Effects of thyroidectomy and starvation on the activity and properties of hepatic carnitine palmitoyltransferase.

1. Hepatic carnitine palmitoyltransferase activity was measured over a range of concentrations of palmitoyl-CoA and in the presence of several concentrations of the inhibitor malonyl-CoA. These measurements were made in mitochondria obtained from the livers of fed and starved (24 h) normal rats and of fed and starved thyroidectomized rats. 2. In the fed state thyroidectomy substantially decreased overt carnitine palmitoyltransferase activity and also decreased both the Hill coefficient and the s0.5 when palmitoyl-CoA concentration was varied as substrate. Thyroidectomy did not appreciably alter the inhibitory effect of malonyl-CoA on the enzyme. 3. Starvation increased overt carnitine palmitoyltransferase activity in both the fed and the thyroidectomized state. In percentage terms this response to starvation was substantially greater after thyroidectomy. In both the hypothyroid and normal states starvation decreased sensitivity to inhibition by malonyl-CoA.     

Interacting effects of L-carnitine and malonyl-CoA on rat liver carnitine palmitoyltransferase.

Malonyl-CoA significantly increased the Km for L-carnitine of overt carnitine palmitoyltransferase in liver mitochondria from fed rats. This effect was observed when the molar palmitoyl-CoA/albumin concentration ratio was low (0.125-1.0), but not when it was higher (2.0). In the absence of malonyl-CoA, the Km for L-carnitine increased with increasing palmitoyl-CoA/albumin ratios. Malonyl-CoA did not increase the Km for L-carnitine in liver mitochondria from 24h-starved rats or in heart mitochondria from fed animals. The Km for L-carnitine of the latent form of carnitine palmitoyltransferase was 3-4 times that for the overt form of the enzyme. At low ratios of palmitoyl-CoA/albumin (0.5), the concentration of malonyl-CoA causing a 50% inhibition of overt carnitine palmitoyltransferase activity was decreased by 30% when assays with liver mitochondria from fed rats were performed at 100 microM-instead of 400 microM-carnitine. Such a decrease was not observed with liver mitochondria from starved animals. L-Carnitine displaced [14C]malonyl-CoA from liver mitochondrial binding sites. D-Carnitine was without effect. L-Carnitine did not displace [14C]malonyl-CoA from heart mitochondria. It is concluded that, under appropriate conditions, malonyl-CoA may decrease the effectiveness of L-carnitine as a substrate for the enzyme and that L-carnitine may decrease the effectiveness of malonyl-CoA to regulate the enzyme.     

Hepatic mitochondrial inner membrane properties and carnitine palmitoyltransferase A and B. Effect of diabetes and starvation.

Intact mitochondria and inverted submitochondrial vesicles were prepared from the liver of fed, starved (48 h) and streptozotocin-diabetic rats in order to characterize carnitine palmitoyltransferase kinetics and malonyl-CoA sensitivity in situ. In intact mitochondria, both starved and diabetic rats exhibited increased Vmax., increased Km for palmitoyl-CoA, and decreased sensitivity to malonyl-CoA inhibition. Inverted submitochondrial vesicles also showed increased Vmax. with starvation and diabetes, with no change in Km for either palmitoyl-CoA or carnitine. Inverted vesicles were uniformly less sensitive to malonyl-CoA regardless of treatment, and diabetes resulted in a further decrease in sensitivity. In part, differences in the response of carnitine palmitoyltransferase to starvation and diabetes may reside in differences in the membrane environment, as observed with Arrhenius plots, and the relation of enzyme activity and membrane fluidity. In all cases, whether rats were fed, starved or diabetic, and whether intact or inverted vesicles were examined, increasing membrane fluidity was associated with increasing activity. Malonyl-CoA was found to produce a decrease in intact mitochondrial membrane fluidity in the fed state, particularly at pH 7.0 or less. No effect was observed in intact mitochondria from starved or diabetic rats, or in inverted vesicles from any of the treatment groups. Through its effect on membrane fluidity, malonyl-CoA could regulate carnitine palmitoyltransferase activity on both surfaces of the inner membrane through an interaction with only the outer surface.     

The effect of malonyl-CoA on overt and latent carnitine acyltransferase activities in rat liver and adipocyte mitochondria.

1. Carnitine palmitoyltransferase and carnitine octanoyltransferase activities were measured in mitochondria at various acyl-CoA concentrations before and after sonication, thus permitting assessment of both overt and latent activities. 2. Overt carnitine palmitoyltransferase in liver and adipocyte mitochondria and overt carnitine octanoyltransferase in liver mitochondria were inhibited by malonyl-CoA. None of the latent activities were affected by this metabolite. 3. 5,5'-Dithiobis-(2-nitrobenzoic acid) stimulated latent hepatic carnitine palmitoyltransferase at low [palmitoyl-CoA]. 4. Starvation (24 h) decreased overt carnitine palmitoyltransferase activity in adipocyte mitochondria, but did not alter the sensitivity of this activity to malonyl-CoA.     

Studies on the activation in vitro of carnitine palmitoyltransferase I in liver mitochondria from normal, diabetic and glucagon-treated rats.

The activation of overt carnitine palmitoyltransferase activity that occurs when rat liver mitochondria are incubated at near-physiological temperatures and ionic strengths was studied for mitochondria obtained from animals in different physiological states. In all instances, it was found to be due exclusively to an increase in the catalytic capacity of the enzyme and not to an increase in affinity of the enzyme for palmitoyl-CoA. The enzyme in mitochondria from fed animals always showed a larger degree of activation than that in mitochondria from starved animals. This was the case even for mitochondria (e.g. from fed diabetic animals) in which the kinetic characteristics of carnitine palmitoyltransferase were more similar to those for the enzyme in mitochondria from starved rats. Glucagon treatment of rats before isolation of the mitochondria did not affect the characteristics either of the kinetic parameters of overt carnitine palmitoyltransferase or of its activation in vitro.     

Carnitine acyltransferase activities in rat liver and heart measured with palmitoyl-CoA and octanoyl-CoA. Latency, effects of K+, bivalent metal ions and malonyl-CoA.

1. Liver carnitine acyltransferase activities with palmitoyl-CoA and octanoyl-CoA as substrates and heart carnitine palmitoyltransferase were measured as overt activities in whole mitochondria or in mitochondria disrupted by sonication or detergent treatment. All measurements were made in sucrose/KCl-based media of 300 mosmol/litre. 2. In liver mitochondria, acyltransferase measured with octanoyl-CoA, like carnitine palmitoyltransferase, was found to have latent and overt activities. 3. Liver acyltransferase activities measured with octanoyl-CoA and palmitoyl-CoA differed in their response to changes in [K+], Triton X-100 treatment and, in particular, in their response to Mg2+. Mg2+ stimulated activity with octanoyl-CoA, but inhibited carnitine palmitoyltransferase. 4. The effects of K+ and Mg2+ on liver overt carnitine palmitoyltransferase activity were abolished by Triton X-100 treatment. 5. Heart overt carnitine palmitoyltransferase activity differed from the corresponding activity in liver in that it was more sensitive to changes in [K+] and was stimulated by Mg2+. Heart had less latent carnitine palmitoyltransferase activity than did liver. 6. Overt carnitine palmitoyltransferase in heart mitochondria was extremely sensitive to inhibition by malonyl-CoA. Triton X-100 abolished the effect of low concentrations of malonyl-CoA on this activity. 7. The inhibitory effect of malonyl-CoA on heart carnitine palmitoyltransferase could be overcome by increasing the concentration of palmitoyl-CoA.     

Observations on the affinity for carnitine, and malonyl-CoA sensitivity, of carnitine palmitoyltransferase I in animal and human tissues. Demonstration of the presence of malonyl-CoA in non-hepatic tissues of the rat.

The requirement for carnitine and the malonyl-CoA sensitivity of carnitine palmitoyl-transferase I (EC 2.3.1.21) were measured in isolated mitochondria from eight tissues of animal or human origin using fixed concentrations of palmitoyl-CoA (50 microM) and albumin (147 microM). The Km for carnitine spanned a 20-fold range, rising from about 35 microM in adult rat and human foetal liver to 700 microM in dog heart. Intermediate values of increasing magnitude were found for rat heart, guinea pig liver and skeletal muscle of rat, dog and man. Conversely, the concentration of malonyl-CoA required for 50% suppression of enzyme activity fell from the region of 2-3 microM in human and rat liver to only 20 nM in tissues displaying the highest Km for carnitine. Thus, the requirement for carnitine and sensitivity to malonyl-CoA appeared to be inversely related. The Km of carnitine palmitoyltransferase I for palmitoyl-CoA was similar in tissues showing large differences in requirement for carnitine. Other experiments established that, in addition to liver, heart and skeletal muscle of fed rats contain significant quantities of malonyl-CoA and that in all three tissues the level falls with starvation. Although its intracellular location in heart and skeletal muscle is not known, the possibility is raised that malonyl-CoA (or a related compound) could, under certain circumstances, interact with carnitine palmitoyltransferase I in non-hepatic tissues and thereby exert control over long chain fatty acid oxidation.     

Differential inhibition of ketogenesis by malonyl-CoA in mitochondria from fed and starved rats.

Rates of ketogenesis in mitochondria from fed or starved rats were identical at optimal substrate concentrations, but responded differently to inhibition by malonyl-CoA. Kinetic data suggest that the K1 for malonyl-CoA is greater in the starved animal. These results indicate that, for the regulation of ketogenesis in the starved state, the lower sensitivity of carnitine palmitoyltransferase to inhibition by malonyl-CoA may be more important than the concentration of malonyl-CoA.     

Time-dependence of inhibition of carnitine palmitoyltransferase I by malonyl-CoA in mitochondria isolated from livers of fed or starved rats. Evidence for transition of the enzyme between states of low and high affinity for malonyl-CoA.

The degree of inhibition of CPT I (carnitine palmitoyltransferase, EC 2.3.1.21) in isolated rat liver mitochondria by malonyl-CoA was studied by measuring the activity of the enzyme over a short period (15s) after exposure of the mitochondria to malonyl-CoA for different lengths of time. Inhibition of CPT I by malonyl-CoA was markedly time-dependent, and the increase occurred at the same rate in the presence or absence of palmitoyl-CoA (80 microM), and in the presence of carnitine, such that the time-course of acylcarnitine formation deviated markedly from linearity when CPT I activity was measured in the presence of malonyl-CoA over several minutes. The initial rate of increase in degree of inhibition with time was independent of malonyl-CoA concentration. CPT I in mitochondria from 48 h-starved rats had a lower degree of inhibition by malonyl-CoA at zero time, but was equally capable of being sensitized to malonyl-CoA, as judged by an initial rate of increase of inhibition identical with that of the enzyme in mitochondria from fed rats. Double-reciprocal plots for the degree of inhibition produced by different malonyl-CoA concentrations at zero time for the enzyme in mitochondria from fed or starved animals indicated that the enzyme in the latter mitochondria was predominantly in a state with low affinity for malonyl-CoA (concentration required to give 50% inhibition, I0.5 congruent to 10 microM), whereas that in mitochondria from fed rats displayed two distinct sets of affinities: low (congruent to 10 microM) and high (less than 0.3 microM). Plots for mitochondria after incubation for 0.5 or 1 min with malonyl-CoA indicated that the increased sensitivity observed with time was due to a gradual increase in the high-affinity state in both types of mitochondria. These results suggest that the sensitivity of CPT I in rat liver mitochondria in vitro had two components: (i) an instantaneous sensitivity inherent to the enzyme which depends on the nutritional state of the animal from which the mitochondria are isolated, and (ii) a slow, malonyl-CoA-induced, time-dependent increase in sensitivity. It is suggested that the rate of malonyl-CoA-induced sensitization of the enzyme to malonyl-CoA inhibition is limited by a slow first-order process, which occurs after the primary event of interaction of malonyl-CoA with the mitochondria.(ABSTRACT TRUNCATED AT 400 WORDS)     

Milk composition of rats feeding restricted litters.

Continuous assays of carnitine palmitoyltransferase were used to study the hysteretic behaviour of the enzyme. When reactions were started by adding mitochondria to complete reaction mixtures, there was a lag in the assay even in the absence of malonyl-CoA. When mitochondria were preincubated with malonyl-CoA in the absence of palmitoyl-CoA, there was a greater lag period in the assay of carnitine palmitoyltransferase, but this lag was less prominent at 37 degrees C than at 30 degrees C. Preincubation of mitochondria with malonyl-CoA did not change the sensitivity of the enzyme to inhibition by malonyl-CoA.     

Effect of malonyl-CoA on the kinetics and substrate cooperativity of membrane-bound carnitine palmitoyltransferase of rat heart mitochondria

The effect of malonyl-CoA on the kinetic parameters of carnitine palmitoyltransferase (outer) the outer form of carnitine palmitoyltransferase (palmitoyl-CoA: L-carnitine O-palmitoyltransferase, EC 2.3.1.21) from rat heart mitochondria was investigated using a kinetic analyzer in the absence of bovine serum albumin with non-swelling conditions and decanoyl-CoA as the cosubstrate. The K0.5 for decanoyl-CoA is 3 microM for heart mitochondria from both fed and fasted rats. Membrane-bound carnitine palmitoyltransferase (outer) shows substrate cooperativity for both carnitine and acyl-CoA, similar to that exhibited by the enzyme purified from bovine heart mitochondria. The Hill coefficient for decanoyl-CoA varied from 1.5 to 2.0, depending on the method of assay and the preparation of mitochondria. Malonyl-CoA increased the K0.5 for decanoyl-CoA with no apparent increase in sigmoidicity or Vmax. With 20 microM malonyl-CoA and a Hill coefficient of n = 2.1, the K0.5 for decanoyl-CoA increased to 185 microM. Carnitine palmitoyltransferase (outer) from fed rats had an apparent Ki for malonyl-CoA of 0.3 microM, while that from 48-h-fasted rats was 2.5 microM. The kinetics with L-carnitine were variable: for different preparations of mitochondria, the K0.5 ranged from 0.2 to 0.7 mM and the Hill coefficient varied from 1.2 to 1.8. When an isotope forward assay was used to determine the effect of malonyl-CoA on carnitine palmitoyltransferase (outer) activity of heart mitochondria from fed and fasted animals, the difference was much less than that obtained using a continuous rate assay. Carnitine palmitoyltransferase (outer) was less sensitive to malonyl-CoA at low compared to high carnitine concentrations, particularly with mitochondria from fasted animals. The data show that carnitine palmitoyltransferase (outer) exhibits substrate cooperativity for both acyl-CoA and L-carnitine in its native state. The data show that membrane-bound carnitine palmitoyltransferase (outer) like carnitine palmitoyltransferase purified from heart mitochondria exhibits substrate cooperativity indicative of allosteric enzymes and indicate that malonyl-CoA acts like a negative allosteric modifier by shifting the acyl-CoA saturation to the right. A slow form of membrane-bound carnitine palmitoyltransferase (outer) was not detected, and thus, like purified carnitine palmitoyltransferase, substrate-induced hysteretic behavior is not the cause of the positive substrate cooperativity.     

Hepatic mitochondrial inner-membrane properties, beta-oxidation and carnitine palmitoyltransferases A and B. Effects of genetic obesity and starvation.

Hepatic mitochondrial carnitine palmitoyltransferase (CPT) properties, beta-oxidation of palmitoyl-CoA and membrane polarization were measured in lean and obese Zucker rats. The Vmax. of the 'outer' carnitine palmitoyltransferase ('CPT-A') increased with starvation, with no change in the Km for either carnitine or palmitoyl-CoA. The Ki for malonyl-CoA increased with starvation in lean rats, but not in obese rats. The Vmax. of the 'inner' enzyme ('CPT-B'), as measured by using inverted submitochondrial vesicles, increased with starvation in obese rats only, with no change in the Km for either carnitine or palmitoyl-CoA. The Ki for malonyl-CoA was 2-5-fold higher in inverted vesicles than in intact mitochondria, and showed no alteration with starvation. The activities of both enzymes correlated positively with each other and with beta-oxidation, and inversely with membrane polarization. Malonyl-CoA had little effect on gross membrane fluidity in the Zucker rat, as reflected by diphenylhexatriene fluorescence polarization. The results indicate that both enzymes are related and respond similarly to alterations in membrane fluidity. Membrane fluidity may provide a mechanism for co-ordinated control of CPT activity on both sides of the mitochondrial inner membrane.     

Preventive effect of clofibrate on carnitine palmitoyltransferase I inhibition and mitochondrial membrane phospholipid depletion induced by galactosamine

We have previously reported that a D-galactosamine injection induces a decrease of carnitine palmitoyltransferase I activity correlated with a depletion of total phospholipid content in the mitochondrial membrane. The impact of a short-term clofibrate treatment on these membrane alterations is investigated, i.e., the kinetic properties of carnitine palmitoyltransferase I, including its sensitivity to malonyl-CoA and mitochondrial membrane content of the various phospholipids. A 4-day clofibrate treatment increases by 42% the apparent Km value of carnitine palmitoyltransferase I for palmitoyl-CoA, while the sensitivity of the enzyme to malonyl-CoA appears slightly decreased. Simultaneously, the cardiolipin content is increased by 70% in the mitochondrial membrane, whereas the phosphatidylethanolamine and phosphatidylcholine contents remain almost unaffected. This 4-day clofibrate treatment prevents the inhibition of carnitine palmitoyltransferase I activity subsequent to galactosamine administration but induces an increase in the apparent Km value for palmitoyl-CoA and a decrease of the sensitivity of the enzyme to malonyl-CoA. The contents of phospholipids which are decreased by galactosamine (phosphatidylcholine, -21%; phosphatidylethanolamine, -29%; cardiolipin, -40%) regain the control values when galactosamine administration is preceded by a clofibrate treatment. The data suggest that the clofibrate treatment counteracts the inhibition of activity of carnitine palmitoyltransferase I through the maintenance of mitochondrial membrane integrity.     

Involvement of hysteretic effects in the inhibition of carnitine palmitoyltransferase by malonyl-CoA.

Carnitine palmitoyltransferase in its normal mitochondrial environment behaves as a hysteretic enzyme, exhibiting slow changes in reaction rate after the addition of oleoyl-CoA or malonyl-CoA. Reaction rates become constant after a short time, but the sensitivity of the enzyme from fed rats to the inhibition by malonyl-CoA remains much greater than that of starved rats.     

Carnitine palmitoyltransferase. Activation by palmitoyl-CoA and inactivation by malonyl-CoA

Extraction of rat liver mitochondria twice with 0.5% Triton X-100 in a salt-free medium leaves less than 10% of the carnitine palmitoyltransferase membrane bound. The remaining membrane-bound enzyme is inhibited virtually completely by 10 microM malonyl-CoA. Preincubation of the extracted membranes with palmitoyl-CoA and salts (KCI) for several minutes activates the enzyme and makes it increasingly insensitive to malonyl-CoA. Addition of malonyl-CoA to the preincubation reverses this desensitization. In albumin-containing media salts also decrease the binding of palmitoyl-CoA to albumin and stimulate carnitine palmitoyltransferase by increasing substrate availability in free solution. The reverse reaction shows accelerated desensitization by palmitoylcarnitine and resensitization by malonyl-CoA.     

Some aspects of fatty acid oxidation in isolated fat-cell mitochondria from rat.

Mitochondrial were prepared from fat-cells isolated from rat epididymal adipose tissues of fed and 48 h-starved rats to study some aspects of fatty acid oxidation in this tissue. The data were compared with values obtained in parallel experiments with liver mitochondria that were prepared and incubated under identical conditions. 2. In the presence of malonate, fluorocitrate and arsenite, malate, but not pyruvate-bicarbonate, facilitated palmitoyl-group oxidation in both types of mitochondria. In the presence of malate, fat-cell mitochondria exhibited slightly higher rates of palmitoylcarnitine oxidation than liver. Rates of octanoylcarnitine oxidation were similar in liver and fat-cell mitochondria. Uncoupling stimulated acylcarnitine oxidation in liver, but not in fat-cell mitochondria. Oxidation of palmitoyl- and octanoyl-carnitine was partially additive in fat-cell but not in liver mitochondria. Starvation for 48 h significantly decreased both palmitoylcarnitine oxidation and latent carnitine palmitoyltransferase activity in fat-cell mitochondria. Starvation increased latent carnitine palmitoyltransferase activity in liver mitochondria but did not alter palmitoylcarnitine oxidation. These results suggested that palmitoylcarnitine oxidation in fat-cell but not in liver mitochondria may be limited by carnitine palmitoyltransferase 2 activity. 3. Fat-cell mitochondria also differed from liver mitochondria in exhibiting considerably lower rates of carnitine-dependent oxidation of palmitoyl-CoA or palmitate, suggesting that carnitine palmitoyltransferase 1 activity may severely rate-limit palmitoyl-CoA oxidation in adipose tissue.     

Binding of malonyl-CoA to isolated mitochondria. Evidence for high- and low-affinity sites in liver and heart and relationship to inhibition of carnitine palmitoyltransferase activity.

[14C]Malonyl-CoA bound to intact mitochondria isolated from rat liver and heart in a manner consistent with the presence of two independent classes of binding sites in each tissue. The binding characteristics for mitochondria obtained from fed male rats were: for heart, KD(1) = 11-18nM, KD(2) = 30 microM, N1 = 7pmol/mg of protein, N2 = approx. 660pmol/mg of protein; for liver, KD(1) = 0.1 microM, KD(2) = 5.6 microM, N1 = 11pmol/mg of protein, N2 = 165pmol/mg of protein. In the presence of 40 microM-palmitoyl-CoA the characteristics of binding at the high-affinity sites were changed, so that for heart KD(1) = 0.26 microM, with no change in N1 and for liver KD(1) = approx. 2 microM, with N1 increased to approx. 40pmol/mg of protein. Differences between the two tissues in tightness of malonyl-CoA binding at the high-affinity sites explains the considerably greater sensitivity of heart CPT1 (overt form of carnitine palmitoyltransferase) to inhibition by malonyl-CoA [Saggerson & Carpenter, (1981) FEBS Lett. 129, 229-232; McGarry, Mills, Long & Foster (1983) Biochem. J. 214, 21-28]. Starvation (24h) did not change the characteristics of [14C]malonyl-CoA binding to liver mitochondria and did not alter the I50 (concentration giving 50% inhibition) for displacement of [14C]malonyl-CoA by palmitoyl-CoA. Therefore the decreased sensitivity of liver CPT1 to inhibition by malonyl-CoA in starvation [Saggerson & Carpenter (1981) FEBS Lett. 129, 225-228; Bremer (1981) Biochim. Biophys. Acta 665, 628-631] is not explained by differences in malonyl-CoA binding. Percentage occupancy of the high-affinity sites in heart mitochondria by malonyl-CoA correlated closely with percentage inhibition of CPT1 measured under similar conditions. This finding supports the proposal that the high-affinity binding sites are the functional sites mediating inhibition of CPT1 by malonyl-CoA. Similar experiments with liver mitochondria also suggested that the occupancy of high-affinity sites by malonyl-CoA regulates CPT1 activity. 5,5'-Dithiobis-(2-nitrobenzoic acid), which decreased the sensitivity of heart or liver CPT1 to inhibition by malonyl-CoA [Saggerson & Carpenter (1982) FEBS Lett. 137, 124-128], also decreased [14C]malonyl-CoA binding to the high-affinity sites of heart mitochondria. N1 values for [14C]malonyl-CoA binding to high-affinity sites in liver mitochondria were determined in various physiological states which encompassed a 7-fold range of CPT1 maximal activity (fed, starved, pregnant, hypothyroid, foetal). The N1 value did not change in these states.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of starvation and diabetes on the sensitivity of carnitine palmitoyltransferase I to inhibition by 4-hydroxyphenylglyoxylate.

The sensitivity of carnitine palmitoyltransferase I to inhibition by 4-hydroxyphenylglyoxylate was decreased markedly in liver mitochondria isolated from either 48 h-starved or streptozotocin-diabetic rats. These treatments of the rat also decreased the sensitivity of fatty acid oxidation by isolated hepatocytes to inhibition by this compound. Furthermore, incubation of hepatocytes prepared from fed rats with N6O2'-dibutyryl cyclic AMP also decreased the sensitivity, whereas incubation of hepatocytes prepared from starved rats with lactate plus pyruvate had the opposite effect on 4-hydroxyphenylglyoxylate inhibition of fatty acid oxidation. The sensitivity of carnitine palmitoyltransferase I of mitochondria to 4-hydroxyphenylglyoxylate increased in a time-dependent manner, as previously reported for malonyl-CoA. Likewise, oleoyl-CoA activated carnitine palmitoyltransferase I in a time-dependent manner and prevented the sensitization by 4-hydroxyphenylglyoxylate. Increased exogenous carnitine caused a moderate increase in fatty acid oxidation by hepatocytes under some conditions and a decreased 4-hydroxyphenylglyoxylate inhibition of fatty acid oxidation at low oleate concentration, without decreasing the difference in 4-hydroxyphenylglyoxylate inhibition between fed- and starved-rat hepatocytes. Time-dependent changes in the conformation of carnitine palmitoyltransferase I or the membrane environment may be involved in differences among nutritional states in 4-hydroxyphenylglyoxylate-sensitivity of carnitine palmitoyltransferase I.     

Carnitine acyltransferase activities in rat brain mitochondria. Bimodal distribution, kinetic constants, regulation by malonyl-CoA and developmental pattern.

Carnitine palmitoyltransferase and carnitine octanoyltransferase activities in brain mitochondrial fractions were approx. 3-4-fold lower than activities in liver. Estimated Km values of CPT1 and CPT2 (the overt and latent forms respectively of carnitine palmitoyltransferase) for L-carnitine were 80 microM and 326 microM, respectively, and K0.5 values for palmitoyl-CoA were 18.5 microM and 12 microM respectively. CPT1 activity was strongly inhibited by malonyl-CoA, with I50 values (concn. giving 50% of maximum inhibition) of approx. 1.5 microM. In the absence of other ligands, [2-14C]malonyl-CoA bound to intact brain mitochondria in a manner consistent with the presence of two independent classes of binding sites. Estimated values for KD(1), KD(2), N1 and N2 were 18 nM, 27 microM, 1.3 pmol/mg of protein and 168 pmol/mg of protein respectively. Neither CPT1 activity, nor its sensitivity towards malonyl-CoA, was affected by 72 h starvation. Rates of oxidation of palmitoyl-CoA (in the presence of L-carnitine) or of palmitoylcarnitine by non-synaptic mitochondria were extremely low, indicating that neither CPT1 nor CPT2 was likely to be rate-limiting for beta-oxidation in brain. CPT1 activity relative to mitochondrial protein increased slightly from birth to weaning (20 days) and thereafter decreased by approx. 50%.     

Differences in the sensitivity of carnitine palmitoyltransferase to inhibition by malonyl-CoA are due to differences in Ki values

The hepatic carnitine palmitoyltransferase that is present on the outer surface of the mitochondrial inner membrane demonstrates hyperbolic substrate saturation curves with oleoyl-CoA in both fasted and fed rats. However, the addition of malonyl-CoA resulted in sigmoid substrate saturation curves, suggesting that malonyl-CoA induced the cooperative behavior. There was more of the outer carnitine palmitoyltransferase in liver mitochondria derived from fasted rats and that enzyme had a much greater Ki for malonyl-CoA than the enzyme from fed rats, but the Km values were apparently not different. The Dixon plot with mitochondria from fed rats, but not fasted rats, was curved upward, indicating cooperative inhibition by malonyl-CoA. Carnitine palmitoyltransferase of heart mitochondria had a Ki for malonyl-CoA that was much less than that of the liver enzyme and it did not change on fasting. Furthermore, no evidence for cooperative inhibition was found in the heart. The results of these studies indicate that carnitine palmitoyltransferase is not subject to substrate cooperativity and that malonyl-CoA is not a simple competitive inhibitor of this enzyme but inhibits by a mechanism involving cooperative inhibition. The fasting-feeding cycle induces changes in the liver enzyme that alter its affinity for malonyl-CoA without changing its affinity for its acyl-CoA substrate. Carnitine palmitoyltransferase from heart appears to be different from that of liver and is apparently not subject to the same control mechanisms.