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M Fukuyama

Publications and source records attributed to M Fukuyama.

87 records · Page 5Linked to original sources
PubMed1979

Identification of the covalently bound flavin prosthetic group of cholesterol oxidase.

Highly purified preparations of cholesterol oxidase from Schizophyllum commune contain a covalently bound flavin component. A flavin peptide has been obtained by digestion with trypsin-chymotrypsin and purification on a column of phosphocellulose. Digestion with nucleotide pyrophosphatase results in increased fluorescence at pH 3.4 and release of 5'-adenylate, showing that the flavin is in the dinucleotide form. The absorption spectrum of the flavin peptide shows the hypsochromic shift of the second absorption band characteristic of 8 alpha-substituted flavins. The fluorescence at pH 7 is extensively quenched even in the mononucleotide form, with a pKa at pH 5.8 in the flavin peptide and at 5.05 following acid hydrolysis to the aminoacyl flavin level. This suggests that histidine is the amino acid substituted at the 8 alpha position of the flavin and that N(1) of the imidazole ring is the site of attachment. These data, the reduction of the flavin by borohydride, and comparison of the mobilities in high voltage electrophoresis at two pH values with N(1)- and N(3)-histidyl riboflavin and their 2',5'-anhydro forms shows that the prosthetic group of cholesterol oxidase is 8 alpha-[N(1)-histidyl]-FAD.

3-Hydroxysteroid Dehydrogenases↗
PubMed1979

Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin.

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.

3-Hydroxysteroid Dehydrogenases↗
PubMed1977

Analysis of the reactivity of microsomal cytochrome P-450 toward drugs and exogenous ligands.

Three forms of cytochrome P-450 in rat liver microsomes (Comai, K. and Gaylor, J.L. (1973) J. Biol. Chem. 284, 4947-4955) were characterized as two types of P-450 in the membrane-bound state. One was reactive not only toward cyanide but also aniline, aminopyrine, and hexobarbital, and the other was less reactive toward cyanide and rather unreactive toward the drugs. Cyanide titrations of microsomes in the presence and absence of the drugs were performed spectrophotometrically for analysis of the interactions. The affinity of cyanide for the less reactive P-450 was about twenty times less than that for the reactive P-450. About 40% of P-450 in the microsomal membrane was reactive and the rest was the less reactive form. The reactive P-450 involved two forms having different affinities for cyanide. The ratio of their amounts was 1:2. Triton WR-1339 interfered with cyanide binding to the reactive P-450 mainly. The relative amount of each form of P-450 as well as the dissociation constant of cyanide could be estimated by the present method, and the modes of interaction of the membrane-bound P-450 with the drugs and exogenous ligands were deduced.

Aminopyrine↗
PubMed1976

An oxygraphic method for the determination of cholesterol and measurement of the slow oxygen-consuming reactions of hepatic microsomes.

Oxygen-consuming reactions of cholesterol oxidase [EC 1.1.3.6] and microsomes were measured with a galvanic oxygen electrode which was attached to an offset amplifier for sensitive measurement of the reaction processes. The sensitivity of this oxygraphic method for detection of oxygen consumption was ten times greater than that of the usual method. The minimum rate of slow oxygen-consuming reactions which could be estimated was about 5 nmoles of oxygen per min, and the minimum amount of oxygen consumption which could be determined was also about 5 nmoles. An oxygraphic method for direct and rapid determination of cholesterol was demonstrated using one-twentieth the amount of cholesterol oxidase which is used for the colorimetric method. The processes of cyanide-suppressed beta-NADH-dependent oxygen consumption and cyanide-insensitive alpha-NADH-dependent oxygen consumption, which were difficult to follow by the usual method, were followed using a small amount of microsomes (less than 1mg protein/ml). Furthermore, the temporary cessation of alpha-NADH-dependent oxygen consumption caused by ferricyanide and the corresponding oxidation-reduction of reduced cytochrome b5 were followed in the presence of ADP. ADP did not inhibit the oxygen consumption. The results indicate that the oxygen consumption with alpha-NADH is due to electron transfer from alpha-NADH via NADH-cytochrome b5 reductase and cytochrome b5, in which the rate-determining step lies at some reaction after the reduction of cytochrome b5.

Adenosine Diphosphate↗
PubMedDate not supplied

Efficacy and safety of the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor fluvastatin in hyperlipidemic patients treated with probucol.

The objective of this open trial was to investigate the efficacy and safety of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor fluvastatin in hypercholesterolemic patients already receiving probucol. All of the participants had hypercholesterolemia. i.e. serum total cholesterol > or = 220 mg/dl, despite administration of probucol, 500 mg/day, for more than 4 weeks. After this, fluvastatin, 30 mg/day, was added to probucol treatment for 12 weeks. Twenty-seven patients were recruited into this study; all were evaluated for safety, and 22 were evaluated for efficacy. The addition of fluvastatin to the probucol regimen produced a significant further reduction in serum total and low-density lipoprotein cholesterol concentrations (of 18 and 20%, respectively; p < 0.001); these effects were fully established within 4 weeks of treatment and were maintained throughout the treatment. Fluvastatin did not affect the serum high-density lipoprotein cholesterol concentration. Fluvastatin treatment decreased serum triglyceride concentrations slightly in all patients (not significant); in patients with hypertriglyceridemia, triglyceride levels were decreased significantly by 34% (p < 0.01; serum triglycerides > or = 150 mg/dl). In addition, fluvastatin significantly decreased serum apolipoprotein B, C-II, C-III and E levels, whereas serum apolipoprotein A-I and A-II levels were unaffected. One patient complained of slight abdominal discomfort during fluvastatin administration, but relationship to fluvastatin remains unclear. One patient had slight elevation of the serum alanine aminotransferase level, and another patient had an elevated gamma-glutamyl transferase level. The addition of fluvastatin to probucol treatment can be considered to be an effective and well tolerated treatment in hypercholesterolemic patients.

Adult↗
PubMedDate not supplied

Properties of a calicivirus isolated from cats dying in an agitated state.

In June 1993, two of five pet cats kept in Yokohama city in Japan suddenly became agitated and died. Feline calicivirus (FCV) was isolated from them. One strain (FCV-S) was isolated from the spinal cord, lung and tonsil of cat 1, another (FCV-B) from the ileum, medulla oblongata and cervical spinal cord of cat 2, and a third (FCV-SAKURA) from the oral cavity of one of the three surviving cats which showed no clinical signs. These three strains were equally resistant to pH 3.0 and serologically similar to each other, but distinct from strain F9. A genetic analysis, using a 208 base pair fragment from region E of the capsid, showed that FCV-Ari had a 70.4 per cent nucleotide and 77.3 per cent amino acid homology and FCV-F9 had a 68.6 per cent nucleotide and 73.9 per cent amino acid homology with the three strains, indicating that these two strains were genetically distinct from the three new isolates. Unvaccinated cats and cats which had been vaccinated against FCV-F9 developed watery diarrhoea but did not become agitated after the administration of FCV-S. The FCV-S strain did not induce signs of excitability after it was administered orally to specific pathogen-free cats.

Animals↗
PubMedDate not supplied

Shear stress induces not only platelet aggregation but also platelet-tumor cell interaction.

To investigate the interaction between platelets and tumor cells under well-defined flow conditions, the effect of tumor cells on platelet aggregation induced by shear stress was studied using a cone and plate viscometer adapted for measuring transmitted light intensity. Aggregation was markedly enhanced by HMV-1 cells in a cell number-dependent fashion under shear stress of 12 dyne/cm2. Enhancement was not observed at a high shear stress of 108 dyne/cm2. A monoclonal antibody against GPIIb/IIIa, 7E3 completely abolished enhancement of aggregation by HMV-1. Apyrase had similar inhibitory effects. Scanning electronmicroscopy showed that direct contacts of platelets with HMV-1 cells could be demonstrated when platelet-platelet interaction was inhibited by 7E3 or apyrase. These results may indicate that, at a shear stress of 12 dyne/cm2, direct contacts of platelets and HMV-1 cells may trigger enhancement of platelet aggregation.

Blood Flow Velocity↗
PubMedDate not supplied

Change in intracellular calcium ions during shear induced platelet aggregation.

The authors have developed a method to measure intracellular calcium ion concentration ([Ca2+]i) during shear-induced platelet aggregation. A cone and plate viscometer was adapted for continuous recording of both light transmission and fluorescence intensity. Citrated platelet rich plasma was incubated with Indo-1AM at a concentration of 10 microns for 30 min at 37 degrees C, then applied to the albumin density gradient to prepare washed platelets. To Indo-1AM loaded washed platelets, fibrinogen, and von Willebrand factor (vWf) were added, with 1 mM CaCl2. Platelets were then exposed to changing shear stress (6-108 dynes/cm2) for simultaneous measurement of aggregation and [Ca2+]i. [Ca2+]i in resting platelets was estimated as approximately 100 nM. At low shear stress (10-20 dynes/cm2), [Ca2+]i did not change. In contrast, a marked increase in [Ca2+]i was observed concurrent with aggregation at high shear stress (100-108 dynes/cm2). However, no increase was seen in the presence of 1 mM EGTA. The increase was prevented by monoclonal antibodies against GPIb or vWf, which inhibited vWf binding to GPIb. A monoclonal anti-vWf antibody, which inhibited vWf binding to the GPIIb/IIIa complex, did not affect [Ca2+]i increase during high shear induced platelet aggregation. These results suggest that binding of vWf to GPIb may trigger Ca2+ influx.

Blood Flow Velocity↗