Biomedical subjects
Publications and source records attributed to S Fukui.
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Profuse appearance of microbodies was observed in the cells of methanol-utilizing yeasts in connection with the enhanced catalase activity. These microbodies were isolated successfully by means of sucrose gradient centrifugation from the methanol-grown cells of Kloeckera sp. no. 2201. Localization of a flavin-dependent alcohol oxidase as well as characteristic microbody enzymes (catalase and D-amino acid oxidase) were ascertained in the isolated microbodies, whereas formaldehyde and formate dehydrogenases were detected in the cytoplasmic region. Localization of catalase in the isolated microbody was also demonstrated by the cytochemical technique with 3,3'-diaminobenzidine.
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Coenzyme B12 dependent diol dehydrase from Aerobacter aerogenes was immobilized by covalent binding to CNBr-activated Sepharose 4B. The Sepharose-bound enzyme exhibited a markedly high catalytic activity, viz., 75-95% of the specific activity of the original free enzyme. The apoenzyme acquired much greater stability to heat by immobilization. No significant difference between the immobilized and free enzymes was observed in the following properties: the affinity for coenzyme B12; the sensitivity to a sulfhydryl-modifying agent; the absolute requirement for a certain monovalent cation, such as K+, for catalysis; the susceptibility toward oxygen upon incubation with coenzyme B12 in the absence of substrate. These results suggest that the structure and function of the enzyme are not significantly influenced by immobilization on Sepharose. The immobilized enzyme was found to provide a convenient method for a study of ligand interaction with the enzyme. The subunit interaction between two dissimilar subunits, components F and S, was investigated using the component S immobilized on CNBr-activited Sepharose and free component F, and it was demonstrated that the substrate (1,2-propanedoil) promotes the hybrid formation between component F and component S, but K+ alone rather retarded the subunit association to some extent. Na+ markedly weakens the forces which bind the subunits together. The relationship between cobalamin binding and subunit structure is also discussed.
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Yeast pyruvate kinase (ATP : pyruvate 2-O-phosphotransferase EC 2.7.1.40) was classified into three groups based on the interaction with fructose-1,6-bisphosphate. The pyruvate kinases of Saccharomyces cerevisiae and Saccharomyces carlsbergensis were activated by fructose 1,6-bisphosphate in the concentration range tested (up to 10 mM) of the substrate, phosphoenolpyruvate; the enzymes of "fermentative Candida" (Candida tropicalis and Candida utilis) were affected by fructose 1,6-biphosphate only when the substrate concentration was below 2 mM. Although the pyruvate kinase of Candida lipolytica (a yeast belonging to "oxidative Candida") was also affected by fructose 1,6-bisphosphate, the degree of the activation was extremely small as compared with the above four yeasts. The pyruvate kinase of C. tropicalis was inhibited by ATP more strongly in the absence of fructose 1,6-bisphosphate than its presence. In the case of the C. lipolytica enzyme, however, the enzyme was inhibited to a lesser extent by ATP, and fructose 1,6-bisphosphate did not reverse the inhibitory effect of ATP. Time course changes of the enzyme levels in the yeasts grown on glucose and on ethanol indicate that the pyruvate kinases of S. cerevisiae and C. tropicalis can be controlled both by an allosteric mechanism and by changes in the enzyme concentration, although a marked difference was observed in the susceptibility to the allosteric effect by fructose 1,6-biphosphate between these fermentative yeasts. On the other hand, that of C. lipolytica would be controlled only by the latter mechanism.
The binding rate of pyridoxal '5-phosphate (Pxa-P) to apotryptophanase and the dissociation rate of the coenzyme from holotryptophanase were able to be determined by following the enzyme activity in continuous flow reactions on a column of immobilized tryptophanase. When the enzyme activity was assayed continuously in the flow system in the absence of coenzyme added to the reaction mixture, immobilized holotryptophanase lost gradually its initial activity owing to dissociation of coenzyme. The coenzyme dissociation at a given concentration of substrate (tryptophan) followed first-order kineticsmin a low substrate concentration range below the Km value, a more decreased rate constant was obtained for the coenzyme dissociation. This indicates that the coenzyme is more dissociable from the apoenzyme-coenzyme-substrate complex (ECS complex) rather than from the apoenzyme-coenzymecomplex (holoenzyme). Immobilized tryptophanase freed of coenzyme restored rapidly its original activity, when the assay mixture containing a given concentration of substrate and Pxa-P was passed through the immobilized enzyme column. The coenzyme binding at a given coenzyme concentration followed first-order kinetics, but the rate was not first order in regard to the coenzyme concentration. A plot of the reciprocal of the first-order rate constant obtained vs. the reciprocal of the coenzyme binding occurs in a two-step fashion; the first step is rapid and the second step is rate determining. Both the dissociation constant for the first step and the rate constant for the second step were shown to be independent of the substrate concentration. This means that Schiff base formation between Pxa-P and tryptophan in the assay mixture has no effect on the binding of Pxa-P to apoenzyme. The coenzyme dissociation constant at a given substrate concentration was calculated from both the rate constant of the coenzyme binding and the rate constant of the coenzyme dissociation. The values obtained by this method at different substrate concentrations were almost identical with those measured at the corresponding substrate concentrations directly by an ordinary method.
Tryptophanase from Escherichia coli B/qt 7-A and tyrosine phenol-lyase (beta-tyrosinase) from Escherichia intermedia were immobilized on Sepharose 4B by several direct coupling reactions or through pyridoxal 5'-phosphate previously bound to Sepharose. The most active preparation of immobilized tryptophanase was obtained by coupling tetrameric apoenzyme to pyridoxal-P bound on Sepharose at the 6-position through a diazo linkage. This immobilization procedure involves the formation to Schiff base linkage between 4-formyl group of Sepharose-bound pyridoxal-P and the epsilon-amino group of the lysine residue at the active center of one subunit of tetrameric apo-tryptophanase, followed by the fixation of the Schiff base linkage by reduction with NaBH4. In the case of beta-tyrosinase having two catalytic centers, however, this method was not so suitable as the case of tryptophanase. Direct coupling of the apoenzyme to CNBr-activated Sepharose or to a bromoacetyl derivative of Sepharose gave better results. In each case, the affinity for substrate or coenzyme was scarcely influenced by the immobilization. When used repeatedly in a batch system or continuously in a flow system in the absence of added pyridoxal-P, immobilized holo-tryptophanase of holo-beta-tyrosinase gradually lost its original activity; however, supplement of pyridoxal-P to the reaction system restored its initial activity. From the kinetic analyses of these phenomena, the rate constants of coenzyme dissociation from immobilized tryptophanase and beta-tyrosinase were calculated. Upon immobilization, the pH optima of both enzymes shifted 0.5 to 1.0 pH unit to the alkaline side. Both immobilized enzymes showed higher thermal stability and resistance to a denaturing agent such as guinidine-HCl than their free counterpart. Furthermore, the reactivity of sulfhydryl group of beta-tyrosinase, in connection with its coenzyme-binding property, was conveniently studied by use of the immobilized enzyme.
Three new derivatives of vitamin B12,0-2'-succinyl-, 0-5'-succinyl-, and 0-2', 0-5'-disuccinyl-vitamin B12, whose alpha-ribose moieties of the nucleotide ligand are succinylated, were prepared by reaction of the vitamin with succinic anhydride. The first succinylation took place rapidly and almost predominantly on 5'-OH of alpha-ribose, and the second succinylation much more slowly on 2'-OH. From the behaviors in paper electrophoresis and the lability to CH- of cobalt-base bond of 0-2'-succinylated vitamin B12 derivatives, it was suggested that the terminal COOH of the 0-2'-succinyl group forms an inner salt with the imidazole nucleus of 5,6-dimethylbenzimidazole. Monosuccinyland disuccinyl-vitamin B12 by mild acid or base hydrolysis. Heating at 130 degrees for 5 min also led to the complete severance of the succinyl group of 0-5'-succinyl-vitamin B12. None of the three succinly derivatives inhibited the diol dehydrase reaction when added with coenzyme B12, SUggesting that the ability to bind to the apoenzyme is strongly diminished or almost lost by succinylation on 2'- or 5'-OH of alpha-ribose. None of the succinyl vitamin B12 compounds showed either biological activity or anti-vitamin B12 activity when tested with Escherichia coli 215, a methionine-B12 auxotroph. None of them significantly inhibited [3-H] vitamin B12 uptake by E. coli 215 cells. This observation implies that succinyl derivatives of vitamin B12 are hardly incorporated into the cells of E. coli.
Vitamin B(12) production by a newly isolated strain of a methanol-utilizing bacterium was studied. The maximal yield of the vitamin, 2.6 mg/liter of medium was attained by optimization.
The events which occur in the early stages of the mating process of the yeast Rhodosporidium toruloides between strains M-919 (mating type A) and M-1057 (mating type a) were investigated. In preliminary experiments we determined the frequency of mating by two newly designed methods: the liquid culture method and the membrane-filter microculture method. The mating frequencies of strains M-919 and M-1057 were 89% in the liquid culture method and 62% in the membrane-filter microculture method. The early stages in the mating process included the following events: (i) M-919 cells produce constitutively the extracellular inducing substance (A factor), (ii) M-1057 cells receive A factor, and in response to it they form mating tubes and secrete another inducing substance (a factor), (iii) M-919 cells receive a factor, and in response to it they form mating tubes, (iv) mating tubes elongate to the cells or the tubes of mating partner, (v) tips of the growing tubes recognize the opposite mating type cells or their tubes, followed by cell-to-cell fusion.
Nine strains of methanol-utilizing yeasts belonging to the genera Candida, Hansenula, Kloeckera, Pichia, and Torulopsis were examined with respect to the interrelationship between their catalase content and ultrastructure. Methanol-grown cells of all the yeasts tested showed higher catalase activities than the respective ethanol- and glucose-grown cells. In connection with this, occurrence of a specific organelle surrounded by a single-unit membrane ("microbodies") was observed only in the methanol-grown cells. Several morphological differences were observed between the microbodies of methanol-utilizing yeasts and those of hydrocarbon-utilizing yeasts such as Candida tropicalis. That is, microbodies of methanol utilizers were large in size, existed in closely associated forms, and had crystalloid structures. Localization of catalase activity in these microbodies was demonstrated cytochemically by use of 3,3'-diaminobenzidene. Especially, 3,3'-diaminobenzidine reaction product accumulated heavily in crystalloids of yeast microbodies.
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