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K Aki

Publications and source records attributed to K Aki.

At least 19 recordsLinked to original sources
PubMed2000

Functional association between nicotinic acetylcholine receptor and sarcomeric proteins via actin and desmin filaments.

By affinity chromatography utilizing alpha-cobrotoxin from digitonin-solubilized fractions of rabbit skeletal muscle, we found that many proteins are associated with the nicotinic acetylcholine receptor (AChR). In addition to the proteins we previously reported to bind to AChR (including dystrophin-dystrophin-associated protein (DAP) complex, utrophin, rapsyn, and actin; Mitsui et al. [1996] Biochem. Biophys. Res. Commun.224:802-807), alpha-actinin, desmin, myosin, tropomyosin, troponin T, and titin are also identified to be associated with AChR. Alkaline treatment or Triton X-100 solubilization released dystrophin-DAP complex, utrophin, and rapsyn from the AChR fraction, while actin and desmin remained associated. These findings demonstrate that AChR is supported primarily by a submembranous organization of actin and desmin filaments, and is linked to sarcomeric proteins via these filaments. To further investigate whether the association has any functional role, we studied the effect of acetylcoline on ATPase activity of the AChR fraction. Acetylcholine (0.5-4 microM) significantly activated Mg(2+)-ATPase activity of digitonin-solubilized AChR fraction (P < 0.05). Furthermore, we found that desmin as well as actin activated myosin Mg(2+)-ATPase activity. From these findings, it is suggested that desmin and actin form a submembranous organization in the postsynaptic region, and function as mediators of excitation of AChR to the sarcomeric contraction system.

Acetylcholine↗
PubMed1998

Evidence of shallow fault zone strengthening after the 1992 M7.5 landers, california, earthquake

Repeated seismic surveys of the Landers, California, fault zone that ruptured in the magnitude (M) 7.5 earthquake of 1992 reveal an increase in seismic velocity with time. P, S, and fault zone trapped waves were excited by near-surface explosions in two locations in 1994 and 1996, and were recorded on two linear, three-component seismic arrays deployed across the Johnson Valley fault trace. The travel times of P and S waves for identical shot-receiver pairs decreased by 0.5 to 1.5 percent from 1994 to 1996, with the larger changes at stations located within the fault zone. These observations indicate that the shallow Johnson Valley fault is strengthening after the main shock, most likely because of closure of cracks that were opened by the 1992 earthquake. The increase in velocity is consistent with the prevalence of dry over wet cracks and with a reduction in the apparent crack density near the fault zone by approximately 1.0 percent from 1994 to 1996.

Journal Article↗
PubMed1998

L-lactate oxidase from Aerococcus viridans crystallized as an octamer. Preliminary X-ray studies.

Crystals of flavoenzyme L-lactate oxidase from Aerococcus viridans (LOX) have been obtained that diffract to 3.0 A resolution (P2(1)2(1)2(1), a = 118.4 A, b = 138.4 A, c = 194.6 A). Crystallographic studies suggest that the enzyme may exist as an octameric form with non-crystallographic two- and four-fold axes in the center of the octamer. The four-fold axis makes the tetramer tight, and the tetramers lie upon one another by the two-fold axis.

Chromatography, Gel↗
PubMed1997

On the reaction mechanism of L-lactate oxidase: quantitative structure-activity analysis of the reaction with para-substituted L-mandelates.

The rate constants for reduction of the flavoenzyme, L-lactate oxidase, and a mutant (in which alanine 95 is replaced by glycine), by a series of para-substituted mandelates, in both the 2-1H- and 2-2H- forms, have been measured by rapid reaction spectrophotometry. In all cases, significant isotope effects (1H/2H = 3-7) on the rate constants of flavin reduction were found, indicating that flavin reduction is a direct measure of alpha-C-H bond breakage. The rate constants show only a small influence of the electronic characteristics of the substituents, but show a good correlation when combined with some substituent volume parameters. A surprisingly good correlation is found with the molecular mass of the substrate. The results are compatible with any mechanism in which there is little development of charge in the transition state. This could be a transfer of hydride to the flavin N(5) position or a synchronous mechanism in which the alpha-C-H is formally abstracted as a H+ while the resulting charge is simultaneously neutralized by another event.

Binding Sites↗
PubMed1996

Conversion of L-lactate oxidase to a long chain alpha-hydroxyacid oxidase by site-directed mutagenesis of alanine 95 to glycine.

A mutant form of L-lactate oxidase (LOX) from Aerococcus viridans in which alanine 95 was replaced by glycine was constructed as a mimic of L-lactate monooxygenase but proved instead to be a mimic of the long chain alpha-hydroxyacid oxidase from rat kidney. A95G-LOX keeps oxidase activity with L-lactate at the same level as wild type LOX but has much enhanced oxidase activity with longer chain L-alpha-hydroxyacids, alpha-hydroxy-n-butyric acid, alpha-hydroxy-n-valeric acid, etc., and also the aromatic alpha-hydroxyacid, L-mandelic acid. Kinetic analysis of the activity with these substrates indicates that the reduction of the enzyme bound flavin by substrates is the rate-limiting step in A95G-LOX. The affinity of pyruvate for the reduced enzyme is increased, and sulfite binding to the oxidized enzyme is weaker in A95G-LOX than in native enzyme. Wild type LOX stabilizes both the neutral and anionic flavin semiquinones with a pKa of 6.1, but A95G LOX stabilizes only the anionic semiquinone form. These results strongly suggest that the environment around the N5-C4a region of the flavin isoalloxazine ring is changed by this mutation.

Alanine↗
PubMed1996

Induction of dystrophin-associated proteins together with nicotinic acetylcholine receptors by denervation in the absence of dystrophin in skeletal muscles of mdx mice.

We purified the nicotinic acetylcholine receptor from digitonin-solubilized rabbit skeletal muscle by affinity chromatography and detected many proteins linked to AChR, including dystrophin, adhalin, beta-dystroglycan, utrophin, rapsyn, and actin. To determine whether or not AChR links to dystrophin-associated proteins (DAPs) without dystrophin, we studied the effects of denervation on AChR and DAPs in the skeletal muscle of a mdx mouse. Following surgical denervation, the levels of adhalin and beta-dystroglycan dramatically increased at the extrajunctional sarcolemma with AChR, suggesting that their association is independent of dystrophin. Furthermore, the diffuse extrajunctional appearance of adhalin, beta-dystroglycan, and AChR was observed after pharmacological denervation through the subcutaneous administration of succinylcholine. Since the depletion of DAPs and the subsequent disruption of sarcolemmal linkage are believed to be a primary cause of muscle cell necrosis in dystrophinopathies, pharmacological denervation may have some beneficial effect on these diseases.

Amino Acid Sequence↗
PubMed1996

Scale dependence in earthquake phenomena and its relevance to earthquake prediction.

The recent discovery of a low-velocity, low-Q zone with a width of 50-200 m reaching to the top of the ductile part of the crust, by observations on seismic guided waves trapped in the fault zone of the Landers earthquake of 1992, and its identification with the shear zone inferred from the distribution of tension cracks observed on the surface support the existence of a characteristic scale length of the order of 100 m affecting various earthquake phenomena in southern California, as evidenced earlier by the kink in the magnitude-frequency relation at about M3, the constant corner frequency for earthquakes with M below about 3, and the sourcecontrolled fmax of 5-10 Hz for major earthquakes. The temporal correlation between coda Q-1 and the fractional rate of occurrence of earthquakes in the magnitude range 3-3.5, the geographical similarity of coda Q-1 and seismic velocity at a depth of 20 km, and the simultaneous change of coda Q-1 and conductivity at the lower crust support the hypotheses that coda Q-1 may represent the activity of creep fracture in the ductile part of the lithosphere occurring over cracks with a characteristic size of the order of 100 m. The existence of such a characteristic scale length cannot be consistent with the overall self-similarity of earthquakes unless we postulate a discrete hierarchy of such characteristic scale lengths. The discrete hierarchy of characteristic scale lengths is consistent with recently observed logarithmic periodicity in precursory seismicity.

Journal Article↗
PubMed1995

L-lactate oxidase and L-lactate monooxygenase: mechanistic variations on a common structural theme.

Properties of L-lactate oxidase from Aerococcus viridans are described. The gene encoding the enzyme has been isolated. From its cDNA sequence the amino acid sequence has been derived and shown to have high similarity with those of other enzymes catalyzing oxidation of L-alpha-hydroxy acids, including flavocytochrome b2, lactate monooxygenase, glycolate oxidase, mandelate dehydrogenases and a long chain alpha-hydroxy acid oxidase. The enzyme is expressed in Escherichia coli, and is a flavoprotein containing FMN as prosthetic group. It shares many properties of other alpha-hydroxy acid oxidizing enzymes, eg stabilization of the anionic semiquinone form of the flavin, facile formation of flavin-N(5)-sulfite adducts and a set of conserved amino acid residues around the bound flavin. Steady-state and rapid reaction kinetics of the enzyme have been studied and found to share many characteristics with those of L-lactate monooxygenase, but to differ from the latter in quantitative aspects. It is these quantitative differences between the two enzymes which account for the differences in the overall reactions catalyzed. These differences arise from different stabilities of a common intermediate of reduced flavin enzyme and pyruvate. In the case of the monooxygenase this complex is very stable and is the form that reacts with O2 to give a complex in which the oxidative decarboxylation occurs, yielding the products, acetate, CO2, and H2O (Lockridge O, Massey V, Sullivan PA (1972) J Biol Chem 247, 8097-8106). With lactate oxidase, the complex dissociates rapidly, with the result that it is the free reduced flavin form of the enzyme that reacts with O2, to give the observed products, pyruvate and H2O2.

Amino Acid Sequence↗
PubMed1991

Mechanisms of generation of oxygen radicals and reductive mobilization of ferritin iron by lipoamide dehydrogenase.

The oxidase reaction of lipoamide dehydrogenase with NADH generates superoxide radicals and hydrogen peroxide under aerobic conditions. ESR spin trapping using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was applied to characterize the oxygen radical species generated by lipoamide dehydrogenase and the mechanism of their generation. During the oxidase reaction of lipoamide dehydrogenase, DMPO-OOH and DMPO-OH signals were observed. The DMPO-OOH signal disappeared on addition of superoxide dismutase. These results demonstrate that the DMPO-OOH adduct was produced from the superoxide radical generated by lipoamide dehydrogenase. In the presence of dimethyl sulfoxide, a DMPO-CH3 signal appeared at the expense of the DMPO-OH signal, indicating that the DMPO-OH adduct was produced directly from the hydroxyl radical rather than by decomposition of the DMPO-OOH adduct. The DMPO-OH signal decreased on addition of superoxide dismutase, catalase, or diethylenetriaminepentaacetic acid, indicating that the hydroxyl radical was generated via the metal-catalyzed Haber-Weiss reaction from the superoxide radical and hydrogen peroxide. Addition of ferritin to the NADH-lipoamide dehydrogenase system resulted in a decrease of the DMPO-OOH signal, indicating that the superoxide radical interacted with ferritin iron.

Catalase↗
PubMed1990

Superoxide-mediated release of iron from ferritin by some flavoenzymes.

NADH-lipoamide dehydrogenase mobilized iron from ferritin under aerobic conditions. Superoxide dismutase strongly inhibited this mobilization, indicating that the superoxide radical is generated by the enzymatic reaction and release iron from ferritin. Addition of lipoamide as an electron acceptor to NADH-lipoamide dehydrogenase increased the release of iron from ferritin and this release was partially inhibited by superoxide dismutase. Similarly, addition of menadione (2-methyl-1, 4-naphthoquinone) as an electron acceptor to xanthine-xanthine oxidase promoted the release of iron from ferritin and this release was strongly inhibited by superoxide dismutase. These results suggest that dihydrolipoamide and semiquinone of menadione can react with oxygen to form the superoxide radical that mediates release of iron from ferritin.

Animals↗
PubMed1988

Branched-chain amino acid aminotransferase of Escherichia coli: overproduction and properties.

ilvE gene of Escherichia coli was inserted into the region downstream of the tac promotor. As a result, the branched-chain amino acid aminotransferase was overproduced by about a hundred-fold in E. coli W3110. The overproduced aminotransferase was purified from cell extracts about 40-fold to homogeneity. Chemical and physicochemical analyses confirmed that it was a product of the ilvE gene. The enzyme existed in a hexamer with a subunit molecular weight of 34,000; the double trimer model of the enzyme presumed by the previous chemical cross-linking experiments (Lee-Peng, F.-C. et al. (1979) J. bacteriol. 139, 339-345) was supported by electron micrographs. The circular dichroic (CD) spectrum of branch-chain amino acid aminotransferase had double negative maxima at 210 and 220 nm. The alpha-helical content was estimated to be about 40% from the CD spectrum in the region of 200 to 250 nm. The absorption spectrum of the enzyme showed two peaks at 330 and 410 nm. There was no pH-dependent spectral shift. The CD spectrum of the coenzyme, pyridoxal 5'-phosphate, had negative peaks at 330 and 410 nm. These spectral properties of branched-chain amino acid aminotransferase were quite different from those of E. coli aspartate aminotransferase. Each subunit bound approximately 1 mol of pyridoxal 5'-phosphate. A lysyl residue, which forms a Schiff base with the aldehyde group of the pyridoxal 5'-phosphate, was identified in the primary structure of the enzyme.

Amino Acid Sequence↗
PubMed1987

Biochemical characterization of the conduction system of the bovine heart.

The enzyme level profiles of some regulatory enzymes and the isozyme patterns of some marker enzymes in bovine adult specialized, adult ordinary and fetal ordinary heart muscles were examined in order to biochemically characterize specialized heart muscle. The activities of hexokinase, phosphofructokinase and glucose-6-phosphate dehydrogenase in adult specialized heart muscle were significantly higher than those in adult ordinary heart muscle, but were similar to those in fetal ordinary heart muscle. The carnitine content and carnitine acetyltransferase activity in adult specialized heart muscle were lower than those in adult ordinary heart muscle. The isozyme patterns of creatine kinase, fructose-bisphosphate aldolase and pyruvate kinase in adult specialized heart muscle resembled those in fetal ordinary heart muscle. These results indicate that adult specialized heart muscle has the biochemical characteristics of fetal ordinary heart muscle.

Animals↗
PubMed1986

Region of cytochrome c interacting with yeast cytochrome b2: determination with singly modified carboxydinitrophenyl cytochromes c.

The association and reduction reactions of ten different 4-carboxy-2,6-dinitrophenyl (CDNP) horse heart cytochromes c, singly modified at lysines 8, 13, 27, 39, 60, 72, 73, 86, 87, and 99, with Saccharomyces cerevisiae cytochrome b2 were studied to determine the region of cytochrome c interacting with cytochrome b2. In the presence of higher ratios of free cytochrome c to cytochrome b2, native cytochrome c, and the CDNP-lysine 39, 60, and 99 derivatives associated with cytochrome b2 with a binding stoichiometry close to 2:1, while CDNP-cytochromes c modified at lysines 8, 13, 27, 72, 73, 86, and 87 formed only 1:1 complexes. In the presence of lower ratios of free cytochrome c, modifications of lysines 8, 27, 86, and 87 had more inhibitory effects on the association of cytochrome c with cytochrome b2 than modifications of lysines 13, 39, 60, 72, 73, and 99. This tendency was similar to that on removal of free cytochrome c, except in the case of CDNP-lysine 13 and 73 derivatives. The rate of reduction of cytochrome c by cytochrome b2 was decreased by carboxydinitrophenylation of lysines 8, 13, 27, 72, 73, 86, and 87. In contrast, the rate of reduction of cytochrome c was not affected by modifications of lysines 39, 60, and 99. Since lysines 8, 13, 27, 72, 73, 86, and 87 are located on the front surface and lysines 39, 60, and 99 on the back side, and since different effects of modifying lysine residues located on the front surface may be interpreted in terms of effects on the complementary interaction of cytochrome c and cytochrome b2, these results indicate that the region of cytochrome c interacting with cytochrome b2 is located on the front surface of the cytochrome c molecule containing the exposed heme edge.

Animals↗
PubMed1985

Oxidation and reduction of cytochrome c bound to the phosphoprotein phosvitin.

The oxidation-reduction reactions and structural characteristics of phosvitin-bound cytochrome c were examined at various ratios of cytochrome c to phosvitin. At binding ratios below half the maximum, the rate constants for the oxidation reactions with cytochrome c oxidase and ferricyanide and the rate constants for the reduction reactions with cytochrome b2 and ascorbate were low, but at higher ratios these rate constants gradually increased to that of free cytochrome c and, in particular, the rate constant for oxidation by cytochrome c oxidase was raised to two to three times that of the free form. This binding-ratio dependence of the rate constants for the oxidation and reduction reactions was different from that of the net charge of the cytochrome c-phosvitin complex, implying that the negative charges of phosvitin are unlikely to modulate the rates. In contrast, the broadening of the NMR signals for the heme and methionine-80 methyl groups and the conformational transition in the vicinity of the heme moiety on change from the native to the cyanide-bound or urea-denatured form of cytochrome c showed a similar binding-ratio dependence to the rate constants for the oxidation and reduction reactions. Since the conformation and electronic structure in the heme environment of ferric and ferrous cytochromes c were not changed significantly by binding to phosvitin, and since the binding strength of cytochrome c to phosvitin at binding ratios below half the maximum is different from that at higher ratios, these findings suggest that a difference in the movement of cytochrome c in its complex with phosvitin may modulate its oxidation-reduction reactions.

Circular Dichroism↗
PubMed1985

Sodium-induced aggregation of phosphatidic acid and mixed phospholipid vesicles.

Sodium-induced aggregations of sonicated vesicles prepared from synthetic phosphatidic acid and from its 1:1 mixtures with synthetic phosphatidylethanolamine and phosphatidylcholine were studied by turbidimetric measurements. The aggregation reactions were almost completely reversible on change in the Na+ concentration, pH or temperature. The threshold concentrations of Na+ for aggregations of pure dipalmitoylphosphatidic acid vesicles and mixed dipalmitoylphosphatidylethanolamine- and dimyristoylphosphatidylcholine-dipalmitoylphosphatidic acid vesicles were found to be 200, 310 and 550 mM, respectively, at 25 degrees C and pH 7.2. The hydrocarbon chain lengths of phosphatidic acid and phosphatidylethanolamine had little effect on the threshold concentrations. The threshold concentrations for phospholipid vesicles composed of phosphatidic acid alone or its 1:1 mixture with phosphatidylethanolamine were changed by varying either the pH or temperature, while that for phosphatidylcholine-phosphatidic acid vesicles was almost independent of the pH and temperature, implying that aggregation of the latter vesicles is induced by a somewhat different mechanism.

Dimyristoylphosphatidylcholine↗
PubMed1985

Aspartate aminotransferase isozymes from rabbit liver. Purification and properties.

Cytosolic and mitochondrial isozymes of aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase [EC 2.6.1.1] ) were purified to homogeneity from rabbit liver. The rabbit liver isozymes were closely similar to the corresponding isozymes from other sources, including human heart, pig heart, chicken heart, and rat liver, in their molecular weights, absorption spectra, amino acid compositions, isoelectric points, and Michaelis constants for the substrates. The NH2-terminal amino acid sequences of rabbit liver isozymes were identified up to 30 residues, and showed some differences from those of the corresponding isozymes obtained from other animals so far studied.

Amino Acid Sequence↗