The overt carnitine palmitoyltransferase (palmitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21) activity of intact mitochondria from rat heart and liver was found to be resistant to the action of proteases such as Nagarse (subtilisin, EC 3.4.21.14). Nagarse under the same conditions, however, greatly decreased the malonyl-CoA inhibition of carnitine palmitoyltransferase activity, the highaffinity binding of malonyl-CoA to mitochondria, and the ability of malonyl-CoA to shift to the right the sigmoid activity curve of carnitine palmitoyltransferase observed with variations in palniitoyl-CoA concentration. No noticeable effect of Nagarse pretreatment was observed on the binding of octanoylCoA to mitochondria. Subfractionation of liver mitochondria using a combination of swelling, shrinking, and density gradient centrifugation yielded a membrane fraction in which the specific activities of the outer membrane marker enzymes were enriched ¢16-fold together with a near-parallel enrichment of malonyl-CoA-inhibitable carnitine palmitoyltransferase activity. The percent recovery of this carnitine palmitoyltransferase in the outer membrane vesicles also matched that of the known outer membrane markers. The carnitine palmitoyltransferase activity of these out-side-out vesicles became susceptible to added Nagarse only on their cosonication. These findings show that whereas the malonyl-CoA binding site relevant to the inhibition of carnitine palmitoyltransferase is situated on the outer side of the outer membrane, the overt carnitine palmitoyltransferase activity resides on the inner side of the outer membrane.

Recent evidence has shown that the outer, overt, malonyl-CoA-inhibitable carnitine palmitoyltransferase (CPTo) activity resides in the mitochondrial outer membrane [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382]. A comparison of CPTo activity of rat liver mitochondria with the inner, initially latent, carnitine palmitoyltransferase (CPTi) of the mitochondrial inner membrane has revealed that the presence of digitonin and several other detergents inactivates CPTo activity. The CPTi activity, in contrast, was markedly stimulated by various detergents and phospholipid liposomes. These findings explain why in previous studies, which used digitonin or other detergents to expose, separate and purify the CPT activities, the inferences were drawn that (a) the ratio of latent to overt CPT was quite high, (b) both the CPT activities could be ascribed to one active protein recovered, and (c) the observed lack of malonyl-CoA inhibition indicated possible loss/separation of a putative malonyl-CoA-inhibition-conferring protein. Although both CPTo and CPTi were found to catalyse the forward and the backward reactions, CPTo showed greater capacity for the forward reaction and CPTi for the backward reaction. The easily solubilizable CPT, released on sonication of mitoplasts or of intact mitochondria under hypo-osmotic conditions, resembled CPTi in its properties. When octyl glucoside was used under appropriate conditions, 40-50% of the CPTo of outer membranes became solubilized, but it showed limited stability and decreased malonyl-CoA sensitivity. Malonyl-CoA-inhibitability of CPTo was decreased also on exposure of outer membranes to phospholipase C. When outer membranes that had been exposed to octyl glucoside or to phospholipase C were subjected to a reconstitution procedure using asolectin liposomes, the malonyl-CoA-inhibitability of CPTo was restored. A role of phospholipids in the malonyl-CoA sensitivity of CPTo is thus indicated.

The =C chemical shifts of a series of isoflavones having hydroxy, acetoxy, methoxy and methylenedioxy substituents are compared. Some general relationships between substitution patterns and chemical shifts, useful for the identitication of naturally occurring isoflavones, are outlined.

We recently noted the association of carnitine palmitoyltransferase (CPT) activity with a 54 kDa microsomal protein [Murthy and Pande (1993) Mol. Cell Biochem. 122, 133-138] that, based on amino-acid-sequence identity, seemed to be the protein previously described as a 'glucose-regulated protein-58' (GRP58), phosphoinositide-specific phospholipase C, hormone-induced protein-70, endoplasmic-reticulum protein-61 (ERp61), protein disulphide-isomerase, thiol protease, a protein affected in halothane anaesthesia and one that affects renal-tubular functions and the transcriptional activation of the interferon-alpha inducible genes. To ascertain the catalytic identity of this protein unambiguously, we have expressed the corresponding cDNA transiently and stably in human kidney 293 cells as well as in HeLa cells. In each case we found that expression led to an increase in assayable and immunoreactive 54 kDa CPT activity, whereas the protein disulphide-isomerase activity was not increased. In vitro expression in a cell-free transcription and translation system led to the synthesis of a approximately 57 kDa (precursor) protein that was processed to a approximately 54 kDa (mature) protein when microsomes were present; in both these experiments again a large increase in CPT activity was seen. Thus the present data provide compelling evidence that the 54 kDa protein in question is a CPT isoenzyme. It remains to be seen now how the ability of this protein to interconvert acyl-CoA and acylcarnitine would relate to the diverse functions indicated for this protein in vivo.