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1.
(1) The reason for substrate specificity of Sr2+-induced oscillating cation fluxes in isolated rat liver mitochondria was investigated. (2) With succinate as substrate, rotenone prevented oscillation. In this case the mitochondria were only partially able to take up added Sr2+ and did not take up any of the released K+. Addition of substances decreasing the mitochondrial NADHNAD+ ratio (oxaloacetate or acetoacetate) restored the ability for reuptake of K+ and for complete uptake of Sr2+ and, therefore, oscillation. (3) Inhibition of substrate-level phosphorylation by arsenite or uncoupling of substrate-level phosphorylation by arsenate in the presence of oligomycin also suppressed the reuptake of cations. This effect of inhibition of substrate-level phosphorylation on oscillation could be circumvented by addition of ATP in the presence of oligomycin. (4) Prevention of the intramitochondrial regeneration of 2-oxoglutarate from acetyl-CoA and oxaloacetate by fluorocitrate or from endogenous glutamate by aminoxyacetate shortened the time during which oscillation with succinate as substrate could be observed. (5) From the key role of substrate level phosphorylation it is concluded that for the reuptake of K+ and Sr2+ during oscillation, sufficient GTP generation by the succinyl thiokinase (EC 6.2.1.4) reaction is essential. Therefore substrate level phosphorylation seems to be a necessary energy source additional to the respiratory chain. Since the latter process drives the active cation movements, the former may be required for the restoration of a sufficiently low proton conductance of the mitochondrial inner membrane. Oscillation in the absence of exogenous ATP therefore demands 2-oxoglutarate as substrate or the intramitochondrial generation of 2-oxoglutarate for the maintenance of a sufficient GTP production for a longer time.  相似文献   

2.
A quantitative study of H+, K+, Sr2+ and succinate fluxes in Sr2+-induced oscillatory state of rat liver mitochondria is presented. It was shown that oscillation of succinate content in mitochondria occurs synchronously with oscillations of the cation fluxes. Total charge transferred across the membrane by the registered cations and the succinate-anion is equal to zero. Passive H+-influx has been calculated at all stages of the oscillatory cycle. The conclusion is made that electroneutral 2 H+/Sr2+ exchange is periodically induced in mitochondria. A value of (2 ± 0.2) · 10-7 mol Sr2+/min per mg protein. has been determined for Sr2+ by this type of exchange.  相似文献   

3.
Carbohydrate chains of cancer glycoprotein antigens contain major outer changes dictated by tissue-specific regulation of glycosyltransferase genes, the availability of sugar nucleotides, and competition between enzymes for acceptor intermediates during glycan elongation. However, it is evident from recent studies with recombinant mucin probes that the final glycosylation profiles of mucin glycoproteins are mainly determined by the cellular repertoire of glycosyltransferases. Hence, we examined various cancer cell lines for the levels of fucosyl-, beta-galactosyl, beta-N-acetylgalactosaminyl-, sialyl-, and sulfotransferase activities that generate the outer ends of the oligosaccharide chains. We have identified glycosyltransferases activities at the levels that would give rise to O-glycan chains as reported by others in breast cancer cell lines, T47D, ZR75-1, MCF-7, and MDA-MB-231. Most breast cancer cells express Gal-3-O-sulfotransferase specific for T-hapten Gal beta1-->3GalNAc alpha-, whereas the enzyme from colon cancer cells exhibits a vast preference for the Gal beta1,4GlcNAc terminal unit in O-glycans. We also studied ovarian cancer cells SW626 and PA-1 and hepatic cancer cells HepG2. Our studies show that alpha1,2-L-fucosyl-T, alpha(2,3) sialyl-T, and 3-O-Sulfo-T capable of acting on the mucin core 2 tetrasaccharide, Gal beta1,4GlcNAc beta1,6(Gal beta1,3)GalNAc alpha-, can also act on the Globo H antigen backbone, Gal beta1,3GalNAc beta1,3Gal alpha-, suggesting the existence of unique carbohydrate moieties in certain cancer-associated glycolipids. Briefly, our study indicates the following: (i) 3'-Sulfo-T-hapten has an apparent relationship to the tumorigenic potential of breast cancer cells; (ii) the 3'-sulfo Lewis(x), the 3-O-sulfo-Globo unit, and the 3-fucosylchitobiose core could be uniquely associated with colon cancer cells; (iii) synthesis of a polylactosamine chain and T-hapten are favorable in ovarian cancer cells due to negligible sialyltransferase activities; and (iv) a 6'-sialyl LacNAc unit and 3'-sialyl T-hapten appear to be prevalent structures in hepatic cancer cell glycans. Thus, it is apparent that different cancer cells are expressing unique glycan epitopes, which could be novel targets for cancer diagnosis and treatment.  相似文献   

4.
5.
It has been reported (Becker, G.L., Fiskum, G. and Lehninger, A.L. (1980) J. Biol. Chem. 255, 9009-9012) that respiring rat liver mitochondria suspended in KC1 medium containing ATP, Mg2+ and phosphate, maintain a steady state extramitochondrial free Ca2+ concentration of about 0.5 microM. The results reported here show that the addition of the antibiotic X-537A, at concentrations far below those required for ionophorous activity, caused a perturbation in this steady state, lowering the extramitochondrial free Ca2+ concentration by about 0.20 microM. This shift in steady state was clarified by a study of X-537A inhibition of the Ca2+ efflux induced by ruthenium red; a half-maximum effect was observed at approximately 25 nM X-537A. No effect on Ca2+ transport through the influx uniporter was observed. The possibility of a generalized stabilizing action of the antibiotic on the mitochondrial membrane seems to be ruled out by its effectiveness at very low concentrations.  相似文献   

6.
7.
We have investigated the genotoxic effects of 1-(2-hydroxyethyl)-1-nitrosourea (HENU). We have chosen this agent because of its demonstrated ability to produce N7-(2-hydroxyethyl) guanine (N7-HOEtG) and O6-(2-hydroxyethyl) 2′-deoxyguanosine (O6-HOEtdG); two of the DNA alkylation products produced by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). For these studies, we have used the Big Blue Rat-2 cell line that contains a lambda/lacI shuttle vector. Treatment of these cells with HENU produced a dose dependent increase in the levels of N7-HOEtG and O6-HOEtdG as quantified by HPLC with electrochemical detection. Treatment of Big Blue Rat-2 cells with either 0, 1 or 5 mM HENU resulted in mutation frequencies of 7.2±2.2×10−5, 45.2±2.9×10−5 and 120.3±24.4×10−5, respectively. Comparison of the mutation frequencies demonstrates that 1 and 5 mM HENU treatments have increased the mutation frequency by 6- and 16-fold, respectively. This increase in mutation frequency was statistically significant (P<0.001). Sequence analysis of HENU-induced mutations have revealed primarily G:C→A:T transitions (52%) and a significant number of A:T→T:A transversions (16%). We propose that the observed G:C→A:T transitions are produced by the DNA alkylation product O6-HOEtdG. These results suggest that the formation of O6-HOEtdG by BCNU treatment contributes to its observed mutagenic properties.  相似文献   

8.
The changes in the partial pressures of oxygen and carbon dioxide (PO2 and PCO2) during blood circulation alter erythrocyte metabolism, hereby causing flux changes between oxygenated and deoxygenated blood. In the study we have modeled this effect by extending the comprehensive kinetic model by Mulquiney and Kuchel [P.J. Mulquiney, and P.W. Kuchel. Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations: equations and parameter refinement, Biochem. J. 1999, 342, 581–596.] with a kinetic model of hemoglobin oxy-/deoxygenation transition based on an oxygen dissociation model developed by Dash and Bassingthwaighte [R. Dash, and J. Bassingthwaighte. Blood HbO2 and HbCO2 dissociation curves at varied O2, CO2, pH, 2,3-DPG and temperature levels, Ann. Biomed. Eng., 2004, 32(12), 1676–1693.]. The system has been studied during transitions from the arterial to the venous phases by simply forcing PO2 and PCO2 to follow the physiological values of venous and arterial blood. The investigations show that the system passively follows a limit cycle driven by the forced oscillations of PO2 and is thus inadequately described solely by steady state consideration. The metabolic system exhibits a broad distribution of time scales. Relaxations of modes with hemoglobin and Mg2+ binding reactions are very fast, while modes involving glycolytic, membrane transport and 2,3-BPG shunt reactions are much slower. Incomplete slow mode relaxations during the 60 s period of the forced transitions cause significant overshoots of important fluxes and metabolite concentrations – notably ATP, 2,3-BPG, and Mg2+. The overshoot phenomenon arises in consequence of a periodical forcing and is likely to be widespread in nature – warranting a special consideration for relevant systems.  相似文献   

9.
An ATP-dependent mechanism for Ca2+ uptake in human platelet membrane fractions has been identified and characterized. Ca2+ uptake into a membrane fraction is shown to be stimulated at low concentrations of ATP and Ca2+ and to require magnesium ions. Initial rate kinetics, using Eadie-Scatchard analysis, indicated a single class of calcium uptake sites in the presence of ATP, with a Kd for free [Ca2+] of 0.145 μM. Ca2+ uptake in the presence of several ATP concentrations demonstrates that ATP binds to at least two sites, representing high and low affinities of 3.21 and 80.1 μM, respectively. The neuroleptic drug fluphenazine inhibited ATP-stimulated calcium uptake (IC50 = 55 μM), suggesting this ATP-dependent Ca2+ uptake system may provide a useful ion-transport model with which to study neuroleptic therapy in humans.  相似文献   

10.
In an attempt to elucidate the mechanism of initiation of peroxidation in HgCl2-treated erythrocytes, the effect of HgCl2 on methemoglobin-catalyzed lipid peroxidation was studied. It was found that HgCl2 reinforces the prooxidant action of methemoglobin. This effect seems not to be due to dissociation or degradation of the hemoglobin molecule to heme-containing fragments or iron-containing products of low molecular weight. The results obtained indicate that Hg2+ increases the binding of oxy- and methemoglobin to liposomes. A suggestion is made that the acceleration of methemoglobin-catalyzed peroxidation by HgCl2 is mainly due to increased binding of methemoglobin to liposomes. On the basis of these results and the results obtained previously the possible mechanism of initiation of peroxidation in Hg2+-treated erythrocytes is discussed.  相似文献   

11.
The in vitro opioid activities of a series of leucine enkephalin analogs containing a thioamide linkage in place of the peptide bond at various positions of the backbone were determined in mu- and delta-receptor-selective bio- and binding-assays. Thioamide substitution in the 1-2 position resulted in an inactive compound, whereas the same modification in the 2-3 and 4-5 position produced potency enhancement. Most interestingly, the 2-3 modified analog showed a 3 to 5 times higher preference for delta- over mu-receptors than natural leucine enkephalin. These results suggest that subtle backbone modifications can have a profound effect on receptor affinity and selectivity of biologically active peptides.  相似文献   

12.
The erythro form of the lignin model compound 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol (1) was oxidized with laccase/ABTS, lead(IV) tetraacetate (LTA), lignin peroxidase/H2O2, cerium(IV) ammonium nitrate (CAN) and Fenton's reagent. The product profiles obtained with the different oxidants were compared after separation, identification and quantification of the products using HPLC, UV-diode array detector and electrospray ionization mass spectrometry in positive ionization mode. The oxidants generated different product profiles that reflected their different properties. Oxidation with laccase/ABTS resulted almost exclusively in formation of 1-(3,4-dimethoxyphenyl)-3-hydroxy-2-(2-methoxyphenoxy)-1-propanone (2). Oxidation with LTA resulted in more 3,4-dimethoxybenzaldehyde (3) than ketone 2. Lignin peroxidase and CAN gave similar product profiles and aldehyde 3 was the predominant product (only small amounts of ketone 2 were formed). Oxidation with Fenton's reagent resulted in the formation of more aldehyde 3 than ketone 2 but the yields were very low. CAN served as an excellent model for the lignin peroxidase-catalyzed oxidation, while the laccase-mediator system, LTA and Fenton's reagent provided distinctly different product profiles. Erythro-1-(3,4-dimethoxyphenyl)-1,2,3-propanetriol was present among the products obtained on oxidation with LTA, lignin peroxidase, CAN and Fenton's reagent. The differences in redox potential between the oxidants afford an explanation of the diverse product patterns but other factors may also be of importance. The reactions leading to cleavage of the β-ether bond with formation of 1-(3,4-dimethoxyphenyl)-1,2,3-propanetriol (veratrylglycerol) were found to proceed without affecting the configuration at the β-carbon atom.  相似文献   

13.
14.
Nafoxidine and CI-628, two well known antiestrogenic compounds, reduce the stimulating effect of estradiol on the estrogen-binding capacity of the liver chromatin from roosters. In vitro both antiestrogens compete with [3H] estradiol for the binding sites on the liver chromatin. They inhibit the estrogen-induced synthesis of egg yolk proteins (vitellogenin) and fail to induce this estrogen-specific protein synthesis by themselves. They show the ability, however, to increase the estrogen-binding sites on the liver chromatin to some extent.  相似文献   

15.
The aim of our work is to show the importance of the role of hydrophobic bonds in maintaining Mg2+-ATPase or sucrase activity and Na+-coupled d-glucose uptake normal for the brush border of rat enterocytes. The activity of the two enzymes and the d-glucose uptake were therefore measured under the action of n-aliphatic alcohols and related to the fluidity determined by ESR. Three concentrations were used for the first eight alcohols, those of octanol being about 1500-times lower than those of methanol. For each alcohol the d-glucose uptake and the fluidity were linear functions of the logarithm of the concentration, the linear regressions being practically parallel and equidistant. The concentrations (C) of the eight alcohols inhibiting the d-glucose uptake by 80% were similar to those increasing the membrane fluidity by 3%. The linear relationship which existed in both cases between log 1 / C and log P, P being octanol / water partition coefficients of the alcohols, was evidence of great sensitivity to the hydrophobic effect of the alcohols. Only the first alcohols, however, produced any notable inhibition of Mg2+-ATPase and sucrase. Hydrophobic bonds are thus shown to have little influence in maintaining the activity of Mg2+-ATPase and sucrase, but they modulate the Na+-coupled d-glucose uptake.  相似文献   

16.
K Rotti  J Stevens  D Watson  C Longcope 《Steroids》1975,25(6):807-816
Using a rabbit antisera directed against estriol-3-0-carboxy methyl ether complexed to BSA, an immunoassay for estriol (1) was developed. The mean plus or minus SE concentration of estriol in 18 women in days 5-7 of their cycle was 7.9 plus or minus 0.6 pg/ml which was significantly (P less than 0.01) less than the mean value of 11.1 plus or minus 0.8 pg/ml in 15 women in days 20-22 of the cycle. In 3 of 6 women in whom plasma samples were drawn frequently during their cycle, an estriol peak occurred coincident with the estradiol peak. In 3 women from whom plasma was obtained several times during the course of a day estriol levels did not appear to vary significantly. In 8 women who were on oral contraceptives the mean level of estriol was 7.6 plus or minus 1.5 pg/ml. In 8 post-menopausal women the mean level was 6.0 plus or minus 1.2 pg/ml which is significantly (P less than 0.01) less than the mean luteal phase value but not less (P greater than 0.1) than the follicular phase or oral contraceptive user values. We conclude that some of the circulating estriol is directly secreted by the ovary of normal women.  相似文献   

17.
The Mg2+-dependency of Ca2+-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg2+- and Ca2+-buffering ligands. ATP hydrolysis is strongly stimulated by Mg2+ with a Km of 13 μ M in the absence or presence of 1 μ M free Ca2+. At free Mg2+ concentrations of 1 μ M and lower, ATP hydrolysis is Mg2+ -independent, but is strongly stimulated by submicromolar Ca2+ concentrations Km  0.25 μM, Vmax  24 μmol Pi/h per mg protein). The Ca2+-stimulated ATP hydrolysis strongly decreases at higher Mg2+ concentrations. The Ca2+-stimulated Mg2+-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg2+ concentrations calmodulin and trifluoperazine affect the high affinity Ca2+-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca2+-pumping in renal basolaterals and on Ca2+-ATPase in erythrocyte ghosts suggest that the Ca2+-pumping enzyme requires Mg2+. In contrast, a role of the Ca2+-stimulated Mg2+-independent ATP hydrolysis in active Ca2+ transport across basolateral membranes is rather unlikely.  相似文献   

18.
ATP-enriched human red cells display high rates of Ca2+-dependent ATP hydrolysis (16 mmol·litre cells?1·h?1) with a high Ca2+ affinity (K0.5~0.2 μM). The finding suggests a mechanism for regulation of cell Ca2+ levels, involving highly-cooperative stimulation of active Ca2+ extrusion following binding of calmodulin to the (Ca2+ + Mg2+)-ATPase.  相似文献   

19.
An axolemma-rich membrane vesicle fraction was prepared from the leg nerve of the lobster, Homerus americanus. In this preparation Ca2+ transport across the membrane was shown to require a Na+ gradient (Na+-Ca2+ exchange), and external K+ was found to facilitate this Na+-Ca2+ exchange activity. In addition, at high Ca2+ concentrations (20 mM) a Ca2+-Ca2+ exchange system was shown to operate, which is stimulated by Li+. The Na+-Ca2+ exchange system is capable of operating in the reverse direction, with Ca2+ uptake coupled with Na+ efflux. Such a vesicular preparation has the potential for providing useful experimental approaches to study the mechanism of this important Ca2+ extrusion system in the nervous system.  相似文献   

20.
An intracellular enzyme catalyzing the hydrolysis of sucrose-6-phosphate to glucose-6-phosphate and fructose has been identified in extracts of Streptococcusmutans 6715-10. The preparation was purified chromatographically and found to have an apparent molecular weight of 42,000. The enzyme has as a Km for sucrose-6-phosphate of 0.21 mM, a pH optimum of 7.1, is quite stable and requires no added cofactors or metal ions. Sucrose is a competitive inhibitor of sucrose-6-phosphate hydrolysis (Ki = 8. 12 mM). A previously described intracellular invertase copurifies with the enzyme and could not be separated from it by disc gel electrophoresis. It is concluded that intracellular invertase is a sucrose-6-phosphate hydrolase with a low catalytic activity for hydrolysis of sucrose.  相似文献   

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