A carboxypeptidase was isolated from malted barley by affinity chromatography in a yield of approximately 30%. The specific activity was higher than previously obtained for similar enzymes. The enzyme was a dimer where each of the monomers consisted of two peptide chains linked by disulfide bridges. Each monomer contained a single suifhydryl group, which was inaccessible to p-hydroxymercuribenzoate and ELLMAN' S reagent when the enzyme was in its native state. The two peptide chains were separated and the N-terminal sequence of each of them determined. The enzyme contained 6 amino sugar residues and 8% neutral sugar. Isoelectric focusing indicated that the enzyme existed in two forms with pI of 5.65 and 5.73, respectively, but N-terminal sequence determination indicated that they had identical peptide chains. Diisopropyl phosphorofluoridate completely inhibited the enzyme while Hg++ only inhibited the enzyme after deprotonation of an ionizable group on the enzyme with a pK a of approximately 6.7.

Wheat carboxypeptidase II has been isolated from wheat bran by affinity chromatography and its enzymatic properties and amino acid sequence has been determined. The enzyme is a dimer of molecular weight around 110,000 with each monomer composed of two peptide chains, an A-chain and a B-chain, linked by disulfide bridges. The A-chain exists in two forms, one, the A'-chain, being three amino acids shorter at the N-terminus, and consequently two different subunits, i.e. A-B and A'-B, and three different dimers, i.e. A'-B/A'-B, A'-B/A-B and A-B/A-B, are found. These different forms could be separated by ion exchange chromatography due to the A-and A'-chains differing by a single charge.Fragments of the A'-and B-chains, obtained by chemical cleavages with either cyanogen bromide or hydroxylamine and by enzymatic cleavages with either trypsin or S. aureus V8 protease, were sequenced and aligned to give the complete sequences of the two chains. The A'-and B-chains contain 260 and 160 amino acid residues, respectively. Three glycosylated asparagines are found in each chain. Alignment of the A-and B-chains with the corresponding chains of the known carboxypeptidases from germinated barley (malt) showed 95% homology with carboxypeptidase II and 37% homology with carboxypeptidase I. Similarly, the A-chain of wheat carboxypeptidase II exhibits 29% homology with the N-terminal part ofcarboxypeptidase Y and the B-chain exhibits 21% homology with the C-terminal part of carboxypeptidase Y.Like other serine carboxypeptidases wheat carboxypeptidase II exhibits peptidase activity with an acidic pH-optimum and esterase, amidase and peptidyl amino acid amide hydrolase activities with a basic pH optimum. The three different forms of the enzyme hydrolyse peptides with slightly different Km and k~at values but identical kcat/Km values. The specificity of the enzyme is identical to that of carboxypeptidase II from germinated barley: it exhibits a preference for basic and hydrophobic amino acid residues in the Pj and/or P/positions. Abbreviations: Bicine = N,N-bis(2-hydroxyethyl)glycine; BS = benzyl succinic acid; Bz = benzoyl; CABS-Sepharose = N-(e-aminocaproyl)-p-amino-benzylsuccinyl-Sepharose 4 B; Ches = 2(N-cyclohexylamino)ethane sulfonic acid; DPCC = diphenylcarbamyl chloride; EDTA = ethylenediamine tetraacetic acid, sodium salt; FA = furylacryloyl; Hepes = N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid; HPLC = high performance liquid chromatography; Mes = 2-(N-morpholino)ethanesulfonic acid; p-HMB = parahydroxy-mercuribenzoate; PTH = phenylthiohydantoin; TFA = trifluoroacetic acid. The binding site notation is that of SCHECHTER and BERGER (24).

A serine carboxypeptidase isolated from malted barley by affinity chromatography was termed malt carboxypeptidase II to distinguish it from another malt carboxypeptidase previously described (Carlsberg Res. Commun. 48, 217-230 (1983)), henceforth called malt carboxypeptidase I. Our nomenclature is in agreement with the nomenclature formerly suggested by MIKOLA. Malt carboxypeptidase II has a molecular weight of 110,000-120,000. It appears to be a dimer where each monomer is composed of two peptide chains linked by disulfide bridges: one monomer contains an A-chain (34,000) and a B-chain (27,000), the other an A-chain and a C-chain (24,000). The enzyme contains 28 residues ofglucosamine and 15% neutral sugar. The N-terminal sequence of the A-chain was NHe-Ala-Gly-Gly-His-Ala-Ala-Asp-Arg-Ile-Val-while the B-and C-chains appeared to be N-terminally blocked. The amino acid compositions of the B-and C-chains were identical suggesting that their different molecular weights are due to different contents of carbohydrate.Malt carboxypeptidase II is inhibited by diisopropyl phosphorofluoridate and by Hg § It exhibits a strong preference for substrates containing Lys and Arg as C-terminal amino acid residues but it also hydrolyses substrates with hydrophobic amino acid residues in this position.

Staphylococcus aureus strain V8 protease is a serine endopeptidase which cleaves peptide bonds at the carboxyl side of Glu and Asp. Specific cleavage at Glu has previously been achieved in ammonium bicarbonate whereas in sodium phosphate cleavage at both Glu and Asp was observed. However, it is shown here that bicarbonate does not restrict the specificity to Glu‐X bonds, it simply inhibits the enzyme. The degradation of a mixture of oxidized insulin and glucagon proceeds similarly in the two buffers, although faster in phosphate.

Keywords: A m i n o acid sequence, sequence homology, serine carboxypeptidaseThe primary structure of malt carboxypeptidase I has been determined. The enzyme is composed of two peptide chains, an A-and a B-chain, linked by disulphide bridges. Fragments were obtained by chemical cleavages with either cyanogen bromide or hydroxylamine and by enzymatic cleavages with either trypsin, S. aureus V8 protease or proline specific endopeptidase (E.C. 3.4.21.26), sequenced and aligned to give the total sequence of the two chains. The A-and B-chains contain 266 and 148 amino acid residues, respectively. Glycosylated asparagines are found in positions 118 and 232 of the A-chain and position 56 of the B-chain. Alignment of the sequence of the A-chain with the N-terminal part of carboxypeptidase Y revealed 30% homology. Similarly, the B-chain showed 27% homology with the C-terminal part of carboxypeptidase Y. NO homology was observed with other proteins by a computer search of a sequence data base provided by the National Biomedical Research Foundation. A region of the A-chain was found to be identical to the region around the essential seryl residue in position 146 of carboxypeptidase Y.