scholarly journals Carnitine palmitoyltransferase in human erythrocyte membrane. Properties and malonyl-CoA sensitivity

1991 ◽  
Vol 275 (3) ◽  
pp. 685-688 ◽  
Author(s):  
R R Ramsay ◽  
G Mancinelli ◽  
A Arduini
Keyword(s):  
Membrane Lipids ◽  
Fatty Acyl ◽  
Carnitine Palmitoyltransferase ◽  
Membrane Properties ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Inner Membrane ◽  
Malonyl Coa ◽  
Intracellular Enzymes ◽  
Erythrocyte Plasma Membrane ◽  
Triton X

Carnitine palmitoyltransferase located in the erythrocyte plasma membrane is sensitive to inhibition by malonyl-CoA and 2-bromopalmitoyl-CoA plus carnitine. Although this inhibition and other properties suggest similarities to the intracellular enzymes in other tissues, no cross-reaction was observed with antisera to the peroxisomal or to the mitochondrial inner-membrane enzyme. The activity was solubilized by and was stable in Triton X-100, which destroys the enzymes found in microsomes and in the mitochondrial outer membrane. The substrate specificity is broader than for the intracellular enzymes, the activities with stearoyl-CoA (114%) and arachidonoyl-CoA (97%) being equal to that with palmitoyl-CoA, and the activities with linoleoyl-CoA (44%) and erucoyl-CoA (46%) about half that with palmitoyl-CoA. The function of this carnitine palmitoyltransferase is probably to buffer the acyl-CoA present in the erythrocyte for turnover of the fatty acyl groups of the membrane lipids.

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

1986 ◽  
Vol 233 (2) ◽  
pp. 427-433 ◽  
Author(s):  
L J Brady ◽  
C L Hoppel ◽  
P S Brady
Keyword(s):  
Membrane Fluidity ◽  
Carnitine Palmitoyltransferase ◽  
Membrane Properties ◽  
Zucker Rats ◽  
Beta Oxidation ◽  
Inner Membrane ◽  
Membrane Polarization ◽  
Mitochondrial Inner Membrane ◽  
Malonyl Coa ◽  
Obese Rats

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.

scholarly journals Proteinase treatment of intact hepatic mitochondria has differential effects on inhibition of carnitine palmitoyltransferase by different inhibitors

1992 ◽  
Vol 282 (3) ◽  
pp. 909-914 ◽  
Author(s):  
K Kashfi ◽  
G A Cook
Keyword(s):  
Outer Membrane ◽  
Adenylate Kinase ◽  
Citrate Synthase ◽  
Physiological State ◽  
Carnitine Palmitoyltransferase ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Mitochondrial Inner Membrane ◽  
Malonyl Coa ◽  
No Release

Proteolysis of intact mitochondria by Nagarse (subtilisin BPN') and papain resulted in limited loss of activity of the outer-membrane carnitine palmitoyltransferase, but much greater loss of sensitivity to inhibition by malonyl-CoA. In contrast with a previous report [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382], we found that trypsin had no effect on malonyl-CoA sensitivity. Even when 80% of activity was destroyed by trypsin, there was no difference in the malonyl-CoA sensitivity of the enzyme remaining. Trypsin caused release of the intermembrane-space enzyme adenylate kinase, indicating loss of integrity of the mitochondrial outer membrane, whereas Nagarse and papain caused no release of that enzyme. Citrate synthase was not released by any of the three proteinases, indicating no damage to the mitochondrial inner membrane. When we examined the effects of proteolysis on the inhibition of carnitine palmitoyltransferase by a wide variety of inhibitors having different mechanisms of inhibition, we found differential proteolytic effects that were specific for those inhibitors (malonyl-CoA and hydroxyphenylglyoxylate) that have their inhibitory potencies diminished by changes in physiological state. Both of those inhibitors protected carnitine palmitoyltransferase from the effects of proteolysis, but did not inhibit the proteinases directly. Inhibition by two other inhibitors (DL-2-bromopalmitoyl-CoA and N-benzyladriamycin 14-valerate) was not altered by proteinase treatment, even when most of the enzyme activity had been destroyed. Inhibition by glyburide, which is minimally affected by physiological state, was affected only to a slight extent at the highest concentration of trypsin tested. Proteolysis by Nagarse appeared to produce loss of co-operativity in malonyl-CoA inhibition. The effects of proteolysis are discussed and compared with changes in Ki occurring with changing physiological states.

scholarly journals Some differences in the properties of carnitine palmitoyltransferase activities of the mitochondrial outer and inner membranes

1987 ◽  
Vol 248 (3) ◽  
pp. 727-733 ◽  
Author(s):  
M S Murthy ◽  
S V Pande
Keyword(s):  
Phospholipase C ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Mitochondrial Outer Membrane ◽  
Rat Liver Mitochondria ◽  
Outer Membranes ◽  
Mitochondrial Inner Membrane ◽  
Octyl Glucoside ◽  
Malonyl Coa

Recent evidence has shown that the outer, overt, malonyl-CoA-inhibitable carnitine palmitoyltransferase (CPTo) activity resides in the mitochondrial outer membrane [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382]. A comparison of CPTo activity of rat liver mitochondria with the inner, initially latent, carnitine palmitoyltransferase (CPTi) of the mitochondrial inner membrane has revealed that the presence of digitonin and several other detergents inactivates CPTo activity. The CPTi activity, in contrast, was markedly stimulated by various detergents and phospholipid liposomes. These findings explain why in previous studies, which used digitonin or other detergents to expose, separate and purify the CPT activities, the inferences were drawn that (a) the ratio of latent to overt CPT was quite high, (b) both the CPT activities could be ascribed to one active protein recovered, and (c) the observed lack of malonyl-CoA inhibition indicated possible loss/separation of a putative malonyl-CoA-inhibition-conferring protein. Although both CPTo and CPTi were found to catalyse the forward and the backward reactions, CPTo showed greater capacity for the forward reaction and CPTi for the backward reaction. The easily solubilizable CPT, released on sonication of mitoplasts or of intact mitochondria under hypo-osmotic conditions, resembled CPTi in its properties. When octyl glucoside was used under appropriate conditions, 40-50% of the CPTo of outer membranes became solubilized, but it showed limited stability and decreased malonyl-CoA sensitivity. Malonyl-CoA-inhibitability of CPTo was decreased also on exposure of outer membranes to phospholipase C. When outer membranes that had been exposed to octyl glucoside or to phospholipase C were subjected to a reconstitution procedure using asolectin liposomes, the malonyl-CoA-inhibitability of CPTo was restored. A role of phospholipids in the malonyl-CoA sensitivity of CPTo is thus indicated.

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

1985 ◽  
Vol 232 (2) ◽  
pp. 445-450 ◽  
Author(s):  
L J Brady ◽  
L J Silverstein ◽  
C L Hoppel ◽  
P S Brady
Keyword(s):  
Membrane Fluidity ◽  
Diabetic Rats ◽  
Carnitine Palmitoyltransferase ◽  
Membrane Properties ◽  
Inner Membrane ◽  
Fed State ◽  
Treatment Groups ◽  
Mitochondrial Inner Membrane ◽  
Malonyl Coa ◽  
Carnitine Palmitoyltransferase Activity

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.

Cold acclimation increases carnitine palmitoyltransferase I activity in oxidative muscle of striped bass

1994 ◽  
Vol 266 (2) ◽  
pp. R405-R412 ◽  
Author(s):  
K. J. Rodnick ◽  
B. D. Sidell
Keyword(s):  
Cold Acclimation ◽  
Striped Bass ◽  
Fatty Acyl ◽  
Thermal Acclimation ◽  
Carnitine Palmitoyltransferase ◽  
Malonyl Coa ◽  
Red Muscle ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I ◽  
Long Chain

The effect of thermal acclimation on the activity of carnitine palmitoyltransferase I (CPT I), the rate-limiting enzyme for beta-oxidation of long-chain fatty acids, was determined in oxidative red muscle of striped bass (Morone saxatilis) acclimated at 5 or 25 degrees C. As observed in mammalian tissues, malonyl-CoA potently inhibited CPT I activity of mitochondria. Inhibition by malonyl-CoA required inclusions of both bovine serum albumin (BSA) and palmitoyl-CoA in the reaction media. Because BSA binds long-chain fatty acyl-CoAs, this observation suggests that free fatty acyl-CoAs may disrupt mitochondrial membranes and affect the CPT I protein. Cold acclimation increased citrate synthase activity 1.6-fold and total CPT activity 2-fold in homogenates of red muscle; free carnitine increased 62%, and specific activity of CPT I in mitochondria increased 2-fold. No differences were observed between cold- and warm-acclimated fish in substrate-binding properties of CPT I at an assay temperature of 15 degrees C, as judged by the Michaelis constant (Km) for carnitine (0.11 +/- 0.02 vs. 0.13 +/- 0.02 mM) or inhibition of CPT I, as determined by the half-maximal inhibition concentration (IC50) for malonyl-CoA (0.14 +/- 0.05 vs. 0.09 +/- 0.03 microM). Thermal sensitivity of CPT I (Q10 = 2.91 +/- 0.12 vs. 3.02 +/- 0.20) and preference of CPT I for different long-chain fatty acyl-CoA substrates (16:1-CoA = 16:0-CoA > 18:1-CoA) were not altered by thermal acclimation.(ABSTRACT TRUNCATED AT 250 WORDS)

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

1987 ◽  
Vol 241 (3) ◽  
pp. 751-757 ◽  
Author(s):  
P S Brady ◽  
A K Dunker ◽  
L J Brady
Keyword(s):  
Carnitine Palmitoyltransferase ◽  
Dimethyl Sulphoxide ◽  
Inner Membrane ◽  
Phosphatidylcholine Liposomes ◽  
Aqueous Buffer ◽  
Mitochondrial Inner Membrane ◽  
Membrane Enzyme ◽  
Malonyl Coa ◽  
Assay Conditions

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.

scholarly journals Different sites of inhibition of carnitine palmitoyltransferase by malonyl-CoA, and by acetyl-CoA and CoA, in human skeletal muscle

1987 ◽  
Vol 245 (1) ◽  
pp. 205-209 ◽  
Author(s):  
S Zierz ◽  
A G Engel
Keyword(s):  
Skeletal Muscle ◽  
Human Subjects ◽  
Fold Increase ◽  
Inhibitory Effect ◽  
Carnitine Palmitoyltransferase ◽  
Inhibitory Effects ◽  
Acetyl Coa ◽  
Malonyl Coa ◽  
Normal Human ◽  
Triton X

The inhibition of carnitine palmitoyltransferase (CPT, EC 2.3.1.21) by malonyl-CoA, acetyl-CoA and free CoA was studied in sonicated skeletal-muscle homogenates from normal human subjects and from five patients with a mutant CPT [Zierz & Engel (1985) Eur. J. Biochem. 149, 207-214]. (1) Malonyl-CoA, acetyl-CoA and CoA were competitive inhibitors of CPT with palmitoyl-CoA. (2) Acetyl-CoA and CoA inhibited normal and mutant CPT to the same degree, whereas malonyl-CoA inhibited mutant CPT more than normal CPT. (3) Triton X-100 abolished the inhibition of normal CPT by malonyl-CoA, but not by acetyl-CoA or CoA. Triton X-100 by itself caused loss of activity of the mutant CPT. (4) In the concentration range 0.1-0.4 mM, the inhibitory effects of any two of the three inhibitors were synergistic. (5) The inhibitory constants (Ki) for acetyl-CoA and CoA were close to 45 microM. The Ki for malonyl-CoA was 200-fold lower, or 0.22 microM. Addition of 40 microM-acetyl-CoA or CoA resulted in a 3-fold increase in the Ki for acetyl-CoA. Addition of 20 microM-CoA resulted in a 3-fold increase in the Ki for acetyl-CoA. (6) The findings indicate that acetyl-CoA and CoA can inhibit CPT at the catalytic site or a nearby site which is different from that at which malonyl-CoA inhibits CPT. (7) The fact that small changes in the concentration of acetyl-CoA and CoA can antagonize the inhibitory effect of malonyl-CoA suggests that these compounds could modulate the inhibition of CPT by malonyl-CoA.

scholarly journals Activity of carnitine palmitoyltransferase in mitochondrial outer membranes and peroxisomes in digitonin-permeabilized hepatocytes. Selective modulation of mitochondrial enzyme activity by okadaic acid

1992 ◽  
Vol 287 (2) ◽  
pp. 487-492 ◽  
Author(s):  
M Guzmán ◽  
M J H Geelen
Keyword(s):  
Okadaic Acid ◽  
Carnitine Palmitoyltransferase ◽  
Mitochondrial Outer Membrane ◽  
Short Term ◽  
Experimental Conditions ◽  
Outer Membranes ◽  
Palmitate Oxidation ◽  
Physiological Relevance ◽  
Malonyl Coa ◽  
Selective Modulation

A procedure is described for the rapid measurement of the activity of mitochondrial-outer-membrane carnitine palmitoyltransferase (CPTo) and peroxisomal carnitine palmitoyltransferase (CPTp) in digitonin-permeabilized hepatocytes. CPTo activity was determined as the tetradecylglycidate (TDGA)-sensitive malonyl-CoA-sensitive CPT activity, whereas CPTp activity was monitored as the TDGA-insensitive malonyl-CoA-sensitive CPT activity. Under these experimental conditions, the respective contributions of CPTo and CPTp to total hepatocellular malonyl-CoA-sensitive CPT activity were 74.6 and 25.4%, which correlated well with the values of 76.9 and 23.1% for the respective contributions of the mitochondrial and the peroxisomal compartment to total hepatocellular palmitate oxidation. The sensitivity of CPTo to inhibition by malonyl-CoA was very similar to that of CPTp; thus 50% inhibition of CPTo and CPTp activities was achieved with malonyl-CoA concentrations of 2.6 +/- 0.5 and 3.0 +/- 0.4 microM respectively. Short-term incubation of hepatocytes with the phosphatase inhibitor okadaic acid (i) increased the activity of CPTo and the rate of mitochondrial palmitate oxidation, (ii) decreased the affinity of CPTo for palmitoyl-CoA substrate, and (iii) decreased the sensitivity of CPTo to inhibition by malonyl-CoA. By contrast, neither the properties of CPTp nor the rate of peroxisomal palmitate oxidation were changed upon incubation of cells with okadaic acid. Results indicate therefore that CPTo, but not CPTp, may be regulated by a mechanism of phosphorylation/dephosphorylation. The physiological relevance of these findings is discussed.

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

1990 ◽  
Vol 272 (2) ◽  
pp. 421-425 ◽  
Author(s):  
M P Kolodziej ◽  
V A Zammit
Keyword(s):  
Outer Membrane ◽  
Membrane Fluidity ◽  
Rat Liver ◽  
Isoamyl Alcohol ◽  
Membrane Lipid ◽  
Carnitine Palmitoyltransferase ◽  
Mitochondrial Outer Membrane ◽  
Rat Liver Mitochondria ◽  
Malonyl Coa ◽  
Cpt I

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.

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