Masami Soejima

Studies on a new proteolytic enzyme from Achromobacter lyticus M497-1. I. Purification and some enzymatic properties

Achromobacter lyticus M497-1 produces three kinds of alkaline proteases (protease I, II and III) in culture medium along with the bacteriolytic enzyme (Masaki, T., Nakamura, K., Isono, M. and Soejima, M. (1978) Agric. Biol. Chem. 42, 1443--1445). Among these three proteases, Achromobacter protease I (EC 3.4.21.-) shows strict splitting for lysine residues at the carboxyl side of the splitting point. This enzyme was purified through a sequence of benzalkonium chloride treatment, acetone fractionation, CM-cellulose and DEAE-cellulose treatment chromatography on AH-Sepharose 4B and isoelectric focusing method. This form was shown to be homogeneous by polyacrylamide gel electrophoresis and ultracentrifugation analysis. The physicochemical properties of the enzyme were: Mr 30 500; partial specific volume (v), 0.717 ml/g; intrinsic viscosity (nu), 0.0385) dl/g; isoelectric point (pI) 6.9; and E1%1cm at 280 nm, 18.77. The enzyme was composed of 294 residues of amino acid per molecule, with glycine as NH2-terminal and lysine as COOH-terminal amino acids. The optimum pH values with casein, Bz-lys-pNA and Tos-Lys-OMe were 8.5--10.7, 9.0--9.5 and 7.8--8.2, respectively. The enzyme was inhibited by iPr2P-F, PhCH2SO2F and Tos-LysCH2Cl but not by Tos-ArgCH2Cl, EDTA, o-phenanthroline and PCMB.

Studies on a new proteolytic enzyme from Achromobacter lyticus M497-1 II. Specificity and inhibition studies of Achromobacter protease I☆

The unique specificity of Achromobacter protease I for lysine residue was investigated using synthetic and natural substrates, i.e., lysine derivatives, arginine derivatives, lysine vasopressin, substance P, ACTH and insulin. The enzyme cleaved only the -Lys-X- bonds in the above substrates. The binding affinity of alkylamines as determined by Ki was much stronger than that of the corresponding alkylguanidines.

Studies on the induced synthesis of maleate cis-trans isomerase by malonate: Part III. Purification and properties of maleate cis-trans isomerase induced by malonate

Maleate cis-trans isomerase in Alcaligenes faecalis IB–14 was induced by malonate and purified about 100-fold over the crude cell-free extract by treatments of ammonium sulfate fractionation, Sephadex G–100 gel filtration, DEAE-cellulose and DEAE-Sephadex A–50 column chromatography. The preparation was shown to be monodisperse on ultracentrifugal analysis and Svedberg value was found to be 3.84 S.The enzyme was most active at pH value around 8.3 and was stable over the range of pH 5.0 to 7.0 in the presence of dithiothreitol (DTT) for a few weeks, but in the absence of it, the enzyme activity was markedly decreased, especially in the alkaline region. The enzyme activity was inhibited by various sulfhydryl reagents and oxidizing agents, whereas it was not affected by metal chelating agents. The inhibition by Hg2+ and PCMB was overcome by the addition of sulfhydryl compounds such as DTT, 2-mercaptoethanol, l-cysteine and glutathione. It was observed that the enzyme did not require co-factor for its function...

Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate: Part IV. Catabolite Repression and its Reversal of Maleate cis-trans Isomerase

Repression of maleate cis-trans isomerase(maleate isomerase) by carbon sources and its reversal were investigated by using Alcaligenes faecalis IB-14.The formation of maleate isomerase was induced by malonate favorably in a poor medium, whereas it was repressed in a rich medium by carbon sources such as intermediates of TCA cycle. The repression provoked by dl-malate was accompanied with remarkable promotion of the cell growth and with accumulation of a large amount of pyruvate. The enzyme levels of TCA cycle were elevated several times in the dl-malate repressed cells. It was probable to assume that the formation of maleate isomerase was subject to catabolite repression when a rapid and surplus metabolism of dl-malate via TCA cycle was conducted.So, as an approach to reveal the chemical nature of the catabolite moiety, reversal of the catabolite repression was studied. It was demonstrated that the repression provoked by dl-malate was reversed by various cultural conditions as follows; addition of higher ...

Function of the carboxyl group specificity in Achromobacter protease I.

AchromobacterプロテアーゼIの活性中心部位,とくに基質特異性決定部位の性状を解明する手掛りを得る目的で,縮合剤と求核試薬との酵素活性に対する影響,強力な拮抗阻害剤であるω-アミノアルキル基をリガンドとするアフィニティクロマトグラフィーについて実験し,トリプシンと比較検討した. (1)本酵素はEDC単独,または求核試薬(エチレンジアミン)存在下でのEDCによって,トリプシンよりも弱いが明らかに阻害された.また拮抗阻害剤ブチルアミンで酵素を予備処理した後, EDCと求核試薬エチレンジアミンを反応させ,さらに透析することにより不活性化速度が減少した. (2)本酵素のアミノ基を無水酢酸でアセチル化しても,ほとんど酵素活性は影響されなかった. (3)炭素鎖を異にするω-アミノアルキル基をリガンドとするセファロース4B (Seph-NH-(CH2)n-NH2)に対する親和力をトリプシンと比較した結果,本酵素はメチレン基数が増大するとともに次第に強く吸着され, nが8で最大となり, nが8以上ではほぼ同様の強さで吸着された.また本酵素をTos-Lys CH2ClまたはiPr2P-Fによって完全に失活させた酵素はアミノオクチル(AO)セファロース4Bに吸着されなかった.同様に強力な拮抗阻害剤アミルアミン共存下で同様のクロマトグラフィーを行ったが吸着されなかった.

The Effect of Urea and Guanidine Hydrochloride on Activity of Papain

The inactivation of papain brought about by low concentration of urea or guanidine hydrochloride was found to be a non-competitive type and that of cyanate ion to be a mixed type. The results are presented as Lineweaver-Burk plots. These inactivations are not due to a change in the secondary structure of papain molecule as a result of the measurements of optical rotatory dispersion, circular dichroism, and UV-difference spectra.