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The transport of 2-oxoisocaproate into isolated hepatocytes and liver mitochondria of rat was studied using [U-14C]2-oxoisocaproate and the silicone oil filtration procedure. 2-Oxoisocaproate uptake by hepatocytes was composed of: rapid adsorption, unmediated diffusion and carrier-mediated transport. The carrier-mediated transport was strongly inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid and p-chloromercuribenzoate, was less sensitive to alpha-cyano-4-hydroxycinnamate and insensitive to p-chloromercuriphenylsulphonate. Other 2-oxo acids: pyruvate, 2-oxoisovalerate and 2-oxo-3-methylvalerate, were also inhibitory. The kinetic parameters of the carrier-mediated transport were Km 30.6 mM and Vmax 23.4 nmol/min per mg wet wt, at 37 degrees C. It is concluded that at its low, physiological, concentration, 2-oxoisocaproate penetrates the hepatocyte membrane mainly by unmediated diffusion. The uptake of 2-oxoisocaproate by isolated liver mitochondria was partly inhibited by alpha-cyano-4-hydroxycinnamate, the inhibitor of mitochondrial monocarboxylate carrier. The remaining uptake was linearly dependent on 2-oxoisocaproate concentration and represented unmediated diffusion. The carrier-mediated transport exhibited the following kinetic parameters: Km 0.47 mM, Vmax 1.0 nmol/min per mg protein at 6 degrees C; and Km 0.075 mM and Vmax about 8 nmol/min per mg protein at 37 degrees C. 相似文献
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Reinhard Bolli Katarzyna A. Nałecz Angelo Azzi 《Journal of bioenergetics and biomembranes》1986,18(4):277-284
Cytochromec oxidase fromParacoccus denitrificans was homogenously dispersed in Triton X-100. Using gel exclusion chromatography and sucrose gradient centrifugation analysis a molecular weight of the detergent-protein complex of 155,000 was determined. After subtraction of the bound detergent (111 mol/mol hemeaa
3) a molecular weight of 85,000 resulted, which agreed well with the model of a monomer containing two subunits. This monomer showed high cytochromec oxidase activity when measured spectrophotometrically in the presence of Triton X-100 (V
max=85 s–1). The molecular activity, plotted according to Eadie-Hofstee, was monophasic as a function of the cytochromec concentration. AK
m of 3.6×10–6 M was evaluated, similar to theK
m observed in the presence of dodecyl maltoside [Naeczet al. (1985).Biochim. Biophys. Acta
808, 259–272]. 相似文献
66.
Sixty depressed patients and 52 normal controls completed three selfreport inventories: the Symptom Checklist-90 (SCL-90), the Beck Depression Inventory (BDI) and a new Verbal Style Investigation Schedule (VESIS) developed by the first author. The VESIS uses 16 key emotional and physical terms from Western inventories and identified the words and phrases most commonly used by Chinese patients to express these feeling states. Chinese subjects commonly used the key term itself for only 3 or the 16 key terms; they usually preferred to use other words or phrases to express the feeling state. We categorized these Chinese expressions into four styles of verbal expression: Psychological, Somatic, Neutral (i.e., a mixture of psychological and somatic) and Deficient (i.e., lack of expression because of denial or suppression). Three of the 12 key emotional terms of the VESIS (depressed, fearful, and anxiousness) were more commonly expressed in a somatic or neutral mode than the other key emotional terms. The key terms suicidal interest and being punished were more commonly expressed in a deficient style than other key emotional terms. The somatic factor score of the SCL-90 was not correlated with increased somatic expression of emotional states; thus patients who have multiple somatic complaints are not more likely to express emotions somatically. The hypothesis of somatization is discussed in light of this study. 相似文献
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Although acute alterations in Ca2+ fluxes may mediate the skeletal responses to certain humoral agents, the processes subserving those fluxes are not well understood. We have sought evidence for Ca2+-dependent ATPase activity in isolated osteoblast-like cells maintained in primary culture. Two Ca2+-dependent ATPase components were found in a plasma membrane fraction: a high affinity component (half-saturation constant for Ca2+ of 280 nM, of 13.5 nmol/mg per min) and a low affinity component, which was in reality a divalent cation ATPase, since Mg2+ could replace Ca2+ without loss of activity. The high affinity component exhibited a pH optimum of 7.2 and required Mg2+ for full activity. It was unaffected by potassium or sodium chloride, ouabain or sodium azide, but was inhibited by lanthanum and by the calmodulin antagonist trifluoperazine. This component was prevalent in a subcellular fraction which was also enriched in 5′-nucleotidase and adenylate cyclase activities, suggesting the plasma membrane as its principal location. Osteosarcoma cells, known to resemble osteoblasts in their biological characteristics and responses to bone-seeking hormones, contained similar ATPase activities. Inclusion of purified calmodulin in the assay system caused small non-reproducible increases in the Ca2+-dependent ATPase activity of EGTA-washed membranes. Marked, consistent calmodulin stimulation was demonstrated in membranes exposed previously to trifluoperazine and then washed in trifluoperazine-free buffer. These results indicate the presence of a high affinity, calmodulin-sensitive Ca2+-dependent ATPase in osteoblast-like bone cells. As one determinant of Ca2+ fluxes in bone cells, this enzyme may participate in the hormonal regulation of bone cell function. 相似文献
70.
N,N′-Dicyclohexylcarbodiimide (DCCD) inhibits the activity of ubiquinol-cytochrome c reductase in the isolated and reconstitued mitochondrial cytochrome b-c1 complex. DCCD inhibits equally electron flow and proton translocation (i.e., the ratio is not affected) catalysed by the enzyme reconstituted into phospholipid vesicles. The inhibitory effects are accompanied by structural alterations in the polypeptide pattern of both isolated and reconstituted enzyme. Cross-linking was observed between subunits V (iron-sulfur protein) and VII, indicating that these polypeptides are in close proximity. A clear correlation was found between the kinetics of inhibition of enzymic activity and the cross-linking, suggesting that the two phenomena may be coupled. Binding of [14C]DCCD was also observed, to all subunits with the isolated enzyme and preferentially to cytochrome b with the reconstituted vesicles; in both cases, however, it was not correlated kinetically with the inhibition of the enzymic activity. 相似文献