title: Inhibition of mitochondrial creatine kinase activity by D-2-hydroxyglutaric acid in cerebellum of young rats.
abstract: D-2-Hydroxyglutaric aciduria (DHGA) is a neurometabolic disorder biochemically characterized by tissue accumulation and excretion of high amounts of D-2-hydroxyglutaric acid (DGA). Although the affected patients have predominantly severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In previous studies we have demonstrated that DGA, at concentrations as low as 0.25 mM, significantly decreased creatine kinase activity and other parameters of energy metabolism in cerebral cortex of young rats. In the present study, we investigated the effect of DGA (0.25-5 mM) on total creatine kinase (tCK) activity, as well as on CK activity in cytosolic (Cy-CK) and mitochondrial (Mi-CK) preparations from cerebellum of 30-day-old Wistar rats in order to test whether the inhibitory effect of DGA on CK was tissue specific. We verified that tCK (22% inhibition) and Mi-CK (40% inhibition) activities were moderately inhibited by DGA at concentrations of 2.5 mM and higher, in contrast to Cy-CK, which was not affected by the acid. Kinetic studies revealed that the inhibitory effect of DGA was noncompetitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by preincubation of the homogenates with reduced glutathione, suggesting that the inhibition of CK activity by DGA is possibly mediated by modification of essential thiol groups of the enzyme. Our present results therefore demonstrate a relatively weak inhibitory effect of DGA on cerebellum Mi-CK activity, as compared to that provoked in cerebral cortex, and may possibly be related to the neuropathology of DHGA, characterized by cerebral cortex abnormalities.
authors.affiliation: Department of Biochemistry, Institute of Basic Sciences and Health, UFRGS, Porto Alegre, RS, Brazil.
authors.forename: Cleide G
authors.initials: CG
authors.lastname: da Silva
authors.forename: Ana Rubia F
authors.initials: AR
authors.lastname: Bueno
authors.forename: Rafael B
authors.initials: RB
authors.lastname: Rosa
authors.forename: Carlos S
authors.initials: CS
authors.lastname: Dutra Filho
authors.forename: Clovis M D
authors.initials: CM
authors.lastname: Wannmacher
authors.forename: Angela T S
authors.initials: AT
authors.lastname: Wyse
authors.forename: Moacir
authors.initials: M
authors.lastname: Wajner
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology; D002531:Cerebellum / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure; D002839:Chromans / Q000494:pharmacology; D003402:Creatine Kinase / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000494:pharmacology; D005977:Glutarates / Q000737:chemistry / Q000494:pharmacology*; D005978:Glutathione / Q000494:pharmacology; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects* / Q000201:enzymology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D009928:Organ Specificity; D051381:Rats; D017208:Rats, Wistar; D013237:Stereoisomerism
chemical_list: D000975:Antioxidants; D002839:Chromans; D016166:Free Radical Scavengers; D005977:Glutarates; D007527:Isoenzymes; C019417:alpha-hydroxyglutarate; D019001:Nitric Oxide Synthase; D003402:Creatine Kinase; D005978:Glutathione; D001205:Ascorbic Acid; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; D019331:NG-Nitroarginine Methyl Ester