Transverse-plane topography of long-chain acyl-CoA synthetase in the mitochondrial outer membrane.
Transverse-plane topography of mitochondrial outer-membrane long-chain acyl-CoA synthetase was investigated using proteases as probes for exposure of crucial domains, i.e. domains containing the active site or otherwise required for enzymatic activity. Incubation of intact mitochondria with the nonspecific proteases proteinase K and subtilisin resulted in a time-dependent loss of 90% or more of the long-chain acyl-CoA synthetase activity compared to control incubations. The integrity of the outer membrane before and during this treatment was shown by cytochrome c oxidase latency as well as the stability of adenylate kinase activity in the presence of protease. After a 15-min incubation in these conditions, site-specific proteases such as trypsin and chymotrypsin had only a limited inhibitory effect (29 and 58% loss of activity, respectively); however, treatment of hypotonically disrupted mitochondria with these proteases resulted in increased (71 and 77%, respectively) loss of activity. Exposure of trypsin-sensitive crucial domains on the inner surface of the membrane was directly demonstrated by incubation of trypsin-loaded outer-membrane vesicles. Together, these results suggest that mitochondrial long-chain acyl-CoA synthetase is a transmembrane enzyme, possessing crucial domains on both sides of the outer membrane. However, the cytosolic exposure of the enzyme does not appear to be affected by a change in the medium ionic strength as seen previously for other outer-membrane enzymes. In an experiment investigating the topography of the active site of the enzyme, an immobilized substrate analog, desulfo-CoA-agarose, was preincubated with intact mitochondria. This resulted in up to a 42% loss of the activity of long-chain acyl-CoA synthetase, consistent with a cytosolic exposure for at least the CoA-binding domain of the active site.
Hesler
C B
CB
Department of Biological Sciences, St. John's University, Jamaica, New York 11439.
Olymbios
C
C
Haldar
D
D
D000818:Animals; D003066:Coenzyme A Ligases / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013367:Submitochondrial Particles / Q000201:enzymology*; D014357:Trypsin / Q000494:pharmacology
D008565:Membrane Proteins; D010447:Peptide Hydrolases; D014357:Trypsin; D003066:Coenzyme A Ligases