Iraj Ghadiminejad

Regulation of mitochondrial carnitine palmitoyl transferases from liver and extrahepatic tissues

Developments in our understanding of the complex CPT enzyme system over the past ten years have been reviewed. Liver CPT1, which is probably distinct from that in several extrahepatic tissues, is subject to up- or down-regulation of its activity and kinetic properties with changing physiological state. Evidence is now accumulating to support the notion that the catalytic and malonyl-CoA-binding entities of CPT1 are separate polypeptides.

Carnitine palmitoyltransferase (CPT2) from liver mitochondrial inner membrane becomes inhibitable by malonyl-CoA if reconstituted with outer membrane malonyl-CoA binding protein.

A soluble extract was obtained on treatment of rat liver mitochondrial outer membranes with cholate which bound (14C]malonyl‐CoA but was essentially free of carnitine palmitoyltransferase (CPT) activity. Extraction of mitochondrial inner membranes with cholate readily solubilized a CPT activity which was insensitive to malonyl‐CoA. Combination of these two extracts caused the CPT derived from inner membranes to become inhibitable by malonyl‐CoA.

Neonatal carnitine palmitoyltransferase-2 deficiency: a case presenting with myopathy

Mitochondria were isolated from liver, heart and skeletal muscle of a 34-day-old female infant who died from a myopathic illness. Muscle biopsy showed lipid accumulation and no obvious pathology in any other organ. Enzymatic analysis of skeletal muscle extracts revealed normal activities of the markers pyruvate dehydrogenase and citrate synthase. Malonyl-CoA-sensitive carnitine palmitoyltransferase (CPT1) was detected but malonyl-CoA-insensitive carnitine palmitoyltransferase (CPT2) appeared to be absent. Quantitative immunoblotting revealed the presence of a normal abundance of CPT2 protein in the patients muscle. It is concluded that enzymically inactive CPT2 protein was present.

Physiological state and the sensitivity of liver mitochondrial outer membrane carnitine palmitoyltransferase to malonyl-coa. correlations with assay temperature, salt concentration and membrane lipid composition

1. Liver mitochondrial outer membranes were pre-exposed to media of low (20 mM phosphate) or high salt concentration (20 mM phosphate + 0.3 M KCl) before assay of carnitine palmitoyltransferase (CPT) at 25 degrees C. 2. With membranes from fed rats, exposure to high salt decreased sensitivity of CPT to malonyl-CoA whereas high salt increased sensitivity of CPT to malonyl-CoA in membranes from 48 hr-fasted rats. These changes were paralleled by alterations in the KD for high affinity binding of [14C]malonyl-CoA to outer membranes. 3. Decreasing the CPT assay temperatures from 25 to 10 degrees C caused qualitatively similar changes to those seen on exposure to high salt. 4. The relative content of sphingomyelin was increased 2-fold and 4-fold in liver mitochondrial outer membranes from fasted and diabetic rats respectively. Fasting had no effect on the content of cholesterol whereas diabetes decreased this by a third.

A proportion of rat liver mitochondrial carnitine palmitoyltransferase can be made activatable by malonyl-CoA

Treatment of rat liver mitochondrial membranes with cholate yields a soluble extract containing carnitine palmitoyltransferase (CPT) activity that is insensitive to malonyl-CoA. As found previously (I. Ghadiminejad and D. Saggerson (1990) FEBS Lett. 269, 406-408), addition of polyethylenen glycol 6000 (PEG 6000) to this extract conferred sensitivity to malonyl-CoA on the CPT. It is now shown that a sub-population of the CPT activity which is sedimentable at 7000 x g after addition of PEG 6000 is activated by malonyl-CoA, whereas the remainder is inhibited by malonyl-CoA. The presence of KCl increases the proportion of the activatable form of CPT. Possible physiological significance of this finding is discussed.

Use of mitochondrial inner membrane proteins and phospholipids to facilitate disengagement of the catalytic and malonyl-CoA binding components of carnitine palmitoyltransferase from liver mitochondrial outer membranes

1. It was shown by Ghadiminejad and Saggerson (1991) that the anionic detergent cholate caused disengagement of the malonyl-CoA binding entity from the catalytic entity of outer membrane carnitine palmitoyltransferase (CPT1). 2. This disengagement was only observed if inner membrane material was present. 3. It is now shown that this effect is mimicked by a CPT-free inner membrane protein fraction together with an inner membrane lipid extract or with individual phospholipids (phosphatidylcholine, phosphatidylethanolamine or diphosphatidylglycerol). 4. The lipids alone have no effect but act synergistically with the inner membrane protein fraction.