Membrane-bound aminopeptidase P from bovine lung. Its purification, properties, and degradation of bradykinin.

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified to apparent homogeneity from bovine lung microsomes. The enzyme was solubilized using phosphatidylinositol-specific phospholipase C (Bacillus thuringiensis), indicating that bovine lung amino-peptidase P is attached to membranes via a glycosylphosphatidylinositol anchor. The enzyme was purified 1900-fold with a yield of 25% by chromatography on decyl-agarose, omega-aminodecyl-agarose, a second decylagarose column, DEAE-Sephacel, and an ultrafiltration step. Native gradient polyacrylamide gel electrophoresis revealed a single stained protein band whose position in the gel corresponded to cleavage of the Arg1-Pro2 bond of bradykinin. The Mr was 360,000 by gel permeation chromatography and 95,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate specificity of aminopeptidase P was determined using approximately 50 peptides with proline in the second position. The enzyme could hydrolyze lower NH2-terminal homologs of bradykinin, including Arg-Pro-Pro, which was used as the routine substrate in a rapid fluorescence assay performed in the absence of added Mn2+. Some peptides having NH2-terminal amino acids other than arginine were also cleaved. Aminopeptidase P appeared to favor peptides that had 2 proline residues or proline analogs in positions 2 and 3 of the substrate. In general, tripeptides having a single proline residue in position 2 were poor substrates. Aminopeptidase P was inhibited by a series of peptides, 3-8 residues long, having an NH2-terminal Pro-Pro sequence. The enzyme was also inhibited by metal-chelating agents, 2-mercaptoethanol (4 mM), p-chloromercuribenzenesulfonic acid, and NaCl at concentrations greater than or equal to 0.25 M. The purified enzyme had a pH optimum of 6.5-7.0 and was most stable in the basic pH range. A role for membrane-bound aminopeptidase P in the pulmonary inactivation of circulating bradykinin is proposed.     

A peptidyl dipeptidase-4 from Pseudomonas maltophilia: purification and properties

A peptidyl dipeptidase-4 (bacterial PDP-4) was purified to near homogeneity from a supernatant of Pseudomonas maltophilia extracellular medium. Bacterial PDP-4 is a single-polypeptide-chain enzyme, 82 kDa, with an alkaline isoelectric point. Peptides susceptible to hydrolysis by bacterial PDP-4 include angiotensin 1, bradykinin, enkephalins, atriopeptin 2, and smaller synthetic peptides. N-acylated tripeptides are hydrolyzed, but free tripeptides are not. A free carboxy terminus is required for hydrolysis. Peptides with ultimate and penultimate Pro residues are not hydrolyzed. The enzyme does not require an anion for activity. Bacterial PDP-4 was inhibited by EDTA and the dipeptide Phe-Arg. Thiorphan was an inhibitor only at levels well above those required for inhibition of neutral metalloendopeptidase (NEP), an enzyme for which thiorphan is specific. A second NEP and thermolysin inhibitor, phosphoramidon, did not inhibit bacterial PDP-4. The potent angiotensin-converting enzyme inhibitor lisinopril was not inhibitory. Bacterial PDP-4 is distinguished from a similar enzyme from Escherichia coli, which is not susceptible to EDTA inhibition, and one from Corynebacterium equi, which hydrolyzes free tripeptides. These data indicate that the bacterial PDP-4 catalytic site is unlike those of other enzymes that function either wholly or in part as peptidyl dipeptidases.     

Isolation and characterization Salmonella typhimurium mutants lacking a tripeptidase (peptidase T).

Salmonella typhimurium contains an enzyme, peptidase T, that hydrolyzes a variety of tripeptides. Specificity studies with a peptidase activity stain after gel electrophoresis of crude cell extracts showed that peptidase T hydrolyzes tripeptides containing N-terminal methionine, leucine, or phenylalanine. Little or no activity could be detected against dipeptides, N-blocked or C-blocked tripeptides, and tetrapeptides. Analysis of reaction products by high-pressure liquid chromatography showed that peptidase T removes the N-terminal amino acid from tripeptides. Mutants lacking peptidase T were isolated by screening microcultures grown in the wells of plastic microtitration plates for hydrolysis of Met-Ala-Ser or Met-Gly-Gly. Mutations (pepT) that eliminate this enzyme were found to be phage P22 cotransducible with purB at approximately 25 map units on the S. typhimurium map. Comparison of the growth properties of mutant and wild-type strains suggests that peptidase T does not function in utilization of tripeptides to provide amino acids during growth.     

Monkey brain arylamidase. II. Further characterization and studies on mode of hydrolysis of physiologically active peptides.

A large-scale purification of monkey brain arylamidase was carried out. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cystine. The amino terminal residue was determined to be alanine by dansylation. The enzyme was activated by sulfhydryl compounds. Dithiothreitol was more effective than beta-mercaptoethanol. Bestatin competitively inhibited the enzyme activity and the Ki value was calculated to be 2.5 x 10(-7) M, which was of the same order as that of puromycin. The inhibitions by puromycin and bestatin were reversible. The enzyme hydrolyzed di-, tri-, and oligopeptides including physiologically active peptides. Of physiologically active peptides, enkephalins and Met-Lys-bradykinin, which possess a neutral amino acid at the N-terminal position, were more rapidly hydrolyzed by the enzyme. Peptides such as LH-RH and TRH, which possess a pyrrolidonecarboxylyl group at the N-terminal position, and substance P and bradykinin, which possess a proline residue adjacent to the N-terminal residue, were not hydrolyzed by the enzyme. The Km values for various peptides indicate that the enzyme has higher affinity for oligopeptides than di- and tripeptides. The aminopeptidase activity of the enzyme was also competitively inhibited by puromycin and bestatin. Analyses of the hydrolysis products of various peptides by the dansylation method indicate that the enzyme has both kinin-converting activity and angiotensinase activity.     

The fate of extracellular glutathione in the rat.

When intravenously administered to rats, [U-14C]glycine-labelled GSSG, GSH and its analogue ophthalmic acid were rapidly removed from the blood. In perfusion studies with isolated liver, however, the compounds did not enter the liver tissue. Thus, uptake by this tissue is obviously not responsible for the removal of gamma-glutamyl tripeptides from the blood. Instead, rapid hydrolysis of the tripeptides was observed. The undegraded tripeptides were only detected in the blood immediately after administration. Within tissue the degradation product glycine accounted for all the radioactivity. After intravenous injection of the labelled tripeptides the radioactivity accumulated first in the kidney, as shown by autoradiographic studies and chemical analysis of different tissues. The hydrolysis of the gamma-glutamyl tripeptides decreased markedly after the renal arteries were clamped. These observations strongly suggest that renal tissue is the principal site of the degradation of the tripeptides. Inhibition studies and experiments with isolated renal tubules revealed that gamma-glutamyl transpeptidase catalyses the fast hydrolysis of the extracellular peptides. The results indicate that, when entering the extracellular space, glutathione and its analogues are completely hydrolysed and must be resynthesized after reuptake of the constituent amino acids. It is concluded that the degradation occurs mainly on the luminal surface of the renal brush-border membrane and that gamma-glutamyl transpeptidase is a glutathionase acting on extracellular glutathione.     

The proteolytic system of Lactobacillus sanfrancisco CB1: purification and characterization of a proteinase, a dipeptidase, and an aminopeptidase.

A cell envelope 57-kDa proteinase, a cytoplasmic 65-kDa dipeptidase, and a 75-kDa aminopeptidase were purified from Lactobacillus sanfrancisco CB1 sourdough lactic acid bacterium by sequential fast protein liquid chromatography steps. All of the enzymes are monomers. The proteinase was most active at pH 7.0 and 40 degrees C, while aminopeptidase and dipeptidase had optima at pH 7.5 and 30 to 35 degrees C. Relatively high activities were observed at the pH and temperature of the sourdough fermentation. The proteinase is a serine enzyme. Urea-polyacrylamide gel electrophoresis of digest of alpha s1- and beta-caseins showed differences in the pattern of peptides released by the purified proteinase and those produced by crude preparations of the cell envelope proteinases of Lactobacillus delbrueckii subsp. bulgaricus B397 and Lactococcus lactis subsp. lactis SK11. Reversed-phase fast protein liquid chromatography of gliadin digests showed a more-complex peptide pattern produced by the proteinase of Lactobacillus sanfrancisco CB1. The dipeptidase is a metalloenzyme with high affinity for dipeptides containing hydrophobic amino acids but had no activity on tripeptides or larger peptides. The aminopeptidase was also inhibited by metal-chelating agents, and showed a broad N-terminal hydrolytic activity including di- and tripeptides. Km values of 0.70 and 0.44 mM were determined for the dipeptidase on Leu-Leu and the aminopeptidase on Leu-p-nitroanilide, respectively.     

Racemization in the synthesis of polytripeptide models of a collagen.

Racemization in the synthesis of tripeptide intermediates and their polymers was investigated, using L -amino acid oxidase. Stepwise investigation of peptide intermediates showed no racemization during peptide coupling steps or deprotection of benzyl esters by hydrogenolysis. Saponification of one of the methyl esters produced some racemization. Preparation of active esters from N-protected tripeptide acids containing optically active C-terminal amino acid, with one exception, produced racemization. The fractionated polymers were found to contain less racemized amino acids than the crude products or starting monomeric tripeptides, indicating that the racemized sequences gave rise to lower molecular-weight oligomers. The sequences investigated were -Pro-Pro-Ala-, -Ala-Pro-Pro-, -Val-Pro-Pro-, -Pro-Pro-Leu-, -Pro-Gly-Leu-, -Pro-Gly-Phe-, -Pro-Gly-Val-, -Gly-Val-Pro-, -Phe-Pro-Gly-, -Leu-Pro-Gly-, and Ile-Pro-Gly-.     

Examination of methodology for the synthesis of cyclic thioether peptide libraries derived from linear tripeptides.

Work was undertaken to examine methodology for the cyclization of linear tripeptides on the solid phase via intramolecular S-alkylation using the Multipin(trade mark) Solid-Phase Peptide Synthesis platform. While previous work had shown that this chemistry could be used to efficiently cyclize linear tetrapeptide libraries, application of this synthetic strategy to the model linear tripeptide sequence Leu-Ser-Lys resulted in significant cyclic dimer formation. Ultimately, it was found that the addition of a large excess of lithium in the form of LiCl to the cyclization solution, significantly reduced cyclic dimer formation affording highly pure crude cyclic monomer. The application of this modified cyclization protocol to the preparation of cyclic peptide libraries was successfully demonstrated with the synthesis of a 20-membered library 4{1-20} based on the linear tripeptide sequence Leu-Xxx-Lys in which the position Xxx was varied with the standard 20 proteogenic residues.     

Sensitivity to nikkomycin Z in Candida albicans: role of peptide permeases.

The uptake of tritiated nikkomycin Z, a potent inhibitor of chitin synthetase, is mediated by a peptide transport system in Candida albicans. Kinetic transport assays with radioactive di- and tripeptides and competition studies suggest that two distinct systems operate in this yeast. Nikkomycin Z was transported through one of these systems, common to di- and tripeptides. A peptide transport-deficient mutant was isolated on the basis of its resistance to nikkomycin Z. The mutant lost most of its capacity to take up dipeptides but simultaneously increased its ability to transport tripeptides. These results indicate that C. albicans handles peptides through multiple transport systems and adjusts their expression to environmental conditions.     

Synthesis and evaluation of a new series of peptide-based endothelin receptor antagonists.

Novel peptide-based endothelin (ET) receptor antagonists were designed and synthesized in our laboratory. BQ-485, HIM-CO-Leu-d-Trp-d-Trp-OH, was selected as the leading compound. The primary structures of these new tripeptides were ABO-CO-Leu-d-Trp-d-AA(X)-OH. The introduction of unnatural aromatic amino acids into these tripeptides was useful in the structure-activity relationship studies. Among the 20 tripeptides, 16 of them showed high activities against the contraction of rat aortic smooth muscles induced by ET-1.     

Isolation and characterization of an arginine ester hydrolase from Bothrops jararacussu venom which induces contractions of the isolated rat uterus.

The isolation and partial characterization of a serine protease with arginine ester hydrolase activity from Bothrops jararacussu snake venom are described. The purification procedure consisted of a gel filtration of the crude venom on Sephadex G-75 followed by an ion-exchange chromatography of the active fraction on DEAE-cellulose and a rechromatography on Bio-Rex 70 resin. The esterase fraction (DI-III), M(r) = 25,000 by SDS-PAGE, showed proteolytic activity on fibrinogen and casein. After 2 hr incubation, the A alpha and B beta chains of fibrinogen were intensely hydrolysed, while the gamma chain kept apparently intact, even after 20 hr of incubation. In spite of that, DI-III did not clot fibrinogen. DI-III induced edema in the rat paw. Although unable to release bradykinin, it induced contractions of the isolated rat uterus. DI-III did not catalyse the hydrolysis of bradykinin. Its arginine ester hydrolase activity was completely inhibited by diisopropyl fluorophosphate after 1 hr incubation, but not by phenylmethylsulfonyl fluoride under the same conditions.     

Influence of different tripeptides on the stability of the collagen triple helix. II. An experimental approach with appropriate variations of a trimer model oligotripeptide

A collagen model peptide comprising three covalently cross-linked chains (Ala-Gly-Pro)₈ with a stable triple-helix conformation was utilized as the constant part of elongated model peptides of different composition. The tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, Gly-Pro-Pro, Gly-Pro-Ser, Gly-Ala-Hyp, Gly-Phe-Hyp, Gly-Glu-Hyp, Gly-Ala-Lys, and Gly-Pro-Phe were coupled at the N-terminal to the cross-linked peptide. The transition temperatures determined by CD measurements are higher for the peptides containing the Gly-X-Hyp sequences followed by those with the Gly-Pro-Y sequences. In experiments with combinations of two different tripeptides coupled to the constant part of the cross-linked model peptides higher transition temperatures were observed if the sequence of helix-promoting tripeptides was not interrupted.     

Preferred conformations of a linear RGD tripeptide.

Conformational study of RGD tripeptides in the nonhydrated and hydrated states was carried out using an empirical potential function ECEPP/3 and the hydration shell model in order to investigate preferred conformations and factors responsible for their stability. RGD tripeptides in the nonhydrated and hydrated states can be interpreted as existing as an ensemble of feasible conformations rather than as a single dominant conformation from the analysis of distributions of backbone conformations, hydrogen bonds and beta-turns. The different distributions of conformations for the neutral and zwitterionic RGD tripeptides in both states may indicate that the conformation of the RGD tripeptide is liable to depend on solvent polarity and pH values. beta-Turn populations for the neutral tripeptide in both states are reasonably consistent with NMR measurements on linear RGD-containing peptides. The degradation of RGD tripeptide seems to be attributed mainly to the hydrogen bonds between the Asp side-chain and the backbone of Asp residue or C-terminal NHMe group, rather than to the flexible backbones of Gly and Asp residues.     

Hydrated electron-initiated main-chain scission in peptides: an e.s.r. and spin-trapping study.

The reactions of hydrated electrons (eaq-) with 19 tripeptides were investigated. Hydrated electrons were produced by gamma-radiolysis of aqueous peptide solutions containing sufficient sodium formate to remove hydroxyl radicals and hydrogen atoms. t-Butanol was also used to scavenge hydroxyl radicals. The short-lived radicals formed by the reactions of eaq- with the peptides were spin-trapped with t-nitrosobutane to form stable nitroxide radicals and identified by e.s.r. spectroscopy. The tripeptides studied contained two glycine residues. Following the addition of eaq- to tripeptides, C-N bond scission was observed at three sites. Cleavage occurred between the nitrogen of the ammonium group and the alpha-carbon and between the nitrogen of the peptide linkage and the adjoining alpha-carbons. The radicals corresponding to each of these three types of scission were identified. From a comparison of the radical yields of the reaction of eaq- with ala, (ala)2, and poly-DL-ananine with an average degree of polymerization of 1800, it was shown that eaq-, can react with many carbonyl groups of poly-DL-alanine, leading to main-chain scission. Analogous reactions of eaq- with proteins and enzymes may be expected to lead to loss of biological activity.     

Crystal Structures of an Oligopeptide-Binding Protein from the Biosynthetic Pathway of the β-Lactamase Inhibitor Clavulanic Acid

Clavulanic acid (CA) is a clinically important β-lactamase inhibitor that is produced by fermentation of Streptomyces clavuligerus. The CA biosynthesis pathway starts from arginine and glyceraldehyde-3-phosphate and proceeds via (3S,5S)-clavaminic acid, which is converted to (3R,5R)-clavaldehyde, the immediate precursor of (3R,5R)-CA. Open reading frames 7 (orf7) and 15 (orf15) of the CA biosynthesis cluster encode oligopeptide-binding proteins (OppA1 and OppA2), which are essential for CA biosynthesis. OppA1/2 are proposed to be involved in the binding and/or transport of peptides across the S. clavuligerus cell membrane. Peptide binding assays reveal that recombinant OppA1 and OppA2 bind di-/tripeptides containing arginine and certain nonapeptides including bradykinin. Crystal structures of OppA2 in its apo form and in complex with arginine or bradykinin were solved to 1.45, 1.7, and 1.7 Å resolution, respectively. The overall fold of OppA2 consists of two lobes with a deep cavity in the center, as observed for other oligopeptide-binding proteins. The large cavity creates a peptide/arginine binding cleft. The crystal structures of OppA2 in complex with arginine or bradykinin reveal that the C-terminal arginine of bradykinin binds similarly to arginine. The results are discussed in terms of the possible roles of OppA1/2 in CA biosynthesis.     

Effect of some fragments of peptide hormones on the content of biogenic monoamines from mouse brain]

The effect of some tripeptides, which are fragments of peptide hormones, and their analogs on the content of biogenic monoamines (BM) from albino mice brain was studied. It was found that thyroliberin, melanostatin and the C-terminal tripeptide of gonadoliberin activate the dophaminergic (DA-ergic) system in the forebrain of mice treated with reserpine or haloperidol, whereas the C-terminal tripeptide of gastrin acts as a synergic blocker of the DA receptors. The N-terminal tripeptides (with and without the amido group) do not affect the content of BM. No effect of the tripeptides was observed in intact animals. It is assumed that the agonistic or antagonistic effect of the tripeptides on BM is due to certain structural peculiarities of the tripeptides, e.g. the presence of the C-terminal amido group and their endogenous nature.     

Identification of a novel inflammatory stimulant of chondrocytes. Early events in cell activation by bradykinin receptors on pig articular chondrocytes.

The inflammatory peptide bradykinin stimulated a rapid and transient increase in cytoplasmic [Ca2+] in primary pig chondrocytes, as measured by the fluorescent indicator dye Fura-2. This increase occurred in the absence of extracellular Ca2+, indicating a mobilization from intracellular stores. The elevation in intracellular [Ca2+] was mediated by authentic bradykinin receptors, since it was blocked by the specific bradykinin antagonist [beta-(2-thienyl)-L-Ala5,8,D-Phe7]bradykinin. Activation of chondrocytes by bradykinin induced a concentration-dependent [ED50 (dose for half-maximal response) approximately 40 nM] accumulation of inositol monophosphate in the presence of LiCl and a concentration-dependent increase in production of prostaglandin E2. The generation of the secondary mediator prostaglandin E2 was a biologically relevant output response induced by bradykinin, but chondrocyte responses, such as the rate of entry into DNA synthesis, the rate and pattern of new protein synthesis and the rate of synthesis and resorption of cartilage proteoglycan, were unaltered by bradykinin treatment. Chondrocytes were also shown to be activated by two pharmacological mediators of cytosolic [Ca2+] elevation, i.e. the ionophore A23187 and thapsigargin, which both produced alterations in protein synthesis which were mimicked by bradykinin. Thus Ca2+-sensitive pathways exist which are not functionally responsive to a Ca2+-mobilizing and inositol phosphate-generating hormone, potentially indicating other routes of regulation. These results call attention to bradykinin and related peptides as another class of inflammatory mediators which may regulate physiological and pathological chondrocyte metabolism.     

A new 1-aminocyclopropane-1-carboxylic acid-conjugating activity in tomato fruit.

A new conjugate, 1-(gamma-L-glutamylamino)cyclopropane-1-carboxylic acid (GACC), of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is identified. The only previously identified conjugate of ACC is 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC). GACC, not MACC, was the major conjugate formed by crude protein extracts of tomato (Lycopersicon esculentum Mill cv Ailsa Craig) fruit pericarp and seeds incubated with [14C]ACC. GACC was resolved from [14C]ACC and [14C]MACC by reversed-phase C18 thin-layer chromatography and subsequently detected and quantified using a radioisotope-imaging system. Proteins precipitated from crude extracts failed to catalyze formation of GACC unless the supernatant was added back. Reduced glutathione, but not other reducing agents, replaced the crude supernatant. When [35S-cysteine]glutathione and [3H-2-glycine]glutathione were used as substrates, neither radiolabeled glycine nor cysteine from the glutathione tripeptide was incorporated into GACC. Oxidized glutathione, S-substituted glutathione, and di- and tripeptides having an N-terminal gamma-L-glutamic acid, but lacking cysteine and glycine, also served as substrates for GACC formation. Peptides lacking the N-terminal gamma-L-glutamic acid did not serve as substrates. Acid hydrolysis of GACC yielded ACC, suggesting that GACC is an amide-linked conjugate of ACC. Taken together, these results indicate that GACC is 1-(gamma-glutamylamino)cyclopropane-1-carboxylic acid and that its formation is catalyzed by a gamma-glutamyltranspeptidase. Gas chromatography-mass spectrometry analysis of the N-acetyl dimethyl ester of GACC confirmed this structure.     

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2

An aminopeptidase was purified from cell extracts of Lactococcus lactis subsp. cremoris AM2 by ion-exchange chromatography. After electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulfate, one protein band was detected. The enzyme was a 300-kilodalton hexamer composed of identical subunits not linked by disulfide bridges. Activity was optimal at 40 degrees C and pH 7 and was inhibited by classical thiol group inhibitors. The aminopeptidase hydrolyzed naphthylamide-substituted amino acids, as well as dipeptides and tripeptides. Longer protein chains such as the B chain of insulin were hydrolyzed, but at a much slower rate. The Michaelis constant (K(m)) and the maximal rate of hydrolysis (V(max)) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate l-histidyl-beta-naphthylamide. Amino acid analysis showed that the enzyme contained low levels of hydrophobic residues. The partial N-terminal sequence of the first 19 residues of the mature enzyme was determined. Polyclonal antibodies were obtained from the purified enzyme, and after immunoblotting, there was no cross-reaction between these antibodies and other proteins in the crude extract.     

Absorption and plasma kinetics of collagen tripeptide after peroral or intraperitoneal administration in rats

Collagen tripeptide (CTP) is a collagen-derived compound containing a high concentration of tripeptides with a Gly-X-Y sequence. In this study, the concentrations and metabolites of CTP were monitored in rat plasma after its administration. We performed a quantitative analysis using high-performance liquid chromatography tandem mass spectrometry according to the isotopic dilution method with stable isotopes. We confirmed that the tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Ala-Hyp were transported into the plasma. Dipeptides, which are generated by degradation of the N- or C-terminus of the tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Ala-Hyp, were also present in plasma. The plasma kinetics for peroral and intraperitoneal administration was similar. In addition, tripeptides and dipeptides were detected in no-administration rat blood. The pharmacokinetics were monitored in rats perorally administered with Gly-[³H]Pro-Hyp. Furthermore, CTP was incorporated into tissues including skin, bone, and joint tissue. Thus, administering collagen as tripeptides enables efficient absorption of tripeptides and dipeptides.