Cell-free synthesis of a putative precursor to the rat liver mitochondrial glycerol-3-phosphate dehydrogenase.
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).
Garrib
A
A
Department of Biochemistry, University of Western Ontario, London, Canada.
McMurray
W C
WC
D000818:Animals; D002474:Cell-Free System; D004792:Enzyme Precursors / Q000235:genetics*; D005993:Glycerolphosphate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification; D036341:Intercellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D012156:Reticulocytes / Q000378:metabolism; D014757:Viper Venoms / Q000302:isolation & purification* / Q000494:pharmacology
D004792:Enzyme Precursors; D036341:Intercellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010455:Peptides; D012333:RNA, Messenger; D014757:Viper Venoms; C057986:echistatin; D011061:Poly A; D012313:RNA; D005993:Glycerolphosphate Dehydrogenase