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1.
Glycerate kinase (ATP: D-glycerate 2-phosphotransferase EC 2.7.1.31) is a key enzyme of glyconeogenesis from serine via hydroxypyruvate. A differential centrifugation of rat liver homogenate and an analysis of the particle fraction by sucrose density gradient centrifugation indicated that 72% and 26% of glycerate kinase are present in mitochondria and cytosol, respectively. A study on the intramitochondrial localization of the enzyme suggested that the mitochondrial glycerate kinase was present in inner membrane and/or matrix. It was found that dietary protein selectively induced mitochondrial glycerate kinase. This result suggested that mitochondrial glycerate kinase had a physiological function for gluconeogenesis from serin. However, the metabolic significance of the cytoplasmic enzyme was still unclear. The properties of solubilized-mitochondrial and cytosolic glycerate kinases were compared. However, no difference between the two enzymes could be found in the kinetic properties, thermal stability, molecular size or electrochemical properties. These results suggested that both enzymes originate from common genetic information. In order to elucidate the regulatory mechanism of the intracellular distribution of glycerate kinase in rat liver, the responses of mitochondrial and cytosolic glycerate kinases to an alteration of dietary protein were studied. The result suggested that an alteration of dietary protein content may regulate the distribution and the translocation of glycerate kinase to mitochondria and cytosol as well as the total amount of glycerate kinase. 相似文献
2.
Yasuo Kitagawa Hirokazu Katayama Etsuro Sugimoto 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,582(2):260-275
Glycerate kinase (ATP : D-glycerate 2-phosphotransferase EC 2.7.1.31) is a key enzyme of gluconeogenesis from serine via hydroxypyruvate. A differential centrifugation of rat liver homogenate and an analysis of the particle fraction by sucrose density gradient centrifugation indicated that 72% and 26% of glycerate kinase are present in mitochondria and cytosol, respectively. A study on the intramitochondrial localization of the enzyme suggested that the mitochondrial glycerate kinase was present in inner membrane and/or matrix. It was found that dietary protein selectively induced mitochondrial glycerate kinase. This result suggested that mitochondrial glycerate kinase had a physiological function for gluconeogenesis from serine. However, the metabolic significance of the cytoplasmic enzyme was still unclear. The properties of solubilized-mitochondrial and cytosolic glycerate kinases were compared. However, no difference between the two enzymes could be found in the kinetic properties, thermal stability, molecular size or electrochemical properties. These results suggested that both enzymes originate from common genetic information. In order to elucidate the regulatory mechanism of the intracellular distribution of glycerate kinase in rat liver, the responses of mitochondrial and cytosolic glycerate kinases to an alteration of dietary protein were studied. The result suggested that an alteration of dietary protein content may regulate the distribution and the translocation of glycerate kinase to mitochondria and cytosol as well as the total amount of glycerate kinase. 相似文献
3.
Yasuo Kitagawa Etsuro Sugimoto 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,582(2):276-282
Intracellular localization of D-glycerate dehydrogenase (D-glycerate : NAD+ oxidoreductase, EC 1.1.1.29), one of the enzymes of the pathway for gluconeogenesis from serine via hydroxypyruvate, was studied by differential centrifugation. Almost all enzyme activity was found in cytosol. Since the major activities of two other enzymes, serine : pyruvate aminotransferase (EC 2.6.1.51) and glycerate kinase (ATP : D-glycerate 2-phosphotransferase, EC 2.7.1.31), of the pathway via hydroxypyruvate are localized in mitochondrial inner membrane and/or matrix, the possible localization of D-glyceratedehydrogenase in mitochondria was examined. Detailed analysis of mitochondrial fraction prepared by differential centrifugation indicated that rat liver mitochondria do not contain any D-glycerate dehydrogenase activity. Based on these results, a cooperative connection between mitochondria and cytosol in gluconeogenesis from serine via hydroxypyruvate is proposed. Possible mechanisms for transport of intermediates of the pathway via hydroxypyruvate across the mitochondrial membranes are also discussed. 相似文献
4.
Deoxyguanosine kinase (dGK) initiates the salvage of purine deoxynucleosides in mitochondria and is a key enzyme in mitochondrial DNA precursor synthesis. The active form of the enzyme is a 60-kDa protein normally located in the mitochondrial matrix. Here we describe the subcellular distribution of dGK during apoptosis in human epithelial kidney 293 cells and human lymphoblast Molt-4 cells. Immunological methods were used to monitor dGK as well as other mitochondrial proteins. Surprisingly, dGK was found to relocate to the cytosolic compartment at a similar rate as cytochrome c, a mitochondrial intermembraneous enzyme known to enter the cytosol early in apoptosis. The redistribution of dGK from the mitochondria to the cytosol may be of importance for the activation of apoptotic purine nucleoside cofactors such as dATP and demonstrates that mitochondrial matrix proteins may selectively leak out during apoptosis. 相似文献
5.
Cyclic AMP-dependent protein kinase in mitochondria and cytosol from different-sized follicles and corpora lutea of porcine ovaries 总被引:1,自引:0,他引:1
cAMP-dependent protein kinase was examined in mitochondria and cytosol prepared from different-sized antral follicles and corpora lutea of porcine ovaries. In all ovarian tissues examined except small follicles, protein kinase-specific activity was significantly higher in mitochondria than in cytosol, with the highest to lowest activities being found in medium (4-6 mm) follicles, large (7-12 mm) follicles, corpora lutea, and small (1-3 mm) follicles, respectively. Using the photoaffinity analogue [32P]8-N3cAMP, two major cAMP binding proteins with Mr = 47,000 (the apparent regulatory subunit of protein kinase Type I) and 54,000-56,000 (Type II) were found in all ovarian preparations. Type II was predominant in the cytosol of all ovarian samples, with the cytosolic Type I to Type II ratio increasing from approximately 0.05 in small and medium follicles top approximately 0.20 in large follicles and corpora lutea. In contrast, ovarian mitochondrial preparations contained relatively more Type I than did cytosol, with the mitochondrial Type I to Type II ratio increasing from approximately 0.50 in small and medium follicles to 0.88 in large follicles and 2.96 in corpora lutea. Also, mitochondrial [4-14C]cholesterol conversion and 3 beta-hydroxysteroid dehydrogenase/isomerase activities increased with follicle size and luteinization. These results suggest that Type I may play a role in the regulation of ovarian mitochondrial steroidogenesis. 相似文献
6.
Enzymatically inactive forms of acetyl-CoA carboxylase in rat liver mitochondria. 总被引:4,自引:2,他引:2
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Biotinyl proteins were labelled by incubation of SDS-denatured preparations of subcellular fractions of rat liver with [14C]methylavidin before polyacrylamide-gel electrophoresis. Fluorographic analysis showed that mitochondria contained two forms of acetyl-CoA carboxylase [acetyl-CoA:carbon dioxide ligase (ADP-forming) EC 6.4.1.2], both of which were precipitated by antibody to the enzyme. When both forms were considered, almost three-quarters of the total liver acetyl-CoA carboxylase was found in the mitochondrial fraction of liver from fed rats while only 3.5% was associated with the microsomal fraction. The remainder was present in cytosol, either as the intact active enzyme or as a degradation product. The actual specific activity of the cytosolic enzyme was approx. 2 units/mg of acetyl-CoA carboxylase protein while that of the mitochondrial enzyme was about 20-fold lower, indicating that mitochondrial acetyl-CoA carboxylase was relatively inactive. Fractionation of mitochondria with digitonin showed that acetyl-CoA carboxylase was associated with the outer mitochondrial membrane. The available evidence suggests that mitochondrial acetyl-CoA carboxylase represents a reservoir of enzyme which can be released and activated under lipogenic conditions. 相似文献
7.
Lipoamide dehydrogenase (LADase) was purified to homogeneity from rat liver mitochondria, and the intracellular distribution and biosynthesis of the LADase were investigated with antibody prepared against the purified enzyme. 1) LADase activity was mostly found in mitochondria; the activity in cytosol was about one-tenth of that in mitochondria. 2) LADase in the crude mitochondrial and cytosolic extracts and the purified LADase were immunologically identical as judged from the Ouchterlony double diffusion test. These LADases were indistinguishable from each other on immunochemical titration; i.e., the amount of LADase precipitated by a fixed amount of the anti-LADase antibody was the same for all the preparations. However, cytosolic LADase activity was inhibited by the antibody more strongly than mitochondrial LADase activity. 3) Two min after intravenous injection of [35S]methionine, more radioactivity was incorporated into cytosolic LADase than into the mitochondrial enzyme in the liver. This result suggests that localization of LADase in the cytosolic fraction is not an artifact due to leakage from mitochondria during homogenization of rat liver. 4) LADase was synthesized predominantly on free ribosomes, which indicates that LADase is synthesized on cytoplasmic ribosomes and translocated into mitochondria just as other mitochondrial proteins are. 5) After cell-free protein synthesis with post-mitochondrial supernatant, radioactivity immunoprecipitated with anti-LADase antibody was detected as a major peak with the same molecular weight as the purified LADase. 相似文献
8.
Purification and properties of deoxyadenosine kinase from rat liver mitochondria. I. Purification and physical properties 总被引:1,自引:0,他引:1
Deoxyadenosine kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76, AdR kinase) from rat liver mitochondria has been partially purified and compared with partially purified AdR kinase from the cytosol of the same biological material. Some physical properties of both enzymes, including molecular weight, gel electrophoresis and gel isoelectric focusing were investigated and considerable differences between these data for mitochondrial and cytosol AdR kinase were found. 相似文献
9.
To characterize and compare the thymidine (TdR) and deoxycytidine (CdR) kinase isozymes of uninfected and herpesvirus-infected cells: (i) the subcellular distribution of the isozymes has been studied; (ii) a specific assay for CdR kinase has been devised; (iii) the TdR kinase isozymes have been partially purified; and (iv) the purified enzymes have been analyzed by disc polyacrylamide gel electrophoresis, isoelectric focusing, and glycerol gradient centrifugation and by substrate competition and dCTP inhibition studies. The results indicate that there are interesting individual differences with respect to nucleoside acceptor specificity between the cytosol and mitochondrial pyrimidine deoxyribonucleoside kinases of uninfected cells and between the enzymes induced by different herpesviruses. In the cytosol of uninfected mouse, chicken, and owl monkey kidney cells, two different proteins, TdR kinase F and CdR kinase 2, catalyze the phosphorylations of TdR and CdR, respectively. TdR kinase F does not phosphorylate CdR, nor does CdR kinase 2 phosphorylate TdR. A second TdR kinase isozyme present in HeLa(BU25) mitochondria (TdR kinase B) also lacks CdR phosphorylating activity. In contrast, a genetically distinctive deoxypyrimidine kinase (TdR kinase A) of mouse, human, and chick mitochondria catalyzes the phosphorylation of both TdR and CdT. Three herpesviruses, marmoset herpesvirus and herpes simplex virus types 1 and 2, induce in the cytosol fraction of LM(TK-) mouse cells isozymes which share common properties with mitochondrial TdR kinase A, including the ability to catalyze the phosphorylation of both TdR and CdR. However, the herpesvirus-induced deoxypyrimidine kinases differ from mitochondrial TdR kinase A with respect to sedimentation coefficient, sensitivity to dCTP inhibition, and antigenic determinants. The herpesvirus-specific and the mitochondrial deoxypyrimidine kinases exhibit a preference for TdR over CdR as nucleoside acceptor. Pseudorabies virus and herpesvirus of turkeys induce cytosol TdR kinases resembling the other herpesvirus-induced TdR kinases in several properties, but like cellular TdR kinase F, the pseudorabies virus and herpesvirus of turkeys TdR kinases lack detectable CdR phosphorylating activities. Finally, a marmoset herpesvirus nutant resistant to bromodeoxyuridine, equine herpesvirus type 1, and Herpesvirus aotus induces neither TdR nor CdR phosphorylating enzymes during productive infections. 相似文献
10.
H J Geuze J W Slot K Yanagibashi J A McCracken A L Schwartz P F Hall 《Histochemistry》1987,86(6):551-557
In order to study the distribution of mitochondrial cytochromes P-450 in porcine adrenal glands, the glands of anesthetized pigs were fixed in situ. Polyclonal antibodies against two cytochromes P-450, i.e., C27 side-chain cleavage enzyme and 11 beta-hydroxylase, were used to study the distribution of these enzymes in cryosections of the adrenal cortex. Ultrathin cryosections were evaluated by both protein-A/gold/silver immunocytochemistry and immunoelectron microscopy using double labeling with protein-A/colloidal-gold. At light microscopy, the two cytochrome P-450 enzymes were found to be broadly distributed in both the fasciculata and glomerulosa zones of the adrenal cortex. Quantitative immunoelectron microscopy revealed that both enzymes were localized only in mitochondria, in which they were present on the inner aspects of the inner mitochondrial membrane. Both cytochromes P-450 were demonstrable in all of the mitochondria examined, and statistical evaluation of the ratios of the two enzymes present in individual mitochondria yielded a normal distribution curve. Since no evidence was found for the preferential localization of either enzyme in a special population of mitochondria, we conclude that all mitochondria of the adrenal cortex contain both enzymes. We discuss implications of these findings with respect to the regulation of steroidogenesis. 相似文献
11.
Phorbol 12-myristate 13-acetate (PMA) induces time-dependent changes in protein kinase C subcellular distribution and enzymatic activity in the human osteosarcoma cell line SaOS-2. Short (less than 60 min) incubations with PMA caused decreased cytosolic enzyme activity and a concomitant increase in particulate protein kinase; after 3 h, particulate protein kinase C activity also declined to reach less than 10% of basal activity by 24 h (Krug, E., and Tashjian, Jr., A. H., (1987) Cancer Res. 47, 2243-2246). In order to determine whether the loss in enzyme activity was due to decreased enzyme protein, Western blot analyses were performed using a polyclonal antibody against protein kinase C raised in rabbits. This approach confirmed the previously reported time-related changes: 80-kDa immunoreactive protein kinase C initially translocated from the cytosol to the particulate cell fraction and later disappeared completely from the particulate fraction. Loss of protein kinase C enzymatic activity thus results from actual loss of the 80-kDa protein; we found no evidence for generation of a calcium/phospholipid-independent protein kinase C-like form of the enzyme. Membrane association was confirmed by immunoprecipitation experiments using [35S]methionine-labeled cells. Brief exposure to PMA caused a marked loss in the [35S]methionine-labeled cytosolic protein kinase C band and an increase in the labeled particulate band. Protein kinase C immunoprecipitated from cells treated with PMA for 14 h displayed an increase in [35S]methionine label despite a greater than 80% loss of enzyme activity. The high specific radioactivity of the remaining 80-kDa protein leads us to conclude that long term treatment with PMA causes an increase in the rate of protein kinase C synthesis accompanied by a still greater increase in the rate of enzyme degradation in SaOS-2 cells. 相似文献
12.
Adenosine 3':5'-monophosphate-dependent protein kinase present in membrane fractions of bovine brain, heart, liver, and muscle was solubilized with Triton X-100. Certain properties of the membrane-derived enzyme were compared with those of two adenosine 3':5'-monophosphate-dependent protein kinases present in the cytosol fractions from each of the same tissues. The properties studied included chromatographic behavior on DEAE-cellulose columns, specificity with respect to substrate proteins, and sensitivity to NaCl and Triton X-100. The membrane-derived enzyme from each tissue had properties similar to those of the membrane-derived enzyme from each of the other tissues. Moreover, the cytosol enzymes from each tissue had properties similar to those of the corresponding enzymes in the cytosol from each of the other tissues. However, for any given tissue, the properties of the membrane-derived enzyme differed from those of the cytosol enzymes, possibly reflecting different functional roles for the membrane-bound and cytosol enzymes. 相似文献
13.
HeLa(BU25), a mutant subline of HeLa S3 cells, contains mitochondrial thymidine (dT) kinase, despite a marked deficiency in the dT kinase activity of the “cytosol” (high-speed supernatant) cell fraction. The HeLa(BU25) mitochondrial dT kinase differs from the “cytosol” enzyme of parental HeLa S3 cells in sedimentation coefficient, ability to utilize ribonucleoside 5′-triphosphates other than ATP as phosphate donors, sensitivity to inhibition by dCTP, and in disc polyacrylamide gel electrophoretic (disc PAGE) patterns. Two dT kinase activities [relative mobilities (Rm) of 0.4 and 0.6–0.7] were detected after disc PAGE of HeLa(BU25) mitochondrial extracts and both activities migrated more rapidly than the typical cytosol enzyme (Rm = 0.2) of dT kinase-positive human cells. The 0.6 to 0.7-Rm dT kinase of HeLa(BU25) mitochondria, but not the 0.4-Rm activity, utilized GTP and UTP, as well as ATP, as phosphate donors. HeLa S3 mitochondrial fractions contained the 0.6–0.7 Rm and the 0.4-Rm activities, and in addition, a “cytosol-like” 0.2-Rm activity. The 0.6 to 0.7-Rm dT kinase of HeLa S3 mitochondria utilized either UTP or ATP as phosphate donors, but the 0.4- and 0.2-Rm dT kinases utilized only ATP. Similarly, the HeLa S3 cytosol dT kinase efficiently utilized ATP, but not UTP, as a phosphate donor. 相似文献
14.
Biosynthesis of rat liver pyruvate kinase. Measurement of enzyme lifetime and the rate of synthesis at weaning. 总被引:2,自引:2,他引:0
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Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of immunoprecipitates of liver cytosol with anti-(L-type pyruvate kinase) serum revealed proteins of mol.wt. 56 000 and 42 000 in addition to the heavy and light chains. The ratio of the 56 000 mol.wt. to the 42 000 mol.wt. protein increased under dietary conditions that resulted in an increase in the apparent specific activity of hepatic pyruvate kinase. The 42 000 mol.wt. protein was removed from immunoprecipitates if the liver cytosol was partially purified by pH precipitation and (NH4)2SO4 fractionation before addition of the antiserum. This technique may be used to analyse the formation of pure L-type pyruvate kinase in liver. By using H14CO3-labelling, the t1/2 of L-type pyruvate kinase was estimated as 75 +/- 1.7 h in post-weaned high-carbohydrate-diet-fed rats. Before weaning there was little immunoreactive pyruvate kinase in rat liver cytosol. Induction began between 6 and 24 h after weaning and reached a maximum value 120 h after weaning. When clearly enhanced total pyruvate kinase activity was first observed at 24 h post-weaning, the apparent specific activity of hepatic pyruvate kinase was considerably lower than the specific activity of the pure isolated enzyme. When the induction of L-type pyruvate kinase was monitored by the incorporation of L-[4,5-3H]leucine, the maximum rate of synthesis occurred 24--48 h after weaning. After this period synthesis declined, indicating a relatively slow turnover of the enzyme once the enzyme concentration was established in the liver. 相似文献
15.
Leanza L Ferraro P Reichard P Bianchi V 《The Journal of biological chemistry》2008,283(24):16437-16445
Mitochondrial deoxynucleoside triphosphates are formed and regulated by a network of anabolic and catabolic enzymes present both in mitochondria and the cytosol. Genetic deficiencies for enzymes of the network cause mitochondrial DNA depletion and disease. We investigate by isotope flow experiments the interrelation between mitochondrial and cytosolic deoxynucleotide pools as well as the contributions of the individual enzymes of the network to their maintenance. To study specifically the synthesis of dGTP used for the synthesis of mitochondrial and nuclear DNA, we labeled hamster CHO cells or human fibroblasts with [(3)H]deoxyguanosine during growth and quiescence and after inhibition with aphidicolin or hydroxyurea. At time intervals we determined the labeling of deoxyguanosine nucleotides and DNA and the turnover of dGTP from its specific radioactivity in the separated mitochondrial and cytosolic pools. In both cycling and quiescent cells, the import of deoxynucleotides formed by cytosolic ribonucleotide reductase accounted for most of the synthesis of mitochondrial dGTP, with minor contributions by cytosolic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. A dynamic isotopic equilibrium arose rapidly from the shuttling of deoxynucleotides between mitochondria and cytosol, incorporation of dGTP into DNA, and degradation of dGMP. Inhibition of DNA synthesis by aphidicolin marginally affected the equilibrium. Inhibition of DNA synthesis by blockage of ribonucleotide reduction with hydroxyurea instead disturbed the equilibrium and led to accumulation of labeled dGTP in the cytosol. The turnover of dGTP decreased, suggesting a close connection between ribonucleotide reduction and pool degradation. 相似文献
16.
Glycolytic enzymes associate dynamically with mitochondria in response to respiratory demand and support substrate channeling 总被引:5,自引:0,他引:5
Graham JW Williams TC Morgan M Fernie AR Ratcliffe RG Sweetlove LJ 《The Plant cell》2007,19(11):3723-3738
In Arabidopsis thaliana, enzymes of glycolysis are present on the surface of mitochondria and free in the cytosol. The functional significance of this dual localization has now been established by demonstrating that the extent of mitochondrial association is dependent on respiration rate in both Arabidopsis cells and potato (Solanum tuberosum) tubers. Thus, inhibition of respiration with KCN led to a proportional decrease in the degree of association, whereas stimulation of respiration by uncoupling, tissue ageing, or overexpression of invertase led to increased mitochondrial association. In all treatments, the total activity of the glycolytic enzymes in the cell was unaltered, indicating that the existing pools of each enzyme repartitioned between the cytosol and the mitochondria. Isotope dilution experiments on isolated mitochondria, using (13)C nuclear magnetic resonance spectroscopy to monitor the impact of unlabeled glycolytic intermediates on the production of downstream intermediates derived from (13)C-labeled precursors, provided direct evidence for the occurrence of variable levels of substrate channeling. Pull-down experiments suggest that interaction with the outer mitochondrial membrane protein, VDAC, anchors glycolytic enzymes to the mitochondrial surface. It appears that glycolytic enzymes associate dynamically with mitochondria to support respiration and that substrate channeling restricts the use of intermediates by competing metabolic pathways. 相似文献
17.
Ornithine aminotransferase (OAT) from rat liver mitochondria was purified to homogeneity. A monospecific antiserum against the enzyme protein was prepared in rabbits. Immunotitrations were performed on OAT present in crude mitochondrial extracts obtained from the livers and kidneys of rats in several hormonal and dietary states. No evidence was found for the existence of an immunologically reactive but enzymatically inactive form of OAT. The relative rate of enzyme synthesis in vivo was studied by pulselabeling rats with [4, 5-3H]leucine, isolating the enzyme protein by immunoprecipitation, and dissociating the immunoprecipitates on sodium dodecyl sulfate-acrylamide gels. Nine hours after a single subcutaneous injection of a glucagon oil emulsion, a 3-fold increase in OAT activity and a 12-fold increase in the synthetic rate of the enzyme were observed. Serine dehydratase activity increased on a time-course very similar to that of OAT following glucagon injection. These increases occurred only on low (0–12.5%) protein diets. At higher levels of dietary protein (30% and up), no further stimulation of OAT synthesis by glucagon was observed. Administration of actinomycin D within the first 2 h after glucagon injection resulted in an inhibition of OAT induction. When the administration of the antibiotic was delayed until 4 h after glucagon, no inhibition of OAT induction was observed. Glucose repression of the glucagon induction of the enzyme in hepatic mitochondria was demonstrated to be the result of a rapid inhibition of OAT synthesis. 相似文献
18.
We have previously shown rat liver mitochondrial glycerol-3-phosphate acyltransferase (mtGAT), which catalyzes the first step in de novo glycerolipid biosynthesis, is stimulated by casein kinase 2 (CK2) and that a phosphorylated protein of approximately 85 kDa is present in CK2-treated mitochondria. In this paper, we have identified the (32)P-labeled 85-kDa protein as mtGAT. We have also investigated whether the phosphorylation of mtGAT is because of CK2. Mitochondria were treated with CK2 and [gamma-(32)P]GTP as the phosphate donor. Autoradiography, Western blot, and immunoprecipitation results showed mtGAT was phosphorylated by CK2. Next, we incubated mitochondria with CK2 and either ATP or GTP, in the presence of heparin, a known inhibitor of CK2. Heparin inhibited CK2-induced stimulation of mtGAT activity; this inhibition resulted in decreased (32)P-labeling of mtGAT. Additionally, mitochondria were treated with CK2 and [gamma-(32)P]ATP in the presence of staurosporine (a serine/threonine protein kinase inhibitor), genistein (a tyrosine kinase inhibitor), and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB, a CK2 inhibitor). Only DRB, the CK2 inhibitor, greatly reduced the amount of (32)P-incorporation into mtGAT by CK2. Finally, isolated mitochondrial outer membrane was incubated with cytosol in the presence of [gamma-(32)P]GTP; (32)P-labeled mtGAT was detected. Collectively, these data suggest that CK2 phosphorylates mtGAT. The impact of our results in the regulation of mtGAT and other anabolic processes is discussed. 相似文献
19.
Autophagic-lysosomal and mitochondrial sequestration of [14C]sucrose. Density gradient distribution of sequestered radioactivity 总被引:3,自引:0,他引:3
[14C]Sucrose, introduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, was sequestered by sedimentable subcellular particles during incubation of the cells at 37 degrees C. The sedimentation characteristics of particle-associated [14C]sucrose were different from the lysosomal marker enzyme acid phosphatase, suggesting an involvement of organelles of greater size than the average lysosome. Isopycnic banding in isotonic metrizamide/sucrose density gradients resolved two major peaks of radioactivity: a light peak (1.08-1.10 g/ml) coinciding with lysosomal marker enzymes, and a dense peak (1.15 g/ml), coinciding with a mitochondrial marker enzyme. The dense peak was preferentially associated with large-size particles having the sedimentation properties of mitochondria, and it was resistant to the detergent digitonin at a concentration which extracted all of the radioactivity in the light peak. Similarly the autophagy inhibitor 3-methyladenine prevented accumulation of [14C]sucrose in the light peak, while the radioactivity in the dense peak was unaffected. We therefore tentatively conclude that the light peak represents autophagic sequestration of [14C]sucrose into lysosomes (and probably autophagosomes) while the dense peak represents a mitochondrial uptake unrelated to autophagy. 相似文献
20.
Carnitine acetyltransferase was isolated from yeast Saccharomyces cerevisiae with an apparent molecular weight of 400,000. The enzyme contains identical subunits of 65,000 Da. The Km values of the isolated enzyme for acetyl-CoA and for carnitine were 17.7 microM and 180 microM, respectively. Carnitine acetyltransferase is an inducible enzyme, a 15-fold increase in the enzyme activity was found when the cells were grown on glycerol instead of glucose. Carnitine acetyltransferase, similarly to citrate synthase, has a double localization (approx. 80% of the enzyme is mitochondrial), while acetyl-CoA synthetase was found only in the cytosol. In the mitochondria carnitine acetyltransferase is located in the matrix space. The incorporation of 14C into CO2 and in lipids showed a similar ratio, 2.9 and 2.6, when the substrate was [1-14C]acetate and [1-14C]acetylcarnitine, respectively. Based on these results carnitine acetyltransferase can be considered as an enzyme necessary for acetate metabolism by transporting the activated acetyl group from the cytosol into the mitochondrial matrix. 相似文献