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K Koike

Publications and source records attributed to K Koike.

At least 865 records · Page 48Linked to original sources
PubMed1977

Template specificity of rat mitochondrial DNA polymerase.

Mitochondrial DNA polymerase was purified 2300-fold over isolated mitochondria from rat liver. Template-primer specificities of this enzyme were investigated. Activated DNA was satisfactorily used as an active template-primer, but both native and denatured DNAs showed a slight activity. Synthetic polynucleotide, poly(dA) - oligo(dT)10 was found to have a high efficiency under the same condition for activated DNA. When the closed-circular, nicked and gapped Co1E1 DNAs were employed as a template-primer, the enzyme could only utilize the gapped DNA, indicating that the displacement synthesis was not catalyzed by the enzyme itself. The enzyme also copied poly(A) - oligo(dT)10 in high efficiency at pH 7.5 in the presence of MnCl2. Such RNA-directed DNA polymerase activity of the enzyme was further characterized. Cofractionated endouclease activity was completely separated from the enzyme by glycerol gradient centrifugation.

Animals↗
PubMed1977

Mitochondrial DNA polymerase from rat liver.

A DNA-dependent DNA polymerase from rat liver mitochondria was partially purified and characterized. Mitochondrial DNA polymerase has been found to be quite different from other DNA-dependent DNA polymerases alpha and beta present in the rat liver in the following points: elution patterns in a DEAE-cellulose column chromatography, sedimentation coefficients determined by the glycerol gradient centrifugation in the presence of high salt, and sensitivities to N-ethylmaleimide, ethidium bromide and KCl.

Animals↗
PubMed1976

Mode of extension of the daughter strands in the replication of closed circular mitochondrial DNA in vitro.

1. The properties of nascent DNA in the replicative closed circular mitochondrial DNA were examined in the in vitro system of discontinuous replication, using newborn rat liver mitochondria containing endogeneous DNA templates and enzymes. 2. The nascent DNA associated with the closed circular DNA fraction was found to be of two types; one class consisted of the fragments, and the other of the higher molecular-weight DNAs. Data from pulse and chase experiments indicate that the fragments were initially synthesized and subsequently converted into both heavy and light strands of the higher molecular-weight DNAs in an asymmetrical mode. 3. DNA - DNA hybridization experiments revealed that half of the fragments at least were copies of complementary parts of the parental DNA. 4. Based on the present in vitro data, a tentative structure of the replicating region and its expansion are discussed.

Animals↗
PubMed1976

Effect of guanidine hydrochloride on the holo- and apo-enzymes of pig heart lipoamide dehydrogenase.

1. The effect of guanidine hydrochloride (GuHCl) on pig heart lipoamide dehydrogenase [NADH: lipoamide oxidoreductase, EC 1.6.4.3.] was investigated by means of enzymatic activity and optical measurements (CD, absorption, and fluorescence spectra). The activity of the enzyme decreased on increasing the concentration of GuHCl and the enzyme was completely inactivated in 2.0 M GuHCl. 2. The contents of alpha-helix, beta, and unordered forms in lipoamide dehydrogenase were estimated to be 34, 14, and 52%, respectively. On increasing the concentration of GuHCl, the content of alpha-helix in lipoamide dehydrogenase decreased, whereas the content of the beta form hardly changed. 3. The native lipoamide dehydrogenase showed absorption, CD, and fluorescence spectra characteristic of bound FAD in the visible region, suggesting hydrophobic interaction between the protein moiety and FAD chromophore. The absorption, CD, and fluorescence spectra of the enzyme in 2.0 M GuHCl were similar to those of free FAD in the buffer, suggesting the release of FAD from the protein moiety. 4. The protein fluorescence spectrum of lipoamide dehydrogenase had a maximum at 350 nm blue-shifted by 8 nm from that of tryptophan in aqueous solution. The maximum of the enzyme in 2.0 M GuHCl was red-shifted to 357 nm. This suggests exposure of tryptophan residues to a polar environment. The maximum, 352nm, of the apoenzyme shifted to 350 nm on addition of FAD. These results show that the conformation in the microenvironment of some tryptophan residues in lipoamide dehydrogenase is affected by the dissociation-association of FAD. 5. The contents of alpha-helix, beta, and unordered forms in the apoenzyme were estimated to be 35, 8, and 57%, respectively. These values are similar to those of the native holoenzyme. The alpha-helical structure in the apoenzyme molecule was more sensitive to GuHCl than that in the holoenzyme. FAD and two hydrophobic probes, 8-anilinonaphthalene-1-sulfonate (ANS) and 4 benzolamido-4'-aminostilbene-2,2'-disulfonate (MBAS), which can bind to the apoenzyme, stabilized the alpha-helical structure in the apoenzyme molecule.

Animals↗
PubMed1976

Interaction of hydrophobic probes with the apoenzyme of pig heart lipoamide dehydrogenase.

The interaction of hydrophobic probes, 8-anilinonaphthalene-1-sulfonate (ANS) and 4-benzoylamido-4'-aminostilbene-2, 2'-disulfonate (MBAS), with pig heart lipoamide dehydrogenase [NADH: lipoamide oxidoreductase, EC 1.6.4.3] was investigated. When ANS or MBAS was mixed with the apoenzyme of lipoamide dehydrogenase, the fluorescence quantum yield, of each dye was enhancedd markedly and the emission maxima concurrently shifted to the blue. The quantum yield, 0.038, of ANS bound to the apoenzyme, calculated from the corrected emission spectrum, was eight times higher than that in buffer solution, and the value, 0.0090, for bound MBAS was eighteen times higher than that in buffer solution. Moreover, the absortion bands of both ANS and MBAS shifted to the red upon binding with the apoenzyme. A general feature of the absorption spectra of these dyes observed on changing the solvent from polar to apolar was a red shift of the absorption bands. These results indicate that ANS or MBAS bound to the apoenzyme of lipoamide dehydrogenase is situated in a hydrophobic region of the apoenzyme molecule. It was found that 2 moles of each dye was bound per mole of the apoenzyme, which contains two polypeptide chains. The dissociation constants for the ANS- and MBAS-apoenzyme complexes were estimated to be 1.03X10(-5) and 1.54X10(-5) M, respectively. The enhanced fluorescence of both dyes bound to the apoenzyme decreased linearly upon adding FAD and disappeared at about 2 moles of FAD per mole of the apoenzyme. This suggests that both ANS and MBAS were displaced from their binding sites on the apoenzyme by FAD. The protein fluorescence spectrum of the apoenzyme had a maximum at 352 nm, which was blue-shifted by 6 nm from that of tryptophan in the buffer. Upon binding ANS or MBAS, the maximum of the protein fluorescence of the apoenzyme returned to 350 nm for the holoenzyme, and the fluorescence intensity decreased. Thus, the conformation around some tryptophan residues was affected by the binding of the dyes. When guanidine hydrochloride (GuHCl) was added to the ANS-apoenzyme complex solution, the enhanced fluorescence due to the bound ANS decreased and the emission maximum concurrently shifted to the red. Further, the maximum of the protein fluorescence of the apoenzyme shifted to the red, indicating the exposure of some tryptophan residues buried in an apolar region of the apoenzyme. Thus the binding of ANS to the apoenzyme was inhibited by protein denaturation due to GuHCL. In contrast, the holoenzyme of lipoamide dehydrogenase did not bind ANS or MBAS at all.

Anilino Naphthalenesulfonates↗
PubMed1976

Properties and subunit structure of pig heart pyruvate dehydrogenase.

Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (alpha and beta), with estimated molecular weights of 41,000 (alpha) and 36,000 (beta), respectively, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as alpha2beta2. The content of right-handed alpha-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively. The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5 X 10(-5) M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and alpha-ketobutyrate, but it also showed some activity with alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuribenzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.

Amino Acids↗
PubMed1976

[Protective effect of schizophyllan on Pseudomonas aeruginosa infection of mouse (author's transl)].

Effects of schizophyllan (SPG) and several antibiotics in combination against experimental Pseudomonas aeruginosa infection were examined. (1) Whereas the increase in protective effect of SPG was not observed in combination with carbenicillin or sulbenicillin, remarkable effects could be recognized in combination with dibekacin or gentamicin (GM). The combined therapy with SPG and GM proved to be most excellent. (2) The time course for changes of viable number of Ps. aeruginosa in various organs of mice revealed that the multiplication of the infected bacteria was more markedly inhibited in mice treated with SPG and GM in combination than those treated with each alone. (3) The increase in bactericidal activity of the peritoneal exudate cells was more remarkably demonstrated in the combined treatment with SPG and GM than with each alone, showing the rapid clearance of bacteria from the peritoneal cavity in the former. (4) The microscopic estimation demonstrated that the increase in both phagocytic index and intracellular digestion of bacteria by cultivated macrophages was larger in combination of SPG and GM than each alone. (5) The measurement of viable number of bacteria in cultivated macrophages by the plate-count method also showed that the ingestion and bactericidal activity within the cells were much higher in combined treatment with SPG and GM than each alone.

Animals↗
PubMed1976

Structure, assembly and function of mammalian alpha-keto acid dehydrogenase complexes.

An attempt has been made to provide a description of the mammalian alpha-keto acid dehydrogenase multienzyme complexes, especially with respect to the structure, assembly and function of PDC and OGDC based on the investigation carried out in this laboratory over the last 14 years. Special efforts have been focused on the purification, characterization, and stoichiometric resolution into component enzymes, and the reassembling process to produce these multienzyme complexes. Studies of the macroorganization and the mechanism of regulation of these multienzyme complexes still remains a most challenging question.

Acetyltransferases↗
PubMed1975

Abnormal synthesis of mitochondrial DNA in the presence of N-methyl-N-nitro-N-nitrosoguanidine in vitro.

1. The in vitro effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on mtDNA synthesis was studied using isolated newborn rat liver mitochondria. 2. From the kinetics of the incorporation of [3H]thymidine into the acid-insoluble material, MNNG neither stimulated nor inhibited the DNA synthesizing activity of mitochondria. The activity observed in the presence of MNNG was inhibited by N-ethylmaleimide and actinomycin D. 3. By the band velocity sedimentation in CsCl/ethidium bromide, the properties of the nascent mtDNA formed in the presence of MNNG were analyzed. The nascent DNA-containing molecule was not found in the closed-circle fraction, and essentially detected in the open-circle fraction. This change of the template was blocked by N-ethylmaleimide but not by actinomycin D, suggesting a conversion of the closed-circular template to the open-circular one by single-strand cleavage(s). From the band sedimentation in alkaline CsCl, the number of nascent higher molecular DNAs was increased but the molecules were all of relatively lower molecular weight. On the other hand, the formation of nascent fragments was inhibited. 4. The alkaline CsCl equilibrium centrifugation analysis revealed that the nascent DNA synthesized in the presence of MNNG consisted of both light and heavy components. 5. Present results suggest that MNNG exerts its effect on the mtDNA synthesis by modifying the intrinsic mechanism of discontinuous synthesis, since the conversion of the template DNA molecule from the closed- to open-circular form and the continuous polymerization of the nascent higher-molecular DNA on such a relaxed template were characteristic events in vivo.

Animals↗
PubMed1975

A kinetic study of the alpha-keto acid dehydrogenase complexes from pig heart mitochondria.

The kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease complexes from pig heart mitochondria were studied at pH 7.5 and 25 degrees. A three-site ping-pong mechanism for the actin of both complexes was proposed on the basis of the parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvate concentration for various levels of CoA and a level of NAD+ near its Michaelis constant value. Rate equations were derived from the proposed mechanism. Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reaction are: 2-oxoglutarate, 0.220 mM; CoA, 0.025 mM; NAD+, 0.050 mM. Those of the pyruvate dehydrogenase complex are: pyruvate, 0.015 mM; CoA, 0.021 mM; NAD+, 0.079 mM. Product inhibition studies showed that succinyl-CoA or acetyl-CoA was competitive with respect to CoA, and NADH was competitive with respect to NAD+ in both overall reactions, and that succinyl-CoA or acetyl-CoA and NADH were uncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for succinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These results are consistent with the proposed mechanisms.

Acetyl Coenzyme A↗