InhA-like protease secreted by Bacillus sp. S17110 inhabited in turban shell

A strain producing a potent protease was isolated from turban shell. The strain was identified as Bacillus sp. S17110 based on phylogenetic analysis. The enzyme was purified from culture supernatant of Bacillus sp. S17110 to homogeneity by ammonium sulfate precipitation, SP-Sepharose, and DEAE-Sepharose anion exchange chromatography. Protease activity of the purified protein against casein was found to be stable at pH 7 to pH 10 and around 50 degrees . Approximately 70% of proteolytic activity of the enzyme was detected either in the presence of 100 mM SDS or Tween 20. The enzyme activity was enhanced in the presence of Ca2+, Zn2+, Mg2+, but was inhibited by EDTA, indicating that it requires metal for its activity. The purified enzyme was found to be a monomeric protein with a molecular mass of 75 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The purified enzyme was analyzed through peptide fingerprint mass spectra generated from matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and a BLAST search, and identified as immune inhibitor A (inhA) deduced from nucleotide sequence of B. cereus G9241. Since InhA was identified as protease that cleave antibacterial proteins found in insect, inhA-like protease purified from Bacillus sp. S17110 might be pathogenic to sea invertebrates.     

Partial characterization of an endogenous inhibitor of a calcium-dependent form of insulin protease

Insulin protease activity has resisted high-yield purification to homogeneity, due to its low amount in tissues, its instability, and its erratic recovery from several types of chromatography. This report outlines the preliminary characterization of a naturally-occurring insulin protease inhibitor that accounts for some of these problems in rat skeletal muscle. In these experiments, inhibitory activity was assayed by its effect upon hydrolysis of 125I-(A14)-insulin by the partially purified insulin protease activity of rat skeletal muscle cytosol. During Sephadex G-200 chromatography of cytosol at pH 7.5, inhibitory activity copurifies with insulin protease activity, and the incomplete resolution of the two activities contributes to the impression that insulin protease exists in distinct 180,000-dalton and 80,000-dalton forms. By contrast, during DEAE-Sephacel chromatography of cytosol at pH 7.5, inhibitory activity and insulin protease activity are resolved by eluting the resin with 50 mM NaCl and 200 mM NaCl, respectively. Post-DEAE-Sephacel inhibitor has an Mr(app) of 67,000 daltons or 80,000-120,000 daltons, as determined by high-performance liquid chromatography or Sephadex G-150 chromatography, respectively. Post-DEAE-Sephacel insulin protease activity exhibits a Km for insulin of 15 nM and resides in a 200,000-dalton neutral thiol protease which requires 50 micromolar calcium for its maximum insulin-degrading activity. The inhibitor reduces the enzyme's activity reversibly, nonprogressively, and non-competitively with respect to insulin, but it does not alter the enzyme's sensitivity to calcium ion. These observations suggest that calcium and an endogenous protease inhibitor may influence cellular degradation of insulin via previously unrecognized effects upon cytosolic insulin protease activity.     

Purification and properties of a serine protease from Pseudomonas matophilia.

The extracellular protease of Pseudomonas maltophilia was partially purified by ammonium sulfate precipitation and chromatography on Sephadex G-75 and Bio-rex 70. Gel electrophoresis revealed minor impurities. The enzyme exhibited the following properties: (i) molecular weight, 35,000; (ii) A see article; 10.8; (iii) isoelectric point, 9.3; (iv) pH optimum, 10.0; (v)s20, w equal 3.47. The enzyme was rapidly inactivated by ethylenediaminetetracetate, but activity could be partially restored with divalent cations. Of those tested, Ca2+, Sr2+, Ba2+, Co2+, Cu2+, Mg2+, and Zn2+ were all effective. Both phenylmethylsulfonylfluoride and diisopropylfluorophosphate were powerful inhibitors of protease activity, but L-1-tosylamide-2-phenylethylchloromethyl ketone, iodoacetic acid, and iodoacetamide were without effect. The enzyme hydrolyzed the esters N-acetyl-L-tyrosine ethyl ester and alpha-N-benzoyl-L-arginine ethyl ester (BAEE) with Km values of 10.4 and 3.4 mM, respectively. The hydrolysis of BAEE was also inhibited by phenylarsonic acids. The kinetics of inhibition by m-nitrophenylarsonate were of the mixed type, and the K1 was 1.8 mM. The data followed a theoretical curve for a 1:1 enzyme-inhibitor complex with a dissociation constant of 1.8 mM. Inhibition by m-nitrophenylarsonate was pH dependent and followed a theoretical curve for the titration of a protonated group with a pKa of 7.0.     

Proteolysis of skeletal muscle adenylate cyclase. Destruction and reconstitution of fluoride and guanylnucleotide sensitivity

Adenylate cyclase was measured in skeletal muscle plasma membranes incubated with subtilisin. Under specific conditions the protease preferentially inactivated fluoride and guanylnucleotide sensitivity. Following protease treatment, membranes were solubilized with Lubrol 12A9 and subjected to ion-exchange chromatography. Adenylate cyclase was eluted with 200 mM NaCl; the enzyme recovered was completely unresponsive to either NaF or guanylyl imidodiphosphate. Responsiveness to the two ligands was restored by adding a heart fraction in which basal activity had been destroyed by heating at 40 degrees C or by adding a soluble skeletal muscle fraction in which basal activity had been largely destroyed by N-ethylmaleimide. The solubilized subtilisin-treated skeletal muscle preparation may serve as a source of catalytic activity for the study and purification of regulatory factors for adenylate cyclase.     

Proteolysis of skeletal muscle adenylate cyclase Destruction and reconstitution of flouride and guanylnucleotide sensitivity

Adenylate cyclase was measured in skeletal muscle plasma membranes incubated with subtilisin. Under specific conditions the protease preferentially inactivated flouride and guanylnucleotide sensitivity. Following protease treatment, membranes were solubilized with Lubrol 12A9 and subjected to ion-exchange chromatography. Adenylate cyclase was eluted with 200 mM NaCl; the enzyme recovered was completely unresponsive to either NaF or guanylyl imidodiphosphate. Responsiveness to the two ligands was restored by adding a heart fraction in which basal activity had been destroyed by heating at 40°C or by adding a soluble skeletal muscle fraction in which basal activity had been largely destroyed by N-ethylmaleimide. The solubilized subtilisin-treated skeletal muscle preparation may serve as a source of catalytic activity for the study and purification of regulatory factors for adenylate cyclase.     

Zinc metalloprotease activity in the cement precursor secretion of the barnacle, Chthamalus fragilis Darwin

Adult barnacles, Chathamalus fragilis, were removed carefully from the leaves and stems of marsh grass and floated base-up in algae-supplemented sea water. During the next 24-72 h, the animals secreted onto their exposed bases, a fluid, the cement precursor secretion (CPS), aliquots of which were collected in glass micropipets and pooled. The concentration of protein in pooled samples of CPS averaged 1.5 g +/- 0.42 protein/l of secretion. Protease activity was expressed as A(492) units/h/g of CPS protein. Aliquots of 5-45 l of pooled CPS samples, incubated in the presence of FTC-casein at 37 degrees C for 24 h, exhibited 8.31 x 10(-5) +/- 1.55 x 10(-5) DeltaA(492) units/hr/g of protein on average. Protease activity was optimized by the addition of 10 mM Ca(++) ions. Activity was detectable over a broad pH range, but was optimal around pH 8. Protease activity was inhibited up to 40% in the presence of 2.7 mM ethylenediaminetetraacetic acid (EDTA) and up to 97% in the presence of 2.5 mM 1,10-phenanthroline (OP) in the presence of 10 mM Ca(++) ions. Although low concentrations of Zn(++) ions (10 M) had little effect on protease activity, higher concentrations of Zn(++) ions (50 M to 15 mM Zn(++)) inhibited CPS protease activity. Protease activity was not inhibited by 1 mM phenylmethylsulfonylfluoride (PMSF), nor by 28 M E64, nor by 20 M leupeptin. At the present time, the protease activity in the barnacle CPS may best be characterized as a Ca-stimulated Zn-metalloprotease.     

A Zn2+-dependent and histone H1-specific protease in sea urchin sperm

Protease activity was extracted from sea urchin sperm with 1% Triton X-100 and partially purified by DEAE-cellulose and Sephadex G-100 chromatography. The enzyme preferentially degraded histone H1, while showing only a weak activity toward other histones. Heat-denatured casein and bovine serum albumin were not digested by this enzyme under the present experimental conditions. This protease hydrolyzed only Boc-Val-Leu-Lys-MCA among various peptidyl-MCAs. The optimal pH ranged from 7 to 11. Its molecular weight was about 41,000. Among various known inhibitors of proteases, only omicron-phenanthroline effectively inhibited the activity. The enzyme was stimulated by Zn2+ or Co2+. It was inactivated by omicron-phenanthroline but could be reactivated by the addition of Zn2+ or Co2+. Therefore, this protease seems to be a metalloprotease dependent on Zn2+ or Co2+. The insensitivity of this enzyme to phosphoramidon and its very restricted substrate specificity suggest that this enzyme is very different from other metalloproteases described hitherto.     

Purification and properties of phosphatidylglycerophosphate synthetase from mammalian liver mitochondria

The enzyme which catalyzes the synthesis of phosphatidylgly cerophosphate from an-glycerol-3-phosphated and cytidine diphosphate diacylglycerol was released from rat or pig liver mitochondrial membranes by extraction with Triton X-100 or Nonidet P-40. The detergent-extracted enzyme, like the activity of intact mitochondria, did not require added cations or lipids. The Triton extracts were fractionated by column chromatography on Bio-Gel A-1.5. The fractions obtained from the columns exhibited little activity in the standard assay system unless divalent cations were included. Additional stimulation (about twofold) was observed in the presence of added phospholipids. The cation requirement of the purified enzyme was relatively nonspecific with Mg2+, Ba2+, or Ca2+ providing maximal activity in the 10mM range. Either Mn2+ or Co2+ were stimulatory at somewhat lower concentrations but higher concentrations were inhibitory. Other cations such as Cd2+, Zn2+,Hg2+, or Cu2+ were ineffective as cofactors, and in the presence of Mg2+ inhibited the reaction at concentrations greater than 0.5 mM. The phospholipik stimulation was obtained specifically with phosphatidylethanolamines from natural or synthetic sources. Other diacylglycerophosphatides or lysophosphatides including lysophosphatidylethanolamine were ineffective.     

Purification and characterization of a Cl- -activated aminopeptidase from bovine skeletal muscle

To elucidate the mechanisms involved in the increase in free amino acids during postmortem storage of meat, a novel aminopeptidase was purified from bovine skeletal muscle by ammonium sulfate fractionation and successive chromatographies such as DEAE-cellulose, Sephacryl S-200, Hydroxyapatite, Phenyl-Sepharose, and Hi-Trap affinity column chromatography. The molecular mass of the enzyme was found to be 58 kDa on SDS-PAGE. This enzyme had optimum pH at around 7.5, and preferably hydrolyzed Ala-beta-naphthylamide (-NA) in amino acid-NAs. The activity was strongly inhibited by phenylmethansulfonyl fluoride (PMSF) and bestatin, suggesting that it is to be classified as a serine protease. Moreover, the activity was enhanced by chloride and nitrate ions, which is the most remarkable property of this enzyme. The enzyme appeared to be involved in the increase in free amino acids during postmortem storage of meat.     

Heat-stable protease from the marine sponge Geodia cydonium.

A protease from the marine sponge Geodia cydonium was purified from an aqueous extract by gel filtration and anion-exchange chromatography. A 200-kDa proteolytically active band was obtained when the enzyme was analyzed in gelatin-copolymerized zymograms. The enzyme was also able to degrade casein, bovine collagen, and the synthetic substrate alpha-N-benzoyl-D-arginine p-nitroanilide (BAPNA). Optimal conditions for proteolytic activity were achieved in the presence of 10 mM CaCl2 and within the pH range 7.0 to 8.5. The protease showed an extraordinary heat resistance. The enzyme activity was inhibited by phenylmethylsulphonyl fluoride (PMSF) and N-tosyl-lysine chloromethyl ketone (TLCK), suggesting that the enzyme belongs to the group of serine-type proteases. We propose that the protease is involved in sponge collagen catabolism.     

Purification and characterization of the fibrinolytic enzyme from Agkistrodon halys halys venom

By Q-sepharose column ion-exchange chromatography, alkyl-sepharose column hydrophobic chromatography the purified fibrinogenolytic enzyme was obtained from Agkistrodon halys halys venom. It is a single peptide-chain with molecular weight about 28 kDa. It was founded that this enzyme cleaved A alpha-chain of fibrinogen, pH-optimum was determined in the range of 7.5-8.0. Its fibrinogenolytic activity was estimated 15.6 mM fibrinogen/min per mg protein; caseinolytic activity was estimated 7.5 c.u., and amidolytic activity was 0.325 mM pNA/min/mg and 0.175 mM pNA/min/mg for S2238 and S2251 respectively; K(m) was 5.6 mM. The enzyme activity was inhibited by DFP and benzamidine. These results suggest that the enzyme is serine protease. It inhibited the platelet-aggregation.     

Purification and Characterization of a Cl−-Activated Aminopeptidase from Bovine Skeletal Muscle

To elucidate the mechanisms involved in the increase in free amino acids during postmortem storage of meat, a novel aminopeptidase was purified from bovine skeletal muscle by ammonium sulfate fractionation and successive chromatographies such as DEAE-cellulose, Sephacryl S-200, Hydroxyapatite, Phenyl-Sepharose, and Hi-Trap affinity column chromatography. The molecular mass of the enzyme was found to be 58 kDa on SDS–PAGE. This enzyme had optimum pH at around 7.5, and preferably hydrolyzed Ala-β-naphthylamide (-NA) in amino acid-NAs. The activity was strongly inhibited by phenylmethansulfonyl fluoride (PMSF) and bestatin, suggesting that it is to be classified as a serine protease. Moreover, the activity was enhanced by chloride and nitrate ions, which is the most remarkable property of this enzyme. The enzyme appeared to be involved in the increase in free amino acids during postmortem storage of meat.     

Studies on new intracellular proteases in various organs of rat. 3. Control of group-specific protease under physiological conditions.

1. To study the role of group-specific protease in enzyme degradation, alternation of its activity under various physiological conditions was examined. 2. Studies on the distribution of group-specific protease in various organs of rats showed high activity in skeletal muscle and the muscle layer of small intestine, and rather low activity in liver. The activity varied in different muscles, but red muscle tended to have higher activity than white muscle. Activity was much lower in the muscles of the stomach and colon than in those of the small intestine. 3. Group-specific protease in skeletal muscle increased under various dietary conditions (starvation, protein-free diet or high protein diet), but the activities in the muscle layer of the small intestine and liver were not greatly influenced by dietary conditions. None of the hormones tested (i.e. hydrocortisone, glucagon, insulin, growth hormone and estrogen) influenced the activity of group-specific protease in liver. 4. The level of group-specific protease in skeletal muscle was increased markedly fifteen days after denervation, with a reciprocal decrease in the level of muscle phosphorylase, which is a good substrate of the protease. 5. Liver protease activity appeared in the late suckling period. The activity in skeletal muscle was high at the time of birth and attained the adult level 3 weeks after birth. The activity in the muscle layer of the small intestine did not change after birth. Thus the mechanism for evoking these three specific proteases during development are apparently different. The activity of liver protease began to decrease approximately 12 h after partial hepatectomy and reached a minimum after about 72 h. Recovery of the protease activity was very slow and activity had not returned to the normal value 7 days after the operation. This observation seems to be consistent with the fact that there is little or no protease activity in liver in the neonatal period.     

Purification, characterization and localization of serine protease of Morris hepatoma 8999.

1. A serine protease of hepatoma 8999, isolated in the mitochondrial fraction, was purified and crystallized. The purified enzyme was apparently homogeneous on ultracentrifugal analysis and polyacrylamide disc gel electrophoresis. The ratio of absorbance at 280 nm and 260 nm, A280/A260, was 1.90 and its absorption coefficient, A280 1% was 10.5 cm-1 estimated from dry weight measurements. Its S20, w value was 2.23 S and its molecular weight was estimated to be 24000 +/- 1000. The enzyme contained twice as much lysine, arginine and histidine as chymotrypsinogen did, but had a very similar amino acid composition to serine protease from skeletal muscle. Its isoelectric point was pH 10.6. 2. The substrate specificity of the enzyme was the same as that of chymotrypsin A. Its Km and kcat values for N-acetyl-L-tyrosine ethyl ester, N-acetyl-L-phenylalanine ethyl ester and N-acetyl-L-tryptophan ethyl ester were 0.35 mM and 10.69 s-1, 0.38 mM and 10.7 s-1, and 0.11 mM and 11.8 s-1, respectively. Its activity was completely inhibited by phenylmethylsulfonyl fluoride and partially inhibited with tosylphenylalanine chloromethyl ketone. 3. The enzyme was shown to be located in different granules from the intracellular particules (light and heavy mitochondrial fraction) by sucrose density gradient centrifugation, and it was stained in mast cells of the hepatoma 8999 by the immunofluorescent technique. 4. Serine protease is present in different amounts in various organs of rat and the enzyme from hepatoma 8999 gave a single band that fused completely with those of the enzymes from skeletal muscle, heart, liver and kidney, respectively, on Ouchterlony double-diffusion analysis using antiserum to the crystalline enzyme of hepatoma 8999, but the enzyme from small intestine did not react with the antiserum.     

Staphylococcus aureus adenosine triphosphatase: inhibitor sensitivity and release from membrane.

Homogeneous preparations of cytoplasmic membrane isolated from Staphylococcus aureus 6538P exhibited membrane-associated adenosine triphosphatase (ATPase) activity. Membrane ATPase activity was activated by divalent cations (4.0 mM: Mg2+ greater than Mn2+ greater than Co2+ greater than Zn2+), and ATP was hydrolyzed more readily than other nucleoside triphosphates and phosphorylated substrates. The pH optimum for the membrane ATPase was 6.5. The ATPase could not be released from the membrane by differential osmotic treatments, but detergent treatment effectively solubilized active enzyme. The nonionic detergent Triton X-100 (1%) released a protein with ATPase activity, after substrate-dependent staining in polyacrylamide gels, that differed slightly in electrophoretic migration when compared to the active enzyme solubilized with sodium dodecyl sulfate (0.1%). Membrane-associated ATPase activity was inhibited by N,N'-dicyclohexylcarbodiimide (0.001 to 1 mM) and NaF (50% inhibition at 5 mM NaF). Azide and trypsin inhibited activity, whereas ouabain had a slight inhibitory effect. Diethylstilbestrol showed appreciable activation of the membrane ATPase over the range employed (0.001 to 1 mM).     

Stability and Specificity of the Cell Wall-Associated Proteinase from Lactococcus lactis subsp. cremoris H2 Released by Treatment with Lysozyme in the Presence of Calcium Ions

The cell wall-associated proteinase from Lactococcus lactis subsp. cremoris H2 (isolate number 4409) was released from the cells by treatment with lysozyme, even in the presence of 50 mM calcium chloride. Cell lysis during lysozyme treatment was minimal. The proteinase activity released by lysozyme treatment fractionated on ion-exchange chromatography as three main forms, the molecular masses of which were determined by gel exclusion chromatography and polyacrylamide gel electrophoresis. Two of the enzyme forms released, 137 and 145 kDa, were the same as those released by incubation of cells in calcium-free phosphate buffer. In the presence of calcium, lysozyme treatment also resulted in the release of a 180-kDa enzyme molecule. The total proteinase activity released by lysozyme treatment (in the presence or absence of calcium) was not only greater than that released by phosphate buffer but was also greater than that initially detectable on the surface of whole cells, suggesting an unmasking of enzyme on the cell surface. The presence of calcium during release treatment resulted in increased stability of the crude enzyme preparations. For the proteinase preparation released by using lysozyme with 50 mM CaCl₂, the half-life of proteinase activity at 37°C was 39 h, compared with 0.22 h for the calcium-free phosphate buffer-released preparation. In all cases, maximum stability was observed at pH 5.5. Comparison of β-casein hydrolysis by the three forms of the enzyme showed that the products of short-term (5- to 30-min) digestions were very similar, although subtle differences were detected with the 180-kDa form.     

Studies on the utilization and mobilization of lipid in skeletal muscles from streptozotocin-diabetic and control rats.

Several aspects of lipid metabolism in the soleus and diaphragm muscles of streptozotocin-diabetic and control rats were investigated. The triglyceride content of both muscles was elevated in the diabetic state and the presence of increased intracellular lipid was confirmed by electron microscopy. In vitro glucose and palmitate oxidation studies showed that both types of muscle from the diabetic animals metabolized more fat than did the soleus and diaphragm from control rats. While isoproterenol alone produced a significant lipolytic response in both the soleus and diaphragm from control and diabetic animals, there was no difference in the percent increase in fatty acids released from muscles of diabetic rats compared to controls. However, the absolute difference was greater when the diaphragms were compared. Muscles from experimental and control animals showed a marked reduction in the amount of free fatty acids released in response to insulin. In addition, in the presence of the hormone, both the absolute and percent isoproterenol-stimulated increases in fatty acids were significantly greater for both diaphragm and soleus muscles from diabetic rats. The effects of insulin, isoproterenol, and the combination of these two hormones on the amount of glycerol released into the incubation medium were similar to those found on free fatty acid release. The results of these experiments show that there is an apparent increase in fat utilization in skeletal muscle of diabetic rats. Furthermore, measurements of triglyceride concentration and the enhanced response to isoproterenol stimulation in the muscles from these animals suggests that they may have an increased capacity for mobilization of intracellular lipids. Finally, in the diabetic state, both the soleus and diaphragm appear to demonstrate an increased response to the antilipolytic effect of insulin as measured by the decreased amount of fatty acid released into the incubation medium, the percent change also being significant for the soleus muscle.-Stearns, S. B., H. M. Tepperman, and J. Tepperman. Studies on the utilization and mobilization of lipid in skeletal muscles from streptozotocin-diabetic and control rats.     

Separation and Some Properties of Two Intracellular beta-Glucosidases of Sporotrichum (Chrysosporium) thermophile

Intracellular, inducible beta-glucosidase from the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile (ATCC 42464) was fractionated by gel chromatography or isoelectric focusing into components A and B. Enzyme A (molecular weight 440,000) had only aryl-beta-glucosidase activity, whereas enzyme B (molecular weight 40,000) hydrolyzed several beta-glucosides but had only low activity against o-nitrophenyl-beta-d-glucopyranoside (ONPG). Both enzymes had temperature optima of about 50 degrees C. The pH optimum was 5.6 for enzyme A and 6.3 for enzyme B, respectively. The K(m) (ONPG) value for enzyme A was 0.5 mM, and the corresponding values for enzyme B were 0.18 mM (ONPG) and 0.28 mM (cellobiose). Enzyme B, when tested with ONPG, showed substrate inhibition at a substrate concentration above 0.4 mM which could be released by cellobiitol and other alditols. Enzyme A was isoelectric at pH 4.48, and enzyme B was isoelectric at pH 4.64. Several inhibitors were tested for their action on the activity of enzymes A and B. Both enzymes were found to be concomitantly induced in cultures with either cellobiose or cellulose as carbon source.     

Post-proline dipeptidyl-aminopeptidase from synaptosomal membranes of guinea-pig brain. A possible role for this activity in the hydrolysis of His-ProNH2, arising from the action of synaptosomal membrane pyroglutamate aminopeptidase on thyroliberin

In this paper we report that while 55% of the total post-proline dipeptidyl-aminopeptidase activity in guinea-pig brain is associated with the soluble fraction of the cells, the remaining activity is widely distributed throughout the particulate fractions. A significant portion of this particulate activity is, however, associated with a synaptosomal membrane fraction. The specific activity of this enzyme rose as the synaptosomal membrane fraction was prepared from a synaptosomal fraction and had previously risen at the synaptosomal fraction was prepared from a postmitochondrial pellet. The synaptosomal membrane post-proline dipeptidyl-aminopeptidase was released from the membrane by treatment with Triton X-100 and partially purified by chromatography on Sephadex G-200. By contrast with the soluble enzyme the partially purified solubilised synaptosomal membrane post-proline dipeptidyl-aminopeptidase was not inhibited by 1.0 mM p-chloromercuribenzoate, 1.0 mM N-ethylmaleimide or 0.5 mM puromycin but was inhibited by 0.5 mM bacitracin. The partially purified solubilised enzyme was capable of releasing His-Pro from His-Pro-Val, His-Pro-Leu, His-Pro-Phe and His-Pro-Tyr and of releasing Gly-Pro from Gly-Pro-Ala but could not release Arg-Pro from Arg-Pro-Pro or from Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (bradykinin). It was also unable to release Pro-Pro from Pro-Pro-Gly or Glp-Pro from Glp-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-MetNH2 (eledoisin). Using [Pro-3H]thyroliberin we show that the membrane-bound enzyme converts His-ProNH2, produced by the action of the synaptosomal membrane pyroglutamate aminopeptidase, to His-Pro thus competing with the spontaneous cyclisation of His-ProNH2 to His-Pro diketopiperazine. Purified preparations of synaptosomal membrane pyroglutamate aminopeptidase were used to generate His-ProNH2, which could then be converted to His-Pro by the presence of the partially purified synaptosomal membrane post-proline dipeptidyl-aminopeptidase. This preparation was free of contaminating post-proline cleaving endopeptidase, carboxypeptidase P, aminopeptidase P, prolyl carboxypeptidase or proline dipeptidase.