共查询到20条相似文献,搜索用时 31 毫秒
1.
We investigated the uptake of biphenyl by the psychrotolerant, polychlorinated biphenyl (PCB)-degrader, Pseudomonas sp. strain Cam-1 and the mesophilic PCB-degrader, Burkholderia sp. strain LB400. The effects of growth substrates, metabolic inhibitors, and temperature on [14C]biphenyl uptake were studied. Biphenyl uptake by both strains was induced by growth on biphenyl, and was inhibited by dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), which are metabolic uncouplers. The Vmax and Km for biphenyl uptake by Cam-1 at 22 degrees C were 5.4 +/- 1.7 nmol x min(-1) x (mg of cell protein)(-1) and 83.1 +/- 15.9 micromol x L(-1), respectively. The Vmax and Km for biphenyl uptake by LB400 at 22 degrees C were 3.2 +/- 0.3 nmol x min(-1) x (mg of cell protein(-1)) and 51.5 +/- 9.6 micromol x L(-1), respectively. At 15 degrees C, the maximum rate for biphenyl uptake by Cam-1 and LB400 was 3.1 +/- 0.3 nmol x min(-1) x (mg of cell protein)(-1) and 0.89 +/- 0.1 nmol x min(-1) x (mg of cell protein)(-1), respectively. Thus, the maximum rate for biphenyl uptake by Cam-1 at 15 degrees C was more than 3 times higher than that for LB400. 相似文献
2.
Grotjohann N Kowallik W Huang Y Schulte in den Bäumen A 《Zeitschrift für Naturforschung. C, Journal of biosciences》2000,55(3-4):203-212
Axenic mycelia of the ectomycorrhizal basidiomycete, Suillus bovinus, were grown in liquid media under continuous aeration with compressed air at 25 degrees C in darkness. Provided with glucose as the only carbohydrate source, they produced similar amounts of dry weight with ammonia, with nitrate or with alanine, 60-80% more with glutamate or glutamine, but about 35% less with urea as the respectively only exogenous nitrogen source. In crude extracts of cells from NH4(+)-cultures, NADH-dependent glutamate dehydrogenase exhibited high aminating (688 nmol x mg protein(-1) x min(-1)) and low deaminating (21 nmol x mg protein(-1) x min(-1)) activities. Its Km-values for 2-oxoglutarate and for glutamate were 1.43 mM and 23.99 mM, respectively. pH-optimum for amination was about 7.2, that for deamination about 9.3. Glutamine synthetase activity was comparatively low (59 nmol x mg protein(-1) x min(-1)). Its affinity for glutamate was poor (Km = 23.7 mM), while that for the NH4+ replacing NH2OH was high (Km = 0.19 mM). pH-optimum was found at 7.0. Glutamate synthase (= GOGAT) revealed similar low activity (62 nmol x mg protein(-1) x min(-1)), Km-values for glutamine and for 2-oxoglutarate of 2.82 mM and 0.28 mM, respectively, and pH-optimum around 8.0. Aspartate transaminase (= GOT) exhibited similar affinities for aspartate (Km = 2.55 mM) and for glutamate (Km = 3.13 mM), but clearly different Km-values for 2-oxoglutarate (1.46 mM) and for oxaloacetate (0.13 mM). Activity at optimum pH of about 8.0 was 506 nmol x mg protein(-1) x min(-1) for aspartate conversion, but only 39 nmol x mg protein(-1) x min(-1) at optimum pH of about 7.0 for glutamate conversion. Activity (599 nmol x mg protein(-1) x min(-1)), substrate affinities (Km for alanine = 6.30 mM, for 2-oxoglutarate = 0.45 mM) and pH-optimum (6.5-7.5) proved alanine transaminase (= GPT) also important in distribution of intracellular nitrogen. There was comparatively low activity of the obviously constitutive enzyme, urease, (42 nmol x mg protein(-1) x min(-1)) whose substrate affinity was rather high (Km = 0.56 mM). Nitrate reductase proved substrate induced; activity could only be measured after exposure of the mycelia to exogenous nitrate. Routes of entry of exogenous nitrogen and tentative significance of the various enzymes in cell metabolism are discussed. 相似文献
3.
Rohdich F Lauw S Kaiser J Feicht R Köhler P Bacher A Eisenreich W 《The FEBS journal》2006,273(19):4446-4458
The ispC gene of Arabidopsis thaliana was expressed in pseudomature form without the putative plastid-targeting sequence in a recombinant Escherichia coli strain. The recombinant protein was purified by affinity chromatography and was shown to catalyze the formation of 2C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate at a rate of 5.6 micromol x min(-1) x mg(-1) (k(cat) 4.4 s(-1)). The Michaelis constants for 1-deoxy-D-xylulose 5-phosphate and the cosubstrate NADPH are 132 and 30 microm, respectively. The enzyme has an absolute requirement for divalent metal ions, preferably Mn2+ and Mg2+, and is inhibited by fosmidomycin with a Ki of 85 nm. The pH optimum is 8.0. NADH can substitute for NADPH, albeit at a low rate (14% as compared to NADPH). The enzyme catalyzes the reverse reaction at a rate of 2.1 micromol x min(-1) x mg(-1). 相似文献
4.
Charoenphandhu N Limlomwongse L Krishnamra N 《Canadian journal of physiology and pharmacology》2006,84(5):555-563
Prolactin has recently been shown to directly stimulate 2 components of the active duodenal calcium transport in female rats, i.e., solvent drag-induced and transcellular-active calcium transport. Since the basolateral Na(+)/K(+)- and Ca(2+)-ATPases, respectively, play important roles in these 2 transport mechanisms, the present study aimed to examine the direct actions of prolactin on the activities of both transporters in sexually mature female Wistar rats. The results showed that 200, 400, and 800 ng/mL prolactin produced a significant increase in the total ATPase activity of duodenal crude homogenate in a dose-dependent manner within 60 min (i.e., from a control value of 1.53 +/- 0.13 to 2.29 +/- 0.21 (p < 0.05), 2.68 +/- 0.19 (p < 0.01), and 3.92 +/- 0.33 (p < 0.001) micromol Pi x (mg protein)(-1) x min(-1), respectively). Activity of Na+/K+-ATPase was increased by 800 ng/mL prolactin from 0.17 +/- 0.03 to 1.18 +/- 0.29 micromol Pi x (mg protein)(-1) x min(-1) (p < 0.01). Prolactin at doses of 400 and 600 ng/mL also significantly increased the activities of Ca(2+)-ATPase in crude homogenate from a control value of 0.84 +/- 0.03 to 1.75 +/- 0.29 (p < 0.05), and 2.30 +/- 0.37 (p < 0.001) micromol Pi x (mg protein)(-1) x min(-1). When the crude homogenate was purified for the basolateral membrane, the Na(+)/K(+)-ATPase activities were elevated 10-fold. In the purified homogenate, 800 ng/mL prolactin increased Na(+)/K(+)-ATPase activity from 1.79 +/- 0.38 to 2.63 +/- 0.44 micromol Pi x (mg protein)(-1) x min(-1) (p < 0.05), and Ca(2+)-ATPase activity from 0.08 +/- 0.14 to 2.03 +/- 0.23 micromol Pi x (mg protein)(-1) x min-1 (p < 0.001). Because the apical calcium entry was the first important step for the transcellular active calcium transport, the brush border calcium uptake was also investigated in this study. We found that, 8 min after being directly exposed to 800 ng/mL prolactin, the brush border calcium uptake into the duodenal epithelial cells was increased from 0.31 +/- 0.02 to 0.80 +/- 0.28 nmol x (mg protein)(-1) (p < 0.05). It was concluded that prolactin directly and rapidly enhanced the brush border calcium uptake as well as the activities of the basolateral Na(+)/K(+)- and Ca(2+)-ATPases in the duodenal epithelium of female rats. These findings explained the mechanisms by which prolactin stimulated duodenal active calcium absorption. 相似文献
5.
Openreading frame mj0608 of the Methanococcus jannaschii genome, recognized by its sequence similarity to that of the gene coding for class C inorganic pyrophosphatase in Bacillus subtilis, was cloned and over-expressed in Escherichia coli. The protein was purified and characterized by SDS-PAGE, M(r), and N-terminal sequence. Under suitable conditions it catalyzed the specific hydrolysis of PPi at about 600 micromol x min(-1) x mg(-1) at 25 degrees C, and at 8000 micromol x min(-1) x mg(-1) at 85 degrees C. Therefore this protein is a specific inorganic pyrophosphatase. The activities of Mg(2+), Mn(2+), Co(2+), and Zn(2+) ions as cofactors for hydrolysis of PPi were compared at pH 7.5 and 9.0. Unlike the class C pyrophosphatase of B. subtilis, this enzyme required no prior activation by low concentrations of Mn(2+) or Co(2+) ions. However, prior exposure to these ions afforded striking protection against inhibition by sodium fluoride, to which the enzyme was otherwise very sensitive. 相似文献
6.
The electrogenic NADH:Q oxidoreductase from the enterobacterium Klebsiella pneumoniae transports Na(+) ions. The complex was purified with an increase of the specific Na(+) transport activity from 0.2 micromol min(-1) mg(-1) in native membrane vesicles to 4.7 micromol min(-1) mg(-1) in reconstituted enzyme specimens. The subunit pattern resembled that of complex I from Escherichia coli, and two prominent polypeptides were identified as the NuoF and NuoG subunits of complex I. During purification the typical cofactors of complex I were enriched to yield approximately 17 nmol mg(-1) iron, 24 nmol mg(-1) acid-labile sulfide, and 0.79 nmol mg(-1) FMN in the purified sample. The enzyme contained approximately 1.2 nmol mg(-1) Q6 and 1.5 nmol mg(-1) Q8. The reduction of ubiquinone by NADH was Na(+)-dependent, which indicates coupling of the chemical and the vectorial reaction of the pump. The Na(+) activation profile corresponded to the Hill equation with a Hill coefficient K(H)(Na(+)) = 1.96 and with a half-maximal saturation at 0.33 mm Na(+). The reconstituted complex I from Klebsiella pneumoniae catalyzed deamino-NADH oxidation, Q1 reduction, and Na(+) translocation with specific activities of 2.6 units mg(-1), 2.4 units mg(-1), and 4.7 units mg(-1), respectively, which indicate a Na(+)/electron stoichiometry of one. 相似文献
7.
Properties of the superoxide-generating oxidase of B-lymphocyte cell lines. Determination of Michaelis parameters. 总被引:4,自引:0,他引:4
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The capacity of three B-lymphocyte cell lines to generate superoxide (O2.-) was examined. The Burkitt lymphoma lines P.3HR-1 and Jijoye gave no response to phorbol 12-myristate 13-acetate (PMA) at 100 ng/ml but produced up to 0.35 nmol of O2.-/min per mg of protein when stimulated with 5 micrograms of PMA/ml; the cell line RPMI 1788 produced Nitro Blue Tetrazolium-positive responses to low PMA concentrations and approx. 0.4 nmol of O2.-/min per mg of protein at 5 micrograms of PMA/ml. Each cell line contained approx. 10 pmol of low-potential cytochrome b (cytochrome b-245)/mg of protein. Homogenates of PMA-activated cells gave 10-20-fold greater rates of O2.- produced per mg of protein. The Km for NADPH varied between approx. 250 microM for P3.HR-1 and RPMI 1788 cell lines and 30.5 +/- 6.5 microM for the Jijoye cell line; the Km values for NADH were higher. Determination of intracellular NADPH concentration showed that this might limit the rate of O2.- production since in each cell line it was at or below the Km concentration. 相似文献
8.
A UDP-glucose pyrophosphorylase (UGPase) gene from Acetobacter xylinum BRC5 has been cloned, sequenced, and expressed in Escherichia coli. The gene consists of 867 nucleotides and encodes a polypeptide of 289 amino acid residues with a calculated molecular mass of 31,493 Da. The amino acid sequences of the enzyme showed an 85.8% identity to those of an enzyme from A. xilinum ATCC 23768. A polyhistidine-UGPase fusion enzyme was expressed and purified from the transformed E. coli. The enzyme showed a 35,620-Da single protein band on SDS/PAGE and an about 160,000-Da protein band on 8-16% pore-gradient polyacrylamide gel, indicating the enzyme may be a tetramer or pentamer composed of four or five identical subunits. Kinetic analysis of the enzyme showed a typical Michaelis-Menten substrate saturation pattern, from which Km and Vmax were calculated to be 3.22 mM and 175.4 micromol x min(-1) x mg(-1) for UDP-glucose and 0.24 mM and 69.4 micromol x min(-1) x mg(-1) for PPi, respectively, required Mg2+ for maximal activity, and was inhibited by free pyrophosphate. Computer-aided comparison of the Acetobacter enzyme sequence with those of other bacterial enzymes found significant similarities among them and predicted that Lys84 is a catalytically important residue. Lys84 in the enzyme, which was also conserved in other bacterial enzyme sequences, was replaced by arginine or leucine. The K84R mutant enzyme was successfully expressed in E. coli and showed enzyme activity (63% of the wild-type enzyme activity), but K84L was not isolated in stable form. These results suggest that Lys84 is significant in not only catalysis but also maintenance of the active structure. 相似文献
9.
Fryer RM Eells JT Hsu AK Henry MM Gross GJ 《American journal of physiology. Heart and circulatory physiology》2000,278(1):H305-H312
We examined the role of the sarcolemmal and mitochondrial K(ATP) channels in a rat model of ischemic preconditioning (IPC). Infarct size was expressed as a percentage of the area at risk (IS/AAR). IPC significantly reduced infarct size (7 +/- 1%) versus control (56 +/- 1%). The sarcolemmal K(ATP) channel-selective antagonist HMR-1098 administered before IPC did not significantly attenuate cardioprotection. However, pretreatment with the mitochondrial K(ATP) channel-selective antagonist 5-hydroxydecanoic acid (5-HD) 5 min before IPC partially abolished cardioprotection (40 +/- 1%). Diazoxide (10 mg/kg iv) also reduced IS/AAR (36.2 +/- 4.8%), but this effect was abolished by 5-HD. As an index of mitochondrial bioenergetic function, the rate of ATP synthesis in the AAR was examined. Untreated animals synthesized ATP at 2.12 +/- 0.30 micromol x min(-1) x mg mitochondrial protein(-1). Rats subjected to ischemia-reperfusion synthesized ATP at 0.67 +/- 0.06 micromol x min(-1) x mg mitochondrial protein(-1). IPC significantly increased ATP synthesis to 1.86 +/- 0.23 micromol x min(-1) x mg mitochondrial protein(-1). However, when 5-HD was administered before IPC, the preservation of ATP synthesis was attenuated (1.18 +/- 0.15 micromol x min(-1) x mg mitochondrial protein(-1)). These data are consistent with the notion that inhibition of mitochondrial K(ATP) channels attenuates IPC by reducing IPC-induced protection of mitochondrial function. 相似文献
10.
Mouse Cyp4a isoforms: enzymatic properties, gender- and strain-specific expression, and role in renal 20-hydroxyeicosatetraenoic acid formation 总被引:1,自引:0,他引:1
Muller DN Schmidt C Barbosa-Sicard E Wellner M Gross V Hercule H Markovic M Honeck H Luft FC Schunck WH 《The Biochemical journal》2007,403(1):109-118
AA (arachidonic acid) hydroxylation to 20-HETE (20-hydroxyeicosatetraenoic acid) influences renal vascular and tubular function. To identify the CYP (cytochrome P450) isoforms catalysing this reaction in the mouse kidney, we analysed the substrate specificity of Cyp4a10, 4a12a, 4a12b and 4a14 and determined sex- and strain-specific expressions. All recombinant enzymes showed high lauric acid hydroxylase activities. Cyp4a12a and Cyp4a12b efficiently hydroxylated AA to 20-HETE with V(max) values of approx. 10 nmol x nmol(-1) x min(-1) and K(m) values of 20-40 microM. 20-Carboxyeicosatetraenoic acid occurred as a secondary metabolite. AA hydroxylase activities were approx. 25-75-fold lower with Cyp4a10 and not detectable with Cyp4a14. Cyp4a12a and Cyp4a12b also efficiently converted EPA (eicosapentaenoic acid) into 19/20-OH- and 17,18-epoxy-EPA. In male mice, renal microsomal AA hydroxylase activities ranged between approx. 100 (NMRI), 45-55 (FVB/N, 129 Sv/J and Balb/c) and 25 pmol x min(-1) x mg(-1) (C57BL/6). The activities correlated with differences in Cyp4a12a protein and mRNA levels. Treatment with 5alpha-dihydrotestosterone induced both 20-HETE production and Cyp4a12a expression more than 4-fold in male C57BL/6 mice. All female mice showed low AA hydroxylase activities (15-25 pmol x min(-1) x mg(-1)) and very low Cyp4a12a mRNA and protein levels, but high Cyp4a10 and Cyp4a14 expression. Renal Cyp4a12b mRNA expression was almost undetectable in both sexes of all strains. Thus Cyp4a12a is the predominant 20-HETE synthase in the mouse kidney. Cyp4a12a expression determines the sex- and strain-specific differences in 20-HETE generation and may explain sex and strain differences in the susceptibility to hypertension and target organ damage. 相似文献
11.
Postnatal ontogeny of kinetics of porcine jejunal brush border membrane-bound alkaline phosphatase,aminopeptidase N and sucrase activities 总被引:1,自引:0,他引:1
Fan MZ Adeola O Asem EK King D 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2002,132(3):599-607
Our objectives were to determine postnatal changes in the maximal enzyme activity (V(max)) and enzyme affinity (K(m)) of jejunal mucosal membrane-bound alkaline phosphatase, aminopeptidase N and sucrase using a porcine model which may more closely resemble the human intestine. Jejunal brush border membrane was prepared by Mg(2+)-precipitation and differential centrifugation from pigs of suckling (8 days), weaning (28 days), post-weaning (35 days) and adult (70 days) stages. p-Nitrophenyl phosphate (0-8 mM), L-alanine-p-nitroanilide hydrochloride (0-28 mM) and sucrose (0-100 mM) were used in alkaline phosphatase, aminopeptidase N and sucrase kinetic measurements. V(max) of alkaline phosphatase was the lowest in the adult (4.27 micromol.mg(-1) protein.min(-1)), intermediate in the suckling (9.75 micromol.mg(-l) protein.min(-l)) and the highest in the weaning and post-weaning stage (12.83 and 10.40 micromol.mg(-l) protein.min(-l)). K(m) of alkaline phosphatase was high in the suckling and weaning stages (5.14 and 9.93 mM) and low in the adult (0.66 mM). V(max) of aminopeptidase N was low in the suckling (7.04 micromol.mg protein(-1).min(-1)) and high in the post-weaning stage (13.36 micromol.mg(-l) protein.min(-l)). K(m) of aminopeptidase N was the highest in the two weaning stages (2.96 and 3.39 mM), intermediate in the adult (2.33 mM) and the lowest in the suckling stage (1.66 mM). V(max) of sucrase increased from the suckling to the adult (0.48-1.30 micromol.mg(-l) protein.min(-l)). K(m) of sucrase ranged from 11.19 to 16.57 mM. There are dramatic postnatal developmental changes in both the maximal enzyme activity and enzyme affinity of jejunal brush border membrane-bound alkaline phosphatase, aminopeptidase N and sucrase in the pig. 相似文献
12.
McFalls EO Baldwin D Marx D Fashingbauer P Ward HB 《American journal of physiology. Endocrinology and metabolism》2000,278(1):E96-E102
2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) may be used to predict glucose kinetics when the factor relating differences in transport and phosphorylation between compounds remains constant ("lumped constant"). It is not clear whether hyperemia alters that factor. In anesthetized swine, myocardial FDG uptake was estimated by positron emission tomography, during an intracoronary infusion of either adenosine, ATP, or bradykinin (40 microg x kg(-1) x min(-1), 40 microg x kg(-1) x min(-1), and 2 nmol x kg(-1) x min(-1), respectively; n = 6 for all groups). In controls during normal perfusion (n = 6), FDG uptake was 0.78 +/- 0.32 micromol x g(-1) x min(-1), whereas glucose uptake by Fick was 0.71 +/- 0.25 micromol x g(-1) x min(-1) (r = 0.73; P < 0.05). Adenosine increased blood flow from 1.29 +/- 0.43 to 4.80 +/- 2.19 ml x g(-1) x min(-1) (P < 0.05) and glucose uptake from 1.16 +/- 1.10 to 3.35 +/- 2.12 micromol x g(-1) x min(-1) (P < 0.05), whereas FDG uptake in the hyperemic region was lower than remote regions (0.46 +/- 0.29 and 0.95 +/- 0.55 micromol x g(-1) x min(-1), respectively; P < 0.05). In the ATP and bradykinin groups, blood flow increased four- and twofold, respectively, with no net change in glucose uptake. FDG uptake in the hyperemic region was also significantly lower than remote regions. For all animals, the ratio of blood flow in the hyperemic region relative to remote region was inversely proportional to the ratio of FDG uptake in the same regions (r(2)=0.73; P < 0.001). Because nitric oxide elaboration during hyperemia could potentially alter substrate preference and FDG kinetics, six additional swine were studied during maximal adenosine before and after intracoronary N(G)-monomethyl-L-arginine (1.5 mg/kg). Inhibition of nitric oxide had no effect on either regional myocardial substrate uptake or FDG accumulation. In conclusion, hyperemia decreased regional myocardial FDG uptake relative to normally perfused regions and this effect on the lumped constant was independent of nitric oxide. 相似文献
13.
M Gregor J Mejsnar A Janovská J Zurmanová O Benada B Mejsnarová 《Physiological research / Academia Scientiarum Bohemoslovaca》1999,48(1):27-35
The aim of this study was to evaluate myofibrillar creatine kinase (EC 2.7.3.2) activity on the background of the effect of substrate channeling by myosin ATPase and to compare it with creatine kinase (CK) activity of whole skinned fibers. In order to assess CK activity, skinned fibers were prepared from the rat psoas major muscles defined by light microscopy. The activity in permeabilized fibers after treatment with saponin, Triton X-100 and Ca(2+)-free medium reached 2.80, 6.97 and 3.32 micromol ATP min(-1) mg(-1) protein, respectively, when a coupled enzyme assay system with external hexokinase and glucose-6-phosphate dehydrogenase was used. Transmission electron microscopy (TEM) revealed a possible interference among activities of sarcolemmal, sarcoplasmic, myofibrillar and mitochondrial CK from persisting structures. For evaluation of the myofibrillar CK itself, a pure myofibrillar fraction was prepared. Fraction purity was confirmed by TEM and by enzymatic assays for marker enzymes. Two procedures, i.e. the coupled enzyme assay and the evaluation of phosphocreatine (PCr) concentration before and after the CK reaction, were used for measurement of CK activity in this fraction. The procedures resulted in 3.2 nmol ATP min(-1) mg(-1) protein and 7.6 nmol PCr min(-1) mg(-1) protein, respectively. These alternative approaches revealed a discrepancy between the reacting portions of PCr by more than 50 %, which provides information about the size of the effect, generally described as substrate channeling. 相似文献
14.
Serotonin uptake and metabolism was studied in ependymal primary cultures. Serotonin uptake was facilitated by two different systems, one of which was the neuronal serotonin transporter SERT, exhibiting a Vmax value of 3.8+/-0.1 pmol x min(-1) x (mg protein)(-1) and an apparent Michaelis-Menten constant of 0.41+/-0.03 microM. The main product of metabolism was 5-hydroxyindole acetic acid, which resulted from the action of monoamine oxidase A. This enzyme showed a maximal rate of 0.85+/-0.02 nmol x min(-1) x (mg protein)(-1) and a Michaelis-Menten constant of 78+/-5 microM. Ependymal cells were able to dispose of extracellular serotonin with initial rates of approximately 600 pmol x min(-1) x (mg protein)(-1) and of 4 pmol x min(-1) x (mg protein)(-1) when challenged with 500 microM and 1 microM extracellular serotonin, respectively. Ependymal cells are concluded to facilitate the "sink" action of the CSF by removing waste compounds upon passing of the fluid from the parenchymal extracellular space into the ventricular system. 相似文献
15.
K Saarem J I Pedersen S Tollersrud 《Comparative biochemistry and physiology. B, Comparative biochemistry》1978,61(4):485-490
1. Mitochondria isolated from the kidneys of rachitic pigs have been shown to contain an active 25-hydroxyvitamin D3-1 alpha-hydroxylase. From these mitochondria a cytochrome P-450 has been solubilized with a specific content of 0.02-0.04 nmol/mg protein. 2. In the presence of a bovine adrenal NADPH-ferredoxin reductase, bovine adrenal ferredoxin and NADPH, the cytochrome P-450 supported the formation of 1,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3. 3. The hydroxylation reaction was linear with time up to 40 min, and with the amount of enzyme up to 0.03 nmol cytochrome P-450. The pH optimum for the reaction was 7.4, and the apparent Km was 3 x 10(-10) mol/mg protein. 4. The results show that 25-hydroxyvitamin D3 is metabolized in mammals by the same enzyme system as has been demonstrated in birds. 相似文献
16.
The purified glucoamylase of the thermophilic mold Thermomucor indicae-seudaticaehad a molecular mass of 42 kDa with a pI of 8.2. It is a glycoprotein with 9-10.5% carbohydrate content, which acted optimally at 60 degrees C and pH 7.0, with a t(1/2) of 12 h at 60 degrees C and 7 h at 80 degrees C. Its experimental activation energy was 43 KJ mol(-1) with temperature quotient (Q(10)) of 1.35, while the values predicted by response surface methodology (RSM) were 43 KJ mol(-1) and 1.28, respectively. The enzyme hydrolyzed soluble starch at 50 degrees C (K(m) 0.50 mg mL(-1) and V(max) 109 micromol mg(-1) protein min(-1)) and at 60 degrees C (K(m) 0.40 and V(max) 143 micromol mg(-1) protein min(-1)). The experimental K(m) and V(max) values are in agreement with the predicted values at 50 degrees C (K(m) 0.45 mg mL(-1) and V(max) 111.11 micromol mg(-1) protein min(-1)) and at 60 degrees C (K(m) 0.36 mg mL(-1)and V(max) 142.85 micromol mg(-1) protein min(-1)). An Arrhenius plot indicated thermal activation up to 60 degrees C, and thereafter, inactivation. The enzyme was strongly stimulated by Co(2+), Fe(2+), Ag(2+), and Ca(2+), slightly stimulated by Cu(2+) and Mg(2+), and inhibited by Hg(2+), Zn(2+), Ni(2+), and Mn(2+). Among additives, dextran and trehalose slightly enhanced the activity. Glucoamylase activity was inhibited by EDTA, beta-mercaptoethanol, dithiothreitol, and n-bromosuccinimide, and n-ethylmaleimide inhibited its activity completely. This suggested the involvement of tryptophan and cysteine in catalytic activity and the critical role of disulfide linkages in maintaining the conformation of the enzyme. The enzyme hydrolyzed around 82% of soluble starch and 65% of raw starch (K(m) 2.4 mg mL(-1), V(max) 50 micromol mg(-1) protein min(-1)), and it was remarkably insensitive to glucose, suggesting its applicability in starch saccharification. 相似文献
17.
beta-mannanase (EC 3.2.1.78) from Bacillus subtilis SA-22 was purified successively by ammonium sulfate precipitation, hydroxyapatite chromatography, Sephadex G-75 gel filtration and DEAE-52 anion-exchange chromatography. Through these steps, the enzyme was concentrated 30.75-fold with a recovery rate of 23.43%, with a specific activity of 34780.56 u/mg. Molecular weight of the enzyme was determined to be 38 kD by SDS-PAGE and 34 kD by gel filtration. The results revealed that the optimal pH value for the enzyme was 6.5 and the optimal temperature was 70 degrees C. The enzyme is stable between pH 5 to 10. The enzyme remained most of its activity after a treatment of 4 h at 50 degrees C, but lost 25% of activity at 60 degrees C for 4 h, lost 50% of activity at 70 degrees C for 3 h. The enzyme activity was strongly inhibited by Hg2+. The Michaelis constants (Km) were measured as 11.30 mg/mL for locust bean gum and 4.76 mg/mL for konjac powder, while Vmax for these two polysaccharides were 188.68 (micromol x mL(-1) x min(-1)) and 114.94 (micromol x mL(-1) x min(-1)), respectively. 相似文献
18.
Irene E. Stith Salil K. Das 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,714(2):250-256
Development of mitochondrial and microsomal choline phosphotransferase in the fetal guinea pig lung was investigated. The activity in fetal mitochondria was more than twice of that in fetal microsomes. However, in adult lung, the enzyme was distributed mostly in microsomes. In fetal lung, both the mitochondrial and microsomal enzyme activity was greatest at approx. 81% of the total gestation period (55 days). The specific activity in the microsomal fraction then declined until term, but increased again in the 24-h newborn from 1.0 to 2.3 nmol/min per mg protein. The activity in the mitochondrial fraction declined after 61 days (2.8 nmol/min per mg) to a minimal level at term (0.6 nmol/min per mg). Although the enzyme activity decreased from day 55 (1.2 nmol/min per mg), the amount of phosphatidylcholine gradually increased between day 55 and term. 相似文献
19.
R K Berge 《Biochimica et biophysica acta》1979,574(2):321-333
A long-chain acyl-CoA hydrolase from rat liver microsomes has been purified by solvent extraction and gel chromatography to homogeneity as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The enzyme was a monomer of molecular weight 59 000. In a sucrose gradient it sedimented at 4.3 S. The isoelectric point, pI was 6.9, and the Stokes radius was approx. 31 A. The enzyme hydrolyzed long-chain fatty acyl-CoA (C7--C18) with maximum activity for palmitoyl-CoA. Bovine serum albumin activation of the enzyme was related to the ratio acyl-CoA/bovine serum albumin, and at high ratios, acyl-CoA inhibited the enzyme activity. Disregarding the substrate inhibition, an apparent Km of 65 nmol/mg protein or 1-10(-7) M and a V of 750 nmol/mg protein per min were calculated. The enzyme was inhibited by p-hydroxymercuribenzoate and N-ethylmaleimide. Reactivation by means of dithiothreitol was not complete. 相似文献
20.
Shin JS Torres TP Catlin RL Donahue EP Shiota M 《American journal of physiology. Regulatory, integrative and comparative physiology》2007,292(4):R1381-R1390
Effect of stimulation of glucokinase (GK) export from the nucleus by small amounts of sorbitol on hepatic glucose flux in response to elevated plasma glucose was examined in 6-h fasted Zucker diabetic fatty rats at 10 wk of age. Under basal conditions, plasma glucose, insulin, and glucagon were approximately 8 mM, 2,000 pmol/l, and 60 ng/l, respectively. Endogenous glucose production (EGP) was 44 +/- 4 micromol x kg(-1) x min(-1). When plasma glucose was raised to approximately 17 mM, GK was still predominantly localized with its inhibitory protein in the nucleus. EGP was not suppressed. When sorbitol was infused at 5.6 and 16.7 micromol x kg(-1) x min(-1), along with the increase in plasma glucose, GK was exported to the cytoplasm. EGP (23 +/- 19 and 12 +/- 5 micromol x kg(-1) x min(-1)) was suppressed without a decrease in glucose 6-phosphatase flux (145 +/- 23 and 126 +/- 16 vs. 122 +/- 10 micromol x kg(-1) x min(-1) without sorbitol) but increased in glucose phosphorylation as indicated by increases in glucose recycling (122 +/- 17 and 114 +/- 19 vs. 71 +/- 11 microl x kg(-1) x min(-1)), glucose-6-phosphate content (254 +/- 32 and 260 +/- 35 vs. 188 +/- 20 nmol/g liver), fractional contribution of plasma glucose to uridine 5'-diphosphate-glucose flux (43 +/- 8 and 42 +/- 8 vs. 27 +/- 6%), and glycogen synthesis from plasma glucose (20 +/- 4 and 22 +/- 5 vs. 9 +/- 4 mumol glucose/g liver). The decreased glucose effectiveness to suppress EGP and stimulate hepatic glucose uptake may result from failure of the sugar to activate GK by stimulating the translocation of the enzyme. 相似文献