Date of Award

11-1-1991

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Abstract

Three guanine nucleotide-utilizing enzymes--nucleoside diphosphate kinase (NDPK), succinate thiokinase (STK), and phosphoenolpyruvate carboxykinase (PEPCK)--were studied in mitochondrial matrix preparations from rabbit heart and liver. Previous investigation by others has shown results consistent with the apparent absence of NDPK in mitochondrial matrix as well as with the existence of distinct ATP and GTP-utilizing succinate thiokinases in several species. Possible interrelationships between NDPK and STK in rabbit heart and between NDPK, STK, and PEPCK in rabbit liver were investigated.Indirect evidence for the existence of a NDPK activity in the matrix compartment of mitochondria from rabbit heart and liver was obtained via assay for ATP-linked STK activity. GDP in micromolar concentrations stimulated an apparent ATP-linked STK activity approximately 4-fold above the control with no added GDP. However, apparent ATP-linked STK activity was 6 to 8-fold higher than NDPK activity measured via HPLC-based assay. The findings were consistent with a NDPK activity in heart mitochondrial matrix involved in substrate channeling with a GTP-linked STK activity. Evidence for complex formation between GTP-linked STK and as NDPK activity was provided by glycerol density gradient centrifugation, with the apparent complex migrating with a molecular weight of approximately 150,000 daltons. A NDPK activity in liver mitochondrial matrix may also be involved in substrate channeling, but with PEPCK in addition to GTP-linked STK. Evidence for a separate ATP-linked STK in rabbit heart and liver mitochondria is therefore not supported, and it is proposed that the apparant ATP-linked STK activity observed in these tissues may be due to the combination of an endogenous NDPK activity acting in concert with a GTP-linked STK in the presence of catalytic amounts of GDP.Pigeon breast muscle mitochondria were also examined for these activities. In contrast to the other tissues examined, it was found that mitochondria from this tissue source appeared to contain relatively high levels of ATP-linked STK activity. However, a separate GTP-linked activity appeared also to be present giving rise to an ATP-STK/GTP-STK ratio of approximately 3. Evidence for a distinct GTP-linked STK activity was obtained in spite of relatively high levels of NDPK activity.In addition, purification procedures for rabbit cytosolic and mitochondrial PEPCK as well as for rabbit STK were developed. The final enzyme preparations from the three-step PEPCK procedures and from the single-step STK procedure were nearly homogeneous upon analysis by SDS-PAGE. The STK purified by this procedure was found to be entirely GTP-specific.

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