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V A Zammit

Publications and source records attributed to V A Zammit.

At least 73 records · Page 4Linked to original sources
PubMed1992

Triacylglycerol accumulation and secretion in hepatocyte cultures. Effects of insulin, albumin and Triton WR 1339.

We have investigated the possibility that the apparent inhibition of very-low-density lipoprotein (VLDL)-triacylglycerol secretion by the addition of insulin to rat hepatocyte cultures may result from insulin-mediated enhancement of hepatic lipase secretion and, consequently, of extracellular triacylglycerol hydrolysis. We have, therefore, studied the effects of the inhibitor of lipase activity, Triton WR 1339, on the secretion of triacylglycerol by cultured rat hepatocytes. Incubation of hepatocyte cultures with increasing concentrations of Triton WR 1339 increased the accumulation of acylglycerol in the medium, suggesting that, in normal incubations, a substantial rate of degradation of secreted triacylglycerol does occur and that it results in an under-estimation of the rate of triacylglycerol secretion. However, Triton did not counteract the inhibitory effects of insulin, suggesting that the observed increased activity of hepatic lipase induced by the hormone cannot account for the inhibition of acylglycerol accumulation in the medium that occurred in the presence of insulin. BSA increased the accumulation of triacylglycerol in culture media by about 2-fold and also decreased the activity of hepatic lipase by 80%. A causative relationship between these two effects was supported by the further observation that Triton abolished the effects of BSA on triacylglycerol accumulation in the medium. The implications of these data for the validity of the use of Triton for the study of hepatic rates of triacylglycerol production in vivo and of secretion by hepatocytes in vitro are discussed.

Albumins↗
PubMed1992

Direct demonstration that increased phosphorylation of 3-hydroxy-3-methylglutaryl-CoA reductase does not increase its rate of degradation in isolated rat hepatocytes.

Increased phosphorylation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase has been suggested to target the protein towards an increased rate of degradation. Our previous observations [Zammit & Caldwell (1990) Biochem. J. 269, 373-379] suggested that, although Ca(2+)-mobilizing hormones and other effectors can alter both the phosphorylation state of the enzyme and its total activity in isolated rat hepatocytes, there appears to be no causal correlation between the two parameters. In the present paper we set out to make direct measurements of the specific rate of degradation of 35S-labelled HMG-CoA reductase in hepatocytes treated with agents that produced very marked and prolonged increases in the degree of phosphorylation of the protein, through different mechanisms. Okadaic acid (which inhibits phosphatases 1 and 2A), fructose (which increases cellular AMP through its metabolism to fructose 1-phosphate) and the Ca2+ ionophore A23187 (which also raises cellular AMP through an unknown mechanism) were all unable to alter the rate of HMG-CoA reductase degradation. We conclude that the basal rate of degradation of HMG-CoA reductase is unaffected by its phosphorylation state and that a transiently increased degree of phosphorylation cannot be the mechanism through which mevalonate increases the rate of degradation of the enzyme in rat hepatocytes and other cell types.

Adenosine Monophosphate↗
PubMed1992

Selective labelling of hepatic fatty acids in vivo. Studies on the synthesis and secretion of glycerolipids in the rat.

1. We describe a method for the selective labelling of hepatic fatty acids in the rat in vivo. It relies on (i) the rapid and preferential uptake of cholesteryl ester from chylomicron and/or very-low-density-lipoprotein remnants by the liver [Holder, Zammit & Robinson (1990) Biochem. J. 272, 735-741] (without prior exchange of the ester to other lipoproteins in the plasma), and (ii) the very short half-life of the cholesteryl ester in the liver. The 14C-labelled fatty acid moiety generated by cholesteryl ester hydrolysis was shown to be utilized by the liver for glycerolipid synthesis in a very similar pattern to that demonstrated for exogenous fatty acids by isolated cultured hepatocytes in previous studies. 2. Starvation (24 h) was shown to decrease the proportion of fatty acid utilized for glycerolipid synthesis, but to result in a proportionately smaller effect on incorporation into phospholipid. This was accompanied by a decrease in the fraction of synthesized triacylglycerol that was secreted by the liver. 3. Streptozotocin-diabetes did not affect the phospholipid/triacylglycerol ratio, but resulted in a small, but significant, decline in the fraction of triacylglycerol secreted by the liver. 4. In both starved and diabetic animals fatty acid esterification to the glycerol moiety constituted a smaller proportion of the total disposal of label. 5. These findings appear to validate the present method for the selective labelling of liver fatty acids in vivo in a non-invasive manner. Other possible uses for the method are suggested.

Animals↗
PubMed1992

Development and characterization of a polyclonal antibody against rat liver mitochondrial overt carnitine palmitoyltransferase (CPT I). Distinction of CPT I from CPT II and of isoforms of CPT I in different tissues.

The [3H]tetradecylglycidyl-CoA (TDG-CoA)-binding protein (Mr approx. 88,000) of purified outer membranes from rat liver mitochondria was identified by SDS/PAGE. The region in which it migrated was shown to contain another protein which stained strongly with periodic acid-Schiff reagent and could be removed from membrane extracts by incubation with Sepharose-concanavalin A. Amounts of TDG-CoA-binding protein were prepared from lectin-treated extracts using preparative SDS/PAGE and used to raise a polyclonal antibody in a sheep. The IgG fraction purified from this anti-serum reacted strongly with a protein of Mr approximately 88,000 on Western blots, and much more weakly with two other proteins of Mr approximately 76,000 and Mr approximately 53,000 in extracts of rat liver mitochondrial outer membranes. The crude IgG fraction and immunopurified IgG both removed carnitine palmitoyltransferase (CPT) I activity from very pure outer membrane extracts, suggesting that the TDG-CoA-binding protein against which the antiserum was raised also expresses CPT I activity. This was confirmed by the demonstration of a strong positive correlation between CPT I activity and the amount of immunoreactive protein of Mr approximately 88,000 in mitochondria prepared from rats in different physiological states. By contrast, the antibody did not react with CPT II either in mitochondria or in purified form. Similarly, an anti-(CPT II) antibody did not cross-react with CPT I on Western blots, proving conclusively that CPT I and CPT II are immunologically distinct proteins, as well as being of different functional molecular sizes [Zammit, Corstophine & Kelliher (1988) Biochem. J. 250, 415-420]. Immunoblots of mitochondrial proteins obtained from different tissues indicated that, of the rat tissues tested, only kidney cortex mitochondria contain the same isoform of CPT I as that in liver. Heart, skeletal muscle and brown adipose tissue mitochondria contain a slightly smaller isoform which was only weakly reactive with anti-(rat liver CPT I) antibody, indicating that these tissues contain a molecularly quite distinct isoenzyme. This would explain the previous observations that CPT I in these tissues has markedly different kinetic characteristics from the isoenzyme present in liver mitochondria.

Acyl Coenzyme A↗
PubMed1991

Rapid decrease in the expression of 3-hydroxy-3-methylglutaryl-CoA reductase protein owing to inhibition of its rate of synthesis after Ca2+ mobilization in rat hepatocytes. Inability of taurolithocholate to mimic the effect.

The mechanisms through which Ca2+ mobilization in rat hepatocytes results in the loss of total activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase [Zammit & Caldwell (1990) Biochem. J. 269, 373-379] were investigated. The loss of total activity was shown to be paralleled by an equal loss of immunoreactive HMG-CoA reductase protein after exposure of hepatocytes to optimal concentrations of vasopressin plus glucagon for 40 min. This loss of enzyme protein was due to an inhibition of enzyme synthesis; the rate of degradation was unaffected. Other Ca(2+)-mobilizing conditions (phenylephrine, glucagon, vasopressin added singly and A23187) also resulted in graded inhibition of synthesis of HMG-CoA reductase. These effects were accentuated by omission of Ca2+ from the cell incubation medium, suggesting that it is the depletion of an intracellular InsP3-sensitive pool of Ca2+ to which synthesis of HMG-CoA reductase is sensitive. In agreement with this we found that t-butylhydroxybenzoquinone, which inhibits the activity of the Ca(2+)-ATPase of the endoplasmic-reticular membrane, mimicked the action of Ca(2+)-mobilizing hormones. However, taurolithocholate, which transiently mobilizes Ca2+ from the same pool, was ineffective. All these effects on HMG-CoA reductase were accompanied by parallel inhibition of 35S incorporation from [35S]methionine into total protein, suggesting that inhibition of reductase synthesis formed part of a generalized response of the hepatocyte to Ca2+ mobilization. Inhibition of the rate of synthesis of HMG-CoA reductase was, however, more responsive to Ca2+ mobilization in the absence of added Ca2+ from the extracellular medium. The concentrations of vasopressin required to elicit the inhibition of synthesis of HMG-CoA reductase were of the same order as those that elicited activation of glycogen phosphorylase in hepatocytes.

Animals↗
PubMed1991

Estradiol increases the secretion of hepatic lipase by rat hepatocyte cultures.

Hepatic lipase (EC 3.1.1.3) is synthesized and secreted by parenchymal hepatocytes and binds to endothelial cells of liver sinusoids. The present study shows that the activity of hepatic lipase secreted by hepatocyte cultures from male rats in increased approx. 6-fold after 10 h culture with 10 microM 17 beta-estradiol. The stimulatory effect of 17 beta-estradiol is biphasic and declines at higher concentrations. In hepatocytes from male rats: progesterone, unlike 17 beta-estradiol, had only a small stimulatory effect when present as the sole hormone and a small inhibitory effect in the presence of 17 beta-estradiol, while testosterone and dexamethasone had no effect. Hepatocyte cultures from female rats had a higher basal rate of hepatic lipase secretion than cells from male rats and showed a smaller stimulation by 17 beta-estradiol. These results suggest that 17 beta-estradiol might regulate the secretion of hepatic lipase by hepatocytes, and presumably the activity of the enzyme at either the endothelial surface of the liver sinusoids or at extrahepatic sites.

Animals↗
PubMed1991

Regulation of fatty acid metabolism and gluconeogenesis by growth hormone and insulin in sheep hepatocyte cultures. Effects of lactation and pregnancy.

Primary monolayer hepatocyte cultures derived from non-mated, pregnant and lactating sheep were used to investigate the interactions between the effects of growth hormone and insulin on (i) the partitioning of fatty acid metabolism between oxidation and esterification, and (ii) the rate of gluconeogenesis. In hepatocytes from lactating sheep the rates of gluconeogenesis, ketogenesis and very-low-density lipoprotein secretion were approx. 2-fold higher than in cells from non-mated or pregnant animals. There was no apparent difference in the rates of fatty acid uptake between the three groups of sheep cells. Growth hormone stimulated gluconeogenesis only in hepatocytes from non-mated sheep. It has no effect on the flux of fatty acid towards ketone body formation. Growth hormone inhibited intracellular accumulation of acylglycerol from exogenous fatty acid. Insulin alone had no such effect, but it blunted the effect of growth hormone when the two hormones were present together. The data suggest that major differences may exist between ruminants and non-ruminants in the response of liver metabolism both to lactation per se and to the effects of growth hormone and insulin.

Animals↗
PubMed1991

Sexual dimorphism in the preferential secretion of unsaturated lysophosphatidylcholine by rat hepatocytes but no secretion by sheep hepatocytes.

(1) Rat and ovine hepatocytes were incubated in monolayer culture with various fatty acids to determine their effects on the composition of the lysophosphatidylcholine that was secreted. (2) No lysophosphatidylcholine was detected in the medium from the ovine hepatocytes even though these cells were hormonally responsive and they secreted phosphatidylcholine and triacylglycerol in very-low-density lipoprotein. (3) Lysophosphatidylcholine was readily detected in the incubation medium of rat hepatocytes. The predominant fatty acids in this lipid were unsaturated. Stearate and arachidonate contributed 15 and 34%, and 24 and 26% of the total fatty acids when hepatocytes from male and female rats were used, respectively. The relative proportions of stearate and arachidonate in the phosphatidylcholine secreted from the hepatocytes were 20 and 14%, and 28 and 21% for the males and females, respectively. The equivalent values for stearate and arachidonate for phosphatidylcholine in the hepatocytes were 18 and 17% and 33 and 22% for male and female rats. These results provide further indications of sex differences in hepatic phospholipid metabolism and extend this to the secretion of phosphatidylcholine and lysophosphatidylcholine. (4) The addition of 1 mM stearate to the incubation medium did not significantly decrease the proportion of arachidonate in the lysophosphatidylcholine obtained from the hepatocytes of the male rats. However, the relative proportion of arachidonate was decreased in incubations that contained 1 mM oleate or linoleate. (5) The results provide evidence that the preferential secretion of unsaturated lysophosphatidylcholine by the liver may provide a system for transporting unsaturated fatty acids and choline to other organs in non-ruminant animals. However, this mechanism may not operate for ruminants.

Animals↗
PubMed1991

The roles of different protein kinases and of calmodulin in the effects of Ca2+ mobilization on 3-hydroxy-3-methylglutaryl-CoA reductase activity in isolated rat hepatocytes.

The roles of protein kinase C, Ca2+/calmodulin-dependent protein kinase and AMP-activated protein kinase in the phosphorylation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase induced by Ca2(+)-mobilizing conditions in isolated hepatocytes were investigated. Only partial evidence for the involvement of AMP-activated kinase was found. Antagonism of calmodulin action prolonged the decrease in expressed/total activity ratio induced by vasopressin plus glucagon. Protease inhibitors active against Ca2(+)-dependent cytosolic proteases or lysosomal proteolysis did not attenuate the loss of total HMG-CoA reductase induced by glucagon plus vasopressin, but calmodulin antagonists largely prevented this effect.

Adenosine Monophosphate↗
PubMed1990

The preferential uptake of very-low-density lipoprotein cholesteryl ester by rat liver in vivo.

The removal from the blood and the uptake by the liver of injected very-low-density lipoprotein (VLDL) preparations that had been radiolabelled in their apoprotein and cholesteryl ester moieties was studied in lactating rats. Radiolabelled cholesteryl ester was removed from the blood and taken up by the liver more rapidly than sucrose-radiolabelled apoprotein. Near-maximum cholesteryl ester uptake by the liver occurred within 5 min of the injection of the VLDL. At this time, apoprotein B uptake by the liver was only about 25% of the maximum. Maximum uptake of the injected VLDL apoprotein B label was not achieved until at least 15 min after injection, by which time the total uptakes of cholesteryl ester and apoprotein B label were very similar. The results suggest that preferential uptake of the lipoprotein cholesteryl ester by the liver occurred before endocytosis of the entire lipoprotein complex. The fate of the injected VLDL cholesteryl ester after its uptake by the liver was also monitored. Radiolabel associated with the hepatic cholesteryl ester fraction fell steadily from its early maximum level, the rate of fall being faster and more extensive when the fatty acid, rather than the cholesterol, moiety of the ester was labelled. By 30 min after the injection of VLDL containing [3H]cholesteryl ester, over one-third of the injected label was already present as [3H]cholesterol in the liver. When VLDL containing cholesteryl [14C]oleate was injected, a substantial proportion (about 25%) of the injected radiolabelled fatty acid appeared in the hepatic triacylglycerol fraction within 60 min: very little was present in the plasma triacylglycerol fraction at this time.

Animals↗
PubMed1990

Sensitivity of inhibition of rat liver mitochondrial outer-membrane carnitine palmitoyltransferase by malonyl-CoA to chemical- and temperature-induced changes in membrane fluidity.

We have tested the possibility that alterations in the fluidity of the outer membrane of rat liver mitochondria could result in changes in the sensitivity of overt carnitine palmitoyltransferase (CPT I) to malonyl-CoA [Zammit (1986) Biochem. Soc. Trans. 14. 676-679]. The sensitivity of CPT I to malonyl-CoA inhibition was measured by using highly purified mitochondrial outer membranes prepared from fed or 48 h-starved rats in the presence and absence of agents that increase membrane fluidity by perturbing membrane lipid order [benzyl alcohol, isoamyl alcohol (3-methylbutan-l-ol) and 2-(2-methoxyethoxy)ethyl-8-(cis-2-n-octylpropyl)octanoate (A2C)]. All these agents resulted in marked decreases in the ability of malonyl-CoA to inhibit CPT I. This effect was accompanied by a modest increase in the absolute activity of CPT I in the absence of malonyl-CoA when the short-chain alcohols were used, but not when A2C was used, suggesting that the effect of increased membrane fluidity to decrease the malonyl-CoA sensitivity of CPT I may occur independently from other actions that may affect more directly the active site of the enzyme. In confirmation of the potential importance of fluidity changes, we showed that a marked increase in sensitivity of CPT I to malonyl-CoA could be produced when assays were performed at lower temperatures than those normally employed. These observations are discussed in the context of the slowness of the changes in CPT I sensitivity to malonyl-CoA inhibition that are induced by physiological perturbations.

Alcohols↗
PubMed1990

Changes in the properties of cytosolic acetyl-CoA carboxylase studied in cold-clamped liver samples from fed, starved and starved-refed rats.

We have used the cold-clamping technique to study the changes in acetyl-CoA carboxylase activity that occur in the cytosolic and mitochondrial fractions of the liver of fed, starved and starved-refed rats. No evidence was found for a role of the mitochondrial enzyme as a pool from which cytosolic carboxylase could be replenished upon refeeding of starved rats. Starvation for 24 h or 48 h induced changes in the expressed (assayed at 20 mM-citrate), total (citrate- and phosphatase-treated) and citrate-independent activities of cytosolic carboxylase, as well as in its Ka for citrate. The expressed/total activity ratio was low even in the fed state and was depressed further by starvation. The effects of refeeding occurred in two phases: an acute phase (approx. 1 h) in which the starvation-induced changes in Ka and expressed/total activity ratio were rapidly reversed, and a prolonged slow phase in which the two parameters attained values that were lower and higher, respectively, than those in the normal fed state. Refeeding also resulted in a gradual increase in citrate-independent activity of acetyl-CoA carboxylase. An additional marked increase in this activity occurred only in 48 h-starved-refed rats between 24 h and 48 h of refeeding. These findings are discussed in terms of the observed time courses of changes in lipogenic rates that occur in vivo in starved-refed rats and of the possible molecular mechanisms involved.

Acetyl-CoA Carboxylase↗
PubMed1990

Conditions that result in the mobilization and influx of Ca2+ into rat hepatocytes induce the rapid loss of 3-hydroxy-3-methylglutaryl-CoA reductase activity that is not reversed by phosphatase treatment.

We investigated the effects of conditions that induce Ca2+ mobilization from intracellular stores and Ca2+ influx into hepatocytes on the expressed and total (fully dephosphorylated) activities of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Vasopressin and phenylephrine when added alone had small or negligible effects on the phosphorylation state of the enzyme, as judged from the expressed/total activity ratio. However, when added in combination with glucagon, they elicited appreciable increases in the phosphorylation of the enzyme. Glucagon on its own had no effect either on phosphorylation state or on total HMG-CoA reductase activity during 40 min of incubation. Under conditions of sustained Ca2+ influx (i.e. vasopressin or phenylephrine plus glucagon), there was a marked loss of total HMG-CoA reductase activity. This effect was more pronounced when vasopressin was used; 50% of the enzyme activity was lost within 40 min. The involvement of Ca2+ in these effects was verified directly by the use of ionophore A23187. Its addition to hepatocytes resulted both in a very pronounced increase in the phosphorylation state of the enzyme and in the loss of 50% of the total activity within 30 min. There was no correlation between the ability of any set of conditions to increase the phosphorylation of the enzyme and the subsequent loss of total HMG-CoA reductase activity. The latter parameter appeared to be directly related, however, to the maintenance of prolonged Ca2+ influx, as indicated by the continued activation of glycogen phosphorylase, measured in the same cells. The lack of a causal relationship between increased phosphorylation and loss of total activity was demonstrated directly by studies in which okadaic acid was used to induce phosphorylation of HMG-CoA reductase in hepatocytes by inhibition of phosphatase 1 and 2A activities. This was not accompanied by any loss of total enzyme activity. Neither did okadaic acid enhance the loss of reductase induced by A23187 when the two agents were added together. It is concluded that altered Ca2+ fluxes in hepatocytes in vivo, under conditions of acute or chronic stress (such as may be associated with trauma or diabetes respectively), may be involved in the regulation of the expression of HMG-CoA reductase activity through alteration of enzyme concentration in the liver.

Animals↗
PubMed1990

An improved method for the analysis of data from radiation-inactivation studies.

This paper addresses statistical issues in the estimation of protein molecular weight using radiation-inactivation assays. In particular it considers experiments in which a number of internal standards are used to supplement or replace accurate measurement of the applied doses of radiation. A mathematical model is proposed which allows the use of the standard technique of maximum-likelihood estimation to estimate the unknown molecular weight without knowledge of the applied doses and, in contrast to previous methods, allows the construction of confidence intervals.

Animals↗
PubMed1990

Re-evaluation of the interaction of malonyl-CoA with the rat liver mitochondrial carnitine palmitoyltransferase system by using purified outer membranes.

1. The interaction of malonyl-CoA with the outer carnitine palmitoyltransferase (CPT) system of rat liver mitochondria was re-evaluated by using preparations of highly purified outer membranes, in the light of observations that other subcellular structures that normally contaminate crude mitochondrial preparations also contain malonyl-CoA-sensitive CPT activity. 2. In outer-membrane preparations, which were purified about 200-fold with respect to the inner-membrane-matrix fraction, malonyl-CoA binding was largely accounted for by a single high-affinity component (KD = 0.03 microM), in contrast with the dual site (low- and high-affinity) previously found with intact mitochondria. 3. There was no evidence that the decreased sensitivity of CPT to malonyl-CoA inhibition observed in outer membranes obtained from 48 h-starved rats (compared with those from fed animals) was due to a decreased ratio of malonyl-CoA binding to CPT catalytic moieties. Thus CPT specific activity and maximal high-affinity [14C]malonyl-CoA binding (expressed per mg of protein) were increased 2.2- and 2.0-fold respectively in outer membranes from 48 h-starved rats. 4. Palmitoyl-CoA at a concentration that was saturating for CPT activity (5 microM) decreased the affinity of malonyl-CoA binding by an order of magnitude, but did not alter the maximal binding of [14C]malonyl-CoA. 5. Preincubation of membranes with either tetradecylglycidyl-CoA or 2-bromopalmitoyl-CoA plus carnitine resulted in marked (greater than 80%) inhibition of high-affinity binding, concurrently with greater than 95% inhibition of CPT activity. These treatments also unmasked an effect of subsequent treatment with palmitoyl-CoA to increase low-affinity [14C]malonyl-CoA binding. 6. These data are discussed in relation to the possible mechanism of interaction between the malonyl-CoA-binding site and the active site of the enzyme.

Acyl Coenzyme A↗
PubMed1989

Purification and characterization of the AMP-activated protein kinase. Copurification of acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA reductase kinase activities.

1. We have purified the AMP-activated protein kinase 4800-fold from rat liver. The acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase kinase activities copurify through all six purification steps and are inactivated with similar kinetics by treatment with the reactive ATP analogue fluorosulphonylbenzoyladenosine. 2. The final preparation contains several polypeptides detectable by SDS/polyacrylamide gel electrophoresis, but only one of these, with an apparent molecular mass of 63 kDa, is labelled using [14C]fluorosulphonylbenzoyladenosine. This is also the only polypeptide in the preparation that becomes significantly labelled during incubation with [gamma 32P]ATP. This autophosphorylation reaction did not affect the AMP-stimulated kinase activity. 3. In the absence of AMP the purified kinase has apparent Km values for ATP and acetyl-CoA carboxylase of 86 microM and 1.9 microM respectively. AMP increases the Vmax 3-5-fold without a significant change in the Km for either protein or ATP substrates. 4. The response to AMP depends on the ATP concentration in the assay, but at a near-physiological ATP concentration the half-maximal effect of AMP occurs at 14 microM. Studies with a range of nucleoside monophosphates and diphosphates, and AMP analogues showed that the allosteric activation by AMP was very specific. ADP gave a small stimulation at low concentrations but was inhibitory at high concentrations. 5. These results show that the AMP-activated protein kinase is the major HMG-CoA reductase kinase detectable in rat liver under our assay conditions and that it is therefore likely to be identical to previously described HMG-CoA reductase kinase(s) which are activated by adenine nucleotides and phosphorylation. The AMP-binding and catalytic domains of the kinase are located on a 63-kDa polypeptide which is subject to autophosphorylation.

AMP-Activated Protein Kinases↗
PubMed1989

Target size analysis by radiation inactivation of carnitine palmitoyltransferase activity and malonyl-CoA binding in outer membranes from rat liver mitochondria.

The functional molecular sizes of the protein(s) mediating the carnitine palmitoyltransferase I (CPT I) activity and the [14C]malonyl-CoA binding in purified outer-membrane preparations from rat liver mitochondria were determined by radiation-inactivation analysis. In all preparations tested the dose-dependent decay in [14C]malonyl-CoA binding was less steep than that for CPT I activity, suggesting that the protein involved in malonyl-CoA binding may be smaller than that catalysing the CPT I activity. The respective sizes computed from simultaneous analysis for molecular-size standards exposed under identical conditions were 60,000 and 83,000 DA for malonyl-CoA binding and CPT I activity respectively. In irradiated membranes the sensitivity of CPT activity to malonyl-CoA inhibition was increased, as judged by malonyl-CoA inhibition curves for the activity in control and in irradiated membranes that had received 20 Mrad radiation and in which CPT activity had decayed by 60%. Possible correlations between these data and other recent observations on the CPT system are discussed.

Acyl Coenzyme A↗