Makoto Yamasaki

Studies on the mechanism of protein synthesis incorporation of ethionine into α-amylase of Bacillus subtilis

1. 1. α-Amylase containing ethionine was separated in crystalline form from the culture medium of the methionine-requiring mutant of Bacillus subtilis. 2. 2. The sedimentation, viscosity and electrophoretical mobility of α-amylase containing ethionine were compared with those of normal enzyme. The molecular weight of α-amylase was estimated as 55,000. 3. 3.The peptides containing methionine and ethionine were separated by column chromatography from a tryptic hydrolysate of α-amylase containing ethionine, and the amino acid composition of these peptides was estimated. 4. 4. It was concluded that ethionine can be incorporated into the normal peptide-bond sequence of α-amylase, and that α-amylase containing ethionine has the same physico-chemical properties and enzyme activity as those of normal protein.

Mechanism of interaction of bovine trypsin with human α1-antitrypsin

Abstract 1. 1. Human α1-antitrypsin and bovine β-trypsin form a 1:1 stoichiometric complex which cannot be dissociated with denaturing and reducing agents. 2. 2. With hydrazine treatment this complex (mol. wt 74 000) is degraded to proteins with molecular weights of 48 000 and 23 000. 3. 3. α1-Antitrypsin also forms a stable complex with bovine α-trypsin. In this complex, crosslinking is found to occur between α1-antitrypsin and the B chain of α-trypsin. These results suggest the formation of an acyl bond between the carbonyl carbon of the inhibitor and the γ-oxygen of Ser-183 of the enzyme. 4. 4. When the inhibitor is allowed to react with an excess amount of trypsin the complex is degraded progressively to proteins of mol. wts 63 000, 54 000 and 50 000. 5. 5. It is indicated that this inhibition is not a temporary one, even when the degradation of the complex has occurred with an excess amount of trypsin. 6. 6. The selective hydrolysis at acid pH which is observed in many other reactions of inhibitors is not detected.

Properties and Functions

Nucleoprotamine dissolves in aqueous solution of NaCl (1–2 M) or ammonium sulfate to give a viscous solution. In such solutions it largely dissociates into DNA and protamine, but upon dilution (e.g., to 0.14 M NaCl) they combine to form white fibrous precipitates [Watanabe and Suzuki, 1951 (3)].

Specificity of acid proteinase a from Aspergillus niger var. Macrosporus towards B-chain of performic acid oxidized bovine insulin

1. A comparative study on the mode of action of two highly purified acid endopeptidases (EC 3.4.-) from Aspergillus niger var. macrosporus, acid proteinase A and B, on the B-chain of performic acid oxidized insulin was performed, putting emphasis on the quantitative analysis of the effects of enzyme A. Acid proteinase A behaved very specifically towards the substrate and hydrolyzed four peptide bonds exclusively: three major sites, where hydrolysis proceeded rapidly and almost completely, Asn3-Gln4, Glu13-Ala14, and Tyr26-Thr27; and a minor one, Gly20-Glu21, at which hydrolysis was much slower. 2. The effects of four protease inhibitors, pepstatin, diazoacetyl-D,L-norleucine methyl ester/Cu(II), di-isopropyl phosphorofluoridate, and 1,2-epoxy-3-(p-nitrophenozy) propane on acid proteinases A and B were studied. Acid proteinase A preparations, treated with the former two inhibitors, were used to establish that the major sites of attack were really affected by enzyme A and not by contaminating proteinase B.

Nucleotide sequence of the promotor and NH2-terminal signal peptide region of Bacillussubtilis α-amylase gene cloned in pUB110

Abstract The nucleotide sequence of the promotor and NH2-terminal signal peptide region of the α-amylase gene derived from the α-amylase hyperproducing strain B . subtilis NA64 was determined. DNA sequences of the NH2-terminal region of the mature α-amylase, 41 amino acid residues of the signal peptide, a Shine-Dalgarno sequence (AGGAG), a potential RNA polymerase recognition site (TTGAAA), and a potential Pribnow box (AAGTAA) were identified. The DNA sequence was quite different from that of the α-amylase gene of B . amyloliquefaciens .

Hyperproductivity of extracellular α-amylase by a tunicamycin resistant mutant of Bacillussubtilis

Abstract Several tunicamycin resistant mutants were obtained from Bacillus subtilis NA64. One of them, B7 strain produced a 5-fold larger amount of α-amylase than NA64 did. Only the amount of α-amylase, among excreted proteins, was enhanced. Genetic analyses by transformation suggested that a single mutation in B7 induced both resistance to tunicamycin and hyperproductivity of extracellular α-amylase.

The effective use of affinity chromatography for the study of complex formation of bovine carboxypeptidase B with basic and aromatic amino acid analogues

Abstract The affinity chromatography method was applied to the study of the binding of carboxypeptidase B to basic and aromatic amino acid analogues. This method revealed that these two kinds of ligands occupy different sites on the enzyme and the affinity of its binding site(s) for aromatic substrates or inhibitors was profoundly influenced by the occupation of the other site(s) by ϵ-aminocaproic acid.

Isolation, purification and some chemical properties of an acid carboxypeptidase from Aspergillus niger var. Macrosporus.

Acid carboxypeptidase (peptidyl-L-amino-acid hydrolase, EC 3.4.16.1) was purified to a homogeneous state from the water extracts of Koji cultures of Aspergillus niger var. macrosporus. The molecular weight of the enzyme was determined to e 136 000 by sedimentation equilibrium method. The denatured specimen of the enzyme exhibited a molecular weight of 60 000 in the sedimentation equilibrium in 6 M guanidinium chloride, suggesting that the native enzyme is composed of two identical subunits. However, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the enzyme showed an anomalous Ferguson plot, which may account for the inconsistent values of apparent molecular weights obtained by this method. The acid carboxypeptidase was found to be an acidic glycoprotein (pI, 4.1), composed of 955 amino acid, 140 mannose, 14 galactose and 30 glucosamine residues/molecule.

Analysis of the fibrin-polymerizing reaction using sodium dodecylsulfate-agarose gel electrophoresis

1. A new method of sodium dodecylsulfate gel electrophoresis, sodium dodecyl-sulfate-agarose gel electrophoresis, was developed. The new electrophoresis was shown to have a high and efficient resolving power for fibrin polymers and was also applicable to other proteins. 2. By sodium dodecylsulfate-agarose gel electrophoresis, the fibrin polymerizing reaction with activated fibrin stabilizing factor was analyzed in detail. The cross-linking between the gamma-chains was indicated to occur at an intermolecular level. The solubility of polymerized fibrin was suggested to be related mainly to the formation of the crosslinks between gamma-chains.

Enzymatic properties of an acid carboxypeptidase from Aspergillus niger var. macrosporus

Acid carboxypeptidase (peptidyl-L-amino-acid hydrolase, EC 3.4.16.1) isolated from Aspergillus niger var. macrosporus was investigated in regard to its kinetic parameters for two synthetic substrates. The optimum pH of peptidase activity toward Z-Glu-Tyr-OH was pH 3.0 Km and kappa cat values were 4.0 . 10(-3) M and 270 s-1 at pH 3.0 and 30 degrees C. The optimal pH of esterase activity toward Bz-Arg-OEt was pH 5.2 Km and kappa cat for esterolytic activity were 6.1 . 10(-4) M and 1500 s-1 at pH 5.0 and 30 degrees C. The enzyme released expected amino acids sequentially from the carboxyl ends of S-beta-aminoethylated ribonuclease A and the B-chain of oxidized insulin, demonstrating carboxypeptidase activity of the enzyme. The enzyme was inactivated by diisopropylphosphorofluoridate and phenyl-methanesulfonyl fluoride. In the reaction with [14C]diisopropylphosphorofluoridate, the amount of [14C]diisopropylphosphoryl group incorporated into the enzyme in complete inactivation was estimated as 2 mol/mol enzyme.