The structure and regulation of mitochondrial L-3- glycerophosphate dehydrogenase

L-3-glycerophosphate dehydrogenase (EC 1.1.99.5) ws purified from Triton X-100 solubilised pig brain mitochondria by chromatography on hydroxylapatite followed by preparative isoelectric focusing or preparative SDS polyacrylamide gel electrophoresis. The relative molecular mass, flavin content and reactivity towards various electron acceptors were examined. An antiserum was raised against the protein and this was shown to immuneprecipitate L-3-glycerophosphate - dichlorophenolindophenol oxidoreductase activity and to identify a single protein of Mr 76,000 in both pig brain and rat liver mitochondria by immuneblotting. The structure of L-3-glycerophosphate dehydrogenase was studied in isolation and in rat liver mitochondria using the techniques of limited proteolysis and labelling with hydrophilic and hydrophobic probes. The enzyme was shown to be transmembranous and predominantly located at the matrix face of the inner mitochondrial membrance. Cross-linking studies showed the enzyme to be oligomeric. The effect of thyroid hormone on L-3-glycerophosophate dehydrogenase was investigated in rat liver. Hyperthyroidism caused an increase in mitochondrial enzyme activity and hypothyroidism a decrease. These changes were paralleled by specific changes in enyzme mass as shown by immuneblotting. In a cell-free translation system, the enzyme was synthesised as a protein of Mr 76,000 Da. Synthesis of the enzyme was greater from mRNA purified from hyperthyroid rats than from euthyroid rats but was also increased from mRNA prepared from hypothyroid rats. The import of the cell-free translation product into rat liver mitochondria was demonstrated.