Steen Bech Sørensen

Fragmentation of proteins by S. aureus strain V8 protease. Ammonium bicarbonate strongly inhibits the enzyme but does not improve the selectivity for glutamic acid.

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.

Primary structure and enzymatic properties of carboxypeptidase II from wheat bran

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 of carboxypeptidase 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 kcat 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 P1 and/or P′1 positions.

Primary structure of carboxypeptidase III from malted barley

The primary structure of malt carboxypeptidase III has been determined. The enzyme is a single N-terminally blocked polypeptide chain containing 411 amino acid residues. The sequence of these amino acid residues was deduced from analysis of fragments of the polypeptide chain obtained by chemical cleavages with either cyanogen bromide or hydroxylamine and by enzymatic cleavages with either trypsin, S. aureus V8 protease or proteinase A from yeast. A glycosylated asparagine was found in position 71.The determined sequence was 97% homologous with the amino acid sequence derived from the nucleotide sequence of a gene coding for a wheat protein postulated to be a carboxypeptidase. The malt carboxypeptidase III sequence showed 34% homology with the amino acid sequence of the single-chain carboxypeptidase Y, and about 25% homology with the combined A- and B-chains of malt carboxypeptidase I and II as well as wheat carboxypeptidase II.

Isolation of carboxypeptidase II from malted barley by affinity chromatography

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 byMikola. 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 of glucosamine and 15% neutral sugar. The N-terminal sequence of the A-chain was NH2-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.

Fractionation and characterization of beer proteins

A combination of large scale gel filtration with preparative isoelectric focusing and ion exchange chromatography has been used for fractionation of beer proteins. From gel filtration on Sephadex G-150, a high molecular weight fraction, eluting close to the void volume of the column, a fraction appearing corresponding to a molecular weight of 44,000 and a fraction containing rather low-molecular weight (about 10,000) components were obtained and used for preparative isoelectric focusing in the pH-range 3.5–10. The high-molecular weight fraction was rich in carbohydrate and cross-reacted with yeast antibodies. After preparative isoelectric focusing the amino acid composition of the isolated subfractions resembled that of yeast cell wall components. Preparative isoelectric focusing of the two fractions with molecular weight about 44,000 and 10,000 revealed the presence of two classes of components based on their amino acid composition. One class, which seemed to be present in low concentration in nearly all isolated subfraction, had an amino acid composition resembling that of barley glutelins. In the fraction with molecular weight about 44,000 another class of components—having an amino acid composition resembling barley albumins and globulins—were observed in the region with isoelectric points of about pH 4–5. They cross-reacted immunologically with antibodies against soluble barley proteins. This class of components could also be separated from the glutelin-like constituents by ion exchange chromatography. However, they could not be completely separated from carbohydrate by the fractionation procedures employed. Carbamylation expents demonstrated that approx, half of the ε-amino groups of lysine residues of these proteins were blocked. Furthermore, partial amino acid sequence determination by the Edman procedure revealed a strong heterogeneity with respect to N-terminal amino acid residues. These observations are consistent with a suggestion that a large part of the beer proteins might be polypeptide chains crosslinked by carbohydrates.

Fractionation of protein components from beer by density gradient centrifugation

A protein-rich beer fraction obtained by alcohol precipitation has been fractionated by cesium chloride density gradient centrifugation into three fractions. One with buoyant density 1.27 g·ml−1 contained essentially pure protein, while a fraction with buoyant density 1.37 g·ml−1 contained protein and some carbohydrate, probably covalently bound to the protein. A large fraction with a buoyant density of 1.60 g·ml−1 consisted of carbohydrate.Preparative isoelectric focusing of the protein-containing fractions revealed in both cases a protein peak with pI=4.7 which for the 1.37 g·ml−1 fraction coincided with a carbohydrate peak.

STUDIES ON YEAST FLOCCULATION. COMPARISON OF ENZYMATIC DIGESTS OF FLOCCULENT AND NON-FLOCCULENT CELLS OF SACCHAROMYCES CARLSBERGENSIS

Treatment of a flocculent (strain 201) and a non-flocculent (strain 116) cell suspension of Saccharomyces carlsbergensis with trypsin, followed by β-mercaptoethanol and a second trypsin treatment solubilized approximately 15% of material from both types of cells based on dry weight. The solubilized fractions were resolved by Sephadex gel filtration into high mol. wt. (40,000 to 200,000) and low mol.wt. (less than 4,000) components. High mol.wt. components from the flocculent cells contained more carbohydrate and less protein than the corresponding components isolated from the non-flocculent cells. Furthermore, the polypeptide material from the flocculent cells had a higher content of proline, lysine, and arginine than that from non-flocculent cells. High mol.wt. material contained antigenic determinants similar to those previously found in beer fractions. Yeast-derived antigens were found in all fractions while antigens of barley origin were found only in the tryptic digests.A differential radioactive labelling technique demonstrated minor differences between the components released from flocculent and non-flocculent cell residues upon digestion with pronase.

Studies of Binding Sites in the Subtilisin from Bacillus Lentus by Means of Site Directed Mutagenesis and Kinetic Investigations

The binding site of a proteolytic enzyme may be divided into a number of subsites, each by multiple interactions securing the binding of a single amino acid residue and the proper alignment of the substrate prior to catalysis. The properties of the amino acid residues which constitute a given binding subsite determine which amino acid residue(s) of the substrate may bind and thus, they provide the basis of subsite specificity. The nature of these interactions may be studied by various techniques but it is a prerequisite that the individual subsites are carefully mapped by kinetic investigations using systematic variations of substrate structures. The recent development of highly efficient donor/acceptor pairs for substrates based on intramolecular fluorescence quenching, allowing the use of long peptide substrates spanning the entire binding site, represents a significant improvement in this context.