Eline S. Prado

Intramolecularly quenched fluorogenic tetrapeptide substrates for tissue and plasma kallikreins

Five intramolecularly quenched fluorogenic substrates for arginyl hydrolases with the sequence Abz-Phe-Arg-X-Y-EDDnp (X = Arg or Ser; Y = Val, Pro, or Arg) were synthesized by classical solution methods. Kinetics of their hydrolysis by tissue and plasma kallikreins, trypsin, and thrombin characterized Abz-Phe-Arg-Ser-Arg-EDDnp as a specific and sensitive substrate for the continuous assay of tissue kallikreins while Abz-Phe-Arg-Arg-Pro-EDDnp was the best substrate for human plasma kallikrein. The five peptides were poor substrates for trypsin and resistant to thrombin.

Kinin-converting aminopeptidase from human serum

Abstract A kinin-converting aminopeptidase from human serum (HuPA) which converts kallidin (lysylbradykinin, LBK) to bradykinin (BK) was purified about 900-fold by a three-step procedure involving ammonium sulfate fractionation, continuous loading Sephadex gel electrophoresis, and gel filtration on Sephadex G-200; the final yield was about 7 per cent. A bioassay was developed in which BK formed in the presence of excess LBK or methionyllysylbradykinin (MLBK), pH 7.8, 37°, was separated on carboxymethylcellulose columns and assayed on the isolated guinea pig ileum. L -Aminoacyl-β-naphthylamidase activities of HuPA measured on L -lysine, L -arginine- and L -leucylnaphthylamides (Lys-NA, Arg-NA and Leu-NA) were parallel to the kinin-converting activity in all purification steps. The activity of the purest preparation on LBK and MLBK, measured at the approximate enzyme:substrate molar ratio 1:1500, was respectively 267 and 174 nmoles BK/min/mg of protein. Considering the activity on Met-NA as 100, the activities on other naphthylamides were: Leu-NA, 65; Arg-NA, 46; Lys-NA, 29; Asp-NA, 0; Glu-NA, 0. A few tri- and dipeptides, angiotensin II and its amide, polylysine and polyarginine were poorer substrates. The following substances were inhibitors: 1,10-phenanthroline, puromycin, EDTA and 2,3-dimercapto-l-propanol. 1,10-Phenanthroline or EDTA inhibition could be partially reversed by Co 2+ , Mn 2+ and Zn 2+ . The molecular weight was estimated as 95,000 on Sephadex gel filtration. On agarose gel microelectrophoresis, the enzyme migrated as α 1 -globulin before the electrophoresis step and had a migration intermediate between α 2 - and β -globulin following this step. In this microelectrophoresis, only one band of arylamidase activity was detected on Lys-, Arg- and Leu-NA. The purest preparation was still contaminated with albumin but was free of kininase activity.

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.

Kinin-converting aminopeptidase from human urine partial purification and properties

Abstract 1. 1. An aminopeptidase from human urine, which hydrolyses dipeptides and β-naphthylamides of neutral and basic amino acids and which converts the peptides lysylbradykinin and methionyllysyl-bradykinin into bradykinin, was highly purified by a four-step procedure. 2. 2. The enzyme (mol. wt 100,000) has several similarities with kinin-converting aminopeptidases found in human serum and liver, and is inhibited by 1,10-phenanthroline and puromycin.

Kallidin (lysylbradykinin), the kinin formed from horse plasma by horse urinary kallikrein

Abstract Horse urinary kallikrein when incubated with horse plasma formed kallidin (lysylbradykinin) from the kininogens in the plasma. Horse plasma, like human plasma, was found to contain an aminopeptidase capable of converting kallidin to bradykinin. No evidence, however, could be found that the plasma contained an aminopeptidase capable of converting Met-Lys-bradykinin to kallidin, thus eliminating the possibility that the kallikrein had released Met-Lys-bradykinin which was converted to kallidin during the 1–5 min incubations. The method used for identification of the kinins is rapid, gives a good recovery and requires small amounts of plasma and enzyme.

Proteolysis of salmine by horse urinary kallikrein

Abstract Purified horse urinary kallikrein preparations, free from hemoglobin and casein-splitting activities, were shown to hydrolyze protamine sulfate to a lesser extent than trypsin. This proteolytic activity is not inhibited by ovomucoid, whereas it is arrested by Trasylol. The proteolysis of salmine by this kallikrein parallels its kininogenic (kinin-liberating) activity. It is suggested that the presence of arginyl bonds in salmine may be significant for the observed proteolysis produced by this kallikrein.

Receptor-Mediated Clearance of Tissue Kallireins by Rat Liver

The exsanguinated, isolated and perfused rat liver clears from the perfusate, at comparable rates, some native tissue kallikreins: human and horse urinary as well as hog pancreatic; the clearance rates were dependent on the initial enzyme concentration in the perfusing fluid. Contrary-wise, rat urinary kallikrein was cleared at negligible rates. Neuraminidase pretreatment of these four kallikreins did not alter their clearance rates. Horse urinary kallikrein binding to isolated prefixed hepatocytes was calcium-dependent and inhibited by asialofetuin (but not by fetuin) and some sugars; these characteristics are compatible with the interpretation that this native tissue kallikrein is recognized by the hepatocyte asialoglycoprotein-receptor. It was calculated that there are about 300,000 receptor sites per cell either using perfusion experiments at 4 degrees C or isolated hepatocytes.

Active-site labelling of kallikreins by chloromethylketone derivatives.

Ala-Phe-Lys-CH2-Cl is a chloromethylketone derivative which is able to promote the inhibition of several proteolytic enzymes. In this paper the inhibition of horse urinary and plasmatic kallikreins is described and this inhibition is compared to that produced in human plasma kallikrein. This compound was designed based upon the structure of bradykinin. This enzyme substrate system can provide a model for the study of the interactions between bradykinin and its receptor. The inhibition of the enzymes was achieved both for its esterase and kinin-releasing activities.

Bothropain: A novel cysteine peptidase from plasma of the snake Bothrops jararaca

Abstract 1. 1. Bothropain, a novel cysteine peptidase isolated from Bothrops jararaca plasma, was shown to be a 780 kDa glycoprotein, not dissociated by SDS. 2. 2. Inhibition of bothropain was complete with low M r cysteine proteinase inhibitors but only partial with kininogens. l -trans-epoxysuccinyl- l -leucylamido-(4-guano)-butane was used for the enzyme active site titration. 3. 3. Primary specificity of bothropain for arginine and lysine was demonstrated in the hydrolysis of peptidyl-4-nitroanilides and oligopeptides. Protein substrates for bothropain were not found yet.

Further characterization of bothropain, a cysteine peptidase from the plasma of the snake Bothrops Jararaca

Abstract 1. 1. A new procedure was developed for the isolation of bothropain from Bothrops jararaca plasma. The previously used proteolytic digestion step was abolished and HPLC was introduced for the final purification. 2. 2. Comparison of enzyme prepartions obtained by the two procedures demonstrated a weak proteolysis of bothropain by trypsin, which had no effect on its catalytic properties. 3. 3. The primary specificity of bothropain for basic amino acids was confirmed and extended to the S -benzyl-cysteinyl residue. The enzyme showed a strong specificity for hydrophobic groups at P 2 , with a marked preference for Leu over Phe. No hydrolysis of oxidized insulin B chain by bothropain was detected. Binding of peptidyl diazomethane was also favored by hydrophobic residues at P 2 but a restricted specificity for P 1 was not observed. 4. 4. The catalytic properties, and the inhibition pattern by diazomethanes, indicate a similarity between bothropain and cathepsin L.