Substrate specificities of the membrane-bound and partially purified microsomal acyl-CoA:1-acylglycerol-3-phosphate acyltransferase from etiolated shoots of Pisum sativum (L.) |
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Authors: | Wolfgang Hares Margrit Frentzen |
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Institution: | (1) Institut für Allgemeine Botanik, Universität Hamburg, Ohnhorststrasse 18, 52 Hamburg, Federal Republic of Germany |
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Abstract: | Membrane fractions enriched in rough endoplasmic reticulum and not contaminated with plastidial membranes were isolated from etiolated shoots of Pisum sativum (L.). From these fractions the acyl-CoA:1-acyl-sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.51) was solubilized by extracting the membranes with the zwitterionic detergent 3-(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate at high ionic strength. The subsequent separation of the solubilized fractions on a Mono Q column resulted in a tenfold enriched enzymic activity, which could be stabilized by polyethyleneglycol precipitation. A comparison of the substrate specificities and selectivities of the solubilized, enriched 1-acylglycerol-3-phosphate acyltransferase and the corresponding membrane-bound activity revealed no appreciable difference. Both enzymic forms specifically utilized acyl-CoA thioesters as acyl donors whereas the corresponding acyl-acyl carrier protein thioesters were not used. Furthermore, the membrane-bound as well as the solubilized enriched form showed not only higher activities with 1-oleoylthan with 1-palmitoylglycerol-3-phosphate but also pronounced specificities and selectivities for unsaturated C18-CoA thioesters. Hence, the extraplastidial 1-acylglycerol-3-phosphate acyltransferase which catalyses the formation of phosphatidic acid with an eukaryotic fatty-acid pattern was partially purified.Abbreviations ACP
acyl carrier protein
- CHAPS
3-(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate
- LPA-AT
acyl-CoA:1-acylglycerol-3-phosphate acyltransferase
- PEG
polyethyleneglycol
The authors are grateful to the Deutsche Forschungsgemeinschaft for financial support. We wish to thank Miss Ute Hammer for the analysis of the lipid composition of the microsomal fractions. |
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Keywords: | Acyl-CoA:1-acylglycerol-3-phosphate acyl-transferase Endoplasmic reticulum Eukaryotic fatty acid pattern Membrane protein purification Pisum |
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