The in vitro effects of PR toxin, a toxic secondary metabolite produced by certain strains of Penicillium roqueforti, on the membrane structure and function of rat liver mitochondria were investigated. It was found that the respiratory control and oxidative phosphorylation of the isolated mitochondria decreased concomitantly when the toxin was added to the assay system. The respiratory control ratio decreased about 60% and the ADP/O ratio decreased about 40% upon addition of 3.1 X 10(-5) M PR toxin to the highly coupled mitochondria. These findings suggest that PR toxin impairs the structural integrity of mitochondrial membranes. On the other hand, the toxin inhibited mitochondrial respiratory functions. It exhibited noncompetitive inhibitions to succinate oxidase, succinate-cytochrome c reductase, and succinate dehydrogenase activities of the mitochondrial respiratory chain. The inhibitory constants of PR toxin to these three enzyme systems were estimated to be 5.1 X 10(-6), 2.4 X 10(-5), and 5.2 X 10(-5) M, respectively. Moreover, PR toxin was found to change the spectral features of succinate-reduced cytochrome b and cytochrome c1 in succinate-cytochrome c reductase and inhibited the electron transfer between the two cytochromes. These observations indicate that the electron transfer function of succinate-cytochrome c reductase was perturbed by the toxin. However, PR toxin did not show significant inhibition of either cytochrome oxidase or NADH dehydrogenase activity of the mitochondria. It is thus concluded that PR toxin exerts its effect on the mitochondrial respiration and oxidative phosphorylation through action on the membrane and the succinate-cytochrome c reductase complex of the mitochondria. 相似文献
The administration of insulin and glucose to young Sprague-Dawley rats (125-150 g) resulted in changes in the intracellular distribution and in the turnover rates of delta-aminolevulinic acid synthase (ALAS) activity in the mitochondria and the cytosol. When starved, allylisopropylacetamide (AIA)-induced rats were injected with either insulin or glucose, the percentage of the total ALAS activity found in the cytosol increased from 27% in control animals to 33-40% in insulin-treated and 50% in glucose-treated rats. Similar increases of the ALAS activity in the cytosol were observed after insulin treatment of noninduced, starved animals. Glucose administration also repressed 25-40% of the AIA induction of ALAS as previously reported; however, this effect apparently was not a result of elevated insulin levels, since there was no observed repression of AIA induction after insulin administration. The effects of insulin and glucose on the turnover rates of ALAS activity in the mitochondria and in the cytosol were investigated by observing changes in the half-lives of ALAS activity in the two intracellular compartments. Administration of both insulin and glucose resulted in an increased half-life of ALAS activity in the cytosol from 20.8 to over 100 min, while the mitochondrial half-life was not significantly changed. When insulin was given to either fed, AIA-induced or to starved, noninduced rats, the half-life of the cytosolic ALAS increased from about 14 to 40 min. In contrast to the starved, induced animals, the mitochondrial ALAS half-life in starved, noninduced animals decreased 50%. These results suggest that insulin and glucose treatment may inhibit the translocation of ALAS from the cytosol into the mitochondrial matrix. 相似文献
α-(1→2)--Fucosidase, β--galactosidase and galactose oxidase are sterically hindered by certain types of branching in the oligosaccharide chains. 1) β--Galactosidase will not cleave galactose when the penultimate sugar carries a sialic acid residue as in I. 2) Galactose Oxidase will not oxidize the galactose residue in trisaccharide I but will in II. Moreover, neither galactose nor -acetylgalactosamine, glycosidically bound as in III, is susceptible to oxidation with galactose oxidase until the α-(1→2) linkage between them is cleaved by . 3) α-(1→2)--Fucosidase action is inhibited by or galactosyl residue, as in III and IV. Removal of the terminal sugars makes the fucosyl residue susceptible to fucosidase action. 相似文献
Experiments were performed on mice to investigate the effects of pertussis toxin (PT) on delayed-type hypersensitivity (DTH) to ovalbumin (OA) and on the activity of suppressor T cells on the DTH (DTH-Ts). Mice immunized with alum-precipitated ovalbumin showed a transient DTH, which was determined as footpad swelling which disappeared 2 weeks after immunization. Maximal footpad swelling was observed 24 hr after DTH elicitation. On the other hand, when mice received PT (2 micrograms/mouse) at the time of immunization, the transient DTH became an enhanced and persistent DTH, which persisted for at least 4 weeks. In addition, the time of maximum footpad swelling was delayed from 24 to 48 hr after DTH elicitation. The immune spleen T cells from PT-treated mice showed a persistently high ability to transfer DTH into syngenic naive mice. DTH-Ts was induced in spleens of mice injected iv with OA-coupled syngeneic spleen cells. However, when these mice received PT at the time of suppressor induction, their spleen cells revealed considerably reduced suppressor activity. The activity of DTH-Ts was also reduced when DTH-Ts were either treated in vitro with PT or transferred into PT-injected recipient mice. From these results, interference with the suppressor function of DTH-Ts from PT was considered to be, at least in part, as an enhancing mechanism of DTH. 相似文献
A potent (Ki = 0.01 mM), competitive inhibition of adenylate cyclase activity in particulate fractions of guinea pig lung by 2′O-palmitoyl cyclic AMP has been observed, in striking contrast to the inactivity of cyclic AMP and N6,2′O-dibutyryl cyclic AMP at concentrations of up to 1 mm or more. The possibility that 2′O-palmitoyl cyclic AMP or similar compounds might function as endogenous regulators of the hormonal stimulation of adenylate cyclase activity is discussed. Several 6- and 8- substituted purine 3′,5′-cyclic ribotides also inhibit, probably by direct interaction with enzymatic sulfhydryl groups. A study of the inhibition by purine bases, nucleosides, and 5′ nucleotides suggests that most of the substrate (ATP) binding determinants reside in the nucleoside. The particulate enzyme fractions were found to have lower ATPase activity relative to cyclase activity than cyclase preparations from either guinea pig heart or bronchial smooth muscle. Lung cyclase fractions were maximally stimulated by 5–15 mm Mg2+ in the presence of 1.2 mm ATP as substrate. The percentage of stimulation of cyclase activity by 0.01 mm isoproterenol is dependent on the Mg2+ concentration. Cyclase activity was significantly stimulated not only by the catecholamines (isoproterenol, epinephrine, and norepinephrine) and fluoride ion, but also by prostaglandins E1, E2, and F2α, histamine, and glucagon. 相似文献
The structures of sialo-oligosaccharide alditols as determined by 1H-NMR spectrometry together with methylation analysis did not correspond with those derived previously from quantitative periodate oxidation data alone. Possible causes of the discrepancy were explored in the periodate oxidation methodology. No free sialic acid was released by the acidity of the periodate in the course of oxidation at pH 4.5. The anionic properties of the sialic acid residues were therefore utilized to separate the periodate oxidation products and thereby establish the position of the sialic acid in the oligosaccharide chain. 相似文献
The processing of the high-mannose asparagine-linked oligosaccharides synthesized by first-trimester human placenta has been investigated. Tissue was pulsed for 1 h with [2-3H]mannose and chased for zero, 45, 90, and 180 min in media containing unlabeled mannose. Glycopeptides, prepared by Pronase digestion of the delipidated membrane pellets at each time point, were treated with endo-β-N-acetylglucosaminidase-H to release the high-mannose asparagine-linked oligosaccharides. The largest major processing intermediate isolated was Glc1Man9GlcNAc, which was converted into Man9GlcNAc, and then into Man8GlcNAc, Man7GlcNAc, Man6GlcNAc, and Man5GlcNAc. There was also a minor pathway in which mannosyl residues were removed prior to the glucose. By carrying out the detailed structural characterization of the individual processing intermediates, it was possible to demonstrate that processing of the Man9GlcNAc to Man5GlcNAc proceeded by the nonrandom removal of the α1,2-linked mannosyl residues. Specifically, of 12 possible sequences of removal of the four α1,2-linked mannosyl residues present in Man9GlcNAc, first-trimester human placenta utilized only two of these in the processing of asparagine-linked oligosaccharides. It is suggested that the limited number of processing pathways reflects a high degree of specificity of these reactions in human placenta. 相似文献
The K+-stimulated ATPase activity associated with the purified gastric microsomes from the pig gastric mucosa can be completely inactivated by treatment with 15% ethanol for 60 s at 37 °C but not at 25 °C. Sequential exposure of the microsomes to 15% ethanol at 25 and 37 °C caused the release of 2.9 and 4.3% of the total membrane phospholipids, respectively, consisting entirely of phosphatidyl choline and phosphatidyl ethanolamine. The ethanol-treated (37 °C) membrane had high basal (with Mg2+ as the only cation in the assay mixture) activity, which was further enhanced during reconstitution with phosphatidyl choline or phosphatidyl ethanolamine. The high basal activities could be reduced to the normal control level by assaying the enzyme in presence of the “activator protein,” partially purified from the soluble supernatant of the pig gastric cells. Phosphatidyl choline was somewhat more effective than phosphatidyl ethanolamine in the restoration of the activity of the ethanol-treated enzyme while phosphatidyl serine, phosphatidyl inositol, and sphingomyelin were without any effect. Synthetic phosphatidyl choline with various fatty acid substitutions were tested for their effectiveness in the restoration of the ethanol-inactivated enzyme. The distearoyl (18:0), dioleoyl (18:1), and dilinoleoyl (18:2) derivatives of phosphatidyl choline were almost equally effective while dipalmitoyl (16:0) phosphatidyl choline was somewhat less effective in the reconstitution process. Cholesterol appeared to interfere with phosphatidyl choline in the restoration of the activity of ethanol-treated enzyme. The fatty acid composition of phosphatidyl choline and phosphatidyl ethanolamine extracted by 15% ethanol at 37 °C was clearly different than those of the total microsome. Our data suggest that the phospholipids extracted by 15% ethanol at 37 °C are derived primarily from the immediate lipid environment of the enzyme and ATP together with Mg2+ and K+ help the partially delipidated enzyme to retain the appropriate conformation for the subsequent reconstitution. Furthermore, ethanol appears to either release or inactivate the membrane-associated activator protein, demonstrated to be essential for the K+-stimulated activity of the pig gastric ATPase. 相似文献
The fluorescence probe, 1,6-diphenyl-1,3,5-hexatriene, has been used to investigate the effects of controlled and uncontrolled growth on the dynamic properties of the lipid regions of hepatocyte plasma membranes. DPH was incubated with plasma membranes derived from quiescent and regenerating liver and Morris hepatoma 7777, and the resulting systems were studied by fluorescence polarization spectroscopy. Membranes from the rapidly growing hepatoma exhibited a significantly lower fluorescence polarization than observed in quiescent liver, suggesting the presence of a more fluid membrane lipid domain. Membranes from regenerating liver exhibited a time-dependent increase in membrane fluidity, reaching a maximum 12 h after growth stimulation. A close correspondence between membrane fluidity and the cholesterol-phospholipid ratio was also observed where a decrease in this ratio resulted in a more fluid lipid matrix. These results suggest that cell cycling, as observed in regenerating liver and Morris hepatoma 7777, results in significant increases in membrane fluidity, a property which may play an important regulatory role in various cell functions. 相似文献
A general procedure is described for the isolation of urinary acidic oligosaccharides and glycopeptides resulting from catabolism of glycoproteins. This procedure has been applied to normal urine and to urine from patients with diseases of the metabolism, including mucolipidosis and fucosidosis. 相似文献
The endosperm of the seed of Gleditsia triacanthos contains 4.8% of 85% ethanol-soluble, galactomannan-like oligosaccharides having Man:Gal ratios of 1.5–2.6:1 and an average degree of polymerization of 15. They have a narrow distribution of molecular weights and of ratio of components. The oligosaccharides have the gross structure accepted for the galactomannans, namely, a β-(1→4)-linked d-mannopyranosyl backbone having single stubs of α-(1→6)-linked d-galactopyranosyl groups. Some of the lateral chains contain more than one unit, and a minor proportion of the branches are ended by arabinofuranose or fucopyranose residues. Unusual branching points formed by 3,4-linked d-mannosyl, or 3,6-linked d-galactosyl units, or both, were also found. Despite their low molecular weight, the oligosaccharides form aggregates with a structure similar to that of the aggregates of the related galactomannans, but having a lower association energy. This fact, together with the difficulty of combining with more than one partner (due to the short, central chain), results in an increased solubility and in nonviscous solutions. The 13C-n.m.r. spectrum differentiated clearly the five structural units of the oligosaccharides, namely, the reducing and nonreducing end-chains of the d-mannosyl backbone; substituted and nonsubstitued, internal β-(1→4)-linked mannopyranosyl units of the backbone; and the galactosyl nonreducing end-chain of the lateral chains. The C-4 signal of the (1→4)-linked d-mannose and the C-6 signal of the same, but substituted, units showed splitting into three lines. The first has been attributed to sequence-related heterogeneity, whereas the latter is tentatively explained by assuming that this resonance is sensitive to whether the mannosyl units linked to that residue are also branched, or not. 相似文献
Cycloheptaamylose cinnamate, an intermediate in the hydrolysis of m-nitrophenyl cinnamate by cycloheptaamylose, was isolated in pure form. The deacylation of acyl-cycloamyloses (cinnamate and acetate) catalyzed by noncovalently complexed 6-nitrobenzimidazole (1) was studied. The reaction was enzyme-like. Saturation of acyl-cycloamylose by 1 was observed; the rate and dissociation constants were determined from Lineweaver-Burk plots. The catalyzed reaction rates at neutral pH were two to three times larger than those of the spontaneous reactions for cycloheptaamylose or cyclohexaamylose cinnamate, respectively. The catalytic effect of 1 on the deacylation rate of cyclohexaamylose cinnamate became smaller as the pH of the solution was raised. The deacylation of cyclohexaamylose acetate was followed by nmr spectroscopy, whereas the deacylation of cycloamylose cinnamates was followed by uv spectroscopy and extraction of trans-cinnamic acid with ether. Thermodynamic parameters for the rates of deacylation of cycloamylose cinnamates and dissociation constants of cycloamylose cinnamate-1 complexes were obtained and discussed. 相似文献
Previous work has shown that induction of a high-affinity NADPH-dependent nitrosodimethylamine demethylase (NDMAd) in liver microsomes occurs in rats due to fasting, ethanol consumption, and streptozotocin-induced diabetes. Several lines of observations suggest that this is due to the induction of specific cytochrome P-450 isozymes. Induction of P-450 species by ethanol has also been observed by other investigators. Since each of the above altered metabolic states has in common elevated levels of ketone bodies, the possible role of acetone, a known inducer of NDMAd, in the induction of the demethylase activity was investigated. Levels of endogenous acetone in fasted rats correlated (r = 0.72) with a three- to fourfold increase in NDMAd activity. However, a dose-response experiment showed endogenous levels of acetone to be capable of causing at most 40% of the induction in fasted rats. This suggests that other ketone bodies or factors may have contributed to the induction. The induction of NDMAd by ethanol was enhanced by alcohol dehydrogenase inhibitors pyrazole and acetaldehyde oxime, suggesting that ethanol, rather than its metabolites, was responsible for the induction. 相似文献
A completely iodinated form of ovine prolactin was prepared using lactoperoxidase. The iodination was characterized using gel filtration, electrophoresis and fluorescence analysis. Complete iodination corresponds to a 33% decrease in intrinsic tryptophanyl fluorescence (275348). Each ovine prolactin molecule possesses four iodination sites which cannot be distinguished by kinetic analysis. The receptor binding capacity of the tetraiodoprolactin was also assayed using the particulate fraction from female rat livers. Although the total binding capacity of native and iodoprolactins is indistinguishable, significant differences in receptor binding behavior were observed. 相似文献
The effects of numerous ionophores and inhibitors were tested on methane synthesis, intracellular ATP and potassium concentrations, and the proton motive force of the methanogenic archaebacterium Methanobacterium bryantii. M. bryantii had an internal pH near 6.8 (and hence little ΔpH during growth) with an electrical potential of ?127 mV in growth medium and ?105 mV in a pH 6.5 buffer. The study has identified agents which, in M. bryantii, can effectively cause a decline of intracellular ATP (gramicidin, acetylene) and potassium concentrations (gramicidin, nigericin), inhibit methane synthesis (acetylene, gramicidin, nigericin, triphenylmethylphosphonium bromide), eliminate the electrical potential (high extracellular potassium ion concentrations), and dissipate artificially imposed, inside alkaline, pH gradients (monensin, nigericin, carbonyl cyanide m-chlorophenylhydrazone). Carbonyl cyanide m-chlorophenylhydrazone was generally ineffective in media or buffers reduced with cysteine-sulfide but could be effective in cysteine-free solutions reduced with hydrogen sulfide. 相似文献
The effect of down-regulation on the intracellular pool of insulin receptors and the role of glycosylation in recovery from down-regulation have been studied in fibroblastic cultures from the skin of non-diabetic mice. In control cultures, 55% of the total specific [125I]insulin-binding activity was in the intracellular compartment. Insulin caused a time- and concentration-dependent decrease in the number of cell surface insulin receptors, with no significant change in total insulin receptors. This decrease in surface receptors was accompanied by an increase in the specific binding of [125I]insulin in the intracellular compartment. Removal of insulin from down-regulated cells resulted in a time-dependent increase in the binding of [125I]insulin to surface receptors, reaching 90% of that in controls by 12 h. The recovery of surface insulin receptors after removal of insulin was blocked by incubation of cultures with tunicamycin, but not by cycloheximide. These results indicate that down-regulation of surface insulin receptors by insulin is associated with translocation of receptors into the intracellular pool and suggest that protein glycosylation is important in insulin receptor recycling and externalization. 相似文献
Rats and hamsters, (pre)-treated with copper and cadmium, were used to investigate whether species-differences in renal metallothionein synthesis in response to gold were determined by changes in the kidney concentrations of other metals. The effects of both dietary copper limitation and excess on the renal metabolism of gold also were studied in the rat. In this species, all of the pre-treatments affected the renal concentrations of total and metallothionein-bound copper, but none of them altered either the kidney uptake or thionein-binding of gold. Incorporation of zinc into the metallothionein, which accompanied the binding of gold in this fraction of the kidney, however, was influenced slightly by the pretreatments. In hamsters, pretreatment with cadmium, which increased the concentrations of total and thionein-bound zinc in the kidneys, also did not affect the renal uptake of gold, although it increased significantly the binding of gold to the metallothionein fraction of the renal cytosol. This increased binding of gold also was accompanied by further increases in the zinc and copper contents of the metallothionein; the contents of total and thionein-bound cadmium, however, remained essentially unchanged. Concentrations of copper and zinc in the hamster kidney were not affected significantly by subcutaneous administration of copper alone (five daily doses, each of 3.2 mg Cu/kg body wt.), but were increased when gold was given during the copper-treatment. The concentrations of gold, copper and zinc in the renal metallothionein fraction also were increased under these conditions. From these results it seems that kidney metallothionein synthesis in response to gold may be related to the changes in either the concentration or distribution of zinc, rather than copper. 相似文献
The effect of various factors on hepatic mitochondrial ketogenesis was investigated in the rat. A comparison of three different incubation media revealed that bicarbonate ion inhibited the rate of ketone body production and decreased the ratio of 3-hydroxybutyrate/acetoacetate. The addition of 0.8 mm calcium caused significant inhibition of ketogenesis from both octanoate (40–50%) and palmitate (25–30%) and no change in the ratio of 3-hydroxybutyrate/acetoacetate. In the presence of components of the malate/aspartate shuttle, the inhibition by calcium was 80% or more with both substrates. Experimental alteration of the respiratory state of the mitochondria from state 3 to state 4 was associated with an enhanced rate of ketogenesis. The addition of ketone bodies themselves had marked effects on the rate of ketone body production. Increasing amounts of exogenously added acetoacetate were accompanied by increasing rates of total ketone body production reflecting enhanced 3-hydroxybutyrate synthesis. In the presence of added 3-hydroxybutyrate, there was striking inhibition of ketogenesis. Rotenone, which prevents oxidation of NADH2 via the electron transport chain, almost completely inhibited ketone body synthesis. This inhibition was partially overcome by the addition of acetoacetate which regenerates NAD+ from NADH2 during conversion to 3-hydroxybutyrate. These observations provide evidence for additional sites of metabolic control over hepatic ketogenesis. 相似文献