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1.
Zygosaccharomyces bailii possesses a constitutive malic enzyme, but only small amounts of malate are decomposed when the cells ferment fructose. Cells growing anaerobically on glucose (glucose cells) decompose malate, whereas fructose cells do not. Only glucose cells show an increase in the intracellular concentration of malate when suspended in a malate-containing solution. The transport system for malate is induced by glucose, but it is repressed by fructose. The synthesis of this transport system is inhibited by cycloheximide. Of the two enantiomers l-malate is transported preferentially. The transport of malate by induced cells is not only inhibited by addition of fructose but also inactivated. This inactivation is independent of the presence of cycloheximide. The transport of malate is inhibited by uranyl ions; various other inhibitors of transport and phosphorylation were of little influence. It is assumed that the inducible protein carrier for malate operates by facilitated diffusion. Fructose cells of Z. bailii and cells of Saccharomyces cerevisiae do not contain a transport system for malate.This research was supported in part by a grant from the Forschungsring des Deutschen Weinbaus.  相似文献   

2.
Enzyme activities forming extracellular products from succinate, fumarate, and malate were examined using washed cell suspensions of Pseudomonas fluorescens from chemostat cultures. Membrane-associated enzyme activities (glucose, gluconate, and malate dehydrogenases), producing large accumulations of extracellular oxidation products in carbon-excess environments, have previously been found in P. fluorescens. Investigations carried out here have demonstrated the presence in this microorganism of a malic enzyme activity which produces extracellular pyruvate from malate in carbon-excess environments. Although the three membrane dehydrogenase enzymes decrease significantly in carbon-limited chemostat cultures, malic enzyme activity was found to increase fourfold under these conditions. The regulation of malate dehydrogenase and malic enzyme by malate or succinate was similar. Malate dehydrogenase increased and malic enzyme decreased in carbon-excess cultures. The opposite effect was observed in carbon-limited cultures. When pyruvate or glucose was used as the carbon source, malate dehydrogenase was regulated similarly by the available carbon concentration, but malic enzyme activity producing extracellular pyruvate was not detected. While large accumulations of extracellular oxalacetate and pyruvate were produced in malate-excess cultures, no extracellular oxidation products were detected in succinate-excess cultures. This may be explained by the lack of detectable activity for the conversion of added external succinate to extracellular fumarate and malate in cells from carbon-excess cultures. In cells from carbon-limited (malate or succinate) cultures, very active enzymes for the conversion of succinate to extracellular fumarate and malate were detected. Washed cell suspensions from these carbon-limited cultures rapidly oxidized added succinate to extracellular pyruvate through the sequential action of succinate dehydrogenase, fumarase, and malic enzyme. Succinate dehydrogenase and fumarase activities producing extracellular products were not detected in cells from chemostat cultures using pyruvate or glucose as the carbon source. Uptake activities for succinate, malate, and pyruvate also were found to increase in carbon-limited (malate or succinate) and decrease in carbon-excess cultures. The role of the membrane-associated enzymes forming different pathways for carbon dissimilation in both carbon-limited and carbon-excess environments is discussed.  相似文献   

3.
In the apple variety 'Usterapfel', there are two known genotypes, which differ in malic acid content. One hundred days after full bloom, low-acid fruit (LA-fruit) contained 125 micromolg(-1) dry matter (DW) of malate, while the high-acid genotype (HA-fruit) reached levels up to 627 micromolg(-1) DW. There was no difference in the catalytic activity of enzymes involved in malate metabolism, such as PEPcarboxylase, malate dehydrogenase, and NADP malic enzyme. After [14C]glucose incorporation into the excised tissue of either genotype, the organic acid fraction was labeled to approximately the same extent. Furthermore, uptake of [14C]malate was significantly lower in excised tissue of LA-fruit. These findings suggest that low malate content in LA-fruit is the result of a restricted ability to accumulate malate in apple parenchyma cells. The different ability to accumulate malate had a pronounced effect on overall carbon partitioning. However, the rate of respiration and the rate of malate synthesis was similar in both genotypes. In HA-fruit, the glycolytic flux through pyruvate kinase was increased to compensate for the carbon that accumulated in the vacuole as malate. Since malate storage in the LA-fruit was restricted, it was more easily available for gluconeogenesis, and was correlated with a three-times higher activity of PEPcarboxykinase. LA-fruit showed higher concentrations of ATP, which stimulated Glc6P and fructose-6-phosphate formation. The elevated hexosephosphate content led to an enhanced partitioning of carbon into starch (+40%), hemicellulose (+104%), and sucrose (+40%) in more mature fruit. The activation of carbohydrate synthesis resulted in a significant drop in glucose-1-phosphate (Glc1P). To meet the increased demand for Glc1P, the activities of neutral and acid invertase, hexokinase, and phosphoglucomutase were higher in LA-fruit. Glucose was a more versatile substrate for this metabolic route than was fructose. It was also evident that glycolytic flux in apple was dependent on glucose level, and that the reaction catalysed by phosphoglucomutase contributed to the regulation of carbon partitioning between malate and carbohydrate polymers.  相似文献   

4.
The cytosol and mitochondrial isozymes of bovine brain malic enzyme were studied with respect to their sensitivity towards a series of dicarboxylic acids and sulfhydryl reagents. While no effects were obtained with the dicarboxylic acids in the case of the cytosol enzyme, the activity of the mitochondrial variant was increased considerably when either succinate, 2-mercaptosuccinate, or l-aspartate were tested at low concentrations of l-malate. The activation was associated with a clear decrease in the Hill coefficient for l-malate, and this has been taken as an indication of the presence of an allosteric site on the mitochondrial enzyme. The presence of l-malate or a dicarboxylate anion analog is required at this site in order to achieve optimal velocity. The activators were also effective in increasing the reductive carboxylation of pyruvate by the mitochondrial enzyme and had no effect on the cytosol variant. The two isozymes also showed a clear differential sensitivity to 5,5′-dithiobis(2-nitrobenzoic acid) and Hg2+, since the mitochondrial malic enzyme was inhibited by concentrations of these reagents far below those required in order to achieve an effect on the activity of the malic enzyme found in the cytosol.  相似文献   

5.
d-malate replaced l-malate in supporting both photosynthetic (anaerobic, light) and heterotrophic (aerobic, dark) growth of Rhodopseudomonas capsulata. Growth rates and cell yields were nearly equivalent with both enantiomorphs. Addition of glucose to malate culture media increased the growth rate and doubled the cell yield of heterotrophic cultures, but had little effect on photosynthetic cultures. Aerobically-grown cells showed a higher level of substrate-dependent oxygen uptake with l-malate than with d-malate. This preference for l-malate occured even in cells grown on d-malate. No malic racemase activity was detected in extracts of heterotrophically- or photosynthetically-grown cells.  相似文献   

6.
d-malate replaced l-malate in supporting both photosynthetic (anaerobic, light) and heterotrophic (aerobic, dark) growth of Rhodopseudomonas capsulata. Growth rates and cell yields were nearly equivalent with both enantiomorphs. Addition of glucose to malate culture media increased the growth rate and doubled the cell yield of heterotrophic cultures, but had little effect on photosynthetic cultures. Aerobically-grown cells showed a higher level of substrate-dependent oxygen uptake with l-malate than with d-malate. This preference for l-malate occured even in cells grown on d-malate. No malic racemase activity was detected in extracts of heterotrophically- or photosynthetically-grown cells.  相似文献   

7.
Malate has a number of key roles in the brain, including its function as a tricarboxylic acid (TCA) cycle intermediate, and as a participant in the malate-aspartate shuttle. In addition, malate is converted to pyruvate and CO2 via malic enzyme and may participate in metabolic trafficking between astrocytes and neurons. We have previously demonstrated that malate is metabolized in at least two compartments of TCA cycle activity in astrocytes. Since malic enzyme contributes to the overall regulation of malate metabolism, we determined the activity and kinetics of the mitochondrial and cytosolic forms of this enzyme from cultured astrocytes. Malic enzyme activity measured at 37°C in the presence of 0.5 mM malate was 4.15±0.47 and 11.61±0.98 nmol/min/mg protein, in mitochondria and cytosol, respectively (mean±SEM, n=18–19). Malic enzyme activity was also measured in the presence of several endogenous compounds, which have been shown to alter intracellular malate metabolism in astrocytes, to determine if these compounds affected malic enzyme activity. Lactate inhibited cytosolic malic enzyme by a noncompetitive mechanism, but had no effect on the mitochondrial enzyme. -Ketoglutarate inhibited both cytosolic and mitochondrial malic enzymes by a partial noncompetitive mechanism. Citrate inhibited cytosolic malic enzyme competitively and inhibited mitochondrial malic enzyme noncompetitively at low concentrations of malate, but competitively at high concentrations of malate. Both glutamate and aspartate decreased the activity of mitochondrial malic enzyme, but also increased the affinity of the enzyme for malate. The results demonstrate that mitochondrial and cytosolic malic enzymes have different kinetic parameters and are regulated differently by endogenous compounds previously shown to alter malate metabolism in astrocytes. We propose that malic enzyme in brain has an important role in the complete oxidation of anaplerotic compounds for energy.These data were presented in part at the meeting of the American Society for Neurochemistry in Richmond, Virginia, March 1993  相似文献   

8.
Aminopyrine oxidation was studied in isolated hepatocytes prepared from 24-h-starved mice (i) after induction of the NADPH-generating malic enzyme and glucose-6-phosphate dehydrogenase, but not the mixed function oxygenases by fructose, (ii) after induction of both mixed function oxygenases and NADPH-generating malic enzyme and glucose-6-phosphate dehydrogenase by phenobarbital and (iii) without any pretreatment. Phenobarbital pretreatment, as expected, increased the rate of aminopyrine oxidation of isolated hepatocytes. However, fructose pretreatment also enhanced the rate of N-demethylation of aminopyrine by more than 100% supporting the view that the availability of NADPH is rate limiting in drug oxidation under certain conditions. The role of malic enzyme and glucose-6-phosphate dehydrogenase in the NADPH supply for aminopyrine oxidation was investigated by the addition of two groups of gluconeogenic precursors: lactate or alanine and glycerol or fructose with the simultaneous measurement of glucose synthesis and aminopyrine N-demethylation. There was a clear correlation between the increased rate of aminopyrine oxidation and the decreases of glucose production caused by aminopyrine. Gluconeogenesis in the presence of 1 mM aminopyrine was decreased by 70-80% when alanine or lactate were used as precursors, it was decreased by only 35-40% when glucose production was started from glycerol or fructose; in an accordance with the facts that NADPH generation and gluconeogenesis starting from alanine or lactate share two common intermediates--malate and glucose-6 phosphate--, while there is only one common intermediate--glucose-6 phosphate--if fructose or glycerol are used. Similar results were obtained with the addition of the structurally dissimilar hexobarbital. It is concluded that besides malic enzyme, glucose-6-phosphate dehydrogenase also takes part in NADPH supply for drug oxidation in glycogen-depleted hepatocytes.  相似文献   

9.
1. A high activity of NAD-linked "malic" enzyme was found in homogenates of flight muscle of different species of tse-tse fly (Glossina). The activity was the same as, or higher than, that of malate dehydrogenase and more than 20-fold that of NADP-linked "malic" enzyme. A similar enzyme was found in the flight muscle of all other insects investigated, but at much lower activities. 2. ACa2+-stimulated oxaloacetate decarboxylase activity was present in all insect flight-muscle preparations investigated, in constant proportion to the NAD-linked "malic" enzyme. 3. A partial purification of the NAD-linked "malic" enzyme from Glossina was effected by DEAE-cellulose chromatography, which separated the enzyme from malate dehydrogenase and NADP-linked "malic" enzyme, but not from oxaloacetate decarboxylase. 4. The intracellular localization of the NAD-linked "malic" enzyme was predominantly mitochondrial; latency studies suggested a localization in the mitochondrial matrix space. 5. Studies on the partially purified enzyme demonstrated that it had a pH optimum between 7.6 and 7.9. It required Mg2+ or Mn2+ for activity; Ca2+ was not effective. The maximum rate was the same with either cation, but the concentration of Mn2+ required was 100 times less than that of Mg2+. Acitivity with NADP was only 1-3% of that with NAD, unless very high (greater than 10mM) concentrations of Mn2+ were present. 6. It is suggested that the NAD-linked "malic" enzyme functions in the proline-oxidation pathway predominant in tse-tse fly flight muscle.  相似文献   

10.
1. Two methods of preparing pig heart soluble malate dehydrogenase are described. A slow method yields an enzyme composed of three electrophoretically separable subforms. The more rapid method reproducibly gives a high yield of an enzyme that consists predominantly of the least acid subform. 2. The A(1%) (1cm) of the protein was redetermined as 15 at 280nm. By using this value the enzyme molecule was found to contain two independent and indistinguishable NADH-binding sites in titrations with NADH. 3. No evidence was found for the dissociation of the enzyme in the concentration range 0.02-7.2mum. 4. l-Malate (0.1m) tightened the binding of NADH to both pig and ox heart enzyme (2-fold), but, in contrast with the report by Mueggler, Dahlquist & Wolfe [(1975) Biochemistry14, 3490-3497], did not cause co-operative interactions between the binding sites. 5. Fructose 1,6-bisphosphate had no effect on the binding of NADH to the pig heart enzyme, but with the ox heart enzyme the NADH is slowly oxidized. This slow oxidation explains the ;sigmoidal' binding curves obtained when NADH was added to ox heart soluble malate dehydrogenase in the presence of fructose 1,6-bisphosphate [Cassman (1973) Biochem. Biophys. Res. Commun.53, 666-672] without the postulate of site-site interactions. 6. It is concluded that neither l-malate nor fructose 1,6-bisphosphate could in vivo modulate the activity of soluble malate dehydrogenase and alter the rates of transport of NADH between the cytosol and the mitochondrion. 7. Details of the preparation of soluble malate dehydrogenase have been deposited as Supplementary Publication SUP 50080 (8 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem. J. (1978) 169, 5.  相似文献   

11.
Malate dehydrogenase may interfere with the assay of NAD malic enzyme, as NADH is formed during the conversion of malate to oxaloacetate. During the present study, two additional effects of malate dehydrogenase were investigated; they are evident only if the malate dehydrogenase reaction is allowed to reach equilibrium prior to initiating the malic enzyme reaction. One of these (Outlaw, Manchester 1980 Plant Physiol 65: 1136-1138) might cause an underestimation of NAD reduction by malic enzyme due to the oxidation of NADH during reversal of the malate dehydrogenase reaction. A second effect may result in overestimation of malic enzyme activity, as Mn2+-catalyzed oxaloacetate decarboxylation causes continuing net NADH formation via malate dehydrogenase. These effects were studied by assaying the activity of a partially purified preparation of Amaranthus retroflexus NAD malic enzyme in the presence or absence of purified NAD malate dehydrogenase.  相似文献   

12.
In the biotechnological production of L-lysine and L-glutamate by Corynebacterium glutamicum media based on glucose, fructose or sucrose are typically used. Glutamate production by C. glutamicum was very similar on glucose, fructose, glucose plus fructose and sucrose. In contrast, lysine production of genetically defined C. glutamicum strains was significantly higher on glucose than on the other carbon sources. To test whether malic enzyme or fructose-1,6-bisphosphatase might limit growth and lysine on fructose, glucose plus fructose or sucrose, strains overexpressing either malE which encodes the NADPH-dependent malic enzyme or the fructose-1,6-bisphosphatase gene fbp were generated. Overexpression of malE did not improve lysine production on any of the tested carbon sources. Upon overexpression of fbp lysine yields on glucose and/or fructose were unchanged, but the lysine yield on sucrose increased twofold. Thus, fructose-1,6-bisphosphatase was identified as a limiting factor for lysine production by C. glutamicum with sucrose as the carbon source.  相似文献   

13.
  1. Malic enzyme was induced by malic acid and malo-lactic enzyme was induced by malic acid and glucose in cells of three strains ofLactobacillus casei that were able to grow on malate as carbon source. Two strains ofStreptococcus faecalis formed malic enzyme only, whereas only malo-lactic enzyme was formed by a glucose requiring strain ofStreptococcus lactis.
  2. Given sequential induction, cells ofLactobacillus casei M40 were found to contain malic enzyme and malo-lactic enzyme simultaneously.
  3. Malic enzyme and malo-lactic enzyme have been separated by chromatography on Sephadex G-200. These two enzymes have a different pH optimum, different affinities for substrates, form different end products from malate, and have molecular weights of 120000 and 150000 daltons respectively.
  相似文献   

14.
Gluconeogenesis in the kidney cortex. Effects of d-malate and amino-oxyacetate   总被引:15,自引:13,他引:2  
1. Rat kidney-cortex slices incubated with d-malate alone formed very little glucose. d-Malate, however, augmented gluconeogenesis from l-lactate and inhibited gluconeogenesis from pyruvate and l-malate. 2. d-Malate had little effect on the rate of the tricarboxylic acid cycle with or without other substrates added. 3. d-Malate inhibited the activity of the l-malate dehydrogenase in a high-speed-supernatant fraction from kidney cortex. 4. It was concluded that d-malate inhibited either the operation of the cytoplasmic l-malate dehydrogenase or malate outflow from the mitochondria in the intact kidney-cortex cell. This supports the hypothesis of Lardy, Paetkau & Walter (1965) and Krebs, Gascoyne & Notton (1967) on the role of malate as carrier for carbon and reducing equivalents in gluconeogenesis. 5. Gluconeogenesis from l-lactate in kidney-cortex slices was strongly inhibited by a low concentration (0.1mm) of amino-oxyacetate, whereas glucose formation from pyruvate, malate, aspartate and several other compounds was only slightly affected. 6. High concentrations of l-aspartate largely reversed the inhibition of gluconeogenesis from l-lactate caused by amino-oxyacetate. 7. Amino-oxyacetate inhibited strongly the glutamate-oxaloacetate transaminase in the 30000g supernatant fraction of a kidney-cortex homogenate. The presence of l-aspartate decreased the inhibition of the transaminase by amino-oxyacetate. 8. Detritiation of l-[2-(3)H]aspartate was inhibited by 90% during an incubation of kidney-cortex slices with l-lactate and amino-oxyacetate. 9. Low concentrations (10mum) of artificial electron acceptors such as Methylene Blue and phenazine methosulphate abolished most of the inhibition of gluconeogenesis from l-lactate by amino-oxyacetate. This is interpreted as an activation of net malate outflow from the mitochondria by-passing the inhibited transfer of oxaloacetate. 10. These findings support the concept that transamination to aspartate is involved in the transfer of oxaloacetate from mitochondria to cytosol required in gluconeogenesis from l-lactate.  相似文献   

15.
16.
It has been shown that the conversion of cholesterol to progesterone by human term placental mitochondria incubated in the presence of malate or fumarate was inhibited by hydroxymalonate—an inhibitor of malic enzyme. No inhibition was observed when mitochondria were incubated in the presence of citrate or isocitrate. The degree of inhibition by hydroxymalonate of partly purified NAD(P)-linked malic enzyme activity was identical to that of both malate dependent pyruvate and progesterone formation by intact mitochondria. These data strongly support a previous suggestion that malic enzyme plays an important role in the malate dependent progesterone biosynthesis by human placental mitochondria.  相似文献   

17.
Malic enzyme (S)-malate: NADP+ oxidoreductase (oxaloacetate-decar☐ylating, EC 1.1.1.40) purified from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4, catalyzed the metal-dependent decar☐ylation of oxaloacetate at optimum pH 7.6 at a rate comparable to the decar☐ylation of l-malate. The oxaloacetate decar☐ylase activity was stimulated about 50% by NADP but only in the presence of MgCl2, and was strongly inhibited by l-malate and NADPH which abolished the NADP activation. In the presence of MnCl2 and in the absence of NADP, the Michaelis constant and Vm for oxaloacetate were 1.7 mM and 2.3 μmol·min−1·mg−1, respectively. When MgCl2 replaced MnCl2, the kinetic parameters for oxaloacetate remained substantially unvaried, whereas the Km and Vm values for l-malate have been found to vary depending on the metal ion. The enzyme carried out the reverse reaction (malate synthesis) at about 70% of the forward reaction, at pH 7.2 and in the presence of relatively high concentrations of bicarbonate and pyruvate. Sulfhydryl residues (three cysteine residues per subunit) have been shown to be essential for the enzymatic activity of the Sulfolobus solfataricus malic enzyme. 5,5′-Dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate and N-ethylmaleimide caused the inactivation of the oxidative decar☐ylase activity, but at different rates. The inactivation of the overall activity by p-hydroxymercuribenzoate was partially prevented by NADP singly or in combination with both l-malate and MnCl2, and strongly enhanced by the car☐ylic acid substrates; NADP + malate + MnCl2 afforded total protection. The inactivation of the oxaloacetate decar☐ylase activity by p-hydroxymercuribenzoate treatment was found to occur at a slower rate than that of the oxidative decar☐ylase activity.  相似文献   

18.
Kinetic studies of Morris 7777 hepatoma mitochondrial NAD(P) malic enzyme were consistent with an ordered mechanism where NAD adds to the enzyme before malate and dissociation of NADH from the enzyme is rate-limiting. In addition to its active site, malate apparently also associates with a lower affinity with an activator site. The activator fumarate competes with malate at the activator site and facilitates dissociation of NADH from the enzyme. The ratio of NAD(P) malic enzyme to malate dehydrogenase activity in the hepatoma mitochondrial extract was found to be too low, even in the presence of known inhibitors of malate dehydrogenase, to account for the known ability of NAD(P) malic enzyme to intercept exogenous malate from malate dehydrogenase in intact tumor mitochondria (Moreadith, R.W., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 6215-6221). However, NAD(P) malic enzyme may be able to intercept exogenous malate because according to the present results, it can associate with the pyruvate dehydrogenase complex, which could localize NAD(P) malic enzyme in the vicinity of the inner mitochondrial membrane. The activity levels of some key metabolic enzymes were found to be different in Morris 7777 mitochondria than in liver or mitochondria of other rapidly dividing tumors. These results are discussed in terms of differences among tumors in their ability to utilize malate, glutamate, and citrate as respiratory fuels.  相似文献   

19.
Acetylpyridine NADP replaced NADP in promoting the Mn2+ ion-requiring mitochondrial "malic" enzyme of Hymenolepis diminuta. Disrupted mitochondria displayed low levels of an apparent oxaloacetate-forming malate dehydrogenase activity when NAD or acetylpyridine NAD served as the coenzyme. Significant malate-dependent reduction of acetylpyridine NAD by H. diminuta mitochondria required Mn2+ ion and NADP, thereby indicating the tandem operation of "malic" enzyme and NADPH:NAD transhydrogenase. Incubation of mitochondrial preparations with oxaloacetate resulted in a non-enzymatic decarboxylation reaction. Coupling of malate oxidation with electron transport via the "malic" enzyme and transhydrogenase was demonstrated by polarographic assessment of mitochondrial reduced pyridine nucleotide oxidase activity.  相似文献   

20.
Adrenal cortex mitochondria prepared by a standard method do not exhibit malic enzyme activity. Addition of physiological concentrations of Ca2+ and Mg2+ enables these mitochondria to reduce added NADP+ by malate to form free NADPH. Half-maximum activation of the mitochondrial malic enzyme requires 0.3 mM Ca2+ and 1 mM Mg2+. Solubilized mitochondrial malic enzymes is independent of Ca2+ and has a K M of 0.2 mM for Mg2+. The Ca2+ effect is dependent on an initial period of active Ca2+ uptake which also causes other changes in respiratory properties similar to those observed with mitochondria from other tissues. After Ca2+ accumulation has taken place, free Ca2+, but not additional accumulation, is still required for malic enzyme activity. The requirement for Mg2+ can be met by Mn2+ (1 mM). This concentration of Mn2+ alone yielded only a slight activation of mitochondrial malic enzyme while higher concentrations of Mn2+ alone gave good activation of the mitochondrial malic enzy.e The NADPH generated by the Ca2+-Mg2+ activated malic enzyme effectively supports the 11beta-hydroxylation of deoxycorticosterone, whereas in the presence of malate, or malate plus Mg2+ but absence of Ca2+, the energy linked transhydrogenase supplies all the required NADPH. The activated malic enzyme appears to be more efficient than transhydrogenase in generating NADPH to support 11beta-hydroxylation. Cyanide and azide have been found to inhibit solubilized mitochondrial malic enzyme.  相似文献   

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