Date of Award

12-2001

Level of Access Assigned by Author

Open-Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Advisor

Michael E. Vayda

Second Committee Member

Bruce Sidell

Third Committee Member

Robert Cashon

Abstract

The white muscle of Chuenocephulus aceratus, an Antarctic teleost of the Channicthyidae family, has a compromised glycoiytic capacity and this fish cannot depend on glycolysis for rapid ATP generation For C. aceratus, creatine kinase (CK) and phosphocreatine (PCr) reserves comprise the metabolic pathway that may supplement and overcome this deficiency in energy transduction. Two conditions, low glycolytic capacity and evolution in a chronically cold habitat (-1.86°C), give us reason to believe that C. aceratus muscle CK (MMCK) has been subjected to strong selective pressure. Thus, the hypothesis of this thesis is that MMCK fiom C. aceratus white muscle exhibits a high specific activity. In order to test this hypothesis, MMCK from C. aceratus white (glycolytic) muscle was purified. This revealed that C. aceratus expresses two cytosolic isoforms of MMCK. Mammals, birds, and most fish express only one MMCK. Thermal stability studies indicate that C. aceratus MMCKs exhibit thermal denaturation after a 30 minute incubation at temperatures greater-than 10°C. At O°C, the average apparent Vmax was found to be 3-to-5 times higher than that predicted fiom a Q10 analysis. The estimate of activation enthalpy for C. aceratus MMCK was 7 kcal/mol lower than that of rabbit MMCK and the activation free energy estimate of C. aceratus MMCK was 1 kcal/mol lower. This helps explain C. aceratus MMCK's high catalytic activity at low temperature, which was found to be greater-than eurythermal fish MMCK activities at 25°C. Kinetic constant estimates indicate that C. aceratus MMCKs have an affinity for ADP (Km= 0.06 mM) and PCr (Km= 17 mM) that is similar to what has been reported for other MMCKs. All C. aceratus CK cDNAs were cloned and sequenced, which confirmed that C. aceratus expresses two distinct isoforms of MCK. The tissues that express muscle, brain, and mitochondria1 CK mRNAs were determined. This is a characteristic of each CK isoform and C. aceratus MCK mRNAs were obsevered only in skeletal muscle. The evidence presented in this thesis demonstrates that one or both MMCKs have undergone cold-temperature adaptation.

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