scholarly journals Importance of albumin binding in the assay for carnitine palmitoyltransferase

1983 ◽  
Vol 216 (2) ◽  
pp. 495-498 ◽  
Author(s):  
K McCormick ◽  
V J Notar-Francesco
Keyword(s):  
Enzyme Activity ◽  
Carnitine Palmitoyltransferase ◽  
Albumin Binding ◽  
Biochemical Modification ◽  
Physiological Relevance ◽  
Chain Acyl ◽  
Absolute Quantity ◽  
Substrate Concentrations ◽  
Long Chain ◽  
Do So

Alterations in the long-chain acyl-CoA binding to albumin in the carnitine palmitoyltransferase (CPT) assay appreciably affect the reaction at commonly used substrate concentrations. Since in the CPT assay the latter are typically well below saturation or Vmax. values, the measured enzyme activity depends on both the absolute quantity of albumin in the CPT assay and any biochemical modification of its binding. The present study verifies the striking dependence of the K0.5 for palmitoyl-CoA on albumin and the misleading ‘activation’ of the enzyme by compounds that also avidly bind to albumin. In assessing the intracellular physiological relevance of any modifier of CPT, the effects of protein binding in the assay assume particular importance. Indeed, any compound that alters CPT activity may do so, not directly, but as an assay artifact changing the free or unbound substrate concentrations.

scholarly journals Effect of long-chain fatty acyl-CoA on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cell

1984 ◽  
Vol 220 (2) ◽  
pp. 371-376 ◽  
Author(s):  
S Soboll ◽  
H J Seitz ◽  
H Sies ◽  
B Ziegler ◽  
R Scholz
Keyword(s):  
Liver Cell ◽  
Adenine Nucleotide ◽  
Intact Cell ◽  
Inhibitory Effect ◽  
Fatty Acyl ◽  
Fed State ◽  
Physiological Relevance ◽  
Chain Acyl ◽  
Long Chain

The effect of long-chain acyl-CoA on subcellular adenine nucleotide systems was studied in the intact liver cell. Long-chain acyl-CoA content was varied by varying the nutritional state (fed and starved states) or by addition of oleate. Starvation led to an increase in the mitochondrial and a decrease in the cytosolic ATP/ADP ratio in liver both in vivo and in the isolated perfused organ as compared with the fed state. The changes were reversed on re-feeding glucose in liver in vivo or on infusion of substrates (glucose, glycerol) in the perfused liver, respectively. Similar changes in mitochondrial and cytosolic ATP/ADP ratios occurred on addition of oleate, but, importantly, not with a short-chain fatty acid such as octanoate. It is concluded that long-chain acyl-CoA exerts an inhibitory effect on mitochondrial adenine nucleotide translocation in the intact cell, as was previously postulated in the literature from data obtained with isolated mitochondria. The physiological relevance with respect to pyruvate metabolism, i.e. regulation of pyruvate carboxylase and pyruvate dehydrogenase by the mitochondrial ATP/ADP ratio, is discussed.

Carnitine palmitoyltransferases in pea leaf chloroplasts: partial purification, location, and properties

2000 ◽  
Vol 78 (3) ◽  
pp. 328-335 ◽  
Author(s):  
Christine Masterson ◽  
Clifford Wood
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Carnitine Palmitoyltransferase ◽  
Chloroplast Envelope ◽  
Partial Purification ◽  
Western Blots ◽  
Chain Acyl ◽  
Long Chain ◽  
Inner Envelope

Carnitine palmitoyltransferase (EC 2.3.1.21), an enzyme that catalyses the reversible transfer of activated long-chain acyl groups between CoASH and L-carnitine, has been confirmed in pea leaf chloroplasts. This enzyme is bound to the chloroplast inner envelope membrane and has two isoforms, one bound to the outside (cytosol side) of the inner envelope and one bound to the inside (stromal side) of the inner envelope. Malonyl CoA inhibited the activity of the outer carnitine palmitoyltransferase, while stimulating the activity of the inner isoform and may be a regulator of these enzymes in vivo. Carnitine palmitoyltransferase was solubilized from the chloroplast envelope by detergent treatment and the two isoforms separated by Q-Sepharose anion exchange chromatography. Both proteins were immunochemically observed by probing Western blots of sodium dodecyl sulfate - polyacrylamide gel electrophoresis gels using an anti-beef heart mitochondrial carnitine palmitoyltransferase polyclonal antibody. The monomeric molecular mass of the protein recognized by this antibody was approximately 20 kDa. This 20-kDa protein also bound3H-carnitine. Both isoforms had broad acyl CoA substrate specificities, but showed increased activity with desaturated long-chain acyl CoAs, exhibiting a preference for linolenoyl CoA. A role for carnitine palmitoyltransferase in the shuttling of fatty acids across the chloroplast envelope is suggested.Key words: Pisum sativum, chloroplasts, carnitine palmitoyltransferase, fatty acid metabolism, eukaryotic pathway, membrane transport.

scholarly journals Very Long-Chain Acyl-CoA Dehydrogenase Deficiency: High Incidence of Detected Patients With Expanded Newborn Screening Program

2021 ◽  
Vol 12 ◽  
Author(s):  
Ziga I. Remec ◽  
Urh Groselj ◽  
Ana Drole Torkar ◽  
Mojca Zerjav Tansek ◽  
Vanja Cuk ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Pilot Study ◽  
Genetic Analysis ◽  
Newborn Screening ◽  
Dehydrogenase Deficiency ◽  
Screening Program ◽  
Dried Blood Spots ◽  
Expanded Newborn Screening ◽  
Chain Acyl ◽  
Long Chain

Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is a rare autosomal recessive disorder of fatty acid metabolism with a variable presentation. The aim of this study was to describe five patients with VLCADD diagnosed through the pilot study and expanded newborn screening (NBS) program that started in 2018 in Slovenia. Four patients were diagnosed through the expanded NBS program with tandem mass spectrometry; one patient was previously diagnosed in a pilot study preceding the NBS implementation. Confirmatory testing consisted of acylcarnitines analysis in dried blood spots, organic acids profiling in urine, genetic analysis of ACADVL gene, and enzyme activity determination in lymphocytes or fibroblasts. Four newborns with specific elevation of acylcarnitines diagnostic for VLCADD and disease-specific acylcarnitines ratios (C14:1, C14, C14:2, C14:1/C2, C14:1/C16) were confirmed with genetic testing: all were compound heterozygotes, two of them had one previously unreported ACDVL gene variant each (NM_000018.3) c.1538C > G; (NP_000009) p.(Ala513Gly) and c.661A > G; p.(Ser221Gly), respectively. In addition, one patient diagnosed in the pilot study also had a specific elevation of acylcarnitines. Subsequent ACDVL genetic analysis confirmed compound heterozygosity. In agreement with the diagnosis, enzyme activity was reduced in five patients tested. In seven other newborns with positive screening results, only single allele variants were found in the ACDVL gene, so the diagnosis was not confirmed. Among these, two variants were novel, c.416T > C and c.1046C > A, respectively (p.Leu139Pro and p.Ala349Glu). In the first 2 years of the expanded NBS program in Slovenia altogether 30,000 newborns were screened. We diagnosed four cases of VLCADD. The estimated VLCADD incidence was 1:7,500 which was much higher than that of the medium-chain acyl-CoA dehydrogenase deficiency (MCADD) cases in the same period. Our study also provided one of the first descriptions of ACADVL variants in Central-Southeastern Europe and reported on 4 novel variants.

scholarly journals Identical subcellular distribution of palmitoyl-CoA and arachidonoyl-CoA synthetase activities in human blood platelets

1989 ◽  
Vol 261 (1) ◽  
pp. 71-76 ◽  
Author(s):  
A M Bakken ◽  
M Farstad
Keyword(s):  
Enzyme Activity ◽  
Arachidonic Acid ◽  
Human Blood ◽  
Blood Platelets ◽  
The Other ◽  
Heat Inactivation ◽  
Tubular System ◽  
Chain Acyl ◽  
Human Blood Platelets ◽  
Long Chain

Fractionation of human blood platelets showed that palmitoyl-CoA synthetase and arachidonoyl-CoA synthetase activities had an identical distribution among subcellular fractions. The activity was highest with arachidonic acid as substrate in all fractions, with an enzyme activity of 50 nmol/min per mg of protein, in a ‘dense-tubular-system’-enriched fraction. The ratio activities with arachidonate and palmitate as substrates was about 1.5 in all fractions. Heat inactivation did not distinguish between arachidonoyl-CoA synthetase and a palmitoyl-CoA synthetase. On the other hand, heat inactivation indicated two pools of long-chain acyl-CoA synthetases: one in a mitochondria- and one in the dense-tubular-system-enriched fraction.

Engineering Arabidopsis long-chain acyl-CoA synthetase 9 variants with enhanced enzyme activity

2019 ◽  
Vol 476 (1) ◽  
pp. 151-164 ◽  
Author(s):  
Yang Xu ◽  
Kristian Mark P. Caldo ◽  
Roman Holic ◽  
Elzbieta Mietkiewska ◽  
Jocelyn Ozga ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Fatty Acid ◽  
Amino Acid ◽  
Amino Acid Residue ◽  
Acid Site ◽  
Amino Acid Site ◽  
Acyl Donor ◽  
Evolutionary Analysis ◽  
Chain Acyl ◽  
Long Chain

Abstract Long-chain acyl-CoA synthetase (LACS, EC 6.2.1.3) catalyzes the ATP-dependent activation of free fatty acid to form acyl-CoA, which, in turn, serves as the major acyl donor for various lipid metabolic pathways. Increasing the size of acyl-CoA pool by enhancing LACS activity appears to be a useful approach to improve the production and modify the composition of fatty acid-derived compounds, such as triacylglycerol. In the present study, we aimed to improve the enzyme activity of Arabidopsis thaliana LACS9 (AtLACS9) by introducing random mutations into its cDNA using error-prone PCR. Two AtLACS9 variants containing multiple amino acid residue substitutions were identified with enhanced enzyme activity. To explore the effect of each amino acid residue substitution, single-site mutants were generated and the amino acid substitutions C207F and D238E were found to be primarily responsible for the increased activity of the two variants. Furthermore, evolutionary analysis revealed that the beneficial amino acid site C207 is conserved among LACS9 from plant eudicots, whereas the other beneficial amino acid site D238 might be under positive selection. Together, our results provide valuable information for the production of LACS variants for applications in the metabolic engineering of lipid biosynthesis in oleaginous organisms.

scholarly journals SIRT3 and SIRT5 Regulate the Enzyme Activity and Cardiolipin Binding of Very Long-Chain Acyl-CoA Dehydrogenase

PLoS ONE ◽  
2015 ◽  
Vol 10 (3) ◽  
pp. e0122297 ◽  
Author(s):  
Yuxun Zhang ◽  
Sivakama S. Bharathi ◽  
Matthew J. Rardin ◽  
Radha Uppala ◽  
Eric Verdin ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Chain Acyl ◽  
Long Chain
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