Tadao Hata

The interaction of L-ascorbic acid with the active center of myrosinase.

Only L-ascorbic acid activated plant myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1), whereas ascorbic acid analogs did not. The enzyme protein was conformationally changed by the addition of L-ascorbic acid to the spectrophotometric analysis, approx. 1.5 amino residues appeared on the surface of the enzyme and about 2.3 tryptophan residues were buried in the molecule when 1 mM L-ascorbic acid was added. Optimum temperature for the myrosinase activity was approx. 55 degrees C without L-ascorbic acid, but with L-ascorbic acid it was about 35 degrees C; that for beta-glucosidase activity was the same (55 degrees C) with or without L-ascorbic acid. The effect of chemical modification of the functional groups of myrosinase on the interaction of L-ascorbic acid was investigated and the interaction of L-ascorbic acid with the active center of the enzyme is proposed.

Differences in fatty acid composition among phosphatidylethanolamine, phosphatidylglycerol and cardiolipin of Escherichia coli

Abstract The fatty acid composition of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin of Escherichia coli B during growth at various temperatures was analyzed. Marked differences in the unsaturated fatty acid content, especially the cis-vaccenic acid content, were observed among the three phospholipids during the exponential growth phase at any given temperature. As the cultures progressed to the stationary growth phase, unsaturated fatty acids decreased.

Purification and general characteristics of bacterial myrosinase produced by Enterobacter cloacae.

Myrosinase in cell-free extract of Enterobacter cloacae, no. 506, was purified about 1,000 fold by precipitation with ammonium sulfate, chromatography on CM-Sephadex and gelfiltration on Sephadex G–200 and Sephadex G–100. The enzyme was shown to be homogeneous by chromatography and by ultracentrifuge. Molecular weight obtained by gelfiltration was 61,000 and the sedimentation coefficient was 4.5 S. Maximum activity occurred at pH 6.8, The enzyme was stable in a pH range of 5.0 to 7.0 at temperature below 40°C and for 24 hr. Copper(I) and (II), mercury (II) and ferrous(II) ions strongly inhibited the activity. Sulfhydryl reagents had little effect but EDTA was a strong inhibitor. In contrast to plant myrosinase, this enzyme was inhibited by l-ascorbic acid. Many glucosides and sugars inhibited the enzyme. The relation between bacterial myrosinase and β-gluco-sidase is discussed in comparison to plant and fungous myrosinases. Some comparative properties of bacterial, fungous and plant myrosinases are discussed.

Action of yeast proteinase C on synthetic peptides and poly-α,l-amino acids

Abstract Yeast proteinase C liberates carboxy-terminal amino acids from a wide variety of N-acyl dipeptides which have aromatic, neutral, acidic or basic amino acid in the carboxy-terminal end. Noticeably, carboxy-terminal or internal proline in peptides can also be liberated. The enzyme has esterase activity for N- acetyl- l -tyrosine ethyl ester and amidase activity for carbobenzoxy dipeptide amides. The enzyme rapidly hydrolyses poly-α, l -glutamic acid at pH 4.2, liberating only glutamic acid, while poly- l -lysine and poly- l -proline are never hydrolysed. All these activities are powerfully inhibited by p-chloromercuribenzoate (PCMB) or DFP. Poly- l -lysine inhibits both peptidase activity for carbobenzoxy-Glu-Tyr and esterase activity for acetyl- l -tyrosine ethyl ester.

Effect of changes in fatty acid composition of phospholipid species on the β-galactoside transport system of Escherichia coli K-12

Abstract On lowering the growth temperature of Escherichia coli K-12 from 37 to 17 °C, the cells resumed growth after a lag period of 40 min. During the lag period, the transition points in Arrhenius plots of the preinduced β-galactoside transport system were not changed while the saturated/unsaturated fatty acids ratio decreased gradually in phosphatidylethanolamine, rapidly in phosphatidylglycerol and little in cardiolipin.

Purification of Yeast Proteinases:Part III. Isolation and Physicochemical Properties of Yeast Proteinase A and C

yeast was demonstrated by the chromatography on DEAE-Sephadex A-50, and proteinase A and C were isolated and purified by a relatively simple procedure, which was mainly conducted by repeating the chromatography and alcohol fractionation. The final preparation of proteinase C was found to be homogeneous by various physical criteria and crystallized from alcohol solution. On the other hand, although the preparation of proteinase A also showed homogeneous in chromatographic and ultracentrifugal analyses, the result of elec trophoresis disclosed the heterogeneity of the preparation. As the results of the chemical and physicochemical analyses, both enzymes showed large contents of carbohydrate, higher molecular weights and acidic isoelectric points, which seemed to be characteristic to the

Studies on the Myrosinase in Mustard Seed: Part I. The Chromatographic Behaviors of the Myrosinase and Some of its CharacteristicsPart II. On the Activation Mode of the Myrosinase by L-Ascorbic Acid

In order to clarify whether the myrosinase containes only thioglucosidase, or thioglucosidase and sulfatase, chromatographic behaviors of the myrosinase on cellulose ion exchanger were studied. Myrosinase showed two peaks by the chromatography on TEAE-cellulose at pH 8.5, but the fractionation of the two enzymes was not accomplished. It was concluded that the two peaks was not ascribable to an artifact but the two closely resembled species of myrosinase existing in the sample solution. Chromatographic separation of the two enzymes on DEAE-cellulose resulted in failure. These results confirmed that the myrosinase was a single β-thioglucosidase originally.

Proteinase Inhibitors in Plant Seeds: Part III. Characterization of Two Different Types of Inhibitors Obtained from Japanese Radish Seed (Raphanus sativus)Part IV. Trypsin-inhibitor Complex Formation

The proteinase inhibitors I (R-I) and III (R-III) isolated from Japanese radish seed were characterized in terms of their N-terminal amino acids, amino acid composition and reacting groups. The amino acid composition of two proteins differed from each other, while histidine, methionine and tryptophan contents were all low. N-Terminal amino acids of these inhibitors determined by Edman degradation were the same; valine.By modifying free amino groups in the inhibitors with trinitrobenzenesulfonic acid, R-III was greatly inactivated in proportion to the modification of amino groups, but the activity of R-I was not affected.However, modification of arginyl residues of R-I by cyclohexanedione reduced its activity. These results indicate that R-I is an arginine-type and R-III is a lysine-type inhibitor.

Studies on Fungous Myrosinase: Part I. Production, Purification and Some CharacteristicsPart II. Effects of Various Reagents on Its Enzymatic ActivitiesPart III. On the β-Glucosidase Activity of Fungous Myrosinase and the Relationship of Fungous and Plant Myrosinases to β-Glucosidases

In order to obtain fungous myrosinase, Aspergillus sydowi IFO 4284 was cultured on a medium containing mustard seed extract for 2 weeks. Myrosinase in the broth was purified about 150 fold by precipitation with ammonium sulfate and chromatography on DEAE-cellulose and DEAE-Sephadex. Comparison of thioglucosidase and sulfatase activities of the myrosinase preparation using pH-activity, pH-stability and temperature-stability curves revealed no differences from each other. The chromatograms of the two activities on DEAE-Sephadex showed good agreement. Consequently, the myrosinase produced by Aspergillus sydowi was concluded to be a single β-thioglucosidase, not a mixture of thioglucosidase and sulfatase.The effects of various reagents on Aspergillus sydowi myrosinase were studied.The enzymatic activity was stimulated by cobalt (II), zinc (II) and magnesium ions and inhibited by mercury (II), iron (II) and copper (II) ions. However, metal-complexing agents, SH reagents and diisopropylfluorophosphate showed no...

Proteinase Inhibitors in Plant Seed:Part II. Purification and Some Properties of Three Different Types of Inhibitors from Japanese Radish Seed ( Raphanus sativus )

Three different types of proteinase inhibitors, I, II and III, were fractionated from Japanese radish seed by repeated column chromatographies on SE- and CM-cellulose. The finally purified preparation of inhibitor III was found to be homogeneous by both chromatographic and electrophoretic analyses. All three of them strongly and stoichiometrically inhibited trypsin whereas they showed weak inhibition on other proteinases, such as chymotrypsin, Nagarse and Pronase. From nitrogen content and ultraviolet absorption spectra, each of the inhibitors I and III was confirmed to be a protein. The molecular weights of inhibitors I and III were calculated to be 8000 and 12,000, respectively. These inhibitors were stable at temperatures above 90°C in an acidic pH.