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L L Bieber

Publications and source records attributed to L L Bieber.

At least 19 recordsLinked to original sources
PubMed1992

Conferral of malonyl coenzyme A sensitivity to purified rat heart mitochondrial carnitine palmitoyltransferase.

An immunoaffinity column against the 86-kDa malonyl-CoA-binding protein of beef heart mitochondria was prepared, and the properties of the eluates were compared to those of eluates of an anti-carnitine palmitoyltransferase immunoaffinity column. Both eluates contain seven to eight major proteins with a malonyl-CoA-binding capacity of approximately 5 nmol/mg of protein; in contrast, the eluates from a preimmune IgG column did not contain any of the major proteins. The eluates from both immunoaffinity columns conferred malonyl-CoA sensitivity to purified rat heart mitochondrial carnitine palmitoyltransferase (CPTi/CPT-II). Addition of phospholipids increased the degree of malonyl-CoA inhibition. Doubling the amount of column eluate approximately doubled the malonyl-CoA sensitivity when added to a fixed amount of CPT; i.e., the inhibition increased from 32 to 67%. These results show that CPTi/CPT-II is capable of exhibiting malonyl-CoA sensitivity in the presence of malonyl-CoA-binding proteins. The results do not support the concept that the 86-kDa malonyl-CoA-binding protein is detergent-inactivated carnitine palmitoyltransferase I;rather, they suggest that it is a regulatory subunit of a carnitine palmitoyltransferase complex.

Animals↗
PubMed1992

Effect of etomoxiryl-CoA on different carnitine acyltransferases.

The effects of etomoxiryl-CoA on purified carnitine acyltransferases and on carnitine acyl-transferases of rat heart mitochondria and rat liver microsomes were determined. At nanomolar concentrations, the data agreed with that of other investigators who have shown that etomoxiryl-CoA must be binding to a high affinity site with specific inhibition of mitochondrial carnitine palmitoyltransferase (CPTo). Micromolar amounts of etomoxiryl-CoA inhibited both short- and long-chain carnitine acyltransferases. The concentrations of etomoxiryl-CoA required for 50% inhibition of the different carnitine acetyltransferases and microsomal and peroxisomal carnitine octanoyltransferase were in the low micromolar range. Mixed-type and uncompetitive inhibition kinetics were obtained, depending on the source of purified enzyme. When purified rat heart CPT was incubated with etomoxiryl-CoA, it increased the K0.5 and decreased the Hill coefficient for acyl-CoA. Both proteins and phospholipids of mitochondria and microsomes formed covalent adducts of [3H]etomoxir, with the predominant labeling in phospholipids. None of the purified enzymes formed covalent adducts when incubated with [3H]etomoxiryl-CoA, in contrast to intact mitochondria or microsomes. The major 3H-labeled protein for rat heart mitochondria had a molecular weight of 81,000 +/- 4000, and the major proteins from microsomes had a molecular weight of 51,000-57,000. Malonyl-CoA prevented most of the tritum incorporation into the 81,000 Da protein of mitochondria, but it had little effect on incorporation of tritiated etomoxir into the 51,000-57,000 Da proteins of microsomes. When 50 microM etomoxiryl-CoA was added to microsomes and to mitochondria that had been incubated with radioactive etomoxiryl-CoA, much of the radioactive etomoxir disappeared from the major microsomal proteins, but virtually none was displaced from the mitochondrial protein. Thus, at least two different types of covalent etomoxir complexes were formed. This pulse-chase experiment showed that the mitochondrial protein-etomoxir complex was not turned over, consistent with other data showing that etomoxir inhibited carnitine palmitoyltransferase. In contrast, the major protein-etomoxir complex in microsomes was turned over during the pulse-chase experiment.

Adenosine Triphosphate↗
PubMed1992

Purification of the medium-chain/long-chain (COT/CPT) carnitine acyltransferase of rat liver microsomes.

A procedure for the purification of the rat liver microsomal carnitine octanoyltransferase (COT) that catalyzes the reversible formation of medium-chain and long-chain acylcarnitines from acyl-coenzyme A is described. The K0.5 for L-carnitine is 0.6 mM and the K0.5 for both decanoyl-CoA and palmitoyl-CoA is 0.6 microM. The Vmax with decanoyl-CoA is approximately fourfold greater than the Vmax with palmitoyl-CoA. The enzyme is monomeric, sodium dodecyl sulfate-polyacrylamide gel electrophoresis gives a molecular weight of 50,100, and molecular sieving gives a molecular weight of 54,300. Purified COT does not cross-react with either antimitochondrial carnitine palmitoyltransferase or antiperoxisomal COT antibodies. It also does not form a covalent adduct when incubated with etomoxiryl-CoA. Microsomal COT is a different protein than either mitochondrial carnitine palmitoyltransferase or peroxisomal COT.

Acyl Coenzyme A↗
PubMed1991

Analysis of acylcarnitines as their N-demethylated ester derivatives by gas chromatography-chemical ionization mass spectrometry.

A novel approach to the analysis of acylcarnitines has been developed. It involves a direct esterification using propyl chloroformate in aqueous propanol followed by ion-pair extraction with potassium iodide into chloroform and subsequent on-column N-demethylation of the resulting acylcarnitine propyl ester iodides. The products, acyl N-demethylcarnitine propyl esters, are volatile and are easily analyzed by gas chromatography-chemical ionization mass spectrometry. For medium-chain-length (C4-C12) acylcarnitine standards, detection limits are demonstrated to be well below 1 ng starting material using selected ion monitoring. Well-separated gas chromatographic peaks and structure-specific mass spectra are obtained with samples of synthetic and biological origin. Seven acylcarnitines have been characterized in the urine of a patient suffering from medium-chain acyl-CoA dehydrogenase deficiency.

Acyl-CoA Dehydrogenase↗
PubMed1991

Cardiac carnitine acyltransferase activities in exercised normal and dystrophic hamsters.

Carnitine acyltransferase activities in the hearts of normal and dystrophic, sedentary and swim exercised hamsters were studied, in order to analyze the relationship between carnitine metabolism and exercise in cardiomyopathy. After 12 weeks, the mean specific activities of cardiac carnitine acetyltransferase (CAT), carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) were significantly higher in the dystrophic sedentary group, relative to the normal sedentary group (p less than 0.05). There was no significant effect of exercise on the mean specific activity of the carnitine acyltransferases, compared to the dystrophic or normal sedentary controls. Thus, the improvements in cardiac histopathology due to exercise noted previously are not associated with altered carnitine acyltransferase activity.

Animals↗
PubMed1990

The medium-chain carnitine acyltransferase activity associated with rat liver microsomes is malonyl-CoA sensitive.

The data presented herein show that both rough and smooth endoplasmic reticulum contain a medium-chain/long-chain carnitine acyltransferase, designated as COT, that is strongly inhibited by malonyl-CoA. The average percentage inhibition by 17 microM malonyl-CoA for 25 preparations is 87.4 +/- 11.7, with nine preparations showing 100% inhibition; the concentrations of decanoyl-CoA and L-carnitine were 17 microM and 1.7 mM, respectively. The concentration of malonyl-CoA required for 50% inhibition is 5.3 microM. The microsomal medium-chain/long-chain carnitine acyltransferase is also strongly inhibited by etomoxiryl-CoA, with 0.6 microM etomoxiryl-CoA producing 50% inhibition. Although palmitoyl-CoA is a substrate at low concentrations, the enzyme is strongly inhibited by high concentrations of palmitoyl-CoA; 50% inhibition is produced by 11 microM palmitoyl-CoA. The microsomal medium-chain/long-chain carnitine acyltransferase is stable to freezing at -70 degrees C, but it is labile in Triton X-100 and octylglucoside. The inhibition by palmitoyl-CoA and the approximate 200-fold higher I50 for etomoxiryl-CoA clearly distinguish this enzyme from the outer form of mitochondrial carnitine palmitoyltransferase. The microsomal medium-chain/long-chain carnitine acyltransferase is not inhibited by antibody prepared against mitochondrial carnitine palmitoyltransferase, and it is only slightly inhibited by antibody prepared against peroxisomal carnitine octanoyltransferase. When purified peroxisomal enzyme is mixed with equal amounts of microsomal activity and the mixture is incubated with the antibody prepared against the peroxisomal enzyme, the amount of carnitine octanoyltransferase precipitated is equal to all of the peroxisomal carnitine octanoyltransferase plus a small amount of the microsomal activity. This demonstrates that the microsomal enzyme is antigenically different than either of the other liver carnitine acyltransferases that show medium-chain/long-chain transferase activity. These results indicate that medium-chain and long-chain acyl-CoA conversion to acylcarnitines by microsomes in the cytosolic compartment is also modulated by malonyl-CoA.

Acyl Coenzyme A↗
PubMed1990

Differential excretion of xenobiotic acyl-esters of carnitine due to administration of pivampicillin and valproate.

The fate of supplemental carnitine was studied in human subjects treated with drugs known to cause carnitine deficiency. Six children were treated with pivampicillin and equimolar L-carnitine for 7 days. On the last day of treatment, the plasma levels of total and free carnitine were decreased, but acylcarnitine levels were increased. A 12-fold increase in urinary excretion of acylcarnitines was found; it increased from 188.5 +/- 82.7 to 2218.4 +/- 484.1 mumole/day, and 84% was pivaloylcarnitine. Free carnitine excretion was reduced. Ten epileptic children on chronic valproate treatment received equimolar carnitine for a 2-week period. Plasma carnitine levels were elevated on the last day of treatment. A 3.4-fold increase in urinary acylcarnitines was found, but most of the excreted carnitines were free (64.5-fold increases). These data show that pivalate is readily converted to carnitine esters, in contrast to the limited conversion of valproate to acylcarnitines in humans.

Adolescent↗
PubMed1990

Carnitine ester excretion in pediatric patients receiving parenteral nutrition.

Carnitine plasma concentrations and the excretion of carnitine and individual carnitine esters were determined in 25 children and adolescents with gastrointestinal diseases receiving carnitine-free parenteral nutrition for at least 1 mo using radiochemical and radioisotopic exchange HPLC methods. Children less than 12-y-old usually had carnitine plasma concentrations less than -2 SD from the normal mean for age, whereas patients greater than 12-y-old had carnitine plasma concentrations within the normal range. Age was the only variable to correlate significantly with plasma carnitine concentrations during parenteral nutrition. Free carnitine (FC) excretion was closely correlated with plasma FC concentrations and minimal at values less than 25 mumols/L. The excretion of FC and short-chain acylcarnitines was reduced by an order of magnitude in younger compared with older patients and controls, but the excretion of "other" acylcarnitines was less affected. Some of the latter were tentatively identified using gas-liquid chromatographic and mass spectroscopic techniques as unsaturated and/or branched medium-chain carnitine esters with a carbon chain of C8-C10. The results suggest that FC and short-chain acylcarnitine are conserved by the kidney in nutritional carnitine deficiency but that there may be an obligatory renal excretion of other carnitine esters that contributes to the development of hypocarnitinemia in the younger age group.

Absorption↗
PubMed1989

Carnitine and acylcarnitine levels of human peripheral blood lymphocytes and mononuclear phagocytes.

Lymphocytes and mononuclear phagocytes are essential to host defense, yet little is known about their metabolic requirements. To determine the involvement of carnitine in the intermediary metabolism of these cells, the amounts of free carnitine and individual acylcarnitines were determined for human peripheral blood lymphocytes and mononuclear phagocytes. Lymphocytes from healthy young adults contain 79 +/- 6 and 56 +/- 5 nmol/10(9) cells total and free carnitine, respectively, showing a 29% acylation. When expressed per mg cell protein, they contain 3.41 +/- 0.54 nmol total and 2.44 +/- 0.40 nmol free carnitine. By comparison, phagocytes contained approximately 4-fold more total carnitine per cell (301 nmol/10(9) cells) and had a much higher level of acylation (69%). Acetylcarnitine was the predominant acylcarnitine in both lymphocytes (15.4 nmol/10(9) cells) and phagocytes (98.3 nmol/10(9) cells) accounting for 72-73% of total acylcarnitines. Long-chain acylcarnitines constituted 3-4% of total acylcarnitines. These data suggest that carnitine is involved in the metabolism of both short and long-chain acyl-CoAs within lymphocytes and phagocytes, but its specific roles in these two cell types have not been determined.

Acetylcarnitine↗
PubMed1989

Quantitation of urinary carnitine esters in a patient with medium-chain acyl-coenzyme A dehydrogenase deficiency: effect of metabolic state and L-carnitine therapy.

Urinary carnitine esters were quantitated in an infant with medium-chain acylcoenzyme A dehydrogenase deficiency by means of a highly sensitive and specific radioisotopic exchange high-pressure liquid chromatography method. During fasting, the excretion of free carnitine and of acetylcarnitine, octanoylcarnitine, and hexanoylcarnitine was increased. The fractional tubular reabsorption of free carnitine was decreased, suggesting a renal leak of free carnitine. In the symptom-free, fed state, only minor amounts of free carnitine and of short-chain acylcarnitine, octanoylcarnitine, and hexanoylcarnitine were present in urine, and carnitine loss occurred in the form of "other" carnitine esters not exceeding that of control subjects. During L-carnitine therapy, the excretion of free carnitine, short-chain acylcarnitine, octanoylcarnitine, and hexanoylcarnitine, and particularly of "other" carnitine esters, was increased, suggesting a possible detoxifying effect of administered carnitine that is not confined to the elimination of octanoic and hexanoic acids. The employed method detects very low urinary concentrations of octanoylcarnitine and hexanoylcarnitine (less than 1 mumol/L) characteristic of medium-chain acyl-coenzyme A dehydrogenase deficiency and may be useful in screening for this disease, which has been associated with sudden infant death.

Acetylcarnitine↗
PubMed1988

Quantitation of the effect of L-carnitine on the levels of acid-soluble short-chain acyl-CoA and CoASH in rat heart and liver mitochondria.

The steady state levels of mitochondrial acyl-CoAs produced during the oxidation of pyruvate, alpha-ketoisovalerate, alpha-ketoisocaproate, and octanoate during state 3 and state 4 respiration by rat heart and liver mitochondria were determined. Addition of carnitine lowered the amounts of individual short-chain acyl-CoAs and increased CoASH in a manner that was both tissue- and substrate-dependent. The largest effects were on acetyl-CoA derived from pyruvate in heart mitochondria using either state 3 or state 4 oxidative conditions. Carnitine greatly reduced the amounts of propionyl-CoA derived from alpha-ketoisovalerate, while smaller effects were obtained on the branched-chain acyl-CoA levels, consistent with the latter acyl moieties being poorer substrates for carnitine acetyltransferase and also poorer substrates for the carnitine/acylcarnitine translocase. The levels of acetyl-CoA in heart and liver mitochondria oxidizing octanoate during state 3 respiration were lower than those obtained with pyruvate. The rate of acetylcarnitine efflux from heart mitochondria during state 3 (with pyruvate or octanoate as substrate, in the presence or absence of malate with 0.2 mM carnitine) shows a linear response to the acetyl-CoA/CoASH ratio generated in the absence of carnitine. This relationship is different for liver mitochondria. These data demonstrate that carnitine can modulate the aliphatic short-chain acyl-CoA/CoA ratio in heart and liver mitochondria and indicate that the degree of modulation varies with the aliphatic acyl moiety.

Acyl Coenzyme A↗
PubMed1988

Enzymes of carnitine acylation. Is overt carnitine palmitoyltransferase of liver peroxisomal carnitine octanoyltransferase?

Liver mitochondria prepared by differential centrifugation are contaminated by significant quantities of peroxisomes and microsomal fractions. 'Easily solubilized carnitine palmitoyltransferase' prepared from liver mitochondria is thought to originate from the outer surface of the mitochondrial inner membrane. We have characterized the carnitine palmitoyltransferase activities of freeze-thaw extracts of rat liver mitochondrial preparations. Chromatography on Sephadex G-100 yields two broad peaks of carnitine decanoyltransferase activity: one eluted at the end of the void volume, which can be removed (precipitated) by ultracentrifugation; the second peak represents the soluble activity and is eluted at an Mr near 70,000. The activity in the soluble peak is precipitated by an antibody raised against carnitine octanoyltransferase purified from mouse liver peroxisomes. In contrast, antibody raised against carnitine palmitoyltransferase purified from liver mitochondrial membranes had no effect (P. Brady & L. Brady, personal communication). The carnitine acyltransferase activities of the Mr-70,000 peak in the presence or absence of Tween 20 showed maximum activity with decanoyl-CoA and about one-third of this activity with palmitoyl-CoA, similar to peroxisomal carnitine octanoyltransferase. These data show that 7500 g preparations of liver mitochondria isolated by differential centrifugation are enriched by peroxisomal carnitine octanoyltransferase (approx. 20% of the protein of the fraction is peroxisomal) and indicate that this enzyme may be the one reported as 'overt' or 'easily solubilized' mitochondrial carnitine palmitoyltransferase.

Acylation↗
PubMed1988

Metabolic interaction between skeletal muscle and liver during bacteremia.

To study the effects of bacteremia on skeletal muscle leucine (LEU) metabolism, mongrel dogs were infused with normal saline or Escherichia coli (10(9)/kg). After a bolus dose (3.6 microCi), L(1-carbon 14) LEU (0.3 microCi/min) was infused directly into the isolated, constant-flow, in vivo gracilis muscle. Arteriovenous differences for amino acids, labeled and unlabeled LEU and alpha-ketoisocaproic acid (KIC), and labeled carbon dioxide were measured at ten-minute intervals for one hour. Bacteremia increased the net release of amino acids and total N2 from muscle. Moreover, plasma LEU that was deaminated and released as KIC was increased, and there was also an increase in decarboxylated plasma LEU during bacteremia. Despite the marked increase in KIC release from skeletal muscle during bacteremia, arterial concentrations were not significantly different from those of controls. An unchanged arterial plasma KIC concentration associated with a marked increase in KIC released from skeletal muscle indicates an increase in LEU metabolism, most likely in the liver. Thus, the increased skeletal muscle catabolism is not a futile cycle but rather an essential event to meet the increased metabolic needs of the body during bacteremia.

Amino Acids, Branched-Chain↗
PubMed1988

Effects of octylglucoside and triton X-100 on the kinetics and specificity of carnitine palmitoyltransferase.

The effects of octylglucoside on the substrate specificity, kinetics and aggregation state of purified carnitine palmitoyltransferase (CPT) from beef heart mitochondria were investigated and compared to the effects of Triton X-100. Conditions in which CPT can be assayed in the absence of micelles and albumin, thereby eliminating miceller effects on the kinetic parameters, are described. When octylglucoside is substituted for Triton X-100, the specificity of CPT in the forward direction shifts towards the long-chain acyl-CoAs, and large changes in the kinetic constants are observed. The K0.5 for L-carnitine varied as much as 50-fold, depending on the acyl-CoA and detergent used. At pH 8.0 and 200 microM palmitoyl-CoA, the K0.5 for L-carnitine is 4.9 mM in 12 mM octylglucoside and 0.2 mM in 0.1% Triton X-100. Octylglucoside enhances the activity of CPT with long-chain acyl-CoA and lowers the K0.5 for these substrates. At pH 6.0, the K0.5 for palmitoyl-CoA is 24.2 microM in 0.1% Triton X-100, in contrast to 3.1 microM in 12 mM octylglucoside. Octylglucoside is a competitive inhibitor of CPT with octanoyl-CoA as substrate with a Ki of 15 mM. Nonlinear kinetics for both acyl-CoAs and L-carnitine are observed when the concentration of octylglucoside is reduced to less than half of its critical micellar concentration (cmc). Gel filtration of CPT in octylglucoside below its cmc gives a single protein peak with a molecular mass of ca. 660,000 daltons.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl Coenzyme A↗