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1.
A partially purified preparation of pyridine nucleotide transhydrogenase (E.C. 1.6.1.1.) (energy-independent) has been obtained from membranes of Escherichiacoli by means of deoxycholate extraction and DEAE-cellulose chromatography in the presence of Triton X-100. The enzyme was lipid-depleted by treating with cholate and ammonium sulfate. The preparation was reactivated by various phospholipids, in particular, bacterial cardiolipin and phosphatidyl glycerol. Phosphatidyl ethanolamine, the major phospholipid in the outer membrane of E.coli, was relatively ineffective in stimulating activity. The membrane-bound pyridine nucleotide transhydrogenase is slowly inhibited by N-ethylmaleimide. Protection against inhibition was achieved with NAD+ and NADP+, but NADPH served to accelerate the rate of inhibition.  相似文献   

2.
The micelle-specific palmityl-CoA: monopalmityl-sn-glycerol 3-phosphate palmityltransferase isoenzyme from lactating rabbit mammary gland microsomes is selectively solubilized in Triton X-100 but not Tween 80. Both detergents inactivate the monomer-specific isoenzyme. The possibility that, invivo, physiological surfactants regulate the relative activities of these two isoenzymes is discussed.  相似文献   

3.
A mixture of two pantetheine-free mutant fatty acid synthetases was dissociated and recombined invitro to form a hybrid apoenzyme complex. Invivo the corresponding Saccharomycescerevisiaefas-mutants exhibit interallelic complementation when crossed with each other and the enzyme synthesized in the resulting diploid contains pantetheine and exhibits overall fatty acid synthetase activity. Accordingly, the hybrid apoenzyme formed invitro could be activated to holo-fatty acid synthetase when incubated with coenzyme A and a partially purified yeast cell extract. The enzyme coenzyme A: fatty acid synthetase apoenzyme 4′-phosphopantetheine transferase has thus been identified in yeast. Further studies on the mechanism of fatty acid synthetase holoenzyme formation will now be possible.  相似文献   

4.
5.
An undecaprenyl diphosphate synthase fraction, which was free of other prenyltransferases and was active without the addition of detergent or phospholipid, was obtained by Sephadex G-100 chromatography of cell-free extracts of Micrococcus luteus B-P 26 cells. The addition of small amounts of Triton X-100 to this fraction caused a marked loss of the enzyme activity, but the activity was gradually restored as further detergent was added. When the enzyme fraction was chromatographed on DEAE-cellulose, the synthase was partially purified, but the activity was not detected unless assayed with addition of the detergent or a lipid fraction of this bacterium. Among the three phospholipids isolated from this bacterium, cardiolipin and phosphatidylglycerol had a marked effect in activating lipid-depleted undecaprenyl diphosphate synthase, but O-lysylphosphatidylglycerol, which occurs prominently in this bacterium, had little effect.  相似文献   

6.
When retinol was incubated in the presence of ATP, UDP-galactose, magnesium ions and Triton X-100, with a whole homogenate of rat thyroid as an enzyme source the formation of a retinol containing phosphate compound was observed besides retinol galactoside described before. This phosphate compound is soluble in chloroform-methanol (3:2, vv) and can be separated by chromatography on a column prepared with butanol-washed cellulose powder. The isolated compound was retinol pyrophosphate since it contained retinol and phosphate in a molar ratio of 1:2 shown by double isotopic labelling techniques and was found to be free of galactose.  相似文献   

7.
Inorganic phosphate can be determined either radiometrically or spectrophotometrically after extraction of its complex with molybdate into an organic phase. Triton X-100 interferes with this extraction. Determination of the inorganic phosphate can be carried out in the presence of up to 0.8% (wv) Triton X-100 by modification of the method: After addition of the silicotungstate, the sample is centrifuged, the yellow oily phase removed, and a sufficient amount of silicotungstate added again. Lubrol WX interfered even more drastically than Triton X-100, but the modified method was effective in the presence of 0.04% (wv) Lubrol WX. The method was useful in biochemical assays such as those of ATPase and cyclic nucleotide-dependent phosphodiesterase.  相似文献   

8.
Synthesis and biological activity of 2′-acetyltaxol and 7-acetyltaxol are reported. Activity is measured invivo by cytotoxicity toward the macrophage-like cell line J774.2, and invitro by promotion of microtubule assembly in the absence of exogenous GTP. Addition of an acetyl moiety at C-2′ results in loss of invitro activity but not cytotoxicity. The properties of 7-acetyltaxol are similar to those of taxol in its effects on cell replication and on invitro microtubule polymerization. Therefore a free hydroxyl group at C-7 is not required for invitro activity and this position is available for structural modifications.  相似文献   

9.
A colorimetric method for the determination of orthophosphate in the presence of Triton X-100 and the extent of their mutual interference is presented. Effects of albumin and trichloroacetic acid on the reaction are also examined. The method is based on the very sensitive reaction developed for determination of orthophosphate by complex formation with ammonium molybdate followed by reduction with stannous chloride. The method allows determination of 0.005 μmol of orthophosphate in the presence of up to 0.5% Triton X-100 and as little as 0.3% (vv) Triton X-100 in the absence of phosphate.  相似文献   

10.
Bacillus subtilis membranes can transfer either N-acetylmuramyl-pentapeptide phosphate or N-acetylglucosaminyl phosphate from UMP directly onto undecaprenyl phosphate. Tunicamycin blocks only the latter transfer and inhibits peptidoglycan synthesis by toluenized cells of Bacillus megaterium utilizing added nucleotide sugar precursors or cell wall synthesis by intact cells of B. subtilis. Tunicamycin prevents formation of the cell wall disaccharide lipid intermediate by blocking transfer of N-acetylglucosamine onto undecaprenyl muramyl pentapeptidyl pyrophosphate.  相似文献   

11.
Undecaprenyl pyrophosphate synthetase was partially purified from Lactobacillus plantarum by DEAE-cellulose, hydroxyapatite, and Sephadex G-100 chromatography in Triton X-100. The enzyme has a molecular weight between 53,000 and 60,000. The enzyme demonstrated a fivefold preference for farnesyl pyrophosphate rather than geranyl pyrophosphate as the allylic cosubstrate, whereas dimethylallyl pyrophosphate was not effective as a substrate. Polyprenyl pyrophosphates obtained using either farnesyl or geranyl pyrophosphate as cosubstrate were chromatographically identical. Hydrolysis of these polyprenyl pyrophosphates with either a yeast or liver phosphatase preparation yielded undecaprenol as the major product. Incorporation of radioactive label from mixtures of Δ3-[1-14C]isopentenyl pyrophosphate and Δ3-2R-[2-3H]isopentenyl pyrophosphate into enzymic product indicated that each isoprene unit added to the allylic pyrophosphate substrate has a cis configuration about the newly formed double bond. The removal of detergent from enzyme solutions resulted in a parallel loss in enzyme activity when analyzed with either farnesyl or geranyl pyrophosphate as cosubstrates. Enzymic activity was restored on addition of Triton X-100 or deoxycholate. The enzyme exhibited a pH-activity profile with optima at pH 7.5 and 10.2. It also demonstrated a divalent cation requirement, with Mg2+, Mn2+, Zn2+, and Co2+ exhibiting comparable activities.  相似文献   

12.
Most of the chicken erythrocyte's sphingomyelin is hydrolyzed when the chicken red blood cells are incubated in hypotonie solution at 37 °C.Addition of detergents, such as Triton X-100 or Na-cholate, is essential for hydrolysis of external [3H ]sphingomyelin by the erythrocyte membranes.Pure plasma membranes show relatively high sphingomyelinase activity while no activity could be detected in the soluble fraction of the cells. Mg2+ and Mn2+ activate the enzyme while Ca2+ and EDTA strongly inhibit its activity. The optimal pH of the membrane-bound sphingomyelinase lies between pH 7.0–9.0. The detergents Triton X-100 and Na-cholate, at concentrations of 0.5% (wv) solubilize the membrane-bound enzyme. Human erythrocytes fail to exhibit sphingomyelinase activity.The correlation between the sphingomyelinase activity and its localization is discussed.  相似文献   

13.
The effect of morphine sulfate (MS) on adenylate cyclase (AC) and phosphodiesterase (PDE) activities in the rat striatum was investigated. MS produced a dose-dependent increase in basal AC activity and did not alter sodium fluoride-induced stimulation both invivo (7.5–30 mg/kg, 1 hr pretreatment, i.p.) and invitro (1–100μM). invitro, when submaximal effective concentrations of dopamine and MS were combined, there was an additive effect. However, administration of MS invivo did not alter dopamine-induced stimulation of AC activity. MS, invitro and invivo inhibited PDE activity in a dose-dependent manner only with the high substrate concentration (3.3 × 10−3M cyclic AMP). Preliminary results from this study indicate that morphine affects the cyclic AMP system.  相似文献   

14.
The light-harvesting chlorophyll ab-protein complex has been isolated from barley thylakoids by a rapid, single-step procedure involving adsorption chromatography on controlled-pore glass columns. The Triton X-100-solubilized complex contains a polypeptide of apparent molecular weight, 26,000; the 0.25% Triton X-100 light-harvesting chlorophyll ab-protein has spectral characteristics consistent with its assumed in vivo state. On the same column free chlorophyll and carotenoids have been separated from chlorophyll-protein complex 1, but this complex contained many polypeptides other than those associated with chlorophyll. This method is potentially suitable for the isolation of other thylakoid membrane proteins. It may also be generally applicable for fractionation of intrinsic membrane proteins from other sources and for separation of mixed Triton X-100-lipid micelles.  相似文献   

15.
The invitro biosynthesis of isoprene from DL-mevalonate in the cytosolic fraction of rat liver is described. Evidence is provided suggesting a non-enzymatic formation of isoprene from isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate. Furthermore, the data establish an alternate fate of the mevalonate carbon skeleton providing the first evidence that breath isoprene is linked to cholesterol biosynthesis.  相似文献   

16.
Control guinea pig cardiac myofibrils were isolated in the presence of Triton X-100. Experimental myofibrils, prepared in the presence of Triton X-100, NaF, cyclic AMP and ATP, possessed a reduced myofibrillar ATPase activity. When myofibrils isolated under control conditions were incubated for two hours at 25°C with NaF, ATP and cyclic AMP, the ATPase activity was also decreased; however, the ATPase activity was not reduced as much as that of myofibrils isolated under experimental conditions. Incubation of myofibrils with E. coli aklaline phosphatase and guinea pig heart phosphoprotein phosphatase resulted in an increase in ATPase activity and a decrease in phosphoprotein phosphate. Thus there appeared to be an inverse relationship between myofibrillar ATPase activity and phosphoprotein phosphate content. The results indicated that a protein kinase is associated with the Triton X-100 purified myofibrils and supports the notion that intact myofibrils can exist in at least two catalytic forms.  相似文献   

17.
The antiinflammatory activity of a homologous series of α-alkyl substituted [4-(1-oxo-2-iso-indolinyl)-phenyl]-acetic acid has been assayed by some invitro and invivo tests.These compounds were shown to be particularly active in inhibiting prostaglandin biosynthesis from bovine seminal vesicles, and their potency was seen to increase as the size of the substituents in the side chain increased.The antiinflammatory activity invivo is not correlated with invitro inhibition of PG-synthetase. Discussion of the data takes into account the plasma protein binding and pharmacokinetics of these compounds.  相似文献   

18.
The phospholipid requirement of the (Ca2+ + Mg2+)-ATPase present in a membrane fraction from human platelets was studied using various purified phospholipases. Only those phospholipases, which hydrolyse the negatively charged phospholipids, inhibited the (Ca2+ + Mg2+)-ATPase activity. The ATPase activity could be restored by adding mixed micelles of Triton X-100 and phosphatidylserine or phosphatidylinositol. Micelles with phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine or sphingomyelin could not be used for reconstitution and inhibited the activity of the native enzyme.  相似文献   

19.
G J Smith  P H Pearce  I T Oliver 《Life sciences》1976,19(11):1763-1775
A particulate factor of rat liver is described which interconverts three forms of rat liver cytosolic tyrosine aminotransferase invitro with no alteration of enzyme activity. The factor appears to be a heat- and pH-sensitive lysosomal protein. The interconversion process is stimulated invitro by 2.5 mM MgCl2 and 2.5 mM ATP. Asparate aminotransferase multiple forms are also susceptible to invitro interconversion by the lysosomal factor. The properties of the factor explain several anomalous effects of invitro manipulation on the tyrosine aminotransferase forms which have been reported in the literature and implicate the form interconversion in the degradation of tyrosine aminotransferase.  相似文献   

20.
Synthetic leukotriene B4 (LTB4) and its ω-oxidation products, 20 OH-LT4 and 20 COOH-LTB4, were tested for their ability to induce the aggregation of rat neutrophils invitro, to contract the guinea pig parenchymal strip invitro and to cause vascular permeability changes in rabbit skin invivo. 20 OH-LTB4 had 10, 100 and 20% of the activity of LTB4 in the neutrophil aggregation, parenchymal strip and vascular permeability assays respectively. 20 C00H-LTB4 was inactive invivo and showed <1% of the activity of LTB4invitro. These results show that while ω-oxidation is a route for biological inactivation of LTB4, 20 OH-LTB4 still retains significant biological activity.  相似文献   

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