Characterization of non-cytosolic hexokinase activity in white skeletal muscle from goldfish (Carassius auratus L.) and the effect of cold acclimation.
HK (hexokinase) is an enzyme involved in the first step in the glucose metabolism pathway, converting glucose into G6P (glucose 6-phosphate). Owing to the importance of skeletal muscle for fish swimming and acclimation processes, we used goldfish (Carassius auratus L.) white muscle in order to investigate subcellular distribution and kinetics of HK. In this study, we report that HK activity is predominantly localized in the mitochondrial fraction [NC-HK (non-cytosolic HK)] in goldfish white muscle. Studies of the kinetic parameters revealed that the Km (Michaelis-Menten constant) for glucose was 0.41±0.03 mM and that for mannose was 3-fold lower, whereas the affinity for fructose was too low to be measured. The Km for ATP was 0.88±0.05 mM, whereas no activity was observed when either GTP or ITP was used as a phosphate donor. A moderate inhibition (20-40%) was found for ADP and AMP. Similar to mammalian HK, G6P and glucose analogues were able to promote an inhibition of between 85 and 100% of activity. Here, we found that acclimation of goldfish at 5°C promoted a 2.5-fold increase in NC-HK compared with its counterpart acclimated at 25°C. However, cytosolic HK activity was not altered after thermal acclimation. In summary, our results suggest that the goldfish has a constitutive NC-HK that shows some similarities to mammalian HK-II and, curiously, may play a role in the broad metabolic changes required during the cold acclimation process.
dos Santos
Reinaldo Sousa
RS
Laboratory of Bioenergetics, Institute of Medical Biochemistry, Program of Biochemistry and Cellular Biophysics, Federal University of Rio de Janeiro, Cidade Universitária, Rio de Janeiro, Rio de Janeiro 21941-902, Brazil.
Diniz
Luan Pereira
LP
Galina
Antonio
A
da-Silva
Wagner Seixas
WS
D000064:Acclimatization; D000818:Animals; D003080:Cold Temperature; D006019:Glycolysis; D006054:Goldfish / Q000378:metabolism*; D006593:Hexokinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D018482:Muscle, Skeletal / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology
D006593:Hexokinase