Over-expression and characterization of active recombinant rat liver carnitine palmitoyltransferase II using baculovirus

Abstract

The cDNA encoding rat liver carnitine palmitoyltransferase II (CPT-II) was heterologously expressed using a recombinant baculovirus/insect cell system. Unlike Escherichia coli, the baculovirus-infected insect cells expressed mostly soluble active recombinant CPT-II (rCPT-II). CPT activity from crude lysates of recombinant baculovirus-infected insect cells was maximal between 50 and 72 h post-infection, with a peak specific activity of 100-200 times that found in the mock- or wild-type-infected control lysates. Milligram quantities (up to 1.8 mg/l of culture) of active rCPT-II were chromatographically purified from large-scale cultures of insect cells infected with the recombinant baculovirus. The rCPT-II was found to be: (1) similar in size to the native rat liver enzyme (approximately 70 kDa) as judged by SDS/PAGE; (2) immunoreactive with a polyclonal serum raised against rat liver CPT-II; and (3) not glycosylated. Kinetic analysis of soluble rCPT-II revealed Km values for carnitine and palmitoyl-CoA of 950 +/- 27 microM and 34 +/- 5.6 microM respectively.

Carnitine octanoyltransferase is important for the assimilation of exogenous acetyl-L-carnitine into acetyl-CoA in mammalian cells

“…CPT2-knockout cells also showed a reduced ability to label acetyl-CoA and palmitate under equivalent conditions, but the decrease was not as pronounced as that observed in the CROT-knockout cell line. Previous studies have shown that CPT2 disfavors short-chain substates ( 20 , 21 ). To assess the role of CPT1 in LAC metabolism, etomoxir, an irreversible inhibitor of CPT1 that also inhibits CROT ( 22 ), was also tested and shown to reduce palmitate labeling ( Fig.…”

Acyl-CoA thioesterase-2 facilitates mitochondrial fatty acid oxidation in the liver

“…The elevated ␤ -hydroxybutyrate measured during the day in fed Ad-Acot2 mice suggests that the lower RQ was due, at least in part, to greater hepatic FAO. The liver acyl-carnitine profi le suggests that mitochondrial uptake of FA was increased; the higher levels of long-chain acyl-carnitines may refl ect reversal of CPT2, with reformation of carnitine esters from medium-or long-chain (but not short-chain) acyl-CoAs ( 35,36 ). It should be noted that skeletal muscle Acot2 protein expression was not elevated in Ad-Acot2 mice, suggesting that FA uptake and utilization in skeletal muscle was similar to that for Ad-Ctrl mice; thus skeletal muscle seems unlikely to be a driver of the higher FA utilization in these mice.…”

Isothermal titration calorimetry with micelles: Thermodynamics of inhibitor binding to carnitine palmitoyltransferase 2 membrane protein

“…β‐OG was identified as competitive inhibitor of rCPT‐2: with octanoyl‐CoA as substrate β‐OG had a K i of 15 mM, which is half the CMC of β‐OG [19,20]. In addition, Johnson et al observed “abnormal non‐saturation kinetics with respect to palmitoyl‐CoA” (presumably due to self‐association of palmitoyl‐CoA at high concentrations) [21]. These observations led us to refrain from determining K i values for inhibitors 1–4.…”