Bz-Tyr-Oet

Bz-Tyr-Oet

Detection of a trypsin-like serine protease and its endogenous inhibitor in hake skeletal muscle.[Pubmed:1898057]

Arch Biochem Biophys. 1991 Aug 15;289(1):1-5.

When dialyzed extracts from hake (Merluccius hubbsi) skeletal muscle were chromatographed in DEAE-Sephacel, an alkaline protease (37 degrees C, pH 8.5) and a trypsin inhibitor were isolated. The enzyme showed its maximal activity against azocasein in the range of pH between 7 and 9. The protease was able to hydrolyze the trypsin substrates Bz-Arg-OEt and Tos-Arg-OMe and did not cleave the chymotrypsin substrate Bz-Tyr-Oet. The enzyme was strongly inhibited by several serine protease inhibitors, whereas inhibitors of the other types of proteases scarcely affected it. The protease was able to degrade the major contractile and cytoskeletal constituent proteins of myofibrils and to accumulate acid-soluble products. The protease activity was completely suppressed by the addition of the trypsin inhibitor isolated from the same muscle. These results indicate that hake skeletal muscle contains a trypsin-like serine protease which might be involved in the catabolism of myofibrillar proteins, as well as in the proteolytic events that take place during post mortem storage of fish muscle.

Application of carboxypeptidase C for peptide synthesis.[Pubmed:1366863]

Enzyme Microb Technol. 1990 Nov;12(11):836-40.

Carboxypeptidase C partially purified from the flavedo of citrus fruit by a new, simple procedure was studied as a catalyst for peptide-bond formation. Dipeptides were obtained in high yields (80-95%) with Bz--Tyr--OEt as carboxyl-compound, and amino acid amides and amino acid alkylesters as nucleophiles. To characterize the synthesis reaction, a number of parameters such as pH, excess of the nucleophile, and the molarity of the buffer were evaluated. The yield of dipeptides depends on the side chain of the amino acid alkylester used as the carboxyl component as well as on the N-terminal protecting group. Esterase activity was minimal in the absence of a nucleophile, suggesting a modified mechanism for the synthesis reaction compared to other serine proteases. No secondary hydrolysis of the peptides formed was observed.

Substrate specificity of honeydew melon protease D, a plant serine endopeptidase.[Pubmed:9301107]

Biosci Biotechnol Biochem. 1997 Aug;61(8):1277-80.

The substrate specificity of honeydew melon (Cucumis melo var. inodorus Naud) protease D was studied by the use of synthetic substrates and oligopeptides derived from a protein hydrolyzate. The hydrolysis rates of succinyl-(L-Ala)1-3-p-nitroanilide (Suc-(Ala)1-3-pNA) the hydrolysis rate progressively rose in proportion to the increased chain length. Benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester (Z-Tyr-ONp) and benzoyl-L-tyrosine ethyl ester (Bz-Tyr-Oet) were cleaved by honeydew melon protease D, but benzoyl-L-arginine p-nitroanilide (Bz-Arg-pNA), benzyloxycarbonyl-L-lysine p-nitrophenyl ester (Z-Lys-ONp) and tosyl-L-arginine methyl ester (Tos-Arg-OMe) were not hydrolyzed. Contrary to the results obtained by using synthetic substrates, the carboxyl sides of charged amino acid residues were preferentially cleaved by the enzyme in the oligopeptide substrates. The substrates that had charged or polar amino acids at P2 positions were not cleaved. On the other hand, the non-polar amino acid or proline at P2 were favored for hydrolysis. The information concerning the subsite of protease D was obtained and is useful for synthesis of a good substrate. As it is distinct from molecular mass, the substrate specificity of honeydew melon protease D is most analogous to cucumisin [EC 3.4.21.25] among serine proteases from cucurbitaceous plants.

Improved isolation, stability and substrate specificity of cucumisin, a plant serine endopeptidase.[Pubmed:7576259]

Biotechnol Appl Biochem. 1995 Oct;22(2):215-22.

Cucumisin (EC 3.4.21.25), a serine endopeptidase, was isolated by a simple purification procedure from the prince melon (Cucumis melo ssp. melo, cv. 'Prince Melon'). The enzyme is stable over a wide pH range (4-11) and to heat, 80% of its initial activity remaining even at pH 11.1 and at 60 degrees C for 20 min. The enzyme was inactive at 72 degrees C and pH 8.0, but 38% of the activity remained in the presence of 10% (w/v) glycerol. Caseinolysis by cucumisin indicated full activity in 8 M urea at pH 9.1 and 50 degrees C. Cucumisin was inactivated by treatment with trypsin at 37 degrees C for 24 h, but was not affected by alpha-chymotrypsin. The synthetic substrates benzyloxycarbonyltyrosine nitrophenyl ester (Z-Tyr-ONp) and benzoyltyrosine ethyl ester (Bz-Tyr-Oet) were cleaved, but Z-Lys-ONp and tosylarginine methyl ester (Tos-Arg-OMe) were not cleaved by cucumisin. Oxidized insulin B-chain was hydrolysed by cucumisin at 37 degrees C for 24 h, 21 cleavage sites being detected. Cucumisin could not cleave the C-termini of all the valine residues in the oxidized insulin B-chain molecule.

Purification and characterization of two serine collagenolytic proteases from crab Paralithodes camtschatica.[Pubmed:7953075]

Comp Biochem Physiol Biochem Mol Biol. 1994 Aug;108(4):561-8.

Two enzymes possessing collagenolytic activity were isolated from the hepatopancreas of crab Paralithodes camtschatica by ammonium sulfate fractionation and DEAE-Sepharose chromatography. It was shown that the specific activities of proteases A and C toward insoluble collagen were equal to 400 and 300 Mandl units/mg protein, respectively. The mol. wt of homogenous proteases A and C determined by gradient polyacrylamide gel electrophoresis in the presence of SDS and 2-mercaptoethanol were equal to 30 and 24 kDa, respectively. The isoelectric point values for the enzymes were determined as 2.5 and 2.9. Both enzymes lack carbohydrates. The amino acid compositions of two crab proteases were measured. The optimal conditions for the enzyme catalysis and the catalytic constants for collagenolytic proteases A and C with respect to Bz-Arg-pNA and Bz-Tyr-Oet have been determined. Inhibition data led to classification of the purified enzymes as serine proteases.