chem., 252, 4855–4860, 1977). Pz-peptidase was purified from the culture filtrate of Bacillus licheniformis N22. The purified Pz-peptidase showed a molecular weight of 70,000 in SDS-polyacryl-amide gel electrophoresis and 150,000 in gel filtration. Optimal pH for cleavage of Pz-peptide was 7.8. The Pz-peptidase activity was strongly inhibited by metal chelators such as EDTA and O-phenanthroline. Substrate specificity studies indicated that Pz-peptidase cleaved oligopeptides at the Xaa-Gly site in Xaa-Gly-Pro. However, Pz-peptidase failed to hydrolyze native collage, denatured collagen, hemoglobin and casein.     

Degradation of neurotensin by rabbit brain endo-oligopeptidase A and endo-oligopeptidase B (proline-endopeptidase)

The degradation of neurotensin and D-Tyr11 neurotensin by apparently homogeneous preparations of rabbit brain endo-oligopeptidase A and endo-oligopeptidase B (Proline-endopeptidase) was studied. Peptide fragments were isolated by high performance liquid chromatography and identified by amino acid analysis. Endo-oligopeptidase A cleaved neurotensin at the Arg8-Arg9 bond whereas D-Tyr11 neurotensin was not significantly hydrolysed. Endo-oligopeptidase B cleaved at the carboxyl side of Pro7, Pro10 in neurotensin and at Pro7 in D-Tyr11 neurotensin. The concentration dependent inhibition of neurotensin degradation by bradykinin and vice-versa represents additional evidence that endo-oligopeptidase A cleaves both Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin.     

Purification and characterization of Pz-peptidase B, a neutral metalloendopeptidase from bovine spermatozoa

We have demonstrated previously that the Pz-peptide synthetic substrate is cleaved by two distinct spermatozoal peptidases, Pz-peptidases A and B. To facilitate further investigations, Pz-peptidase B was purified from bovine spermatozoa. The soluble extract from 81 grams of washed epididymal spermatozoa was fractionated by a five-step procedure consisting of anion-exchange, lectin affinity, hydrophobic interaction, chromatofocusing, and gel filtration chromatography. This method yielded 1 mg of 170-fold purified Pz-peptidase B with a 26% recovery. The purified Pz-peptidase B was electrophoretically homogeneous and possessed a monomeric molecular weight of 90,700. Isoelectric focusing revealed microheterogeneity with pIs ranging from 5.02 to 5.09. Pz-peptidase B was irreversibly inactivated at pH 3.5 or below, and activity was reduced at moderate ionic strengths. Hydrolysis of the Pz-peptide was maximal at pH 7.5. Pz-peptidase B was strongly inhibited by a metal chelator and phosphoramidon. Reactivation of metal-depleted enzyme by various metal ions suggested that Pz-peptidase B was a zinc metallopeptidase. Pz-peptidase B hydrolyzed a wide variety of synthetic substrates and physiologically activity peptides at the amino side of hydrophobic amino acids. These results established that Pz-peptidase B should be classified as a neutral metalloendopeptidase. Overall, the properties of Pz-peptidase B were very similar to those of previously described neutral metalloendopeptidases implicated in degradation of regulatory peptides.     

Conversion and inactivation of opioid peptides by rabbit brain endo-oligopeptidase A

The conversion of BAM-12P to Met-enkephalin and the hydrolysis of the Phe-Met and Phe-Leu bonds of met-enkephalin-Arg-Phe and Leu-enkephalin-Arg-Arg, respectively, by rabbit brain endo-oligopeptidase A were demonstrated. Peptide fragments were isolated by high performance liquid chromatography and identified by amino acid analysis. BAM 22P was not hydrolysed by the enzyme. The concentration dependent inhibition of BAM-12P conversion into Met-enkephalin by bradykinin and vice-versa provided additional evidence that endo-oligopeptidase A cleaves both the Phe5-Ser6 bond in bradykinin and the Met5-Arg6 bond of BAM-12P.     

Inhibition of rabbit tissue kininase by anti-(endo-oligopeptidase A) antibodies.

Highly purified rabbit brain endo-oligopeptidase A injected into goats produced, after 60 days of immunization, antisera that specifically inhibit purified rabbit brain endo-oligopeptidase A. An immunoreactive kininase having the same specificity as rabbit brain endo-oligopeptidase A for bradykinin was detected in several rabbit tissues. The highest amount of this immunoreactive kininase was found in the 25000 g supernatant fraction (S fraction) of heart, liver, skeletal muscle, ovary, brain and testis homogenates, corresponding to 89, 86, 78, 59, 56 and 53% respectively of the whole kininase activity found in the S fraction.     

Enkephalin is liberated from metorphamide and dynorphin A1-8 by endo-oligopeptidase A, but not by metalloendopeptidase EC 3.4.24.15.

It has been previously reported that both the cysteinyl-endo-oligopeptidase A and the metalloendopeptidase EC 3.4.24.15 are able to generate enkephalin from a number of enkephalin-containing peptides, including dynorphin A1-8. The present study shows that only endo-oligopeptidase A is able to generate [Leu5]enkephalin and [Met5]enkephalin from dynorphin A1-8 and from metorphamide respectively. It is also shown that endo-oligopeptidase A neither hydrolyses the specific EC 3.4.24.15 substrate alpha-N-benzoyl-Gly-Ala-Ala-Phe p-aminobenzoate, nor is inhibited by the specific EC 3.4.24.15 inhibitor N-[1(RS)-carboxy-2-phenylethyl]-alpha-Ala-Ala-Phe p-aminobenzoate.     

The activities of ''Pz-peptidase'' and ''endopeptidase 24.15'' are due to a single enzyme.

It was found that Pz-peptidase (assayed with 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys) and endopeptidase 24.15 (assayed with benzoyl-Gly-Ala-Ala-Phe-p-aminobenzoate) were co-purified from rat skeletal muscle, were co-eluted in high-resolution gel chromatography and co-existed in a homogeneous preparation of rat testis endopeptidase 24.15. The action of partially purified Pz-peptidase from rat testis on 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg was blocked by an inhibitor of endopeptidase 24.15, and also by a substrate of this enzyme. The partially purified enzyme hydrolysed two substrates of endopeptidase 24.15 with Km values similar to those published previously, and its action on 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys was inhibited by compounds that are considered specific for endopeptidase 24.15. We conclude that the activities previously attributed to two distinct enzymes are due to only one, and that the merging of the two literatures may lead to new lines of research.     

Distribution of Pz-peptidase in bovine epididymal and ejaculated semen

Bovine epididymal or ejaculated semen was fractionated by density gradient centrifugation in Percoll, and seminal components recovered from the gradients were subjected to additional separation and washing steps. This procedure resulted in isolation of four major seminal constituents: particle-free extracellular fluid, washed light particulates, washed cytoplasmic droplets, and washed spermatozoa. When assayed using the Pz-peptide substrate, all the isolated seminal fractions contained substantial Pz-peptidase activity. The extracellular fluid Pz-peptidase was present in soluble form, but Triton X-100 was required for complete extraction of the Pz-peptidase activity from the spermatozoa, cytoplasmic droplets, and light particulates. The greatest Pz-peptidase activities were observed in the cytoplasmic droplet and epididymal sperm extracts, whereas the activities in extracellular fluid, extracts of light particulates, and extracts of ejaculated spermatozoa were relatively low. Most of the Pz-peptidase activity in extracts of epididymal spermatozoa was attributable to cytoplasmic droplets. The specific Pz-peptidase activities found by regression analysis were 6.1 mU/billion attached cytoplasmic droplets and 1.1 mU/billion spermatozoa. These results established that in the bovine, cytoplasmic droplets were the major source of Pz-peptidase activity in semen and that Pz-peptidase was not primarily a spermatozoal enzyme.     

Endo-Oligopeptidase A., a Putative Enkephalin-Generating Enzyme, in the Vertebrate Retina

Endo-oligopeptidase A, EC 3.4.22.19, converts small enkephalin-containing peptides into the corresponding enkephalins in vitro. We investigated the presence of endooligopeptidase A in the retina and its possible colocalization with enkephalins in retinal neurons. The specific activity of endo-oligopeptidase. A found in pigeon retinae (30.3 +/- 7.3 mU/mg, mean +/- standard deviation) was four times higher than in rabbit retinae (7.0 +/- 1.1 mU/mg). The enzyme activity was not modified by EDTA, but it was enhanced by dithiothreitol and inhibited by zinc and 5,5'-dithiobis(2-nitrobenzoic acid). Immunohistochemical experiments with a purified antiserum against rabbit endo-oligopeptidase A revealed labeled neurons in both the inner nuclear layer and the ganglion cell layer of pigeon and rabbit retinae. Double-labeling immunofluorescence experiments demonstrated that about 90% of neurons containing endo-oligopeptidase A-like immunoreactivity also contained [Leu5]-enkephalin-like immunoreactivity. These colocalization results may represent an important step toward the demonstration of the possible involvement of endo-oligopeptidase A in enkephalin generation in vivo.     

The Exquisite Structure and Reaction Mechanism of Bacterial Pz-peptidase A toward Collagenous Peptides: X-RAY CRYSTALLOGRAPHIC STRUCTURE ANALYSIS OF PZ-PEPTIDASE A REVEALS DIFFERENCES FROM MAMMALIAN THIMET OLIGOPEPTIDASE*

Pz-peptidase A, from the thermophilic bacterium Geobacillus collagenovorans MO-1, hydrolyzes a synthetic peptide substrate, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg (Pz-PLGPR), which contains a collagen-specific tripeptide sequence, -Gly-Pro-X-, but does not act on collagen proteins themselves. The mammalian enzyme, thimet oligopeptidase (TOP), which has comparable functions with bacterial Pz-peptidases but limited identity at the primary sequence level, has recently been subjected to x-ray crystallographic analysis; however, no crystal structure has yet been reported for complexes of TOP with substrate analogues. Here, we report crystallization of recombinant Pz-peptidase A in complex with two phosphinic peptide inhibitors (PPIs) that also function as inhibitors of TOP and determination of the crystal structure of these complexes at 1.80–2.00 Å resolution. The most striking difference between Pz-peptidase A and TOP is that there is no channel running the length of bacterial protein. Whereas the structure of TOP resembles an open bivalve, that of Pz-peptidase A is closed and globular. This suggests that collagenous peptide substrates enter the tunnel at the top gateway of the closed Pz-peptidase A molecule, and reactant peptides are released from the bottom gateway after cleavage at the active site located in the center of the tunnel. One of the two PPIs, PPI-2, which contains the collagen-specific sequence, helped to clarify the exquisite structure and reaction mechanism of Pz-peptidase A toward collagenous peptides. This study describes the mode of substrate binding and its implication for the mammalian enzymes.     

Purification and characterization of Pz-peptidase from rabbit muscle

Pz-peptidase was purified from rabbit muscle by acid precipitation of tissue homogenate followed by cation- and anion-exchange chromatography, gel chromatography, and immunoadsorption. In analytical gel chromatography, one single peak of protein with corresponding Pz-peptidase activity was obtained. The enzyme had an apparent Mr of 74,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was eluted at pH 4.8 in chromatofocusing. No metals were detectable in the protein by neutron activation analysis. Purified Pz-peptidase hydrolyzed Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys (Km 7.2 microM) most effectively in the presence of 5 mM 2-mercaptoethanol and 10 mM CaCl2. No inhibition was observed with inhibitors of serine proteinases, aspartic proteinases, or metalloproteinases, apart from some nonspecific reversible inhibition by 1,10-phenanthroline. The activation by Ca2+ was reversed by EDTA. The enzyme was not inhibited by E-64, cystatin, or leupeptin, but was irreversibly inactivated by iodoacetate, iodoacetamide, and N-ethylmaleimide. It was therefore concluded that rabbit muscle Pz-peptidase is not a typical member of any of the four recognized catalytic classes of proteinases, but may be an atypical cysteine endopeptidase. The peptidase was not bound by alpha 2-macroglobulin. No hydrolysis of gelatin or fibronectin by the enzyme was detected, nor was there any activation of latent collagenase.     

Localization of Endo-Oligopeptidase (EC 3.4.22.19) in the Rat Nervous Tissue

The subcellular and regional distribution of endo-oligopeptidase (EC 3.4.22.19), an enzyme capable of generating enkephalin by single cleavage from enkephalin-containing peptides, was determined by an enzymatic assay using metorphamide and by immunochemical techniques in the CNS of the rat. The rat CNS contains a membrane-associated form of endo-oligopeptidase, an enzyme predominantly associated with the soluble fraction of brain homogenates. Subcellular fractionation showed that approximately 17% of the total activity of the enzyme is associated with membrane fractions including synaptosomes. Synaptosomal membranes were prepared from neocortex, striatum, hypothalamus, medulla, spinal cord, and cerebellum. The amount of EC 3.4.22.19 activity solubilized by 3-[( 3-cholamidopropyl]dimethylammonio)-1-propanesulfonate from synaptosomal membranes was similar in neocortex, striatum, and hypothalamus, being three- to 10-fold greater than in spinal cord, cerebellum, and medulla. A polyclonal antibody exhibiting high affinity for endo-oligopeptidase was raised in rabbits against the purified rat brain enzyme and used to localize endo-oligopeptidase by Western blotting and by immunoperoxidase techniques. A strong band corresponding to the Mr of EC 3.4.22.19 was found in solubilized proteins obtained from synaptosomal membranes prepared from hypothalamus, neocortex, and striatum when subjected to Western blotting. The immunohistochemical localization of endo-oligopeptidase indicated that the immunoreactivity was confined to gray matter in regions known to be rich in peptide-containing neurons such as the striatum. In the cerebellum, a region poor in peptides, no staining could be detected. The nonuniform distribution of endo-oligopeptidase in rat brain suggests a role in neurotransmitter processing in the CNS.     

Effect of gonadal steroids on hypothalamus and anterior pituitary endo-oligopeptidase B (proline-endopeptidase) activity in castrated female rats

In the present study we investigated the possible participation of endo-oligopeptidase B (poline-endopeptidase) in the control of gonadotrophin secretion through the control of LH-RH inactivation. This enzyme selectively hydrolyzes the Pro9-Gly10-NH2 peptide bond of LH-RH, thereby inactivating this substance. The enzyme activity was evaluated using a specific colorimetric substrate, i.e., Z-Gly-Pro-SM. Female adult Wistar rats were submitted to castration, experimental situations that are known to produce changes in gonadotrophin secretion. Hypothalamic and pituitary endo-oligopeptidase B activity was shown to be present predominantly in the soluble fraction of the enzyme preparations. The results also indicated that endo-oligopeptidase B activity adult female rat pituitary decreased after castration and increased after administration of estradiol and progesterone to castrated animals. The present results lead us to suggest that anterior pituitary endo-oligopeptidase B may be related to the control gonadotrophin secretion in female rats.     

Thiol-dependent metallo-endopeptidase characteristics of Pz-peptidase in rat and rabbit.

Pz-peptidase was purified from rat testis and rabbit muscle. Zinc was detectable in the rat enzyme. The activity of the enzyme from both species was slowly but completely abolished by EDTA and restored by Zn2+. Free thiol groups were also important for the catalytic activity of rat Pz-peptidase, as previously reported for the rabbit enzyme. We conclude that in both species Pz-peptidase has the characteristics of a thiol-dependent metallo-endopeptidase.