Characterization of rat brain microsomal acyl-coenzyme A ligases: different enzymes for the synthesis of palmitoyl-coenzyme A and lignoceroyl-coenzyme A |
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| Authors: | A Bhushan R P Singh I Singh |
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| Affiliation: | 3. LIMES-Institute, Program Unit Development, Genetics and Molecular Physiology, Molecular Developmental Biology, University of Bonn, Carl-Troll-Strasse 31, Bonn, Germany;4. IMBIO, Molecular Biotechnology, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany;1. Department of Biology, Indian Institute of Science Education and Research (IISER) Tirupati, Tirupati 517507, Andhra Pradesh, India;1. Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA;2. Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 76100, Israel;3. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA;1. Key Laboratory of Bio-Resources and Eco-Environment of the Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, PR China;2. Department of Medical Technology, Affiliated Hospital, Medical College, Hebei University of Engineering, Handan 056002, PR China |
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| Abstract: | Palmitic acid solubilized with Triton WR-1339 was converted to palmitoyl-CoA by microsomal membranes but lignoceric acid solubilized with Triton WR-1339 was not an effective substrate even though the detergent dispersed the same amount of these fatty acids and was also not inhibitory to the enzyme [I. Singh, R. P. Singh, A. Bhushan, and A. K. Singh (1985) Arch. Biochem. Biophys. 236, 418-426]. This observation suggested that palmitoyl-CoA and lignoceroyl-CoA may be synthesized by two different enzymes. We have solubilized the acyl-CoA ligase activities for palmitic and lignoceric acid of rat brain microsomal membranes with Triton X-100 and resolved them into three separate peaks (fractions) by hydroxylapatite chromatography. Fraction A (palmitoyl-CoA ligase) had high specific activity for palmitic acid and Fraction C (lignoceroyl-CoA ligase) for lignoceric acid. Specific activity of palmitoyl-CoA ligase for palmitic acid was six times higher than in Fraction C and specific activity of lignoceroyl-CoA ligase for lignoceric acid was four times higher than in Fraction A. At higher concentrations of Triton X-100 (0.5%), lignoceroyl-CoA ligase loses activity whereas palmitoyl-CoA ligase does not. Lignoceroyl-CoA ligase lost 60% of activity at 0.6% Triton X-100. Palmitoyl-CoA ligase (T1/2 of 4.5 min) is more stable at 40 degrees C than lignoceroyl-CoA ligase (T1/2 of 1.5 min). The pH optimum of palmitoyl-CoA ligase was 7.7 and that of lignoceroyl-CoA ligase was 8.4. Similar to our results with intact membranes, palmitic acid solubilized with Triton WR-1339 was converted to palmitoyl-CoA by palmitoyl-CoA ligase whereas lignoceric acid when solubilized with Triton WR-1339 was not able to act as substrate for lignoceroyl-CoA ligase. Since solubilized enzyme activities for synthesis of palmitoyl-CoA and lignoceroyl-CoA from microsomal membranes can be resolved into different fractions by column chromatography and demonstrate different properties, we suggest that in microsomal membranes palmitoyl-CoA and lignoceroyl-CoA are synthesized by two different enzymes. |
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