Bioactivity studies on new gastrin analogues.

Gastrin antagonists may be useful in the treatment of peptic ulcer disease and gastrointestinal malignancies. The aim of the present study was to synthesize gastrin analogues and test them for their ability to inhibit gastrin-stimulated acid secretion. Five peptides were synthesized: peptide [2], in which methionine was replaced by leucine, and the COOH-terminal amide was replaced by the thiomethylamide; peptide [3], in which the COOH-terminal phenylalanine was removed, and the aspartic acid thioamidated; peptide [5], in which methionine was replaced by leucine, and the peptide bond between leucine and aspartic acid was replaced by a thioamide; peptide [7], in which the bond between leucine and aspartic acid was replaced by a ketomethylene amino bond; and, finally, peptide [8], in which a beta-bend was induced in the COOH-terminal region by the introduction of a D-phenylalanine in place of glycine. The biologic effect of the peptides was tested in the totally isolated, vascularly perfused rat stomach. The peptides were tested in concentrations of 10(-9), 10(-7), and 10(-5) M for agonist activity and together with gastrin 1-17, 5.2 X 10(-10) M, at a concentration of 10(-5) M for antagonist activity. Peptide [2] had full biologic activity but greatly reduced potency, and peptide [7] had a faint biologic activity. None of the peptides showed any antagonist activity.

The behaviour of leucine aminopeptidase towards thionopeptides.

Thionoleucine S-anilide (Leut-anilide), Leut-Gly-OEt and Leut-Phe-OMe were synthesized and shown to be competitive inhibitors of leucine aminopeptidase from pig kidney. The kinetics of inhibition were determined in the presence of leucine 4-methylcoumarin-7-amide as substrate. Although the compounds showed only moderate inhibitory potency, it was found that all were resistant to hydrolysis by the enzyme, in contrast with the reported behaviour of some thionopeptide analogues of substrates for other Zn2+-peptidases such as carboxypeptidase A and angiotensin-converting enzyme.

An improved spectrophotometric assay for leucine aminopeptidase.

A sensitive assay to determine the activity of leucine aminopeptidase (EC 3.4.11.1), using L-leucine thiobenzyl ester as substrate, was developed. Hydrolysis of the ester by leucine aminopeptidase can be monitored in the presence of 5,5-dithiobis-(2-nitrobenzoic acid) by continuous spectrophotometric measurement at 412 nm. Comparison with some amide substrates showed that the thiol ester provides a much more sensitive assay, its specificity constant (Vmax./Km) being some 3000-fold higher than that of leucine p-nitroanilide.

The degradation of bovine and human prothrombin by human polymorphonuclear leukocyte cathepsin G.

Cathepsin G, isolated from human polymorphonuclear leukocytes, was found to effect rapid and specific degradation and biological inactivation of bovine and human prothrombin in the absence of calcium ions with the formation of two peptide fragments from the N-terminal end of the molecule. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the molecular weights of the fragments were 5,000 and 17,500. Proteolysis of prothrombin by cathepsin G was inhibited by calcium ions. Leukocyte proteinases such as cathepsin G may be responsible for haemorrhagic disorders associated with myelocytic leukaemia and septicaemia.

Proteolysis of bovine and human prothrombin and of bovine factor X by rat mast cell proteinase.

The alpha-chymotrypsin-like proteinase from rat peritoneal mast cells (RMCP I) rapidly destroyed the normal clotting activity of purified, calcium-free, bovine prothrombin, human prothrombin and bovine factor X and simultaneously removed similar N-terminal peptides (Mr approximately 5,000) from both prothrombin and the 'light' chain of factor X. The amino acid composition of the peptides agreed with the known composition of the regions of the respective parent molecules where gamma-carboxyglutamic acid residues are situated. Ca2+ ions protected each of the proteins from proteolysis and loss of procoagulant activity. Prolonged incubation in the standard physiological assay medium used for prothrombin or treatment with Echis carinatus venom indicated that the thrombogenic portion of prothrombin survived proteolysis by RMCP I. This restricted proteolysis was confirmed by electrophoretic analysis.

Competitive inhibition of human skin collagenase by N-benzyloxycarbonyl-L-prolyl-L-alanyl-3-amino-2-oxopropyl-L-leucyl-L- alanylglycine ethyl ester.

A 'ketomethylene' peptide, N-benzyloxycarbonyl-L-prolyl-L-alanyl-3-amino- 2-oxopropyl-L-leucyl-L-alanylglycine ethyl ester, was synthesized and shown to be a fairly potent competitive inhibitor of human skin collagenase.

The irreversible inhibition of urokinase, kidney-cell plasminogen activator, plasmin and beta-trypsin by 1-(N-6-amino-n-hexyl)carbamoylimidazole.

1-(N-Amino-n-hexyl)carbamoylimidazole hydrochloride was synthesized and shown to be a potent irreversible inhibitor of human urokinase (EC 3.4.21.31), pig kidney-cell plasminogen activator (EC 3.4.21.-), human plasmin (EC 3.4.21.7) and bovine pancreatic beta-trypsin (EC 3.4.21.4). The kinetics of inhibition of the enzymes were determined by monitoring the hydrolysis of an appropriate fluorogenic substrate. Bovine thrombin and Factor Xa are hardly affected by the inhibitor.

The behaviour of urokinase and porcine kidney cell plasminogen activator towards some synthetic peptides.

The behaviour of human urokinase and porcine kidney cell plasminogen activator towards some synthetic substrates has been investigated. Although N- benzyloxycarbonylglycylglycyl -L-arginine 4-methyl-7- coumarylamide (Z-Gly-Gly-Arg-Amc) (I), glutaryl-Gly-Arg-Amc (II) and Z-Gly-Gly-Arg-Val-OMe (III) were substrates, Boc-Gly-Gly-Arg-Val-Val-Gly-Gly-OEt (IV) and Z-Ala-Pro-Gly-Arg-Val-Val-Gly-Gly-OEt (V) were neither substrates nor inhibitors. Steady-state kinetic parameters for the hydrolysis of (II) and (III) by urokinase and porcine kidney cell plasminogen activator were similar.

A recording viscometer for assaying mammalian collagenase.

A recording viscometer for monitoring the action of mammalian collagenase on soluble collagen is described. For this system, where only one peptide bond is cleaved per subunit, it is shown theoretically that the decrease in viscosity is proportional to the fraction of molecules cleaved. Experimental confirmation was obtained by parallel monitoring of hydrolysis by using the fluorescamine assay of liberated amino groups. The initial velocity of reaction is proportional to substrate concentration and enzyme concentration.

The kinetics of hydrolysis of some synthetic substrates containing neutral hydrophilic groups by pig pepsin and chicken liver cathepsin D.

1. Several peptides containing either of the sequences -Phe(NO2)-Trp- and -Phe(NO2)-Phe- and an uncharged hydrophilic group were synthesized, and the steady-state kinetics of their hydrolysis by pig pepsin (EC 3.4.23.1) and chicken liver cathepsin D (EC 3.4.23.5) were determined. Despite the presence of a hydrophilic group to increase substrate solubility, it was not possible to achieve the condition [S]0 much greater than Km, and, in some cases, only values of kcat./Km could be determined by measuring the first-order rate constant when [S]0 much less than Km. 2. Occupancy of the P2 and P3 sites considerably enhanced the specificity constant, and alanine was more effective than glycine at site P2. 3. The specificity constants for the hydrolysis by pepsin of those substrates in the present series that contain an amino acid residue at site P3 are considerably lower than for comparable substrates containing a cationic group. This difference does not apply to cathepsin D. 4. Hydrolyses with cathepsin D commonly exhibited a lag phase, and a possible explanation for this is given.

Faecal bile acid concentration of patients with carcinoma or increased risk of carcinoma in the large bowel.

Faecal 3-hydroxy bile acids were assayed enzymatically in patients with carcinoma, or at increased risk of developing carcinoma of the large bowel. No rise in bile acid concentration was demonstrated in patients with ulcerative colitis, previously resected adenoma, or resected carcinoma. Patients with carcinoma, before treatment, had faecal bile acid concentrations similar to control values, and surgery did not affect the mean level. These findings cast doubt on the importance of the 3-hydroxy bile acid concentration in the faeces in the pathogenesis of large bowel cancer.

The esterolytic specificity of bovine thrombin and Factor Xa.

Steady state kinetics are compared for the hydrolysis of t-butoxycarbonyl-L-lysine methyl ester and several peptidyl lysine methyl esters catalysed by bovine thrombin and Factor Xa. Thrombin-catalysed reactions have lower Km values and higher kcat/Km values than do reactions catalysed by Factor Xa. Values of kcat are comparable and do not show any particular trend. The best substrate in the present series was t-butoxycarbonylglycylglycyl-L-lysine methyl ester. Thrombin and Factor Xa may possess a hydrophobic region near the P2 binding site which is unfavourable for either asparagine or D-alanine but which readily accommodates glycine, L-alanine or L-phenylalanine. The major improvement in Factor Xa hydrolysis occurred with the occupation of the P2 site by an amino residue while for thrombin the major improvement occurred with the occupation of the P3 site.

The hydrolysis of chemically modified proteins by Factor Xa and thrombin.

Bovine thrombin and Factor Xa were shown to hydrolyse slowly several chemically modified proteins. Both enzymes hydrolyse the proteins at trypsin-susceptible bonds, with arginine, lysine or the synthetically generated S-(beta-aminoethyl)cysteine at the P1 position. Both enzymes, however, cleave at far fewer sites than trypsin. The presence of highly polar groups in the P2 position appears to hinder hydrolysis by Factor Xa or thrombin. The presence of hydrophobic or neutral amino acids around this site may make the site more susceptible to hydrolysis. Differences in the hydrolysis patterns between thrombin and Factor Xa are observed.