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Biomedical subjects

R Hayashi

Publications and source records attributed to R Hayashi.

At least 343 records · Page 19Linked to original sources
PubMed1975

Further confirmation of carboxypeptidase Y as a metal-free enzyme having a reactive serine residue.

The metal content of carboxypeptidase Y was analyzed by the atomic absorption method. After exhaustive dialysis against an EDTA solution, the enzyme showed no loss of activity nor any significant content of metals (Zh,Mg,Ca,Cu,Mn,Ni,Fe, and Co). The activity was, however, rather sensitive to preincubation with various metals. The reactivity of a serine residue of the enzyme was also reevaluated. Diisopropyl fluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF) stoichiometrically and irreversively inhibited the enzyme. The rate of inactivation with DFP was much faster than that for typsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1.], while the rate with PMSF was one-fifteenth of that for chymotrypsin. The pH-dependence of the inactivation by DFP was similar to that of the enzymatic hydrolysis of acetylphenylalanine ethyl ester. The present results indicate that carboxypeptidase Y is free of metals and has a serine residue with a vital role in the catalytic process, though the functional role of this SH group remains to be clarified.

Apoenzymes↗
PubMed1974

Inhibition of thiamine pyrophosphate utilization by thiamine or its monophosphate in Escherichia coli.

The growth of a thiamine pyrophosphate auxotroph of Escherichi coli was inhibited by either thiamine or thiamine monophosphate, and the growth of a thiamine monophosphate auxotroph was inhibited by thiamine. The thiamine pyrophosphate-dependent oxidation of pyruvate was inhibited by thiamine with whole cells of the thiamine pyrophosphate auxotroph but not with cell extracts prepared from the same organism. In addition, the thiamine pyrophosphate uptake of the thiamine pyrophosphate auxotroph was inhibited by either thiamine or thiamine monophosphate. Although the thiamine pyrophosphate uptake of a revertant, selected for prototrophy from the thiamine monophosphate auxotroph, was inhibited by thiamine to an extent comparable to that observed with the thiamine monophosphate auxotroph, its growth was no longer inhibited by thiamine. A possible mechanism for the inhibition by thiamine and thiamine monophosphate in the utilization of thiamine pyrophosphate is discussed.

Anaerobiosis↗
PubMed1972

Biosynthetic pathway of thiamine pyrophosphate: a special reference to the thiamine monophosphate-requiring mutant and the thiamine pyrophosphate-requiring mutant of Escherichia coli.

Two types of mutants of Escherichia coli were isolated, one of which (mutant 70-23-107) responded to thiamine pyrophosphate, and the other (mutant 70-23-102) to thiamine monophosphate and thiamine pyrophosphate. They were produced by further mutation of a thiamine auxotroph of E. coli 70-23 with N-methyl-N'-nitro-N-nitrosoguanidine. The parent organism required thiamine because phosphohydroxymethylpyrimidine kinase activity was lacking in this organism, and hydroxymethylpyrimidine pyrophosphate was not permeable through the cell membrane of E. coli. Thiamine, thiamine monophosphate, and thiamine pyrophosphate were all equally active for the parent, whereas mutants 70-23-102 and 70-23-107 lost their ability to grow on thiamine. Both mutants differed only in the growth response to thiamine monophosphate: the former could grow on thiamine monophosphate, whereas the latter could not. Experimental results with the newly isolated mutants indicate that in E. coli the free form of thiamine is not involved in de novo synthesis of thiamine pyrophosphate, but thiamine monophosphate, an exclusive product formed by the reaction between hydroxymethylpyrimidine pyrophosphate and hydroxyethylthiazole monophosphate, is directly phosphorylated to form thiamine pyrophosphate. Exogenous thiamine, on the other hand, is converted to thiamine pyrophosphate via the intermediate formation of thiamine monophosphate.

Cell-Free System↗