Regulation of carnitine palmitoyltransferase synthesis in spontaneously diabetic BB Wistar rats.

The long-term regulation of hepatic mitochondrial carnitine palmitoyltransferase (CPT) was studied in control, insulin-treated, and untreated spontaneously diabetic BB Wistar rats. The activity of CPT was elevated approximately twofold in the untreated diabetic rats. This corresponded to an approximately equivalent elevation in the immunoreactive CPT activity. mRNACPT was assayed by reticulocyte lysate translation and by dot blot to a CPT oligonucleotide probe. The level of mRNACPT was approximately proportional to the observed CPT activity. A cDNA probe to CPT was developed, and transcriptional activity for CPT was assessed in isolated hepatic nuclei. Again, transcription of CPT mRNA was approximately proportional to the observed activity. We therefore conclude that at least part of the long-term regulation of hepatic CPT in spontaneously diabetic BB Wistar rats is the product of increased de novo synthesis of CPT protein brought about by regulation at the transcriptional level. Additional control of the amount of CPT may be via the regulation of RNA processing and turnover and enzyme insertion into the mitochondrial membrane.

Hepatic and cardiac carnitine palmitoyltransferase activity. Effects of adriamycin and galactosamine.

Carnitine palmitoyltransferase (CPT) activity is located on both the outer and inner sides of the mitochondrial inner membrane and is influenced by the surrounding lipids of the inner mitochondrial membrane. Both adriamycin and galactosamine interact with mitochondrial lipids as a part of their mechanism of toxicity, and thus these agents might be expected to affect CPT activity. Addition of adriamycin to both intact rat liver and heart mitochondria (CPT-A, outer CPT) and inverted submitochondrial vesicles (CPT-B, inner CPT) depressed CPT in the forward direction of reaction (palmitoyl-l-carnitine formation), but the CPT-B activity was more sensitive to the inhibitor. Adriamycin depressed the CPT-A reverse reaction (palmitoyl-CoA formation) to 40% of control, but it had no effect on the CPT-B reverse reaction. In vivo galactosamine administration depressed CPT-A and CPT-B 20-30% and did not affect subsequent action of in vitro adriamycin. Addition of cardiolipin (0.25 to 1.0 mg/assay) increased activity of the CPT-A forward reaction of both control and galactosamine-treated rats, but it did not affect CPT-B activity. The results suggest that CPT-A and CPT-B may be influenced differently by perturbants that affect lipids of the membrane.

Effects of clofibrate and acetylsalicylic acid on hepatic carnitine palmitoyltransferase synthesis.

Clofibrate and acetylsalicylic and have both been reported to increase carnitine palmitoyltransferase (CPT) activity when administered to rats. The purpose of the present study was to determine the mechanism of the increase in CPT activity. Rats (150-200 g) were fed one of the following: chow, chow with 0.5% clofibrate, or chow with 1% acetylsalicylic acid for 2 weeks. At the end of this time, hepatic CPT activity was increased 4-fold over control in the clofibrate group and 3.6-fold over control in the acetylsalicylic acid group. Immunoreactive protein increased 4.0- and 3.6-fold, respectively, over control. Transcription rates of hepatic nuclei were increased 2.8- and 1.9-fold over control in the clofibrate and acetylsalicylic acid groups, and hepatic mRNA levels increased 2.8- and 2.0-fold respectively. These data indicate that increases in CPT activity caused by clofibrate and acetylsalicylic acid administration are due, at least in part, to increased CPT protein, resulting from increased transcription rate and levels of mRNA specific for CPT.

Effect of methylglyoxal bis(guanylhydrazone) on hepatic, heart and skeletal muscle mitochondrial carnitine palmitoyltransferase and beta-oxidation of fatty acids.

Methylglyoxal bis(guanylhydrazone) (MGBG) is an antileukemic agent and a structural polyamine analogue which inhibits S-adenosyl methionine decarboxylase. However, MGBG also produces profound mitochondrial structural damage and inhibition of fatty acid oxidation. Carnitine palmitoyltransferase-A (CPT-A) is located on the outer surface of the inner mitochondrial membrane and is the putative rate-controlling enzyme for mitochondrial long-chain fatty acid oxidation. The present experiments were designed to determine if MGBG inhibits CPT-A. Liver, heart and skeletal muscle mitochondria were isolated from rats following 24 hr of starvation. Measuring the reaction in the direction of palmitoylcarnitine plus CoA formation from palmitoyl-CoA plus carnitine ("forward reaction"), MGBG was competitive with l-carnitine. The MGBG CPT-A Ki values were (mM): liver, 5.0 +/- 0.6 (N = 15); heart 3.2 +/- 1.2 (N = 3); and skeletal muscle, 2.8 +/- 1.0 (N = 3). Lysis of hepatic mitochondria with Triton X-100 yielded a Ki of 4.0 +/- 2.0, which was not significantly different from intact mitochondria or inverted vesicles (4.9 mM). Purified hepatic CPT had a Ki of 4.2 mM. MGBG did not inhibit purified CPT in the "reverse reaction" (palmitoyl-CoA plus carnitine formation from palmitoylcarnitine plus CoA). Spermine and spermidine, which are structurally similar to MGBG, did not inhibit either CPT activity or acid-soluble product formation from 1-[14C]palmitoyl-CoA. MGBG inhibited mitochondrial state 3 oxidation rates of palmitoyl-CoA and palmitoylcarnitine, as well as of glutamate. However, the fatty acid substrates were considerably more sensitive than glutamate to MGBG inhibition. MGBG also increased hepatic mitochondrial aggregation which was reversed by l-carnitine. Fluorescence polarization, using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe, indicated that MGBG increased membrane rigidity in a dose-dependent manner. This effect was not altered by l-carnitine. MGBG also inhibited purified pigeon breast carnitine acetyltransferase (CAT; Ki = 1.6 mM). While MGBG appeared to be competitive with l-carnitine for both CPT and CAT, MGBG also exhibits a number of effects which may be mediated through membrane interaction and which are not reversed by carnitine.

Polybrominated biphenyl (PBB) in the growing pig diet.

Twelve pigs which averaged 13.7 kg were randomly allotted from litters to a corn-soybean meal grower diet containing 0, 20, or 200 ppm of polybrominated biphenyls (PPB). During a 16-week growth trial, average daily gain (kg), average daily feed (kg) and feed/gain for pigs on diets containing 0, 20, or 200 ppm of PBB, respectively, were 0.82, 2.45, 2.99; 0.67, 1.88, 2.79; 0.45, 1.23, 2.70. Mean daily gain differences between all lots were highly significant (p < 0.01). Blood from each pig was withdrawn biweekly through the first 8 weeks of the trial and at 4 week intervals thereafter. Hemoglobin and hematocrit differed significantly only at the 6 weeks bleeding, being reduced in pigs receiving 200 ppm of PBB. Erythrocyte reduced glutathione concentration and glutathione peroxidase activity were not significantly influenced by level of dietary PBB. Serum lactic dehydrogenase activity was significantly higher in control pigs than in either PBB supplemented lots at 16 weeks. There was no significant influence of PBB upon serum glutamic oxaloacetic transaminase, serum alkaline phosphatase or serum creatine phosphokinase. Based on these enzyme assays, PBB produced no evidence of significant necrosis of liver, myocardium, or skeletal muscle. There was no consistent effect of dietary PBB upon total serum protein concentration or electrophoretic profile. Pigs on either level of PBB did not have overt clinical signs of toxicity during the 16-week test period with the exception of a dermatosis on the ventral surface of two of the pigs receiving 200 ppm of PBB. There was a marked increase in liver weight of pigs receiving either level of dietary PBB. Heart, kidney, and adrenals of pigs receiving either level of dietary PBB were heavier as a percent of body weight than that of control pigs. Fat retention of PBB and urinary and fecal PBB excretion were significantly affected by dietary PBB level. Grossly, the glandular portion of the stomach appeared somewhat hyperplastic in pigs on 200 ppm of PBB. Two pigs which had received 200 ppm of PBB were placed on the control diet and over the next 14 weeks normal growth rate occurred. One of these pigs was killed and organ weights were normal. The other pig, a gilt, came into estrus. She was bred and conceived. At the end of gestation, four pigs were born. Three survived and grew normally; the one death at birth examined at gross necropsy did not reveal changes in organ size or other tissue alterations.

The Ca2+ second messenger system and interleukin-1-alpha modulation of hepatic gene transcription and mitochondrial fat oxidation.

Cytokines have been implicated in the modulation of fat metabolism after sepsis. Carnitine palmitoyltransferase (CPT), the regulatory enzyme of hepatic mitochondrial long-chain fatty-acid oxidation, is involved in the control of hepatic fat oxidation in sepsis. Using either H4IIe rat hepatoma cells or rat hepatocytes in primary culture, we tested the hypothesis that interleukin-1-alpha (IL-1 alpha) would modulate CPT transcription (CPT mRNA), CPT translation (35S-methionine CPT protein incorporation), and hepatic mitochondrial oxidation of 1-Carbon 14-labeled (14C) palmitate to ketone bodies (acid soluble products). We showed that IL-1 alpha significantly increased CPT mRNA, 35S-methionine incorporation CPT protein, and hepatic mitochondrial oxidation of 1-14C-palmitate to acid soluble products. We further hypothesized that the Ca2+ second messenger system may play a role in the IL-1 alpha induction of hepatic CPT gene transcription. We showed that either calcium ionophore (A23187) or phorbol myristate acetate increased CPT gene transcription and that either calcium chelation, protein kinase C inhibition (acridine orange), or chronic exposure to phorbol myristate acetate significantly inhibited IL-1 alpha induction of CPT mRNA. We conclude that the IL-1 alpha increases in hepatic mitochondrial fatty-acid oxidation may be, in part, secondary to increased CPT gene transcription and translation and that the Ca2+ second messenger system may play an important role in IL-1 alpha induction of CPT gene transcription.

Sepsis-induced release of interleukin-6 may activate the immediate-early gene program through a hypothalamic-hypophyseal mechanism.

BACKGROUND: The immediate-early gene c-fos has been implicated in transcriptional regulation after sepsis. We test the hypothesis that sepsis-induced central nervous system release of interleukin (IL)-6 regulates hepatic c-fos gene expression. METHODS: Using a stereotaxically placed intracerebral-ventricular (ICV) catheter in rats with and without hypophysectomy, we measured hepatic c-fos protein accumulation after treatment with either IL-6 or vehicle control. Using a rat cecal ligation and puncture (CLP) model, we studied the following groups: (1) sham-CLP, (2) CLP, (3) hypophysectomized sham-CLP, and (4) hypophysectomized CLP and measured hepatic c-fos mRNA. RESULTS: ICV IL-6 treatment increased hepatic c-fos protein in the IL-6-treated group compared with the vehicle-treated group, and hypophysectomy inhibited the ICV IL-6-mediated increase in c-fos protein. After peritoneal sepsis, CLP increased hepatic c-fos messenger RNA compared to either the sham-CLP or the hypophysectomized sham-CLP group, and hypophysectomy before CLP inhibited hepatic c-fos mRNA compared with the CLP group. CONCLUSIONS: ICV IL-6 results in an increase in hepatic fos protein that is mediated through a hypothalamic-hypophyseal mechanism. Peritoneal sepsis results in an increase in hepatic c-fos gene expression that may be, in part, mediated by central nervous system release of IL-6 through a hypothalamic-hypophyseal mechanism.

The possible inhibitory role of the leucine-zipper DNA binding protein c-fos in the regulation of hepatic gene expression after sepsis.

BACKGROUND: The leucine-zipper c-fos has been implicated in the regulation of gene expression. We investigated the possible role of c-fos in the regulation of hepatic gene expression after sepsis. Based on previous data demonstrating that sepsis inhibits hepatic gene expression of carnitine palmitoyltransferase (CPT), we hypothesized that c-fos may play a role in the inhibition of CPT gene expression after sepsis. METHODS: We studied c-fos gene expression after peritoneal sepsis induced by cecal ligation and puncture (CLP) or sham-CLP. To investigate the possible inhibitory role of c-fos on CPT gene transcription, we investigated the effect of c-fos on c-jun-driven CPT promoter-chloramphenicol acyltransferase reporter gene expression in a HepG2 hepatoma cell cotransfection model. To investigate the possible role of cyclic adenosine monophosphate (cAMP) in the regulation of c-fos in vivo, we treated either the sham-CLP group or the CLP group with either vehicle or cAMP. RESULTS: Peritoneal sepsis in the rat model resulted in a four-fold increase in hepatic c-fos mRNA and c-fos protein. In the cotransfection model, c-fos significantly inhibited c-jun-induced chloramphenicol acyltransferase activity. Treatment with cAMP resulted in a 50% decrease in c-fos protein in either the sham-CLP or CLP group. CONCLUSIONS: We conclude that (1) sepsis increases hepatic c-fos transcription and translation, (2) c-fos inhibits c-jun-induced CPT gene expression, and (3) cAMP probably does not directly mediate the increase in c-fos after sepsis.

Pharmacologic action of L-carnitine on hypertriglyceridemia in obese Zucker rats.

Administration of pharmacologic amounts of L-carnitine was studied in the hypertriglyceridemic Zucker rat. When administered subcutaneously, doses from 250 to 2,000 mg/kg/d significantly decreased plasma triglycerides in obese rats over eight to 12 weeks, with no effect on plasma triglycerides in lean rats. Oral doses at the same high levels were not effective in decreasing plasma triglycerides. Triglyceride secretion rate was reduced from 367 micrograms/min to 168 micrograms/min in treated obese rats. Concurrently, liver lipid was increased twofold in obese treated rats, and the livers of these rats showed significant fatty infiltration. The mechanism of action of carnitine in decreasing plasma triglycerides appeared to be via decreased secretion of triglycerides by the liver of obese rats. There was no effect of L-carnitine in lean or obese rats on the following variables: carnitine palmitoyltransferase-A kinetics or malonyl CoA inhibition, mitochondrial or peroxisomal oxidative capacity, lipoprotein lipase in heart, muscle, and adipose, or fecal lipids. The effect of pharmacologic L-carnitine thus appears to be an inhibition of triglyceride synthesis and/or secretion by the liver.

Erythrocyte characteristics in vitamin E-responsive anemia of the owl monkey (Aotus trivirgatus).

To characterize the vitamin E-responsive anemia occurring in owl monkeys (Aotus trivirgatus), osmotic fragility, and H2O2-induced and time-dependent hemolysis, as well as RBC lipid peroxidation, were compared in anemic and nonanemic owl monkeys. Whereas vitamin E serves as a lipid-soluble antioxidant, the glutathione peroxidase system functions in the water-soluble phase of the cell. Thus, activity of glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase, as well as reduced glutathione concentrations in owl monkeys' RBC, were compared with those of rhesus macaques and cebus and squirrel monkeys fed the same diet and maintained under the same management scheme. Osmotic fragility did not differ between anemic and nonanemic owl monkeys. The H2O2-induced and time-dependent hemolysis was approximately 10-fold greater among anemia owl monkeys than among their nonanemic counterparts, and lipid peroxidation values tended to be higher in the anemic monkeys. Owl monkeys, as a species and independent of anemia, exhibited higher RBC peroxidation than did 2 other New World species, cebus and squirrel monkeys. The glutathione peroxidase system was not depressed in owl monkey RBC. The only observed difference in this system was in the glucose-6-phosphate dehydrogenase activity, which was 3- to 6-fold higher in the owl monkey than in the other species, indicating an increased activity of the peroxidase system. Thus, a defect in the glutathione peroxidase system could not be identified.

Interrelationships of erythrocyte glutathione peroxidase system enzymes in the fasted Beagle.

Eight adult female Beagles were fasted for 21 days to investigate responses of the erythrocyte glutathione peroxidase system enzymes. Blood samples were collected once a week. Erythrocyte reduced glutathione was maintained within 10% of base-line value. Hexose monophosphate pathway dehydrogenases showed a significant inverse relationship to glutathione reductase (GR) activity. Further, active GR, as a percentage of total GR, increased as hexose monophosphate pathway dehydrogenase activity fell. A similar relationship between these enzymes has been reported in human glucose-6-phosphate dehydrogenase deficiency. Also, glutathione peroxidase showed an inverse linear response to erythrocytic reduced glutathione. Though the responses of this system are complex, they appear interrelated.

Hepatic carnitine palmitoyltransferase turnover and translation rates in fed, starved, streptozotocin-diabetic and diethylhexyl phthalate-treated rats.

Hepatic carnitine palmitoyltransferase (CPT) turnover was studied in control and in non-ketotic hyperglycaemic streptozotocin-diabetic rats. The degradation constant (kd) and half-life (t1/2) did not appear to be altered by mild diabetes. The hepatic CPT (micrograms/g of liver) was not increased by the mild, non-ketotic, diabetes. However, the total hepatic CPT (micrograms/liver) was 37% greater in the diabetic animals, owing to the increased liver weight. This resulted from a 40% increase in the synthesis constant (ks). Hepatic CPT activity (total detergent-solubilized) and translation rates were measured in fed, starved (48 h), non-ketotic diabetic, ketotic diabetic and diethylhexyl phthalate (DEHP)-treated rats. CPT activity (m units/mg of mitochondrial protein) was not significantly increased with non-ketotic diabetes (44% increase, but non-significant), but was increased approx. 2-fold with starvation and ketotic diabetes, and 3.5-fold with DEHP treatment. CPT expressed as units/liver was increased non-significantly (23%) in non-ketotic and starved rats, similar to the turnover study, but was significantly increased with ketotic diabetes and with DEHP treatment. mRNA-translation activity for CPT was elevated in all states to a somewhat greater extent than was activity. It was concluded that protein synthesis as a product of increased CPT-mRNA translation activity is a major means of long-term regulation.

Action in vivo and in vitro of 2-tetradecylglycidic acid, 2-tetradecylglycidyl-CoA and 2-tetradecylglycidylcarnitine on hepatic carnitine palmitoyltransferase.

The effects of 2-tetradecylglycidic acid (TDGA), TDGA-CoA and TDGA-carnitine were examined in purified hepatic CPT (carnitine palmitoyltransferase) and in hepatic mitochondria and inverted submitochondrial vesicles derived from Sprague-Dawley rats. Since TDGA has been reported as a specific inhibitor of carnitine palmitoyltransferase-A (CPT-A), the focus was on kinetics and inhibition of CPT-A, and the relationship of this key enzyme to beta-oxidation. After administration of TDGA in vivo to overnight-starved rats, the Vmax. of CPT in intact mitochondria and in inverted vesicles (CPT-B) was depressed by 66%. The S0.5 for palmitoyl-CoA and Km for carnitine were unchanged. The I50 (concn. giving 50% inhibition) for malonyl-CoA was significantly increased from 20 to 141 microM in intact mitochondria, but unchanged (199 versus 268 microM) in inverted vesicles. The addition in vitro of TDGA-CoA (0-1.0 microM) gave I50 values of 0.29 and 0.27 microM (S.E.M. = 0.19) in intact mitochondria from fed and 48 h-starved rats, and 0.81 and 1.57 microM (S.E.M. = 0.29) for inverted vesicles derived from fed and starved rats. Addition in vitro of TDGA-carnitine to mitochondria from starved rats yielded an I50 value of 27.7 mM (S.E.M. = 12.2) for L-[methyl-14C]carnitine release from palmitoyl-L-[methyl-14C]carnitine and 0.64 mM (S.E.M. = 0.07) for palmitoyl-L-[methyl-14C]carnitine formation from L-[methyl-14C]carnitine in intact mitochondria. Inverted vesicles were not measurably sensitive to TDGA-carnitine up to 500 microM for the assay of L-[methyl-14C]carnitine release, but were as sensitive as intact mitochondria when inhibition was determined in the direction of palmitoyl-L-[methyl-14C]carnitine formation (I50 = 0.54 +/- 0.07 microM). When TDGA-CoA was added to intact mitochondria, then incubated for 5 min at room temperature and subsequently washed out, Vmax. of CPT decreased from 5.8 to 3.5 (S.E.M. = 0.6) in intact mitochondria, and from 17.2 to 6.3 (S.E.M. = 4.8) in inverted vesicles. The Km for L-carnitine and the S0.5 for palmitoyl-CoA increased 2-fold with TDGA-CoA pretreatment in both intact mitochondria and inverted vesicles. Detergent solubilization (0.05% Triton X-100) resulted in a complete loss of TDGA-CoA sensitivity (up to 1.0 microM measured). Sonicated mitochondria exhibited an I50 of 0.72 +/- 0.03 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of carnitine palmitoyltransferase in vivo by glucagon and insulin.

Carnitine palmitoyltransferase (CPT total) activity and synthesis increase in states where the insulin/glucagon ratio is low, such as starvation and diabetes [Brady & Brady (1987) Biochem. J. 246, 641-646]. However, the effect of glucagon and insulin on CPT synthesis is unknown. The present experiments were designed to determine the effect of glucagon, cAMP [8-(chlorophenylthio) cyclic AMP], and insulin + cAMP on CPT transcription and mRNA amounts over time after injection. The CPT protein that was purified, used to generate antibody, and cloned in these studies was the 68 kDa mitochondrial protein described previously [Brady & Brady (1987) Biochem. J. 246, 641-646; Brady, Feng & Brady (1988) J. Nutr. 118, 1128-1136; Brady & Brady (1989) Diabetes 38, in the press]. Saline-injected control rats exhibited a 2-fold increase in hepatic CPT transcription rate and CPT mRNA over the 5 h experiment from 09:00 to 14:00 h. The effect was most probably due to the fasting state of the rats during the day. Glucagon injection caused an 8-fold increase in transcription rate by 90 min and a 4-fold increase in CPT mRNA by 90-120 min. The cAMP effect had reached a peak by the first time point taken (15 min). Transcription rate was increased 4-fold and CPT mRNA was increased 3-fold at this time. The combination of cAMP + insulin injection did not produce any significant increase in transcription rate or CPT mRNA over the saline-injected controls. CPT mRNA and transcription rate showed a clear dose-response to glucagon injection from 0 to 150 micrograms/100 g body wt. Total CPT activity and immunoreactive CPT were not increased during these experiments. The data indicate that glucagon and insulin interact in control of transcription rate and amount of CPT mRNA, but that increases in CPT immunoreactive protein and activity are temporally delayed. This lag probably relates to the half-life of the CPT protein in vivo, which has been estimated as 2-7 days.

Peroxisomal palmitoyl-CoA oxidation in the Zucker rat.

The effects of 3 or 6 days of starvation on hepatic peroxisomal palmitoyl-CoA oxidation were examined in adult lean and obese Zucker rats. When expressed either per mg of DNA or per total liver, obese rats had almost 2-fold higher oxidation rates than the lean rats. Within 6 days of starvation rates fell by 50% among both phenotypes. When data were expressed per 100 g body wt., lean and obese rats had similar rates, falling from a mean of 0.57 to 0.28 mumol/min per 100 g body wt. within 6 days of starvation. Peroxisomal oxidative changes paralleled mitochondrial beta-oxidative changes.

Hepatic peroxisomal and mitochondrial fatty acid oxidation in the riboflavin-deficient rat.

The effects of riboflavin deficiency on hepatic peroxisomal and mitochondrial palmitoyl-CoA oxidation were examined in weanling Wistar-strain male rats. The specific activities of peroxisomal catalase and palmitoyl-CoA-dependent NAD+ reduction were not affected by up to 10 weeks of riboflavin deficiency. In contrast, the specific activity of mitochondrial carnitine-dependent palmitoyl-CoA oxidation was depressed by 75% at 10 weeks of deficiency. The amount of peroxisomal protein per g of liver was not affected by riboflavin deficiency, whereas, expressed per liver, both riboflavin-deficient and pair-fed controls showed decreased peroxisomal protein compared with controls fed ad libitum. Hepatic mitochondria, but not peroxisomes, were sensitive to riboflavin deficiency.

Characterization of hepatic carnitine palmitoyltransferase. Use of bromoacyl derivatives and antibodies.

Carnitine palmitoyltransferase (CPT) is a mitochondrial-inner-membrane enzyme, with activities located on both the outer and inner sides of the membrane. The inhibition of CPT by bromopalmitate derivatives was studied in intact hepatic mitochondria (representing CPT-A activity, the outer enzyme), in inverted submitochondrial vesicles (representing CPT-B, the inner enzyme), and in purified hepatic CPT. Bromopalmitoyl-CoA had an I50 (concentration giving 50% inhibition of CPT activity) of 0.63 +/- 0.08 microM in intact mitochondria and 2.44 +/- 0.86 microM in inverted vesicles. Preincubation of mitochondria with bromopalmitoyl-CoA decreased V max. for both CPT-A and CPT-B. Sonication decreased sensitivity to bromopalmitoyl-CoA, and solubilization with Triton abolished sensitivity at the concentrations used (0-10 microM). Purified CPT had a bromopalmitoyl-CoA I50 of 353 microM in aqueous buffer, 67 microM in 20% dimethyl sulphoxide, 45 microM in phosphatidylcholine liposomes and 26 microM in cardiolipin liposomes. Increasing [carnitine] at constant bromopalmitoyl-CoA concentrations or increasing [bromopalmitoyl-CoA] in the preincubation resulted in increased inhibition of purified CPT. 2-Tetradecylglycidyl-CoA and malonyl-CoA did not offer measurable protection against bromopalmitoyl-CoA inhibition of the purified CPT, suggesting a different site of interaction of bromopalmitoyl-CoA with CPT. The data suggest that the sensitivity of CPT to bromopalmitoyl-CoA may be modulated by membrane environment and assay conditions.