Conformation between the substrate-binding site and heme of cytochrome P-450 studied by excitation energy transfer

The conformation between the substrate-binding site and heme of cytochrome P-450 was studied by excitation energy transfer. Cytochrome P-450 was obtained from the hepatic microsomes of polychlorinated biphenyl-treated male rats, and ten polycyclic aromatic hydrocarbons were used as substrates. The energy transfer from the substrate to the heme of the enzyme was measured according to the F?rster equation. On the basis of the assumption that the substrates are bound at different positions in the plane of the same substrate-binding site, the position of the heme in relation to the substrate-binding site was determined in solution and in the presence of synthetic phospholipid. The results demonstrated that the distance between the substrate-binding site and the heme of cytochrome P-450 was greater when the enzyme was incorporated into micelles of phospholipid than when in solution, and that the conformational relationship of the substrate-binding site towards the heme was changed by an angle of 21 degrees on incorporation of the enzyme into phospholipid micelles.     

Pathological changes of myocardial cytoskeleton in cardiomyopathic hamster

Immunocytochemical investigation was performed on the cytoskeletal proteins in cardiac tissue of the cardiomyopathic hamster. Male cardiomyopathic UM-X7.1 hamsters at 180 days of age (n=8) and age- and sex-matched normal BIO-RB hamsters (n=8) were used in this study. Immunofluorescence microscopy using monoclonal antibodies against desmin, -actinin, titin, and vincullin was employed. The heart weight to body weight ratio was significantly increased in the heart of cardiomyopathic hamster compared with that of normal hamster. In cardiomyopathic hamster, the left ventricular cavity was markedly dilated. Light microscopically, hypertrophy and atrophy of myocytes and myocardial fibrosis were prominently observed in cardiomyopathic myocardium. Immunocytochemically, desmin, -actinin and titin showed the cross striations along the myofibers in normal myocardium. In contrast, in cardiomyopathic myocardium, desmin was irregularly distributed in myocytes and the amount of desmin was increased. Loss of cross striations of -actinin and titin were frequently observed. Immunofluorescence against vinculin was not significantly altered. We conclude that the alterations of cytoskeletal proteins in myocardial cells may relate to decreased myocardial function in cardiomyopathic hamster failing heart.     

Accumulation pattern of arachin and its subunits in maturation of groundnut seeds

The accumulation pattern of arachin and its subunits in growinggroundnuts was investigated. Soluble proteins were extractedfrom the kernels at twelve different stages of maturation (4–16weeks after pegging). Fractionation showed arachin, conarachinII, 5S and 2S protein components with sucrose gradient centrifugation.Ten weeks after pegging, only 35% of the maximum amount of arachinhad accumulated, whereas conarachin II was 85%, the 5S component89%, and the 2S component 76%. Arachin, however, increased rapidlyin the later stage of maturation. No change in the subunit ratioin arachin during seed growth was observed on the patterns ofsodium dodecyl sulfate-gel electrophoresis and gel isoelectricfocusing in the presence of urea. The ratio of the arachin subunitscontained in urea-extractable fraction of the kernels was constantthroughout seed development and was consistent with the subunitratio in arachin. On the other hand, the arachin subunits inthe free forms, if any, accounted for less than 1% of the associatedarachin subunits. Probably, the arachin subunits synthesizedin equimoles are associated into arachin without individualdeposition and are accumulated as arachin associates in growingseeds. (Received July 17, 1980; )     

A crystallographic investigation on NADPH-adrenodoxin oxidoreductase

Single crystals of NADPH-adrenodoxin oxidoreductase were grown in 50 mM potassium phosphate (pH 7.4) containing 5% glycerol and ammonium sulfate. The crystals are monoclinic, belong to space group P21 and have dimensions of a= 83.4 A, b = 62.6 A, c = 59.3 A, alpha = gamma = 90 degrees, and beta = 107.1 degrees. There is one molecule per asymmetric unit.     

Subcellular Localization of Dystrophin Isoforms in Cardiomyocytes and Phenotypic Analysis of Dystrophin-deficient Mice Reveal Cardiac Myopathy is Predominantly Caused by a Deficiency in Full-length Dystrophin

Duchenne muscular dystrophy (DMD) is an X-linked recessive progressive muscle degenerative disorder that causes dilated cardiomyopathy in the second decade of life in affected males. Dystrophin, the gene responsible for DMD, encodes full-length dystrophin and various short dystrophin isoforms. In the mouse heart, full-length dystrophin Dp427 and a short dystrophin isoform, Dp71, are expressed. In this study, we intended to clarify the functions of these dystrophin isoforms in DMD-related cardiomyopathy. We used two strains of mice: mdx mice, in which Dp427 was absent but Dp71 was present, and DMD-null mice, in which both were absent. By immunohistochemical staining and density-gradient centrifugation, we found that Dp427 was located in the cardiac sarcolemma and also at the T-tubules, whereas Dp71 was specifically located at the T-tubules. In order to determine whether T tubule-associated Dp71 was involved in DMD-related cardiac disruption, we compared the cardiac phenotypes between DMD-null mice and mdx mice. Both DMD-null mice and mdx mice exhibited severe necrosis, which was followed by fibrosis in cardiac muscle. However, we could not detect a significant difference in myocardial fibrosis between mdx mice and DMD-null mice. Based on the present results, we have shown that cardiac myopathy is caused predominantly by a deficiency of full-length dystrophin Dp427.     

Reconstitution of Arachin from Isolated Subunits

Six subunits of arachin were isolated in urea solution. They were then reassociated by removing urea by co-dialysis against 20 mM sodium phosphate buffer (pH 7.9), containing 30% sucrose, 0.1 M> sodium chloride and 7 mM β-mercaptoethanol, without agitation at 25°C. The reconstitution yield was greater than 90%. The reconstituted molecule was indistinguishable from intact arachin in disc electrophoretic mobility, subunit composition, sedimentation behavior depending upon ionic strength, circular dichroism, ultraviolet absorption and fluorescence emission spectra, and stabilities against heating, proteases and guanidine hydrochloride. The reconstituted arachin was, therefore, suggested to be in native state.

On the other hand, we found that co-dialysis of four or five subunits of arachin formed hexamer which contained the corresponding four or five subunits. These hexamers were more labile than intact arachin against heating. These facts suggest that the assembly of all six subunits to a hexamer will most advantage the quaternary structure of arachin.     

The Effects of Proteins and Phospholipids on the Mobility of Wheat Doughs

Penicillium strains (n=394) preserved at NBRC (the NITE Biological Resource Center) were compared as to groupings (11 species-clusters) based on phylogeny and the production of bioactive compounds. The strains in two clusters, of which P. chrysogenum and P. citrinum are representative, showed higher rates of positive strains with multi-biological activities.     

Thermostabilization of ovotransferrin by anions for pasteurization of liquid egg white

The effects of anions on the thermostability of ovotransferrin (oTf) were investigated. The temperature, T(m), causing aggregation of oTf was measured in the presence or absence of anions, and the denaturation temperature, T(m)(DSC), was also determined by differential scanning calorimetry (DSC) in the presence of the citrate anion. We found that some anions (phosphate, sulfate and citrate) raised temperature T(m) of oTf by about 5-7 degrees C. However, neither sodium chloride nor sodium bicarbonate raised T(m) by that much. Temperature T(m) was increased by increasing the concentration of the citrate anion, and was in good agreement with denaturation temperature T(m)(DSC), suggesting that denaturation of the oTf molecules resulted in aggregation of oTf. We also demonstrated that the anions, especially sulfate, repressed the heat-aggregation of liquid egg white.The Van't Hoff plot from the T(m) and DeltaH(d) values revealed that two anion-binding sites were concerned with heat stabilization. These binding sites may have been concerned with sulfate binding (not bicarbonate binding) that is found in the crystal structure of apo-form of oTf, since the bicarbonate anion did not raise T(m).