Subcellular localization and characterization of nucleoside diphosphate kinase in rat retina: effect of diabetes.
Nucleoside diphosphate kinase (NDP kinase) catalyzes the transfer of terminal phosphate from nucleotide triphosphates (e.g. ATP) to nucleotide diphosphates (e.g. GDP) to yield nucleotide triphosphates (e.g. GTP). Since guanine nucleotides play critical role(s) in GTP-binding protein (G-protein)-mediated signal transduction mechanisms in retina, we quantitated NDP kinase activity in subcellular fraction-derived from normal rat retina. A greater than 85% of the total specific activity was present in the soluble fraction, which was stimulated (up to 7 fold) by 2 mM magnesium. NDP kinase exhibited saturation kinetics towards di- and tri-phosphate substrates, and was inhibited by known inhibitors of NDP kinase, uridine diphosphate (UDP) or cromoglycate (CRG). We have previously reported significant abnormalities in the activation of G-proteins in streptozotocin (STZ)-diabetic rat retina (Kowluru et al. Diabetologia 35:624 631, 1992). Since NDP kinase has been implicated in direct interaction with and/or activation of various G-proteins, we quantitated both basal and magnesium-stimulated NDP kinase activity in soluble and particulate fractions of retina derived from STZ-diabetic rats to examine whether abnormalities in G-protein function in diabetes are attributable to alterations in retinal NDP kinase. There was no effect of diabetes either on the basal or the magnesium-activated retinal NDP kinase activity. This study represents the first characterization of NDP kinase activity in rat retina, and suggests that in diabetes, this enzyme may not be rate-limiting and/or causal for the observed alterations in retinal G-protein functions.
Kowluru
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William S. Middleton VA Medical Center, Department of Medicine, University of Wisconsin School of Medicine, Madison, USA.
Kowluru
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RA
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D009701:Nucleoside-Diphosphate Kinase / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000201:enzymology* / Q000648:ultrastructure; D014530:Uridine Diphosphate / Q000494:pharmacology
D008565:Membrane Proteins; D014530:Uridine Diphosphate; D009701:Nucleoside-Diphosphate Kinase; D019204:GTP-Binding Proteins