Two types of cytochrome cd1 in the aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114

Components I and II of cytochrome cd1 which had different spectral features were purified from the aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh 114. Component I showed an absorption maxima at 700 and 406 nm in the oxidized form, and at 621, 552.5, 548 and 416 nm in the reduced form. Component II showed an absorption maxima at 635 and 410 nm in the oxidized form and at 628, 552.5, 548 and 417 nm in the reduced form. The relative molecular mass, Mr, of both cytochromes was determined to be 135,000 with two identical subunits. Components I and II showed pI values of 7.6 and 6.8, respectively. The redox potential of hemes ranged from +234 mV to +242 mV, except for the heme d1 of component I (Em7 = +134 mV). Components I and II showed both cytochrome c oxidase and nitrite reductase activities. Cytochrome c oxidase activity was strongly inhibited by a low concentration of nitrite and cyanide. Erythrobacter cytochromes c-551 and c-552 were utilized as electron donors for the cytochrome c oxidase reaction. The high affinity of cytochrome c-552 to component II (Km = 1.27 microM) suggested a physiological significance for this cytochrome. Erythrobacter cytochromes cd1 are unique in their presence in cells grown under aerobic conditions as compared to other bacterial cytochromes cd1 which are formed only under denitrifying conditions.     

Characterization of crystalline formate dehydrogenase from Candida methanolica

The crystalline formate dehydrogenase from Candida methanolica, which showed the highest specific activity (7.52 U/mg) so far reported, was characterized in detail. The enzyme is a dimer composed of identical subunits, each containing one SH group related to the catalytic activity. The molecular mass of the enzyme is about 82-86 kDa. The Km values were found to be 3.0 mM for formate and 0.11 mM for NAD+. Even if the enzyme was incubated at pH 6.5-9.5 or at 55 degrees C, the activity remained at 100%. Hg2+, Ni2+, NaCN, NaN3 and p-chloromercuribenzoate strongly inhibited the enzyme activity, while the enzyme showed relatively high resistance to various chelating agents. The amino acid composition and some other physicochemical properties of the enzyme were studied. Immunological studies revealed that formate dehydrogenases of methanol-utilizing yeasts immunologically more or less resemble each other, but differ from those of methanol-utilizing bacteria. Furthermore, yeast formate dehydrogenases can be immunologically classified into three types: (a) the Candida type, (b) the Torulopis/Hansenula/Pichia type and (c) the formaldehyde-resistant yeast type. For simple and large-scale preparation of the enzyme for practical use, treatment of cells of C. methanolica with the commercial cationic detergent, 'Benzalkonium' cation, is useful: the total and specific activities of the enzyme are 1.17-fold and 3.10-fold higher than those of the crude cell-free extract, respectively.     

Association of increased intracellular free Ca2+ by platelet-derived growth factor with mitogenesis but not with proteoglycan synthesis in chondrocytes--effect of suramin

The effects of platelet-derived growth factor (PDGF) on the intracellular free Ca2+ concentration [( Ca2+]i) in chondrocytes were studied with a fluorescent Ca2+ indicator, fura 2, and compared with the effects of PDGF on mitogenesis and proteoglycan synthesis. PDGF evoked phasic and then tonic increase in [Ca2+]i dose-dependently in quiescent cultures of chondrocytes, and it also stimulated both DNA and proteoglycan syntheses dose-dependently similar to somatomedins. Suramin, which inhibits the interaction of PDGF with its receptors, caused dose-dependent inhibition of both the PDGF-evoked increase in [Ca2+]i and stimulation of DNA synthesis by PDGF. However, suramin rather enhanced the proteoglycan synthesis induced by PDGF without affecting the basal level of proteoglycan synthesis directly. These results suggest that [Ca2+]i may be an important signal for the action of PDGF on cell proliferation in chondrocytes, and that the initial signal for proteoglycan synthesis is different from that for DNA synthesis induced by PDGF after the activation of PDGF receptor.     

Decay of membrane potential under phosphorylating conditions in chloroplasts with in vivo activated ATPase

(1) The relation between the membrane potential and phosphorylation was studied in chloroplasts rapidly prepared from illuminated spinach leaves (light chloroplasts) and from dark-adapted leaves (dark chloroplasts). Light chloroplasts had a higher ATP hydrolysis activity than dark chloroplasts. (2) In the presence of ADP or ATP, a rapidly decaying phase of the field-indicating 518 nm absorbance change with a half-time of 15 ms became apparent in addition to the slow phase with a half-time of more than 300 ms in either type of chloroplast. Under these conditions, light chloroplasts showed a larger rapid phase than dark chloroplasts. (3) The rapid phase was suppressed by dicyclohexylcarbodiimide and was assumed to reflect the dissipation of membrane potential due to proton movements inside the CF₁-CF₀ ATP synthetase. (4) A model for the proton movement in ATP synthetase is proposed.     

Ornithine transcarbamylase in liver mitochondria

Summary Ornithine transcarbamylase (ornithine carbamoyltransferase, EC 2.1.3.3), the second enzyme of urea synthesis, is localized in the matrix of liver mitochondria of ureotelic animals. The enzyme is encoded by a nuclear gene, synthesized outside the mitochondria, and must then be transported into the organelle. The rat liver enzyme is initially synthesized on membrane-free polysomes in the form of a larger precursor with an amino-terminal extension of 3 400–4 000 daltons. In rat liver slices and isolated rat hepatocytes, the pulse-labeled precursor is first released into the cytosol and is then transported with a half life of 1 2 min into the mitochondria where it is proteolytically processed to the mature form of the enzyme. The precursor synthesized in vitro exists in a highly aggregated form and has a conformation different from that of the mature enzyme. The precursor has an isoelectric point (pI = 7.9) higher than that of the mature enzyme (pI = 7.2).The precursor synthesized in vitro can be taken up and processed to the mature enzyme by isolated rat liver mitochondria. The mitochondrial transport and processing system requires membrane potential and a high integrity of the mitochondria. The transport and processing activities are conserved between mammals and birds or amphibians and is presumably common to more than one precursor. Potassium ion, magnesium ion, and probably a cytosolic protein(s), in addition to the transcarbamylase precursor and the mitochondria, are required for the maximal transport and processing of the precursor.A mitochondrial matrix protease which converts the precursor to a product intermediate in size between the precursor and the mature subunit has been highly purified. The protease has an estimated molecular weight of 108 000 and an optimal pH of 7.5–8.0, and appears to be a metal protease. The protease does not cleave several of the protein and peptide substrates tested. The role of this protease in the precursor processing remains to be elucidated.Rats subjected to different levels of protein intake and to fasting show significant changes in the level of enzyme protein and activity of ornithine transcarbamylase. The dietary-dependent changes in the enzyme level are due mainly to an altered level of functional mRNA for the enzyme. In contrast, during fasting, the increase in the enzyme level is associated with a decreased level of translatable mRNA forthe enzyme.Pathological aspects of ornithine transcarbamylase including the enzyme deficiency and reduced activities of the enzyme in Reye's syndrome are also described. A possibility that impaired transport of the enzyme precursor into the mitochondria leads to a reduced enzyme activity, is proposed.Abbreviation pOTC precursor of ornithine transcarbamylase     

Isolation and characterization of deletion mutants affected in early genes of bacteriophage ?80

Summary When Escherichia coli cells that had been irradiated with ultraviolet light were infected with bacteriophage 80, five major (pE, pB, pA, pC and pD) and two minor (pU and pV) proteins were found to be synthesized during early stages of infection. The genss coding for the five major proteins were mapped on the 80 chromosome using various deletion mutants which lacked the capacity to synthesize some or all the major proteins. The size and positions of all the deletions were determined by gel electrophoresis of EcoRI digests of phage DNA and by electron microscopy of heteroduplexes between DNAs of the deletion and wild-type phage. The five major proteins designated pE(25K), pB(40K), pA(45K), pC(34K) and pD(31K) were shown to be encoded in this order presumably by a single operon that was located at 60.2–67.4% on the 80 genome. These proteins were found to be involved in phage recombination. The absence of pE or pB resulted in a Red^– phenotype and the absence of three proteins (pE, pB and pA) resulted in a Fec^– phenotype. The exact positions of the genes for the minor proteins pU(29K) and pV(26K) have not been determined.     

Insulin and glucagon in rats with islet cell tumors induced by small doses of streptozotocin

Plasma insulin and glucagon responses to oral glucose loading were examined in rats with islet cell tumors induced by a single intravenous injection of streptozotocin (30 or 40 mg/kg body weight). Twenty-four macroscopic and six microscopic tumors occurred in 21 rats. In 15 of 21 tumor-bearing rats, there was exaggerated insulin release in response to oral glucose. Plasma glucose levels did not rise with the oral glucose load and were comparable to those seen in normal animals. Hence these rats are described as having "responsive tumors." In six rats with "nonresponsive tumors" there was no insulin response and the plasma glucose levels rose. No significant differences in plasma levels were observed between the two groups. Nonresponsive tumors as well as responsive tumors contained a significant amount of extractable insulin (17.68 +/- 8.60 and 35.07 +/- 10.05 mg/g wet weight, respectively) and detectable amounts of immunoreactive glucagon (1.47 +/- 0.61 and 2.24 +/- 0.67 micrograms/g wet weight, respectively). These results suggest that a small dose of streptozotocin produces two types of islet cell tumors. One is insulin producing and insulin secreting whereas the other is insulin producing but not insulin secreting.     

Process of attachment of phi X174 parental DNA to the host cell membrane

The phi X174-DNA membrane complex was isolated from Escherichia coli infected with phi X174 am3 by isopycnic sucrose gradient centrifugation followed by zone electrophoresis. The phi X174 DNA-membrane complex banded at two positions, intermediate density membrane fraction and cytoplasmic membrane fraction, having bouyant densities of 1.195 and 1.150 g/ml, respectively. Immediately after infection with phi X147, replicating DNA was pulse-labeled and then the incorporated label was chased. The radioactivity initially recovered in the intermediate density membrane fraction migrated to the cytoplasmic membrane fraction. The DNAs from both complexes sedimented mainly at the position of parental replicative form I (RFI). The phi X174 DNA-membrane complex contained a speficic membrane-bound protein having a molecular weigth of 80,000 which is accumulated in the host DNA-membrane complex. These results suggest that when phi X174 DNA penetrated into cells in the early phase of infection, single-stranded circular DNA was converted to parental RFI at a wall/membrane adhesion region and migrated to the cytoplasmic membrane fraction, where the parental RF could serve as a template in the replication of progeny RF.     

Distribution and molecular form of immunoreactive big endothelin-1 in porcine tissue

In the present study a specific and sensitive radioimmunoassay for big endothelin-1 was developed. Half maximal inhibition of binding of radioiodinated big endothelin-1 was observed at 58 pg/tube and big endothelin-1 was detectable as low as 2 pg/tube. With this assay, the regional distribution of big endothelin-1 was determined in porcine tissue and compared to the distribution of an immunoreactive endothelin. Considerable amount of immunoreactive big endothelin-1 was observed in the aortic intima (0.84 +/- 0.094 pg/mg wet tissue; mean +/- S.D.) and the lung (0.47 +/- 0.055), but there was a low concentration in other tissue including the kidney inner medulla, which has been shown to be abundant in immunoreactive endothelin. Furthermore the molecular form of immunoreactive big endothelin-1 in aortic intima was found to be big ET-1[1-39], but the molecular form of major immunoreactive big endothelin-1 in the lung is big endothelin-1[22-39] with big endothelin-1[1-39] being minor.     

GABAA receptor-mediated increase of cytosolic Ca2+ in isolated bovine adrenal chromaffin cells

We have studied the effects of GABA on cytosolic free Ca2+ concentration ([Ca2+]i) as a means of investigating the role of GABA in adrenal catecholamine (CA) secretion. It was demonstrated that GABA caused an elevation of [Ca2+]i via the GABAA receptor in a concentration-dependent manner, which was well correlated with an increase of 45Ca uptake, an increase of CA release and a depolarization of chromaffin cells assessed with bis-oxonol fluorescence. Since the GABA-induced rise of [Ca2+]i was absolutely dependent on the presence of extracellular Ca2+ and partly sensitive to nifedipine, at least one entry route for Ca2+ facilitated by GABA via a voltage-sensitive Ca2+ channel was suggested. When extracellular Cl- was lowered, GABA-induced CA release, depolarization, and rise of [Ca2+]i were all markedly enhanced. It is possible that GABA plays a modulatory role in the regulation of adrenal CA secretion as a facilitatory modulator.