Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.21 (CPT)
 4,580 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Proteolysis of intact mitochondria by Nagarse (subtilisin BPN') and papain resulted in limited loss of activity of the outer-membrane carnitine palmitoyltransferase, but much greater loss of sensitivity to inhibition by malonyl-CoA. In contrast with a previous report [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382], we found that trypsin had no effect on malonyl-CoA sensitivity. Even when 80% of activity was destroyed by trypsin, there was no difference in the malonyl-CoA sensitivity of the enzyme remaining. Trypsin caused release of the intermembrane-space enzyme adenylate kinase, indicating loss of integrity of the mitochondrial outer membrane, whereas Nagarse and papain caused no release of that enzyme. Citrate synthase was not released by any of the three proteinases, indicating no damage to the mitochondrial inner membrane. When we examined the effects of proteolysis on the inhibition of carnitine palmitoyltransferase by a wide variety of inhibitors having different mechanisms of inhibition, we found differential proteolytic effects that were specific for those inhibitors (malonyl-CoA and hydroxyphenylglyoxylate) that have their inhibitory potencies diminished by changes in physiological state. Both of those inhibitors protected carnitine palmitoyltransferase from the effects of proteolysis, but did not inhibit the proteinases directly. Inhibition by two other inhibitors (DL-2-bromopalmitoyl-CoA and N-benzyladriamycin 14-valerate) was not altered by proteinase treatment, even when most of the enzyme activity had been destroyed. Inhibition by glyburide, which is minimally affected by physiological state, was affected only to a slight extent at the highest concentration of trypsin tested. Proteolysis by Nagarse appeared to produce loss of co-operativity in malonyl-CoA inhibition. The effects of proteolysis are discussed and compared with changes in Ki occurring with changing physiological states.
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PMID:Proteinase treatment of intact hepatic mitochondria has differential effects on inhibition of carnitine palmitoyltransferase by different inhibitors. 155 74

Our earlier work using intact mitochondria and isolated mitochondrial outer membranes confirms the observations of Murthy and Pande that CPT-I is located on the mitochondrial outer membranes and supports the notion that this enzyme has a malonyl-CoA binding domain facing the cytosol and an acyl-CoA binding domain facing the inter membrane space. Our data also suggests that coenzyme A binds at the active site of CPT-I, as does acyl-CoA, 2-bromopalmitoyl-CoA, and (+)-hemipalmitoylcarnitinium, but malonyl-CoA does not bind at that site. Inhibition of CPT-I at the malonyl-CoA binding site by HPG and Ro 25-0187, which have no CoA moiety, contributes to a resolution of this question in that the CoA itself is not essential for the binding of malonyl-CoA to its regulatory site, but the dicarbonyl function which is present in malonyl-CoA, HPG, and Ro 25-0187 is absolutely essential. Our re-evaluation of the topology of hepatic mitochondrial CPT-I confirms the original observations that this enzyme has at least two different binding domains, one domain binding malonyl-CoA, HPG, and Ro-25-187 and the other domain binding acyl-CoA and other inhibitors of CPT-I. Furthermore, the malonyl-CoA binding domain is exposed to the cytosolic face of the membrane. Our data showing that treatment of the intact mitochondria with trypsin causes release of adenylate kinase which indicates that trypsin has damaged the mitochondrial outer membrane, possibly allowing trypsin to enter the intermembrane space and act on CPT from within the outer membrane. Since trypsin's action is limited to arginine and lysine residues, an alternative explanation could be that the portion of the protein domain responsible for malonyl-CoA inhibition may not contain these residues. The latter explanation is plausible, since malonyl-CoA was able to protect against loss of activity and sensitivity to inhibition, but did not protect against loss of adenylate kinase, suggesting that rupture of the outer membrane is not necessarily related to loss of CPT activity. These results suggest that some protein domain that is necessary for CPT activity is exposed on the outer surface of the outer membranes. Therefore, it seems likely that trypsin would have to be able to hydrolyse protein domains of CPT that are inaccessible to Nagarse and papain.
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PMID:Topology of hepatic mitochondrial carnitine palmitoyltransferase I. 1070 25