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Primary cultures of neonatal rat liver cells show an increase in the activity of mitochondrial glycerolphosphate dehydrogenase between the second and tenth day of cultivation. At the end of cultivation the activity level exceeded that of liver tissue in vivo. Replacement of normal serum by hypothyroid serum or addition of triiodothyronine to the medium did not influence significantly the enzyme activity in vitro, in contrast to the very marked effects of thyroid hormones observed in vivo.

We have purified the membrane-intrinsic glycerol-3-phosphate dehydrogenase from both normal and hyperthyroid rat liver mitochondria by extraction with Triton X-100, hydrophobic affinity chromatography, ion exchange chromatography, gel filtration, and FAD-linked Sepharose 4B affinity chromatography. The yields in both cases were over 20%, and purification ranged from 800- to 650-fold in mitochondria from hyperthyroid and normal rats, respectively. The final preparations appeared to be greater than 95% pure by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate. The pure enzyme focused at pH 5.5 and produced a biphasic thermal inactivation plot at 50 degrees C. The holoenzyme was found to have a molecular mass of 250,000 daltons on gel filtration. The subunit molecular mass was found to be 74,000 daltons +/- 3,000 by sodium dodecyl sulfate-gel electrophoresis and high-performance liquid chromatography gel filtration in 0.1% sodium dodecyl sulfate. 1 mol of the holoenzyme preparation contains 1.1 mol of non-heme iron and 0.7-0.9 mol of noncovalently bound FAD. The absorption spectrum has a maximum at 375 nm and a shoulder at 450 nm which is bleached on treatment with sodium dithionite. The enzymatic reaction is competitively inhibited by glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, phosphoenolpyruvate, and phosphoglycolic acid. The apparent Km for DL-alpha-glycerol 3-phosphate and noncovalently bound FAD were found to be 6 mM and 7 microM, respectively.

A rapid three-step procedure is presented for the purification of flavin-linked L-3-glycerophosphate dehydrogenase (E.C. 1.1.99.5.) from rat liver mitochondria. Solubilization of the enzyme is achieved selectively by digitonin, at a detergent-to-protein ratio of 0.7 mg/mg (mitochondrial protein concentration 10 mg/ml). The procedure involves chromatography on hydroxymethyl-hexamethylenediamine-succinyl-hexamethylenediamin e Sepharose 4B, followed by anion exchange chromatography using a FPLC technique. Subunit molecular weight of the enzyme was found to be 77,000 when prepared in the presence of the protease inhibitor phenylmethylsulphonyl fluoride. The Kmapp value for glycerophosphate was not influenced by the purification, and the ability of the enzyme to be activated by Ca2+ was preserved as well.

Antibodies to purified glycerol-3-phosphate dehydrogenase were raised in rabbits and purified from serum by affinity chromatography on enzyme-bound Sepharose columns. RNA from membrane-free polyribosomes, or poly(A)+ RNA (total cellular RNA) of rat liver, was translated in a rabbit reticulocyte protein-synthesizing system in the presence of [35S]methionine, and the glycerol-3-phosphate dehydrogenase synthesized was isolated by immunoprecipitation using the antibody. The in vitro product moved on sodium dodecyl sulfate-polyacrylamide gels as a polypeptide that was about 5,000 daltons larger than the subunit of the mature enzyme (74,000 daltons). Digestion of both the mature and the in vitro newly synthesized forms of the enzyme yielded respective sets of peptide fragments which had similar patterns upon sodium dodecyl sulfate-gel electrophoresis. When the presumptive precursor that had been synthesized in vitro was incubated with isolated intact rat liver mitochondria, it was converted to "mature" subunits that were no longer susceptible to externally added proteases. Import of the presumptive precursor is dependent upon an electrochemical potential across the inner mitochondrial membranes. The mature form of the protein is assembled in its native location (the outer surface of the inner mitochondrial membrane).