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L Juliano

Publications and source records attributed to L Juliano.

At least 109 records · Page 6Linked to original sources
PubMed1997

Characterization of kininogenase activity of an acidic proteinase isolated from human kidney.

An acidic proteinase was purified from human kidney cortex. The enzyme showed a molecular mass of 31 kDa by SDS-PAGE, 36 kDa by gel filtration, and isoelectric points of 5.2 and 6.1. The optimum pH for hydrolysis of bovine hemoglobin was about 3.5. Reverse-phase HPLC analysis of the incubation mixture of the enzyme with human plasma showed the presence of an active peptide on rat uterus muscle with the same retention time as the methionyl-lysyl-bradykinin (MLBK) standard. The specific activities were 2.91 micrograms MLBK equivalent mg-1.min-1 at pH 3.5 and 2.15 micrograms MLBK equivalent mg-1.min-1 at pH 6.0. All the enzymatic activities of this human kidney proteinase were inhibited by pepstatin A. Intramolecularly quenched fluorogenic substrates with amino acid sequences of human kininogen were used to determine the cleavage points. On the N-terminal sequences (Abz-Leu-Met-Lys-Arg-Pro-Eddnp and Abz-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Eddnp) the cleavage occurred at the Leu-Met linkage, and on the C-terminal sequences (Abz-Phe-Arg-Ser-Ser-Arg-Eddnp and Abz-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp) the cleavage occurred at the Arg-Ser linkage. Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp++ + was hydrolyzed by the renal acidic proteinase and yielded the peptide Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (Abz-bradykinin). Kinectic parameters were determined using Abz-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Eddnp (K(m) = 0.69 +/- 0.08 microM; Kcat = 0.052 +/- 0.0095 s-1; Kcat/K(m) = 0.075 +/- 0.005 microM-1.s-1) and Abz-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp (K(m) = 1.56 +/- 0.16 microM; Kcat = 0.0048 +/- 0.0001 s-1; Kcat/K(m) = 0.003 +/- 0.0003 microM-1.s-1). Human liver cathepsin D had no activity on C-terminal sequences and human pepsin hydrolyzed them at the Ser-Ser bond. The results suggest that the renal acid proteinase is distinct from human pepsin and human liver cathepsin D and releases MLBK from human kininogen.

Amino Acid Sequence↗
PubMed1996

A highly selective assay for neutral endopeptidase based on the cleavage of a fluorogenic substrate related to Leu-enkephalin.

An intramolecularly quenched fluorogenic peptide structurally related to Leu-enkephalin, containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-Gly-Gly-D-Phe-Leu-Arg-Arg-Val-EDDnp (Abz-GGDFLRRV-EDDnp), was selectively hydrolyzed at the Arg-Val bond by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) with kinetic parameters (Km = 3 microM, kcat = 127 min-1 and kcatsolidusKm = 42 min-1 microM-1) similar to those of the Leu-enkephalin. The specificity of the NEP assay was demonstrated by incubating Abz-GGDFLRRV-EDDnp with a kidney homogenate or with crude membrane preparations of brain and lung: more than 95% of all products released were the complementary fragments Abz-GGDFLRR and V-EDDnp which were totally inhibited by 1 microM thiorphan, a highly specific NEP inhibitor. The blocked amino- and carboxyl-terminal amino acids protected this substrate against the action of aminopeptidases as well as of carboxypeptidases. Furthermore, D-Phe amino acid also ensured a very good protection of Abz-GGDFLRRV-EDDnp against the action of other tissue endopeptidases distinct from NEP. A continuous fluorometric assay for only 5 min was sufficient to quantify the NEP activity with a minimum sensitivity of 5 ng of purified enzyme or the equivalent enzymatic activity in crude tissue preparations. Therefore, amounts as little as 0.5 ng of enzyme could be quantified employing longer times of incubation.

Amino Acid Sequence↗
PubMed1996

Inhibition of angiotensin converting enzyme and potentiation of bradykinin by retro-inverso analogues of short peptides and sequences related to angiotensin I and bradykinin.

There is pharmacological evidence indicating that, in addition to the inhibition of angiotensin converting enzyme (ACE; EC 3.4.15.1), the potentiation of bradykinin (BK) responses may also involve the BK receptor or some binding site in the structures involved in the contractile response to this peptide. Dipeptides such as Val-Trp and some of its analogues as well as tripeptide homologues, including total and partial retro-inverso peptides, were synthesized and assayed for their ability to inhibit purified guinea pig plasma ACE and to potentiate the action of BK on the isolated ileum of the same species. The peptides containing the P2-P1, P1-P'1, and P'1-P'2 inverted amide bonds inhibited ACE, were resistant to hydrolysis, and, depending on the amino acid composition, some of them potentiated the contractile response to BK while others did not. Des-[Arg1]-BK, which has an intrinsic activity at concentrations higher than 10(-5) M, and the very dissimilar angiotensin I (AI) analogue [Cys5-Cys10]-angiotensin-I-(5-10)-amide, which has no detectable contractile activity, were able to inhibit ACE and potentiate BK. In contrast to these peptides, BPP5a and BPP9a from Bothrops jararaca venom, and Potentiators B and C from Agkistrodon halys blomhoffi venom were more effective as BK potentiators than as ACE inhibitors. In conclusion, we have synthesized and assayed compounds that preferentially inhibit ACE, e.g. retro-inverso tripeptides, or potentiate the response of smooth muscle to BK, e.g. snake venom peptides.

Amino Acid Sequence↗
PubMed1996

Investigation of the substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, through the use of cystatin-derived substrates and inhibitors.

A panel of intramolecularly quenched fluorogenic substrates containing the conserved QVVA and LVG inhibitory sequences of cystatin inhibitors was used to describe the specificity of the major cysteine proteinase of Trypanosoma cruzi (cruzipain or cruzain). This approach was based on the observations that: (1) cruzipain is strongly inhibited by chicken cystatin and rat T-kininogen, two representative members of cystatin families 2 and 3; (2) the QVVA- and LVG-containing substrates are specifically hydrolysed by papain-like proteinases; and (3) the cystatin-like motifs are similar to the proteolytically sensitive sequences in cruzipain that separate the pro-region and/or the C-terminal extension from the catalytic domain. Specificity constants (kcat/Km) were determined and compared with those of mammalian cathepsins B and L from rat liver lysosomes. Cruzipain and the mammalian proteinases cleaved cystatin-derived substrates at the same site, but their specificities differed significantly. Increased specificity for cruzipain was obtained by replacing amino acids at critical positions on both sides of the cleavage sites, especially at position P2'. The specificity constants (k(cat)/Km) obtained for the two substrates with a prolyl residue at P2' (O-aminobenzoyl-QVVAGP-ethylenediamine 2-4-dinitrophenyl and O-aminobenzoyl-VVGGP-ethylenediamine 2-4-dinitrophenyl) were about 50 times higher for cruzipain than for rat cathepsin L and about 100 times higher than for cathepsin B. Diazomethylketone derivatives, based on the non-prime sequence of cystatin-derived substrates, inhibited cruzipain irreversibly, but their inactivation rate constants were considerably lower than those for mammalian cathepsins B and L, confirming the importance of P' residues for cruzipain specificity.

Amino Acid Sequence↗
PubMed1996

Met-Lys-bradykinin-Ser, the kinin released from human kininogen by human pepsin.

This study was carried out to show the site in kininogen, using synthetic substrates, that is cleaved by a purified human pepsin component with a molecular weight of 35,000 daltons. The study used 4 (four) intramolecularly quenched fluorogenic substrates containing N- and C-terminal sequences around the methionyl-lysyl-bradykinin (MLBK) region of kininogen: Abz-LMKRP-Eddnp, Abz-MISLMKRP-Eddnp, Abz-FRSSR-Eddnp and Abz-RPPGFSPFRSSRQ-Eddnp. The hydrolysis on N-terminal sequences Abz-LMKRP-Eddnp and Abz-MISLMKRP-EDDnp occurred at L-M linkage and on C-terminal sequences Abz-FRSSR-Eddnp, and Abz-RPPGFSPFRSSRQ-Eddnp occurred at S-S linkage. The released peptide Abz-RPPGFSPFRS was able to contract rat uterus muscle. The results suggest that Met-Lys-Bradykinin-Ser, should be the peptide released from human kininogen by a purified human pepsin component.

Amino Acid Sequence↗
PubMed1996

Pharmacological characterization of novel tissue kallikrein inhibitors in vivo.

In this study we have investigated the effect of novel tissue kallikreins on the plasma protein exudation induced by porcine pancreatic kallikrein (PPK) in the rabbit skin in vivo. The tissue kallikrein inhibitors here described were synthesized based on analogues of peptide substrates for tissue kallikreins. The intradermal injection of PPK and rabbit urinary kallikrein, but not of rabbit plasma kallikrein, significantly increased the microvascular permeability leading to local oedema formation in the rabbit skin. At the dose of 3-200 nmol/site, the intradermal co-administration of the tissue kallikrein inhibitors Bz-F-F-S-R-EDDnp (Ki = 0.1 microM; ESP5), PAC-F-S-R-EDDnp (Ki = 0.7 microM; ESP6), Bz-F-F-A-P-R-NH2 (Ki = 7.8 microM; ESP8), PAC-F-F-R-P-R-NH2 (Ki = 0.3 microM; ESP9) and Bz-F-F-S-R-NH2 (Ki = 0.3 microM; ESP11) dose-dependently inhibited the plasma protein exudation induced by PPK. The most potent compound was ESP6 (IC25 = 7.8 nmol/site) followed by ESP5 (IC25 = 14.2 nmol/site), ESP8 (IC25 = 25 nmol/site), ESP9 (IC25 = 30 nmol/site) and ESP11 (IC25 = 50.4 nmol/site). The compounds Bz-F-F-R-P-R-NH2 (Ki = 0.5 microM; ESP1), Bz-F-F-pNa (Ki = 0.4 microM; ESP3), Bz-F(NH2)-F-R-P-R-NH2 (Ki = 1.1 microM; ESP7) and Bz-F-F-S-P-R-NH2 (Ki = 4.6 microM; ESP10) had no significant effect on the PPK-induced plasma protein exudation in doses up to 200 nmol/site. ESP6 also inhibited the PPK-induced plasma protein exudation when administered systemically. This compound may constitute a useful tool to further investigate both the physiological and pathological role of tissue kallikreins.

Animals↗
PubMed1996

Liver bradykinin-inactivating-endopeptidase is similar to the metalloendopeptidase (EC 3.4.24.15).

The bradykinin-inactivating-endopeptidase (BIE) removal from rat liver, by perfusing the organ with 0.05% Triton X-100, achieved its maximum at 10 min of perfusion and falls to 50% of the maximum in 30 min, a pattern similar to AST removal. Using an internally quenched fluorescent BK analogue (Abz-RPPGFSPFRQ-EDDnp) we further characterized this enzyme: it is activated by low concentrations of 2-mercaptoethanol, inhibited by p-hydroxymercuribenzoate, o-phenanthroline and EDTA, and is resistant to enalapril, E-64 and PMSF. These results suggest that BIE is a metalloendopeptidase containing a thiol group important for its activity. BIE also hydrolyses the peptides Abz-GGFLRRVQ-EDDnp, Abz-GPQGLAGQ-EDDnp, Abz-FRSVQ-EDDnp, and Abz-ARVRRANSFLQ-EDDnp. All these properties are very similar to those described or assayed by us for EC 3.4.24.15, isolated initially from rat testes and then from several organs of different animals. Both BIE and EC 3.4.24.15: hydrolyze the F5S6 bond of the BK fluorescent substrate; are efficiently inhibited by Orlowski specific inhibitor (CFP-AAF-pAB, Ki 4.4 x 10(-7) M and 1.25 x 10(-7) M, respectively); have the same electrophoretic mobility in SDS-PAGE (Mr 78,000); and are both recognized by three polyclonal antibodies raised against rat testes EC 3.4.24.15. In conclusion, BIE appears to be EC 3.4.24.15.

Animals↗
PubMed1996

Identification of a domain of Trypanosoma cruzi metacyclic trypomastigote surface molecule gp82 required for attachment and invasion of mammalian cells.

Recombinant proteins and synthetic peptides representing various sequences of gp82, a surface glycoprotein of Trypanosoma cruzi metacyclic trypomastigotes implicated in mammalian cell invasion, were used in this study aiming at the identification of the domain(s) of this molecule required for interaction with target cells. Invasion of cultured HeLa cells by metacyclic trypomastigotes was inhibited by about 80% in the presence of native gp82 or the corresponding recombinant construct J18. Inhibition by recombinant proteins J18a and J18b, containing respectively the N-terminal and the C-terminal portions of gp82, was on the order of 30% and 65%. As compared to J18b (amino acids 224-516), the truncated gp82 fragments J18b1 (amino acids 303-516) and J18b2 (amino acids 357-516) displayed lower inhibitory effect (approximately 40% and approximately 15%, respectively). Compatible with these observations, we found that the recombinant protein J18b, but not J18a or J18b2, binds to HeLa cells in a dose-dependent and saturable fashion. Experiments with ten overlapping synthetic peptides, representing the gp82 portion spanning amino acids 224-333, showed that peptides 4 (amino acids 254-273) and 8 (amino acids 294-313) have significant inhibitory activity on HeLa cell invasion by metacyclic forms. All these results indicate that the portion of gp82 required for mammalian cell attachment and invasion is located in the central domain of the molecule.

Amino Acid Sequence↗
PubMed1996

Bleeding time does not predict gastrointestinal bleeding in patients with cirrhosis. The CALC Group. Coagulation Abnormalities in Liver Cirrhosis.

BACKGROUND/AIMS: Bleeding time, a laboratory test which explores primary hemostasis, may be prolonged in cirrhosis, but whether abnormal bleeding time identifies patients with cirrhosis who are at risk of bleeding has never been investigated. The aim of this study was to analyze the relationship between bleeding time and the risk of gastrointestinal bleeding. METHODS: Eighty consecutive patients with liver cirrhosis (47 males, 33 females; age, 60 +/- 9 years; range 31 to 83 years) and esophageal varices were enrolled in the study. RESULTS: In the whole series of patients bleeding time was 11 +/- 6 min; it increased as the degree of liver deficiency increased, from low to severe (p = 0.007). During 14 +/- 9 (median (range): 12 (1-34) months of follow-up, 28 (35%) patients experienced gastrointestinal bleeding. They had a longer bleeding time, higher incidence of previous bleeding, more severe liver failure and larger variceal size than patients who did not bleed. However, multivariate analysis (Cox's model) showed that only previous bleeding, liver failure and variceal size were independently associated with bleeding. Similar data were obtained in patients with moderate-severe liver insufficiency (B and C degree according to Child-Pugh's classification). In patients who had never bled (n = 54), the severity of liver failure and variceal size were independent predictors of bleeding. CONCLUSIONS: This study shows that bleeding time is not a predictor of gastrointestinal bleeding in patients with cirrhosis.

Adult↗
PubMed1996

Kinetic characterization of rat tissue kallikrein using N alpha-substituted arginine 4-nitroanilides and N alpha-benzoyl-L-arginine ethyl ester as substrates.

Hydrolysis of seven N alpha-substituted L-arginine 4-nitroanilides: benzoyl-arginine p-nitroanilide (Bz-Arg-Nan), tosyl-arginine p-nitroanilide (Tos-Arg-Nan), acetyl-leucyl-arginine p-nitroanilide (Ac-Leu-Arg-Nan), acetyl-phenylalanyl-arginine p-nitroanilide (Ac-Phe-Arg-Nan), benzoyl-phenylalanyl-arginine p-nitroanilide (Bz-Phe-Arg-Nan), tosyl-phenylalanyl-arginine p-nitroanilide (Tos-Phe-Arg-Nan), and D-valyl-leucyl-arginine p-nitroanilide (D-Val-Leu-Arg-Nan), and the N alpha-substituted L-arginine ester: benzoyl-arginine ethyl ester (Bz-Arg-OEt), by rat tissue kallikrein was studied throughout a wide range of substrate concentrations. The enzyme showed a bimodal behavior with all the substrates tested except Tos-Arg-Nan. At low substrate concentrations (10 to 170 microM for p-nitroanilides and 50 to 190 microM for Bz-Arg-OEt) the hydrolysis followed Michaelis-Menten kinetics, but at higher substrate concentrations (150 to 700 microM for p-nitroanilides and 200 to 1800 microM for Bz-Arg-OEt) a deviation from Michaelis-Menten kinetics was observed with a significant decrease in hydrolysis rates. At high concentrations of the p-nitroanilide substrates, partial enzyme inhibition was observed, whereas complete enzyme inhibition was observed with Bz-Arg-OEt at high concentration. The kinetic parameters reported here were calculated from data for substrate concentrations range where the enzyme followed Michaelis-Menten behavior. D-Val-Leu-Arg-Nan (Km = 24 +/- 2 microM; Vmax = 10.42 +/- 0.28 microM/min) was the best substrate tested, followed by Ac-Phe-Arg-Nan (Km = 13 +/- 2 microM; Vmax = 3.21 +/- 0.11 microM/min), while Tos-Arg-Nan (Km = 29 +/- 2 microM; Vmax = 0.10 +/- 0.002 microM/min) was the worst of the tested substrates for rat tissue kallikrein. For the hydrolysis of Bz-Arg-OEt (Km = 125 +/- 15 microM; Vmax = 121.3 +/- 7.6 microM/min), the kinetic parameters using a substrate inhibition model can reasonably account for the observed enzyme behavior, with a Ksi value about 13.6 times larger than the estimated Km value.

Animals↗
PubMed1995

Evaluation of the extent of the binding site in human tissue kallikrein by synthetic substrates with sequences of human kininogen fragments.

We have synthesized internally quenched peptides spanning the Met379-Lys380 or Arg389-Ser390 bonds of human kininogen (hkng) that flank lysyl-bradykinin and have studied the kinetics of their hydrolysis by human tissue kallikrein. The kinetic data for the hydrolysis of the Met-Lys bond in substrates with an N-terminal extension showed that interactions up to position residue P10 contribute to the efficiency of cleavage. In contrast, there were no significant variations in the kinetic data for the hydrolysis of substrates with C-terminal extensions at sites P'4 to P'11. A similar pattern was observed for the cleavage of substrates containing an Arg-Ser bond because substrates extended up to residue P6 were hydrolysed with the highest kcat/Km values in the series, whereas those extended to P'11 on the C-terminal side had a lower susceptibility to hydrolysis. Time-course studies of hydrolysis by human and porcine tissue kallikreins of a Leu373 to Ile393 human kininogen fragment containing omicron-aminobenzoic acid (Abz) at the N-terminus and an amidated C-terminal carboxyl group Abz-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg- Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Ile-NH2 (Abz-[Leu373-Ile393]-hkng-NH2) indicated that the cleavage of Met-Lys and Arg-Ser bonds in the same molecule occurs via the formation of independent enzyme-substrate complexes. The hydrolysis of Abz-F-R-S-S-R-Q-EDDnp [where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine] and Abz-M-I-S-L-M-K-R-P-Q-EDDnp by human tissue kallikrein had maximal kcat/Km values at pH 9-9.5 for both substrates. The pH-dependent variations in this kinetic parameter were almost exclusively due to variations in kcat. A significant decrease in kcat/Km values was observed for the hydrolysis of Arg-Ser and Met-Lys bonds in the presence of 0.1 M NaCl. Because this effect was closely related to an increase in Km, it is likely that sodium competes with the positive charges of the substrate side chains for the same enzyme subsites.

Amino Acid Sequence↗
PubMed1995

Purification and characterization of a dynorphin-processing endopeptidase.

Dynorphin B (Dyn B-13, also known as rimorphin) is generated from Dyn B-29 (leumorphin) by the cleavage at a single Arg residue. An enzymatic activity capable of processing at this monobasic site has been previously reported in neurosecretory vesicles of the bovine pituitary and pituitary-derived cell lines. This enzyme termed "the dynorphin-converting enzyme" (DCE) has been purified to apparent homogeneity from the neurointermediate lobe of the bovine pituitary using hydrophobic chromatography on phenyl-Sepharose, preparative isoelectrofocusing in a granulated gel between pH 4 to 6.5, and non-denaturing electrophoresis on 5% polyacrylamide gel. DCE exhibits a pI of about 5.1 and a molecular mass of about 54 kDa under reducing conditions. DCE is a metallopeptidase and exhibits a neutral pH optimum. Specific Inhibitors of soluble metallopeptidases such as enkephalinase (EC 3.4.24.11) or enkephalin generating neutral endopeptidase (EC 3.4.24.15) do not inhibit DCE activity indicating that DCE is distinct from these two enzymes. Cleavage site determination with matrix-assisted laser desorption ionization time of flight (MALDITOF) mass spectrometry shows that DCE cleaves the Dyn B-29 N terminus to the Arg14 generating Dyn B-13 and Dyn B-(14-29). Among other peptides derived from Dyn B-29, DCE cleaves only those peptides that fit the predicted "consensus motif" for monobasic processing. These data are consistent with a broader role for the dynorphin converting enzyme in the biosynthesis of many peptide hormones and neuropeptides by processing at monobasic sites.

Amino Acid Sequence↗
PubMed1995

Characterization of an exocellular serine-thiol proteinase activity in Paracoccidioides brasiliensis.

An exocellular proteinase activity has been characterized in Paracoccidioides brasiliensis culture filtrates. Chromatographic analysis showed that the activity was eluted from an anion-exchange Resource Q column at 0.08-0.1 M NaCl, and by gel filtration near ovalbumin elution, in a single peak. Purification of the proteinase, however, was hampered by the low protein yield, in contrast to the high peptidase activity. Numerous chromogenic peptidyl p-nitroanilide derivatives and internally quenched fluorescent peptides, flanked by Abz (O-aminobenzoyl) and EDDnp (ethylenediaminedinitrophenyl), were tested as substrates. Cleavage was observed with Abz-MKRLTL-EDDnp, Abz-FRLVR-EDDnp, and Abz-PLGLLGR-EDDnp at Leu-Thr, Leu-Val and Leu-Leu/Leu-Gly bonds respectively as determined by isolation of the corresponding fragments by HPLC. Leucine at P1 seemed to be restrictive for the activity of the exocellular enzyme, but threonine (P'1) and leucine (P'2) in Abz-MKRLTL-EDDnp apparently were not essential. Also, a pair of alanines could substitute for lysine (P3) and arginine (P2) in this substrate, with a decrease in the Km values. The exocellular peptidase activity of P. brasiliensis had an optimum pH of > 9.0 and was irreversibly inhibited by PMSF, mercuric acetate and p-hydroxymercuribenzoate. Inhibition of the mercuriate compounds could be partially reversed by Cys/EDTA. E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanido)butene] was a weak and reversible inhibitor, whereas EDTA and pepstatin were not inhibitory. These results suggest that P. brasiliensis exocellular enzyme belongs to the subfamily of SH-containing serine proteinases.

Amino Acid Sequence↗
PubMed1995

Determinants of the unusual cleavage specificity of lysyl-bradykinin-releasing kallikreins.

Kinetic data for the hydrolysis by human tissue kallikrein of fluorogenic peptides with o-aminobenzoyl-Phe-Arg (Abz-FR) as the acyl group and different leaving groups demonstrate that interactions with the S'1, S'2 and S'3 subsites are important for cleavage efficiency. In addition, studies on the hydrolysis of fluorogenic peptides with the human kininogen sequence spanning the scissile Met-Lys bond [Abz-M-I-S-L-M-K-R-P-N-(2,4-dinitrophenyl)ethylenediamine] and analogues with different residues at positions P'1, P'2 and P'3 showed that (a) the presence of a proline residue at P'3 and the interactions with the tissue kallikrein-binding sites S2 to S'2 are determinants of Met-Lys bond cleavage and (b) residues P3, P4 and/or P5 arc important for cleavage efficiency. The substitution of phenylalanine for methionine or arginine in substrates with scissile Met-Lys or Arg-Xaa bonds demonstrated that lysyl-bradykinin-releasing tissue kallikreins also have a primary specificity for phenylalanine. The replacement of arginine by phenylalanine in (D)P-F-R-p-nitroanilide (pNA) produced an efficient and specific chromogenic substrate, (D)P-F-F-pNA, for the lysyl-bradykinin-releasing tissue kallikreins as it is resistant to plasma kallikrein and other arginine hydrolases.

Amino Acid Sequence↗
PubMed1995

Conserved cystatin segments as models for designing specific substrates and inhibitors of cysteine proteinases.

Peptide segments derived from consensus sequences of the inhibitory site of cystatins, the natural inhibitors of cysteine proteinases, were used to develop new substrates and inhibitors of papain and rat liver cathepsins B, H, and L. Papain hydrolyzed Abz-QVVAGA-EDDnp and Abz-LVGGA-EDDnp at about the same rate, with specificity constants in the 10(7) M-1 sec-1 range; cathepsin L also hydrolyzes both substrates with specificity constants in the 10(5) M-1 sec-1 range due to lower k(cat) values, with the Km's being identical to those with papain. Only Abz-LVGGA-EDDnp was rapidly hydrolyzed by cathepsin B, and to a lesser extent by cathepsin H. Peptide substrates that alternate these two building blocks (LVGGQVVAGAPWK and QVVAGALVGGAPWK) discriminate the activities of cathepsins B and L and papain. Cathepsin L was highly selective for cleavage at the G-G bond of the LVGG fragment in both peptides. Papain and cathepsin B cleaved either the LVGG fragment or the QVVAG fragment, depending on their position within the peptide. While papain was more specific for the segment located C-terminally, cathepsin B was specific for that in N-terminal position. Peptidyl diazomethylketone inhibitors based on these two sequences also reacted differently with papain and cathepsins. GlcA-QVVA-CHN2 was a potent inhibitor of papain and reacted with papain 60 times more rapidly (k + 0 = 1,100,000 M-1 sec-1) than with cathepsin L, and 220 times more rapidly than with cathepsin B. Cathepsins B and L were preferentially inhibited by Z-RLVG-CHN2. Thus cystatin-derived peptides provide a valuable frame-work for designing sensitive, selective substrates and inhibitors of cysteine proteinases.

Amino Acid Sequence↗
PubMed1995

Proteolytic specificity of two hemorrhagic factors, LHF-I and LHF-II, isolated from the venom of the bushmaster snake (Lachesis muta muta).

Two hemorrhagic metalloproteinases (LHF-I and LHF-II) were previously isolated from Lachesis muta muta (bushmaster snake) venom. The proteolytic activities of these hemorrhagic factors and of the crude venom were investigated using as substrate the oxidized B-chain of bovine insulin. LHF-II cleaves the Ala14-Leu15 bond of insulin B-chain very rapidly and the Phe24-Phe25, His10-Leu11 and His5-Leu6 more slowly, whereas LHF-I hydrolyzed only the Ala14-Leu15 bond. Both hemorrhagic factors cleaved the Leu-Leu bond in the fluorogenic peptide Abz-Pro-Leu-Gly-Leu-Leu-Gly-Arg-EDDnp. When the insulin B-chain was incubated with crude venom previously treated with 2.5 mM PMSF, the Ala14-Leu15 bond was also rapidly cleaved. In addition, the hemorrhagic activity and the digestion of casein remained unaltered. Both hemorrhagic and proteolytic activities were inhibited when the crude venom was treated with EDTA, confirming that only metalloproteinases are responsible for these activities. The hydrolysis of insulin B-chain and the fluorogenic heptapeptide by these proteinases was found to be in inverse relationship to their hemorrhagic activities.

Amino Acid Sequence↗
PubMed1995

Isolation and characterization of a new bradykinin potentiating octapeptide from gamma-casein.

Peptides that display bradykinin-potentiating activity have been obtained from a number of distinct sources, such as snake venoms, fibrinogen, and casein. This paper describes the isolation and sequencing of a novel bradykinin-potentiating peptide, generated by tryptic hydrolysis of the gamma-casein chain. No homology was found to other known vasoactive or vasopotentiating peptides. The octapeptide Tyr-Pro-Val-Gln-Pro-Phe-Thr-Glu, corresponding to the gamma-casein(114-121) sequence, was isolated from the tryptic hydrolysis of gamma-casein and also synthesized by solid-phase peptide synthesis. Both natural and synthetic peptides had the same retention time in HPLC and displayed a selective potentiating activity on isolated guinea-pig ileum for bradykinin and Lys-bradykinin but were not able to potentiate the effects of Met-Lys-bradykinin, Ile-Ser-bradykinin, angiotensin II, acetylcholine, or histamine. Intravenous injections of bradykinin and of bradykinin-potentiating octapeptide produced a persistent hypotension in conscious rats, a pattern that was not obtained when the octapeptide was replaced by captopril. This bradykinin-potentiating octapeptide is a strong competitive inhibitor of endo-oligopeptidase A (EC 3.4.24.15, formerly EC 3.4.22.19), but it has low inhibitory potency towards angiotensin-converting enzyme (EC 3.4.15.1). Thus, our results suggest that other peptidases in addition to angiotensin-converting enzyme, such as endo-oligopeptidase A, may contribute to the reduction of the effective concentration of bradykinin in the circulation.

Amino Acid Sequence↗