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Baculovirus vectors engineered to contain mammalian cell-active promoter elements have been described as an efficient method for transduction of a broad spectrum of human cell lines at high frequency. In the first large-scale comparative study of secreted protein production using these viral vectors, we have evaluated production of 16 recombinant enzymes--specifically, we exploited these viral vectors, termed 'BacMam' viruses, to drive expression of a panel of proteases selected from all four major mechanistic classes, including secreted, lysosomal, endosomal, and type I transmembrane proteins. To allow a generic purification strategy, coding sequences were truncated to remove transmembrane and/or subcellular retention signals before introduction, in parallel, into a C-terminally Fc-tagged BacMam transfer vector. BacMam viruses were generated and subsequently evaluated for expression of Fc-tagged protein in virus-transduced HEK-F cells. The common Fc-tag enabled single-step affinity purification of secreted recombinant protein from the culture medium. Yields were excellent, with 14 of 16 genes expressed producing 10-30 mg or more purified protein per litre of culture using standardised transduction conditions. At this level, reagent demands for a typical protease high-throughput screen (HTS) could be met from expression cultures as small as 0.1-0.5 L. Our results indicate this expression system offers a highly efficient and scaleable method for production of enzymatically-active secreted proteases and may therefore represent a novel method of protein production for other secreted enzymes with significant advantages over the diverse approaches in current use.

Liver microsomal fractions contain a malonyl-CoA-inhibitable carnitine acyltransferase (CAT) activity. It has been proposed [Fraser, Corstorphine, Price and Zammit (1999) FEBS Lett. 446, 69-74] that this microsomal CAT activity is due to the liver form of carnitine palmitoyltransferase 1 (L-CPT1) being targeted to the endoplasmic reticulum (ER) membrane as well as to mitochondria, possibly by an N-terminal signal sequence [Cohen, Guillerault, Girard and Prip-Buus (2001) J. Biol. Chem. 276, 5403-5411]. COS-1 cells were transiently transfected to express a fusion protein in which enhanced green fluorescent protein was fused to the C-terminus of L-CPT1. Confocal microscopy showed that this fusion protein was localized to mitochondria, and possibly to peroxisomes, but not to the ER. cDNAs corresponding to truncated (amino acids 1-328) or full-length L-CPT1 were transcribed and translated in the presence of canine pancreatic microsomes. However, there was no evidence of authentic insertion of CPT1 into the ER membrane. Rat liver microsomal fractions purified by sucrose-density-gradient centrifugation contained an 88 kDa protein (p88) which was recognized by an anti-L-CPT1 antibody and by 2,4-dinitrophenol-etomoxiryl-CoA, a covalent inhibitor of L-CPT1. Abundance of p88 and malonyl-CoA-inhibitable CAT activity were increased approx. 3-fold by starvation for 24 h. Deoxycholate solubilized p88 and malonyl-CoA-inhibitable CAT activity from microsomes to approximately the same extent. The microsomal fraction contained porin, which, relative to total protein, was as abundant as in crude mitochondrial outer membranes fractions. It is concluded that L-CPT1 is not targeted to the ER membrane and that malonyl-CoA CAT in microsomal fractions is L-CPT1 that is derived from mitochondria, possibly from membrane contact sites.

Liver microsomes contain two carnitine acyltransferase activities. One of these has properties closely corresponding to those of 88 kDa mitochondrial carnitine palmitoyltransferase-1 (CPT-1). Antisera against CPT-1 cross-react with an 88 kDa microsomal protein, suggesting that CPT-1 may be targeted to both microsomal and mitochondrial membranes. However, no experiments using cDNAs corresponding to CPT-1 involving in vitro translation with microsomes or involving in vivo COS-1 cell transfection provided any evidence to support this hypothesis.

A novel hexyl-substituted methylenecyclopropyl acetyl-CoA was tested as an enzyme-specific acyl-CoA dehydrogenase inhibitor. Its CoA ester generated in situ from the carboxylic acid and CoASH, displayed marked differences in inhibition specificity as compared to methylenecyclopropyl acetyl-CoA, consistent with the substrate specificities of the target enzymes. Thus methylenecyclopropyl acetyl-CoA inactivated short-chain-specific acyl-CoA dehydrogenase rapidly, medium-chain-specific acyl-CoA dehydrogenase much more slowly and had no effect on long-chain- or very long-chain-specific acyl-CoA dehydrogenases. The hexyl-substituent on the methylenecyclopropyl ring gave an inhibitor which rapidly inactivated MCAD and LCAD whilst VLCAD was inhibited more slowly and SCAD was essentially unaffected. In some cases (e.g. SCAD and MCPA-CoA) inhibition was accompanied by flavin bleaching. In other cases (e.g. LCAD and C6MCPA) less pronounced bleaching suggests a different chemistry of inhibition.

We have investigated the extent to which membrane environment affects the catalytic properties of the malonyl-CoA-sensitive carnitine acyltransferase of liver microsomal membranes. Arrhenius-type plots of activity were linear in the absence and presence of malonyl-CoA (2.5 microM). Sensitivity to malonyl-CoA increased with decreasing assay temperature. Partly purified enzyme displayed an increased K0.5 (substrate concentration supporting half the maximal reaction rate) for myristoyl-CoA and a reduced sensitivity to malonyl-CoA compared with the enzyme in situ in membranes. Reconstitution with liposomes of a range of compositions restored the K0.5 for myristoyl-CoA to values similar to that seen in native membranes. The lipid requirements for restoration of sensitivity to malonyl-CoA were more stringent. When animals were starved for 24 h the specific activity of carnitine acyltransferase in microsomal membrane residues was increased 3.3-fold, whereas sensitivity to malonyl-CoA was decreased to 1/2.8. When enzymes partly purified from fed and starved animals were reconstituted into crude soybean phosphatidylcholine liposomes there was no difference in sensitivity to malonyl-CoA. When partly purified enzyme from fed rats was reconstituted into liposomes prepared from microsomal membrane lipids from fed animals it was 2.2-fold more sensitive to malonyl-CoA than when reconstituted with liposomes prepared from microsomal membrane lipids from starved animals. This suggests that the physiological changes in sensitivity to malonyl-CoA are mediated via changes in membrane lipid composition rather than via modification of the enzyme protein itself. The increased specific actvity of acyltransferase observed on starvation could not be attributed to changes in membrane lipid composition.

Conditions have been developed for the solubilization of hepatic microsomal carnitine acyltransferase activity in good yield, with excellent long-term stability and with retention of malonyl-CoA sensitivity. Solubilized microsomal carnitine acyltransferase activity can be separated into malonyl-CoA-sensitive and -insensitive activities either by gel filtration on Superdex 200 or by anion-exchange chromatography on Resource Q. On gel filtration the apparent molecular masses of the malonyl-CoA-sensitive and -insensitive activities are approx. 300 kDa and 60 kDa respectively. The malonyl-CoA-sensitive and -insensitive activities have different fatty-acyl-chain-length specificities and different stabilities in the detergent octyl glucoside. Together these findings indicate that the malonyl-CoA-sensitive and -insensitive activities are due to different enzymes. The malonyl-CoA sensitivity of the inhibitable enzyme is markedly increased on reconstitution into soybean L-alpha-lecithin liposomes, demonstrating that phospholipids play a crucial role in the inhibition by this metabolite. Evidence is also provided that the malonyl-CoA-sensitive microsomal carnitine acyltransferase is a different enzyme from the malonyl-CoA-sensitive carnitine palmitoyltransferase found in the mitochondrial outer membrane. The possible physiological role of the two microsomal acyltransferases is discussed.

The sulphonylureas glibenclamide and tolbutamide inhibited carnitine acyltransferase activities in rat liver microsomes. Glibenclamide was a more potent inhibitor than tolbutamide. The effect of tolbutamide on the malonyl-CoA-inhibitable transferase was influenced by the phospholipid/detergent environment whereas the effect of glibenclamide was not. Glibenclamide was a more potent inhibitor of the malonyl-CoA-inhibitable transferase than of the malonyl-CoA-insensitive enzyme. The extent of inhibition of the malonyl-CoA-inhibitable transferase by tolbutamide was similar to its effect on VLDL triacylglycerol secretion as reported by Wiggins and Gibbons [Biochem. J. 284 (1992) 457-462] possibly supporting the suggestion that microsomal carnitine acyltransferases are involved in VLDL triacylglycerol assembly/secretion.

beta-Oxidation of palmitate and tetradecanedioic acid was studied in cell-free extracts of the Gram-positive bacterium Corynebacterium sp. strain 7E1C, and the acyl-CoA ester intermediates formed were analysed by h.p.l.c. beta-Oxidation assays displayed a lag phase before a constant rate of NAD+ reduction was obtained. The length of the lag phase was inversely proportional to the number of units of activity added to assays. This is a characteristic feature of a system of consecutive reactions proceeding via free intermediates. During beta-oxidation of palmitate all the saturated acyl-CoAs from C16 to C8 were detected together with trace amounts of unsaturated and 3-hydroxy-intermediates. The time-course of intermediate formation again indicated a precursor-product relationship indicative of free intermediates being formed. When 3-hydroxyacyl-CoA dehydrogenase was inhibited by completely removing NAD+ from assays, the major acyl-CoAs, detected during palmitate beta-oxidation were palmitoyl-CoA, hexadeca-2-enoyl-CoA and 3-hydroxypalmitoyl-CoA. These compounds also displayed a precursor-product relationship. Under normal assay conditions the acyl-CoA dehydrogenase(s) are the probable rate-limiting enzyme(s) of the beta-oxidation spiral. These results indicate that in cell-free extracts of Corynebacterium sp. strain 7E1C, beta-oxidation proceeds via free acyl-CoA intermediates and is at variance with the concept of substrate channelling or of a 'leaky hose pipe' model as proposed for mitochondrial beta-oxidation in eukaryotic cells. The significant accumulation of chain-shortened acyl-CoA esters is similar to the situation observed for mammalian peroxisomal beta-oxidation.