Photoaffinity labeling of mitochondrial proteins with 2-azido [32P]palmitoyl CoA

AbstractA long-chain fatty acyl CoA photolabel, 2-azido [32P]palmitoyl CoA, was synthesized and its covalent interaction with mitochondrial membrane proteins examined. On binding of 2-azido [32P]palmitoyl CoA to beef heart mitochondria, two polypeptides were primarily labeled, the 30 kDa ADP/ATP carrier and a 41 kDa protein of unknown identity. Car☐yatractyloside and palmitoyl CoA completely protected against labeling of the 30 kDa protein indicating that it was the ADP/ATP carrier. With inverted submitochondrial particles, only the 30 kDa polypeptide was labeled by 2-azido [32P]palmitoyl CoA. The labeling was inhibited by bongkrekic acid and palmitoyl CoA but not car☐yatractyloside, providing evidence that the ADP/ATP carrier was covalently bound from the matrix side of the membrane. In brown adipose tissue mitochondria, 2-azido [32P]palmitoyl CoA photolabeled the ADP/ATP carrier and the 32 kDa uncoupling protein with some minor labeling of 36 and 68 kDa polypeptides. The results indicated that this physiological photolabeling reagent with the azido group on the CoA portion of the molecule interacts like 2-azido ADP with nucleotide binding sites of a number of important enzymes in cell metabolism. Moreover, the evidence strongly supports the hypothesis that long chain fatty acyl CoA esters are natural ligands for key nucleotide binding proteins

Pig liver carnitine palmitoyltransferase. Chimera studies show that both the N- and C-terminal regions of the enzyme are important for the unusual high malonyl-CoA sensitivity

Pig and rat liver carnitine palmitoyltransferase I (L-CPTI) share common K(m) values for palmitoyl-CoA and carnitine. However, they differ widely in their sensitivity to malonyl-CoA inhibition. Thus, pig l-CPTI has an IC(50) for malonyl-CoA of 141 nm, while that of rat L-CPTI is 2 microm. Using chimeras between rat L-CPTI and pig L-CPTI, we show that the entire C-terminal region behaves as a single domain, which dictates the overall malonyl-CoA sensitivity of this enzyme. The degree of malonyl-CoA sensitivity is determined by the structure adopted by this domain. Using deletion mutation analysis, we show that malonyl-CoA sensitivity also depends on the interaction of this single domain with the first 18 N-terminal amino acid residues. We conclude that pig and rat L-CPTI have different malonyl-CoA sensitivity, because the first 18 N-terminal amino acid residues interact differently with the C-terminal domain. This is the first study that describes how interactions between the C- and N-terminal regions can determine the malonyl-CoA sensitivity of L-CPTI enzymes using active C-terminal chimeras

C75 activates malonyl-CoA sensitive and insensitive components of the CPT system

5 pages, 2 figures.-- PMID: 15541339 [PubMed].-- Printed version published Dec 17, 2004.Carnitine palmitoyltransferase I (CPT-I) and II (CPT-II) enzymes are components of the carnitine palmitoyltransferase shuttle system which allows entry of long-chain fatty acids into the mitochondrial matrix for subsequent oxidation. This system is tightly regulated by malonyl-CoA levels since this metabolite is a strong reversible inhibitor of the CPT-I enzyme. There are two distinct CPT-I isotypes (CPT-Iα and CPT-Iβ), that exhibit different sensitivity to malonyl-CoA inhibition. Because of its ability to inhibit fatty acid synthase, C75 is able to increase malonyl-CoA intracellular levels. Paradoxically it also activates long-chain fatty acid oxidation. To identify the exact target of C75 within the CPT system, we expressed individually the different components of the system in the yeast Pichia pastoris. We show here that C75 acts on recombinant CPT-Iα, but also on the other CPT-I isotype (CPT-Iβ) and the malonyl-CoA insensitive component of the CPT system, CPT-II.This project was supported by Grants: SAF2001-2923 from the Ministerio de Ciencia y Tecnología (Programa Nacional de Biomedicina), from the FIS of the Instituto de Salud Carlos III, Red de Centros RCMN from the Ministerio de Sanidad (C03/08), and from the Ajut de Suport als Grups de Recerca de Catalunya 2001SGR 00432.Peer reviewe

Pig liver carnitine palmitoyltransferase. Chimera studies show that both the N- and C-terminal regions of the enzyme are important for the unusual high malonyl-CoA sensitivity

Pig and rat liver carnitine palmitoyltransferase I (L-CPTI) share common K(m) values for palmitoyl-CoA and carnitine. However, they differ widely in their sensitivity to malonyl-CoA inhibition. Thus, pig l-CPTI has an IC(50) for malonyl-CoA of 141 nm, while that of rat L-CPTI is 2 microm. Using chimeras between rat L-CPTI and pig L-CPTI, we show that the entire C-terminal region behaves as a single domain, which dictates the overall malonyl-CoA sensitivity of this enzyme. The degree of malonyl-CoA sensitivity is determined by the structure adopted by this domain. Using deletion mutation analysis, we show that malonyl-CoA sensitivity also depends on the interaction of this single domain with the first 18 N-terminal amino acid residues. We conclude that pig and rat L-CPTI have different malonyl-CoA sensitivity, because the first 18 N-terminal amino acid residues interact differently with the C-terminal domain. This is the first study that describes how interactions between the C- and N-terminal regions can determine the malonyl-CoA sensitivity of L-CPTI enzymes using active C-terminal chimeras