Biosynthesis of Spin-Labelled Lipids and Their Use in the Study of Biological Membranes: Isolation and Characterization of Membrane-Bound Spin-Labelled sn-3-Phosphatidic Acid-2-3H Prepared by Enzymatic Incorporation of Spin-Labelled Stearic Acid into sn-Glycero-3-phosphate-2-3H

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Abstract
Spin-labelled 12-doxylstearic acid was covalently incorporated into sn-glycero-3-phosphate-2-3H yielding sn-3-phosphatidic acid-2-3H, a key intermediate in phospholipid biosynthesis, in a reaction catalyzed by isolated guinea-pig liver microsomes. It was found that at least 17.1% of the obtained sn-3-phosphatidic acid-2-3H contained spin label. Degradation of spin-labelled sn-3-phosphatidic acid-2-3H with the phospholipase A from Crotalus adamanteus yielded both lyso-sn-3-phosphatidic acid-2-3H and free fatty acids which were paramagnetic, establishing that the incorporated 12-doxylstearic acid occupies both sn-1- and sn-2-positions of the biosynthesized sn-3-phosphatidic acid-2-3H.A procedure for the preparation and isolation of biosynthetically spin-labelled radioactive sn-3-phosphatidic acid bound to the microsomal membrane was developed and the concentrations of spin-label and tritium were quantitatively determined. This procedure is believed to have a general application in the study of biological membranes. The electron spin resonance spectra and their quantitation are given and discussed for the spin-labelled sn-3-phosphatidic acid-2-3H in solution and in the membrane-bound form. Some general requirements for covalent spin labelling of biological membranes are discussed.

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