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

DNA topoisomerase I (top1) is a ubiquitous nuclear enzyme. It is specifically inhibited by camptothecin, a natural product derived from the bark of the tree Camptotheca acuminata. Camptothecin and several of its derivatives are presently in clinical trial and exhibit remarkable anticancer activity. The present study is a further investigation of the molecular interactions between the drug and the enzyme-DNA complex. We utilized an alkylating camptothecin derivative, 7-chloromethyl-10,11-methylenedioxycamptothecin (7-ClMe-MDO-CPT), and compared its activity against calf thymus top1 in a DNA oligonucleotide containing a single top1 cleavage site with the activity of its nonalkylating analog, 7-ethyl-10,11-methylenedioxycamptothecin (7-Et-MDO-CPT). In the presence of top1, 7-ClMe-MDO-CPT produced a DNA fragment that migrated more slowly than the top1-cleaved DNA fragment observed with 7-Et-MDO-CPT. Top1 was unable to religate this fragment in the presence of high NaCl concentration or proteinase K at 50 degrees C. This fragment was resistant to piperidine treatment and was also formed with an oligonucleotide containing a 7-deazaguanine at the 5' terminus of the top1-cleaved DNA (base + 1). It was however cleaved by formic acid treatment followed by piperidine. These observations are consistent with alkylation of the +1 base (adenine or guanine) by 7-ClMe-MDO-CPT in the presence of top1 covalent complexes and provide direct evidence that camptothecins inhibit top1 by binding at the enzyme-DNA interface.
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PMID:Interaction of an alkylating camptothecin derivative with a DNA base at topoisomerase I-DNA cleavage sites. 756 32

It has been reported that sodium cholate can separate the catalytic component of carnitine palmitoyltransferase-I (CPT-I) from a putative malonyl-CoA-binding regulatory protein capable of conferring sensitivity to malonyl-CoA on CPT-II. We found that cholate preferentially extracted a contaminating malonyl-CoA-sensitive CPT from mitochondrial inner membranes. When cholate extracts of outer membranes were incubated either with cholate extracts of inner membranes or with osmotically swollen mitochondria, inhibition of CPT by malonyl-CoA was increased. Treatment of intact mitochondria with subtilisin abolished the increased inhibition by malonyl-CoA, suggesting that the outer-membrane CPT-I was responsible for the increased inhibition. Incubation of cholate extracts with proteinase K did not prevent the increased inhibition. Fractionation of the cholate extract indicated the presence of phospholipids. Addition of cardiolipin or phosphatidylglycerol to osmotically swollen mitochondria increased sensitivity of CPT to malonyl-CoA, but several other phospholipids did not. When cardiolipin was added to intact mitochondria from either starved or fed rats, there were large increases in inhibition by malonyl-CoA; sensitivity in mitochondria from starved rats increased to that normally observed with mitochondria from fed rats. These results suggest that phospholipids are responsible for the increased inhibition of CPT by malonyl-CoA with added cholate extracts and that changes in membrane composition may be involved in the physiological regulation of CPT-I.
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PMID:Cholate extracts of mitochondrial outer membranes increase inhibition by malonyl-CoA of carnitine palmitoyltransferase-I by a mechanism involving phospholipids. 819 65

The topology of carnitine palmitoyltransferase I (CPT I) in the outer membrane of rat liver mitochondria was studied using several approaches. 1. The accessibility of the active site and malonyl-CoA-binding site of the enzyme from the cytosolic aspect of the membrane was investigated using preparations of octanoyl-CoA and malonyl-CoA immobilized on to agarose beads to render them impermeant through the outer membrane. Both immobilized ligands were fully able to interact effectively with CPT I. 2. The effects of proteinase K and trypsin on the activity and malonyl-CoA sensitivity of CPT I were studied using preparations of mitochondria that were either intact or had their outer membranes ruptured by hypo-osmotic swelling (OMRM). Proteinase K had a marked but similar effect on CPT I activity irrespective of whether only the cytosolic or both sides of the membrane were exposed to it. However, it affected sensitivity more rapidly in OMRM. By contrast, trypsin only reduced CPT I activity when incubated with OMRM. The sensitivity of the residual CPT I activity was unaffected by trypsin. 3. The proteolytic fragments generated by these treatments were studied by Western blotting using three anti-peptide antibodies raised against linear epitopes of CPT I. These showed that a proteinase K-sensitive site close to the N-terminus was accessible from the cytosolic side of the membrane. No trypsin-sensitive sites were accessible in intact mitochondria. In OMRM, both proteinase K and trypsin acted from the inter-membrane space side of the membrane. 4. The ability of intact mitochondria and OMRM to bind to each of the three anti-peptide antibodies was used to study the accessibility of the respective epitopes on the cytosolic and inter-membrane space sides of the membrane. 5. The results of all these approaches indicate that CPT I adopts a bitopic topology within the mitochondrial outer membrane; it has two transmembrane domains, and both the N- and C-termini are exposed on the cytosolic side of the membrane, whereas the linker region between the transmembrane domains protrudes into the intermembrane space.
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PMID:Topology of carnitine palmitoyltransferase I in the mitochondrial outer membrane. 916 4

The topology of the outer membrane carnitine palmitoyltransferase (CPT I) of rat liver mitochondria was studied systematically using several experimental approaches. Studies with immobilized malonyl-CoA and octanoyl-CoA showed that functionally the active and regulatory sites of CPT I are exposed on the outer (cytosolic) surface of the mitochondrial outer membrane. Anti-peptide antibodies generated against three linear peptide sequences that occur in between and on either side of two hydrophobic, putative transmembrane domains were used to (a) ascertain which were bound by intact mitochondria and mitochondria in which the outer membrane was permeabilized to proteins; and (b) to determine the size of fragments generated by limited proteolysis (by trypsin or proteinase K) of CPT I in intact or outer membrane-ruptured mitochondria. The sizes and immunoreactivity of the proteolytic fragments generated were correlated with the effects of the proteases on CPT I activity and malonyl-CoA sensitivity. The results of all the different approaches suggested the following: (i) CPT I has two transmembrane domains; (ii) both the N- and C-termini are exposed on the cytosolic side of the membrane; (iii) the linker region between the two transmembrane domains protrudes into the intermembrane space; (iv) both the active site and the malonyl-CoA-binding site are exposed on the cytosolic side of the membrane; (v) the amino-terminus of the protein interacts with the C-terminal domain of the protein to maintain the optimal conformation required for activity of the enzyme and its sensitivity to malonyl-CoA.
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PMID:Regulation of mitochondrial outer-membrane carnitine palmitoyltransferase (CPT I): role of membrane-topology. 938 76

Membrane proteins play an important role in cellular function. However, their analysis by mass spectrometry often is hindered by their hydrophobicity and/or low abundance. In this article, we present a method for the mass spectrometric analysis of membrane proteins based on the isolation of the resident membranes, isolation of the proteins by gel electrophoresis, and electroelution followed by enzymatic digestion by both trypsin and proteinase K. With this method, we have achieved 82-99% sequence coverage for the membrane proteins carnitine palmitoyltransferase-I (CPT-I), long-chain acyl-CoA synthetase (LCAS), and voltage-dependent anion channel (VDAC), isolated from rat liver mitochondrial outer membranes, including the transmembrane domains of these integral membrane proteins. This high sequence coverage allowed the identification of the isoforms of the proteins under study. This methodology provides a targeted approach for examining membrane proteins in detail.
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PMID:A targeted proteomic approach for the analysis of rat liver mitochondrial outer membrane proteins with extensive sequence coverage. 1687 2