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Biomedical subjects

G Okada

Publications and source records attributed to G Okada.

At least 55 records · Page 3Linked to original sources
PubMed1981

Multiple species of mammalian S-adenosylmethionine synthetase. Partial purification and characterization.

Two species of S-adenosylmethionine (S-Ado-Met) synthetase (EC 2.5.1.6) exist in rat liver cytosol and a distinct species of the enzyme exists in kidney cytosol. S-Ado-Met synthetases alpha and beta in rat liver cytosol have been partially purified about 200- and 80-fold, respectively. The apparent molecular weight estimated by gel filtration and the sedimentation coefficient are 210 000 and 9 S for S-Ado-Met synthetase alpha and 160 000 and 5.5 S for S-Ado Met synthetase beta. Both enzymes absolutely require Mg2+ and K+ for the activity and are completely inhibited by p-(chloromercuri)-benzoate. Kinetic studies indicate that S-Ado-Met synthetases alpha and beta exhibit negative cooperativity with low S0.5 (ligand concentration required for half-maximal velocity) for L-methionine (17 microM) and ATP (0.5 mM) and positive cooperativity with much higher S0.5 values (S0.5 (L-methionine) = 0.5 mM, S0.5 (ATP) = 2 mM), respectively. The cryoprotectants dimethyl sulfoxide and glycerol markedly lower the S0.5 values of S-Ado-Met synthetase beta without significant effect on Vmax. A single species of S-Ado-Met synthetase has been purified about 1000-fold from rat kidney cytosol. The kidney enzyme, termed S-Ado-Met synthetase gamma, has an apparent molecular weight of 190 000 and a sedimentation coefficient of 7.5 S and is resistant to the inhibition by p-(chloromercuri)benzoate. S-Ado-Met synthetase gamma exhibits slightly negative cooperativity with an apparent S0.5 value for L-methionine of 6 microM and for ATP of 70 microM.

Adenosine Triphosphate↗
PubMed1980

Changes in patterns of S-adenosylmethionine synthetases in fetal and postnatal rat liver.

Three isozymes of S-adenosylmethionine synthetase have been measured in the livers of rats from fetal life to maturity. A greater part of gamma-type enzyme is shown to be present in fetus and is joined by alpha- and beta-types during development. The amount of beta-type enzyme activated by dimethyl sulfoxide is very low in the fetus and rises sharply until about the 15th day after birth. The alpha-type enzyme appears after birth and increases in amount to the tenth day.

Age Factors↗
PubMed1979

The predominantly nonhydrolytic action of alpha amylases on alpha-maltosyl fluoride.

Crystalline alpha amylases from a number of sources utilized alpha-maltosyl fluoride as a glycosyl donor and acceptor at high rates (approximately 10 to approximately 1550 mumol/min/mg of protein, for 30 mM substrate). All enzymes catalyzed conversion of this compound into maltooligosaccharides in preference to causing its hydrolysis. Maltotetraosyl flouride and maltooligosaccharides of d.p. 3 to 6+ accounted for 75--93% (by weight) of early reaction-products. At a late stage, the yield of maltooligosaccharides was 2--5 times that of maltose, with chains as long as 12 D-glucosyl residues formed by one amylase (from Asp. oryzae), which utilized alpha-maltosyl fluoride as a donor and as an acceptor at extremely high rates. These results indicate that alpha amylases have a substantial capacity for binding two molecules of this small substrate in a distinctive way, with the C--F glycosylic bond of one and the free C-4 hydroxyl group of the other located in the region of the enzyme's catalytic groups, therby favoring glycosylation of the suitably positioned acceptor over solvent water. Hydrolysis is assumed to prevail when only a single substrate molecule or segment binds to alpha amylase with a (1 linked to 4)-alpha-D-glucosidic linkage of glycosylic C--F bond positioned at the catalytic center. The present demonstration that glycosyl-transfer reactions can be dominantly expressed by alpha amylases, given an appropriate substrate, illustrates the inadequacy of the usual characterization of these enzymes as hydrolases that produce overwhelming hydrolysis of all substrates.

Amylases↗
PubMed1976

Enzymatic studies on a cellulase system of Trichoderma viride. IV. Purification and properties of a less-random type cellulase.

A cellulase [EC 3.2.1.4] component was purified from a crude cellulase preparation of Trichoderma viride (Meicelase) by consecutive column chromatography procedures, and was designated as cellulase III. The enzyme was homogeneous on polyacrylamide gel disc electrophoresis. The molecular weight of the enzyme was estimated to be about 45,000 by gel filtration. The optimum pH and temperature of the enzyme were pH 4.5-5.0 and 50 degrees, respectively. The enzyme was stable over the range of pH 4.5-7.5 at 4 degrees for 24 hr, and retained 40% of the original carboxymethylcellulose-saccharifying activity after heating at 100 degrees for 10 min. The enzyme was completely inactivated by 1 mM Hg2+, and partially by 1 mM Ag+ and Cu2+. The enzyme was characterized as a less-random type cellulase on the basis of its action on carboxymethylcellulose. The enzyme split cellohexaose, retaining the beta-configuration of the anomeric carbon atoms in the hydrolysis products. The Km values of cellulase III for cellooligosaccharides decreased in parallel with increase of the chain length of the substrates, while Vmax values showed a tendency to increase. The enzyme produced predominantly cellobiose and glucose from various cellulosic substrates as well as from higher cellooligosaccharides. Cellulase III preferentially attacked the aglycone linkage of p-nitrophenyl beta-D-cellobioside. The enzyme was found to catalyze the rapid synthesis of cellotetraose from cellobiose (condensation action).

Carboxymethylcellulose Sodium↗
PubMed1975

Enzymatic studies on a cellulase system of Trichoderma viride. II. Purification and Properties of two cellulases.

Two cellulase [EC 3.2.1.4] components derived from Meicelase, a commercial crude cellulase preparation from Trichoderma viride, were purified by consecutive column chromatography, and were designated as cellulase II-A and cellulase II-B. Cellulases II-A and II-B were each homogeneous on polyacrylamide gel electrophoresis. The molecular weights of cellulases II-A and II-B were 30,000 and 43,000, respectively, on the basis of Sephadex G-100 gel filtration. Both enzymes contained 12-14% carbohydrates (as glucose). Some properties of the purified cellulases were investigated. The optimum pH and temperature for cellulases II-A and II-B were pH 4.5-5.0 and 60 degrees, and pH 4.5-5.0 and 50 degrees, respectively. Both enzymes were stable over the range of pH 5.0-7.0 at 4 degrees for 24 hr. Cellulases II-A and II-B retained 27 and 41% of the original CM-cellulose-saccharifying activities, respectively, after heating at 100 degrees for 10 min. Both enzymes were completely inhibited by some metal ions such as 1 mM Hg-2+, and partially by 1 mM Ag-+ and Cu-2+. However, Mg-2+, Fe-2+, and several other metal ions showed no inhibition at this concentration. The hydrolysis of CM-cellulose by cellulase II-A was more random than that by cellulase II-B.

Animals↗
PubMed1975

Enzymatic studies on a cellulase system of Trichoderma viride. III. Transglycosylation properties of two cellulase components of random type.

Two highly purified cellulases [EC 3.2.1.4], II-A, and II-B, were obtained from the cellulase system of Trichoderma viride. Both cellulases split cellopentaose retaining the beta-configuration of the anomeric carbon atoms in the hydrolysis products at both pH 3.5 and 5.0. The Km values of cellulases II-A and II-B for cellotetraose were different, but their Vmax values were similar and those for cellooligosaccharides increased in parallel with chain length. Both cellulases produced predominantly cellobiose and glucose from various cellulosic substrates as well as from higher cellooligosaccharides. Cellulase II-A preferentially attacked the holoside linkage of rho-nitrophenyl beta-D-cellobioside, whereas cellulase II-B attacked mainly the aglycone linkage of this cellobioside. Both cellulases were found to catalyze the synthesis of cellotriose from rho-nitrophenyl beta-D-cellobioside by transfer of a glucosyl residue, possibly to cellobiose produced in the reaction mixture. They were also found to catalyze the rapid synthesis of cellotetraose from cellobiose, with accompanying formation of cellotriose and glucose, which seemed to be produced by secondary random hydrolysis of the cellotetraose produced. The capacity to synthesize cellotetraose from cellobiose appeared to be greater with cellulase II-B than with cellulase II-A.

Animals↗
PubMed1966

Cellulases of a marine mollusc, Dolabella sp.

1. A crude cellulase extract was prepared from the hepatopancreas of a marine mollusc, Dolabella sp., and partially purified by ammonium sulphate fractionation. 2. The optimum pH values of the partially purified preparation were 6.5 and 8.0 for Walseth cellulose and CM-cellulose respectively. It was most stable at pH6.0 and showed moderate thermostability. 3. The partially purified preparation was subjected to starch-zone electrophoresis, and incompletely resolved into several fractions that contained one or more cellulase components of different substrate specificity. 4. Some of these cellulase fractions showed practically no aryl beta-glucosidase activity and hydrolysed aryl beta-cellobioside with difficulty. From substrates such as higher cello-oligosaccharides, cellodextrin, CM-cellulose, Walseth cellulose and cotton fibre, they produced cellobiose as the major and cellotriose as the minor end products, both of which were resistant to further attack by cellulase. 5. From the slope of the curves of viscosity-reducing power for CM-cellulose, the cellulase components from Dolabella were presumed to be of a ;more-random' or a ;less-random' type in the mode of action. 6. In the hepatopancreas of this mollusc, beta-glucosidases were also present, which hydrolysed cellobiose as well as aryl beta-glucosides. The optimum pH values of these enzymes were about 5.5.

Animals↗
PubMedDate not supplied

Carcinogen-induced de novo methylation in c-myc exon I.

During the response of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylation occurred at the Hpa II site of c-myc exon I, which is located downstream of the P1 initiation site, as evidenced by the assays of Hpa II-PCR. The Hpa II spite of the 5' flanking region did not undergo methylation. UV-irradiation also led to methylation in exon I. The extent of methylation increased depending on the dose of MNNG and UV. The results suggested that methylation takes place in transcriptionally active c-myc responsible for carcinogens and is caused by mechanisms different from that of alkylation in a specific CpG site. Possible contribution of methylation to less repair found in c-myc is discussed.

Carcinogens↗
PubMedDate not supplied

Sensitization and caffeine potentiation of cisplatin cytotoxicity resulting from introduction of wild-type p53 gene in human osteosarcoma.

The present study was performed to investigate whether the introduction of a wild-type p53 gene into human osteosarcoma cells could alter the growth rate and enhance the cytocidal effect of cisplatin (CDDP) and the synergistic antitumor effect of caffeine. The lipofection method was used to transfect a wild-type p53 expression plasmid into the human osteosarcoma cell line, Saos2, which has both p53 alleles deleted. The transfected cells, Saos2/p53, had a reduced growth rate compared with the parental cell line. The colorimetric WST-1 assay demonstrated that Saos2/p53 cells were twice as sensitive to CDDP alone at a 50% inhibition concentration than the parental Saos2 cells. Caffeine significantly potentiated the cytocidal effect of CDDP in the Saos2/p53 cells. Furthermore, the TUNEL assay revealed that following treatment both with CDDP alone and with CDDP combined with caffeine, a higher percentage of the Saos2/p53 cells underwent apoptosis than did the parental Saos2 cells. Therefore the cytocidal effect of CDDP and the synergistic antitumor effect of caffeine are enhanced by the introduction of a wild-type p53 gene into a human osteosarcoma cell line null for p53. This raises the possibility that gene therapy using the p53 gene may prove efficatious for human osteosarcomas lacking p53 and which are resistant to standard chemotherapy.

Apoptosis↗