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Biomedical subjects

Y Mukohata

Publications and source records attributed to Y Mukohata.

54 records · Page 3Linked to original sources
PubMed1985

Purification and properties of a peroxidase from Halobacterium halobium L-33.

A peroxidase was purified from Halobacterium halobium L-33 to an electrophoretically homogeneous state and some of its properties were studied. The enzyme showed an absorption peak at 406 nm in the oxidized form and peaks at 440, 558, and 591 nm in the reduced form. The difference spectrum, reduced + CO minus reduced, of the enzyme showed peaks at 425, 538, and 577 nm and troughs at 444, 562, and 596 nm. These spectral properties were apparently similar to those of "cytochrome a1" except for the occurrence of the peak at 558 nm in the reduced form. The molecular weight of the enzyme was 110,000 and the enzyme possessed one unit of protoheme in the molecule. The activity to oxidize guaiacol in the presence of H2O2 of the peroxidase was about one-twentieth of that of horseradish peroxidase. The enzyme also showed a catalase-activity one-fourth as active as that of liver catalase. The reactions catalyzed by the enzyme were strongly inhibited by KCN.

Amino Acids↗
PubMed1984

Isolation and characterization of halorhodopsin from Halobacterium halobium.

Chromoprotein of a light-driven chloride pump, halorhodopsin (HR), was isolated from Halobacterium halobium L-33, which contains HR and "slowly cycling rhodopsin-like pigment" (SR) but lacks bacteriorhodopsin (BR). The isolation was run in the presence of more than 2 M NaCl, which was required to preserve this halophilic retinal protein. Cell envelope vesicles were washed with Tween-20 to remove 80% of the proteins. The residual membranes were solubilized with 0.5% C12E9, which had little effect on the photochemical activities of HR and SR. HR was purified by passing it through a hydroxyapatite and then a phenyl-Sepharose column in 2 M NaCl and 0.5% C12E9. The absorption maximum of HR was 578 nm and the ratio of absorbance at 280 nm to 580 nm was 1.52. The apparent molecular weight of HR was 20,000 on polyacrylamide gel electrophoresis in the presence of SDS. The characteristic, bilobed CD spectrum of HR in the visible region suggested that HR exists as an oligomer in both its membrane-bound and isolated forms.

Bacteriorhodopsins↗
PubMed1982

[Halorhodopsin].

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Adenosine Triphosphate↗
PubMed1980

Effect of digitonin on photophosphorylation and light-induced H+ uptake in isolated spinach chloroplasts.

The effects of the presence of digitonin in the reaction mixture on photophosphorylation, light-induced H+ uptake, and the size of isolated spinach chloroplasts were studied. Digitonin inactivated photosystem II and dissipated light-induced pH increase without affecting the photophosphorylation driven by photosystem I. Digitonin increased the concentration of NH4Cl required for uncoupling. This effect of digitonin was diminished by valinomycin which barely affects phosphorylation by itself. Although such properties have all been observed with digitonin subchloroplast particles, digitonin at the concentration required for acquiring such properties had little effect on the size of chloroplasts. These results suggest that the characteristic effects of digitonin subchloroplast particles are caused by thylakoid membranes penetrated by digitonin but not by the size of membrane vesicles. Both in chloroplasts with digitonin in the reaction mixture and in digitonin subchloroplast particles, N,N'-dicyclohexylcarbodiimide, purine nucleoside di- and tri-phosphates, and organic bases with low pKa values (pyridine and aniline) made the light-induced pH increase detectable, and this was lost upon the addition of proton ionophores. These results suggest that the lack of the light-induced pH increase by chloroplasts with digitonin in the reaction mixture and by digitonin subchloroplast particles is caused both by a membrane leaky to protons and by the loss of internal buffering capacity of thylakoids. Furthermore, the phosphorylation of chloroplasts with digitonin in the reaction mixture and of digitonin subchloroplast particles is considered to be driven by the proton motive force as is that of chloroplasts without digitonin.

Chloroplasts↗
PubMed1978

Acceleration of the ATPase activity of glycerol-treated muscle fibers by repeated stretch-release cycles.

Glycerol-treated muscle fiber bundles were fixed at their rest length in 50 mM KC1, 2 mM MgC1(2), and 10 micron CaC1(2) at pH 7.8 and 0 degrees C in the presence of sufficient amounts of ATP, creatine kinase, and creatine phosphate. The fiber bundles were stretched linearly with time for 0.3 s at a constant amplitude, suddenly released, then fixed at the rest length for a constant time interval (alpha seconds). The stretch-release cycle was repeated, and the ATPase activity (the rate of ADP liberation) [EC 3.6.1.3] was measured. It was found that: 1. ATPase was activated by repeated stretch-release. As repetitive stretch-release of 1--2% of the rest length caused maximum activation, we usually selected a value of 2.5% of the rest length. The activation of ATPase was found to be a function of the duration, alpha, of the isometric phase after sudden release from stretching. The ATPase activity of fiber bundles was almost unaffected when they were oscillated by a simple stretch-release without an isometric phase after the sudden release (alpha=0). 2. The ATPase activity of oscillated muscle fibers increased with increase in the value of alpha, reached a maximal level, then decreased gradually with further increase of alpha to a value slightly larger than that of static fibers. At 0 degrees C, the value of alpha for the maximum activation was observed at about 2 s, and the maximum activity was about 2.5 times that of static fibers. At 20 degrees C, the alpha value for maximum activation was about 0.5 s, and the maximum activity was about 1.8 times that of static fibers. 3. The time course of ADP liberation after one stretch-release cycle could be easily calculated from the ATPase activity of the summed durations of the isometric phase, alpha, assuming that the ATPase activation was turned off and on by the stretching and release, respectively, and that the state of cross-bridges immediately after the stretch-release was independent of alpha of the cycle. The rate of ADP liberation after stretch-release thus obtained showed a short lag phase, a sigmoidal increase, a decrease to almost zero, then a return to nearly the original level (the rate of static fibers). About 1.3 mol of ATP per mol of myosin was hydrolyzed at both 0 degrees C and 20 degrees C during one cycle of the changes in the rate of ADP liberation.

Adenosine Triphosphatases↗
PubMed1977

Effects of purine nucleotides on photosynthetic electron transport in isolated chloroplasts.

The effects of guanylates and inosinates (and adenylates) on phosphorylation, ferricyanide reduction, and light-induced H+ uptake in spinach chloroplasts were studied. GDP, GTP, IDP, and ITP (but not GMP and IMP) stimulated the light-induced H+ uptake and partially inhibited ferricyanide reduction. Phosphate, arsenate, and phlorizin increased the extent of inhibition by these nucleotides and decreased the values of their apparent dissociation constants for the inhibition process. In the presence of phosphate (or arsenate), restoration of ferricyanide reduction from the level inhibited by guanylates and inosinates was observed as phosphorylation (or arsenylation) proceeded. These results suggest that phosphorylation of GDP and IDP as well as ADP takes place after two steps of nucleotide binding to the chloroplast coupling factor 1. The apparent dissociation constants of GDP and IDP for these two binding steps were estimated to be about 34 and 38 muM for the first and 110 and 160 muM for the second step, respectively (at pH 8.3, 15 degrees C). Above pH9, the ratio (P/delta-e) of the extent of phosphorylation to the increment of electron transport from the basal level measured in the presence of [ATP + Pi] or [ADP + Pi+ phlorizin], became increasingly large. When the electron transport level inhibited by dicyclohexyl-carbodiimide was taken to be the basal activity, the P/delta-e ratio remained almost constant (approximately 1) from pH 7.0 up to 10.

Adenosine Monophosphate↗
PubMed1976

Magnesium ion-induced changes in the binding mode of adenylates to chloroplast coupling factor 1.

The effect of Mg2+ on the binding of adenylates to isolated chloroplast coupling factor 1 (CF1) was studied using CD spectrometry and ultrafiltration. At adenylate concentrations smaller than 100 muM, one mole of CF1 binds three moles of ATP (or ADP) regardless of the presence of Mg2+. In the presence of Mg2+, the first two ATP's bind to CF1 independently with the same binding constant of 2.5 X 10(-1) muM-1, then the third ATP binds with a much higher affinity of 10 muM-1. In the absence of Mg2+, the first ATP binds to CF1 with a binding constant of 2.5 X 10(-1) muM-1 then the other two ATP's bind less easily with the same binding constant of 4.0 X 10(-2) muM-1. The binding mode of ADP to CF1 is quite similar to that of ATP. In the presence of Mg2+, the binding constants of the first two ADP's are both 7.6 X 10(-2) muM-1, that of the third ADP being 4.0 muM-1. In the absence of Mg2+, the binding constant of the first ADP is 7.6 X 10(-2) muM-1, the constants of the other two ADP's both being 4.0 X 10(-2) muM-1. AMP caused a negligible change in CD.

Adenosine Diphosphate↗