Localization and characterization of the mitochondrial isoform of the nucleoside diphosphate kinase in the pancreatic β cell:: Evidence for its complexation with mitochondrial succinyl-CoA synthetase

被引:86
作者
Kowluru, A
Tannous, M
Chen, HQ
机构
[1] Wayne State Univ, Coll Pharm & Allied Hlth Profess, Dept Pharmaceut Sci, Detroit, MI 48202 USA
[2] John D Dingell VA Med Ctr, Beta Cell Biochem Res Lab, Detroit, MI 48201 USA
关键词
pancreatic islet; mitochondria; nucleoside diphosphate kinase; succinyl-CoA synthetase; protein histidine phosphorylation; insulin secretion;
D O I
10.1006/abbi.2001.2710
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Nucleoside diphosphate kinase (NDPK) catalyzes the transfer of terminal phosphates from nucleoside triphosphates to nucleoside diphosphates to yield nucleotide triphosphates. The present study was undertaken to localize and characterize the mitochondrial isoform of NDPK (mNDPK) in the pancreatic beta cell since it could contribute to the generation of mitochondrial nucleotide triphosphates and, thereby, to the mitochondrial high-energy phosphate metabolism of the pancreatic beta cell. Mitochondrial fractions from the insulin-secreting beta cells were isolated by differential centrifugation. mNDPK activity was assayed as the amount of [H-3]GTPgammaS formed from ATPgammaS and [H-3]GDP. Incubation of isolated mitochondrial extracts with either [gamma-P-32]ATP or GTP resulted in the formation [P-32]NDPK, which could be immunoprecipitated by an anti-NDPK serum. mNDPK exhibited saturation kinetics with respect to its nucleoside diphosphate acceptors and nucleoside triphosphate donors and sensitivity to known inhibitors of NDPK (e.g., Uridine diphosphate and cromoglycate). By Western blot analyses, at least three isoforms of NDPK were identified in various subcellular fractions of the beta cell. The nm23-H1 (NDPK-A) was predominantly soluble whereas nm23-H2 (NDPK-B) was associated with the soluble as well as membranous fractions. The mitochondrial isoform of NDPK, nm23-H4, was uniformly distributed in the beta cell mitochondrial subfractions. A significant amount of NDPK (as determined by the catalytic activity and immunological methods) was recovered in the immunoprecipitates of mitochondrial fraction precipitated with an antiserum directed against succinyl-CoA synthetase (SCS), suggesting that NDPK might remain complexed with SCS. We provide the first evidence for the localization of a mitochondrial isoform of the NDPK in the islet beta cell and thus offer a potential mechanism for the generation of intramitochondrial GTP which, unlike ATP, is not transported into mitochondria via the classical nucleotide translocase. Further work will be required to determine the importance of the NDPK/SCS complex to normal beta cell function in the secretion of insulin. (C) 2002 Elsevier Science (USA).
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页码:160 / 169
页数:10
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