Inhibition of creatine kinase activity from rat cerebral cortex by D-2-hydroxyglutaric acid in vitro.
D-2-Hydroxyglutaric acid (DGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as D-2-hydroxyglutaric aciduria (DHGA). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of DGA on total, cytosolic, and mitochondrial creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas cytosolic and mitochondrial activities were measured in the cytosolic and mitochondrial preparations from cerebral cortex. We verified that CK activities were significantly inhibited by DGA (11-34% inhibition) at concentrations as low as 0.25 mM, being the mitochondrial fraction the most affected activity. Kinetic studies revealed that the inhibitory effect of DGA was non-competitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of DGA on tCK activity is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK activity for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA may be related to the neurodegeneration of patients affected by DHGA.
da Silva
Cleide G
CG
Department of Biochemistry, Institute of Basic Sciences and Health, UFRGS, Rua Ramiro Barcelos 2600, Porto Alegre, RS, Brazil.
Bueno
Ana Rúbia F
AR
Schuck
Patrícia F
PF
Leipnitz
Guilhian
G
Ribeiro
César A J
CA
Rosa
Rafael B
RB
Dutra Filho
Carlos S
CS
Wyse
Angela T S
AT
Wannmacher
Clóvis M D
CM
Wajner
Moacir
M
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002540:Cerebral Cortex / Q000187:drug effects / Q000201:enzymology*; D003402:Creatine Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016166:Free Radical Scavengers / Q000494:pharmacology; D005977:Glutarates / Q000494:pharmacology*; D005978:Glutathione / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D010084:Oxidation-Reduction; D051381:Rats; D017208:Rats, Wistar; D014810:Vitamin E / Q000494:pharmacology
D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D005977:Glutarates; D014810:Vitamin E; C019417:alpha-hydroxyglutarate; D019001:Nitric Oxide Synthase; D003402:Creatine Kinase; D005978:Glutathione; D001205:Ascorbic Acid; D019331:NG-Nitroarginine Methyl Ester