F1-ATPase was isolated from yeast . The constituent subunits 1 and 2 were purified by gel permeation chromatography, and their amino acid compositions determined. Both subunits have a similar composition except for cystine, methionine, leucine, histidine, and tryptophan. When F1 is treated for three hours with 5′-p-[3H]fluorosulfonylbenzoyl adenosine in dimethylsulfoxide, 90% of the activity is lost. Disc gel electrophoresis of the modified complex showed that over 90% of the label was associated with subunit 2. A labelled peptide from a digest of subunit 2 was isolated and sequenced. It had the following amino acid sequence: His-Try1-Asp-Val-Ala-Ser-Lys-Val-Gln-Glu, whereby Tyr1 is the modified amino acid residue. This sequence shows homology to other sequences obtained from maize, beef heart, and F1-ATPases. 相似文献
The phosphorylation of RNA polymerase II after isoproterenol stimulation of confluent rat C6 glioma cell cultures has been investigated. Glioma cells were incubated in the presence of Na2H32PO4 and stimulated for 1 hour with the β-adrenergic agonist isoproterenol. The phosphorylation pattern was analyzed after purification of RNA polymerase II by immunoprecipitation, SDS-polyacrylamide gel electrophoresis and autoradiography. Isoproterenol markedly increased [32P]phosphate incorporation into the 214,000 dalton RNA polymerase subunit. Analysis of the phosphate acceptor amino acid revealed the presence of only [32P]phosphoserine. The data demonstrates an isoproterenol-induced structural modification of RNA polymerase II. 相似文献
Superoxide dismutase and catalase were not detected in . and several other species of some of which consume oxygen and secrete H2O2. . in suspension formed O2? in the presence of NADH and flavins and extracts of . formed O2? in the presence of either NADH or NADPH. The lack of superoxide dismutase in . could not be attributed to superoxide dismutase in the complex medium in which the organisms were grown because organisms grown in medium in which the superoxide dismutase had been inactivated by heat still contained undetectable amounts. Mycoplasmas appear to be an exception to the rule that organisms which consume O2 synthesize superoxide dismutase. 相似文献
The plasma membrane-bound penicillinase of has been purified. Amino acid analysis showed no significant differences in composition between the enzyme and exopenicillinase. Enzyme purified from cultures containing H333PO4 or [3H]-glycerol contained 33P or [3H]-glycerol activity and treatment with 8 M urea, 0.2% sodium dodecyl sulfate at 80° C did not remove the 3H-activity from the enzyme protein. Trypsin readily cleaved the glycerol-containing moiety from the enzyme protein, forming enzyme with molecular weight and heat stability like that of the exoenzyme. Phospholipase D and C also produced enzyme resembling the exo-form. 相似文献
An ATPase is demonstrated in plasma membrane fractions of goldfish gills. This enzyme is stimulated by Cl? and HCO3?, inhibited by SCN?.Biochemical characterization shows that HCO3? stimulation () is specifically inhibited in a competitive fashion by SCN? (). The residual Mg2+-dependent activity is weakly is weakly affected by SCN?.In the microsomal fraction chloride stimulation of the enzyme occurs in the presence of HCO3? (); no stimulation is observed in the absence of HCO3?. Thiocyanate exhibits a mixed type of inhibition () towards the Cl? stimulation of the enzyme.Bicarbonate-dependent ATPase from the mitochondrial fraction is stimulated by Cl?, but this enzyme has a relatively weak affinity for this substrate (). 相似文献
incorporates 3H or 14C from 3H- or 14C-labeled ethanolamine into an -alkali soluble, alcohol -insoluble fraction obtained from cell walls. Dansyl ethanolamine was isolated from this alcohol-insoluble fraction following dansylation and hydrolysis. The alcohol-insoluble material was non-dialyzable and contained galactofuranosyl, glucosyl, phosphoryl, amino acyl and variable quantities of uronosyl residues. The lack of detectable quantities of mannosyl residues in this material suggests that the galactofuranosyl-containing cell wall polymer is distinct from the peptidophosphogalactomannan which is obtained from culture filtrates of . (Gander ., (1974) J. Biol. Chem. , 2063). 相似文献
Quercetin inhibited a dog kidney ( preparation without affecting for ATP or for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E2 enzyme conformations, blocked this inhibition, while the K+-phosphatase activity was at least as sensitive to quercetin as the ( activity, all consistent with quercetin favoring E1 conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the for ATP and the for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K+-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E2 conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E1 conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on , for substrate, and for K+, as well as for stimulation of phosphatase activity by both these reagents at low K+ but high Na+ concentrations. 相似文献
Showdomycin inhibited pig brain ( with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na+, K+, Mg2+ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1), (2) , , and none, (3) , and , (4) and , , , and ATP. The highest rate was obtained in the presence of Na+, Mg2+ and ATP. The apparent concentrations of Na+, Mg2+ and ATP for half-maximum stimulation of inhibition () were 3 mM, 0.13 mM and 4μM, respectively. The rate was unchanged upon further increase in Na+ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E1, E2, ES, E1-P and E2-P, i.e., E2 has higher reactivity with showdomycin than E1, while E2-P has almost the same reactivity as E1-P. We conclude that the reaction of ( proceeds via at least four kinds of enzyme form (E1, E2, E1 · nucleotide and EP), which all have different conformations. 相似文献
In order to study the chemical shifts and the cis—trans isomerism of prolyl units neighbouring glycine or other amino acids, 75.4 MHz13C nuclear magnetic resonance (n.m.r.) cross-polarization/magic angel spinning (CP/MAS) spectra of the following solid oligopeptides and sequence polypeptides were measured: Z-Gly-Pro-OH,Z-Gly-Pro-Gly-Gly-OEt,Z-Gly-Pro-Ala-Ala-OMe,(Gly-Pro-Gly)n,(Gly-Pro-Ala)n,(β-Ala-Pro)n and ). Whereas all these oligo- and polypeptided contain exclusively trans X-Pro bonds, both cis and trans peptide bonds were found in a polypeptide prepared by copolymerization of glycine- and in pyridine. On the basis of these model compounds, the 13C n.m.r. CP/MAS spectra of solid elastin allows the following conclusions. Almost all X-Pro bonds assume the trans conformation, most alanine and leucine units form α-helical chain segments, whereas only a small fraction of β-sheet structure is present. A 30.3 MHz 15N n.m.r. CP/MAS spectrum of solid elastin confirms that ~25% of all amino acids assume the α-helical structure. A model of elastin is discussed consisting of an amorphous phase, α-helical chain segments and helical segments of still unknown pitch. 相似文献
ATPase preparations from rat brain, dog kidney, and human red blood cells also catalyze a K+-dependent phosphatase reaction. K+ activation and Na+ inhibition of this reaction are described quantitatively by a model featuring isomerization between E1 and E2 enzyme conformations with activity proportional to E2K concentration: Differences between the three preparations in for K+ activation can then be accounted for by differences in equilibria between E1K and E2K with dissociation constants identical. Similarly, reductions in produced by dimethyl sulfoxide are attributable to shifts in equilibria toward E2 conformations. Na+ stimulation of K+-dependent phosphatase activity of brain and red blood cell preparations, demonstrable with KCl under 1 mM, can be accounted for by including a supplementary pathway proportional to E1Na but dependent also on K+ activation through high-affinity sites. With inside-out red blood cell vesicles, K+ activation in the absence of Na+ is mediated through sites oriented toward the cytoplasm, while in the presence of Na+ high-affinity K+-sites are oriented extracellularly, as are those of the ATPase reaction. Dimethyl sulfoxide accentuated Na+-stimulated K+-dependent phosphatase activity in all three preparations, attributable to shifts from the E1P to E2P conformation, with the latter bearing the high-affinity, extracellularly oriented K+-sites of the Na+-stimulated pathway. 相似文献
, originally introduced as an inadvertent contaminant in solutions used for evaluating the stability of prostaglandins, proved to lead to the rapid disappearance of the cyclopentenone unit of PGA2 (as monitored by circular dichroic spectroscopy). The cyclopentenone unit is converted, in various metabolites, to a 9-keto, 9α or 9β-hydroxy group lacking the ring unsaturation. The major EtoAc-soluble 9-hydroxy metabolite (Compound-I) was shown to be 9α, 15α-dihydroxy-2,3,4,5-tetranor-13--prostenoic acid. Similar tetranor 9-hydroxy metabolites with one additional degree of unsaturation, and with a 9β-hydroxy group, also occur but these have not been fully characterized. Only two of the wide range of 9-keto metabolites are fully characterized by mass spectral (MS) data: 9,15-oxo-2,3,4,5-tetranorprostanoic acid and 9,15-oxo-2,3,4,5-tetranor-13--prostenoic acid. The water soluble metabolites have not been characterized further.The fully characterized metabolites together with MS data from mixtures of minor metabolites indicate that can perform the following transformations: β-oxidation, dehydrogenation at C-15, reduction of the enone carbon-carbon double bonds (both Δ10,11 and Δ13,14), reduction of the 9-ketone, and possibly migration of the cyclopentyl double bond (Δ10,11 → Δ11,12). metabolizes 15-epimeric PGA2 equally readily with the production of similar products. PGA1 affords less 9-keto metabolites with compound I constituting 33% of the product by HPLC analysis. displays some enantioselectivity, PGA2 and 15-epi-PGA2 are each metabolized more rapidly than their enantiomers. Other prostaglandins appear to be less readily metabolized. 相似文献
The synthesis of -4,5,6-trinor-3,7-inter--phenylene-3-oxaprostaglandins oxaprostaglandins of the E1 and F1α series7 from 6-endo-(1-heptenyl)-bicyclo[3:1:0]hexan-3-one (III), is described. Preliminary biological screening data for gerbil colon smooth muscle stimulation, rat blood pressure and substrate specificity toward 15-hydroxyprostaglandin dehydrogenase is presented. Platelet function studies, both and of -4,5,6-trinor-3,7-inter--phenylene-3-oxa-PGE1, methyl ester (VIII) are presented. 相似文献
A 0.5 × 106r RNA found in plastids of the aquatic angiosperm , is synthesized at a much higher rate than any other rapidly labeling RNA species about h after dark-grown plants are transferred to light. The pulse labeling kinetics of the 0.5 × 106r RNA after transfer to light, argue against its involvement in the biogenesis of plant rRNAs. Although poly(A) RNA is found in , poly(A) sequences are not detected in the 0.5 × 106r RNA; yet a sucrose gradient fraction which includes RNA of this r stimulates amino acid incorporation by an cell free extract more than other RNA fractions. The possible involvement of the 0.5 × 106r RNA as a chloroplast messenger is discussed. 相似文献
Aldolase from rabbit muscle covalently binds fructose 1,6 bisphosphate after reduction with NaBH4. The reaction is stereospecific since after acid hydrolysis of the protein only N6(2-deoxy-2-glucitol) L-lysine is isolated. It is suggested that the lysyl amino group of the active site reacts with the of the C-2 group of fructose bisphosphate. 相似文献
The steady-state kinetics of the NADPH + FAD-dependent reduction of nitrate by nitrate reductase from was studied at pH 6.18. At this sub-optimum pH, Vmax was about 83 units × mg protein?1 compared with 225 units × mg protein?1 at pH 7.20. All initial velocity reciprocal plot patterns at pH 6.18 as well as the NADP+/nitrate product inhibition pattern were intersecting. In contrast, the NADP(H)/nitrate plots at pH 7.20 were parallel (Renosto, F. J. Biol. Chem. , 8616, 1981). A major effect of lowering the assay pH was to change the Km for FAD from 0.17 μM at pH 7.20 to 4 μM at pH 6.18. The results suggest that nitrate reductase has a steady-state random kinetic mechanism in which kcat in the forward direction at pH 7.20 (ca. 375 sec?1) is greater that koff for the dissociation of one or more substrates. Several observations suggest that koff for FAD is extremely small at pH 7.20. 相似文献
Pretreatment of Chang liver cells with (0.5 or 1 mM) stimulated Na+-independent uptake of leucine at low concentrations (?1 mM). The stimulatory effect of on the uptake of leucine measured in Na+-replete medium was completely blocked by the addition of (5 mM), which shows that the L system participates in the stimulation. The Na+-dependent uptake of glycine was depressed by pretreatment. The stimulation of the Na+-independent component of leucine uptake continued for at least 30 min after treatment, while the inhibition of glycine uptake was progressive with time and the Na+-dependent uptake of leucine became depressed later, after the treatment. It has been demonstrated that treatment of cells with is capable of increasing the Na+-independent influx of leucine and at the same time slightly decreasing the efflux of it. These results suggest that attacks the Na+-independent system of amino acid transport at the reactive SH groups(s) of relevant protein(s) in favor of specific activation of that system in this cell. 相似文献
The metabolism of the prostaglandin F2α analogues, 15-methyl-Δ4--PGF1α and 16,16-dimethyl-Δ4--PGF1α, has been investigated in the cynomolgus monkey and the human female. The two analogues, tritium labelled in the 9β-position, were administered by intramuscular injections into the monkeys and by subcutaneous injections into the human. Excretion of tritium labelled products were followed in urine (in both species) and feces (in monkeys only) and several metabolites were identified by GC/MS. The analogues were found to be resistant to the 15-hydroxy dehydrogenase and furthermore the degradation by β-oxidation was delayed. About 13% of the given dose of 15-methyl-Δ4--PGF1α was excreted unchanged into urine and feces from the monkey. The corresponding figure for 16,16-dimethyl-Δ4--PGF1α was about 20%. In addition, a large part of the metabolites had the carbon skeleton intact and were only metabolized by ω-oxidation. The relative resistance to degradation of these two analogues is likely to be the basis for their prolonged pharmacological activity. 相似文献
The Michaelis-Menten parameters, and of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na+ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, , decreased with decrease in extracellular Na+ for both α-aminoisobutyric acid and phenylalanine but the for α-aminoisobutyric acid increased markedly as the Na+ concentration fell whereas the for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na+-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na+-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na+-independent but it has a high and is not additive with the Na+-dependent flux. 相似文献
Δ1-Pyrroline-5-carboxylate dehydrogenase (P5CDH) in rat cerebellum was assayed with a simple spectrophotometric method using high-speed supernatants of whole tissue homogenates. Kinetic analysis showed that the enzyme has Km values of 0.83 ± 0.21 mM for Δ1-pyrroline-5-carboxylate and 0.47 ± 0.02 mM for NAD+. Various cations inhibited P5CDH but only at relatively high concentrations. Several amino acids were strongly inhibitory in the order . 相似文献
Immunoglobulins raised against 5,6-dihydro PGI2 crossreact with PGI2. When infused into the rat, these immunoglobulins are capable of I) neutralising the vasodepressor effects (bolus or continuous infusion) of exogenous PGI2, 2) blocking the catabolism of exogenous 3H-PGI2 and prolonging its life-time in the circulation (t approx 60 min) while that of 3H-PGE2 is unaffected, 3) trapping an endogenously produced substance which after extraction from blood and dissociation from the ligand-antibody complex, is immunoreactive with 6-keto PGF1α-specific antiserum. Yet the anti-5,6-dihydro PGI2 immunoglobulins have no effect on resting arterial blood pressure both in the normotensive and spontaneously hypertensive rat. These experiments indicate that endogenously produced PGI2 does not play a significant role in blood pressure control although in combination with other vasodilators it could still participate in the regulation of vascular tone at a local level. 相似文献