G. R. Webster

THE DIFFERENTIATION OF PHOSPHOLIPASE A1 AND A2 IN RAT AND HUMAN NERVOUS TISSUES

—Lipid‐free extracts of rat and human brain have been prepared and shown to contain phospholipase A1 and A2 activities and a lysophospholipase. The phospholipase Aj activity has pH optima of 4·2 and 4·6 in rat and human brain, respectively; it can be partially purified and isolated in high yields by dialysing the extracts at low pH. The purified preparations hydrolyse the ester bond at the 1‐position in lecithin, phosphatidyl‐ethanolamine and phosphatidylserine, but have little or no action on triglyceride or cholesterol ester. An assay system for the enzyme is described.

PHOSPHOLIPASE A ACTIVITIES IN NORMAL AND SECTIONED RAT SCIATIC NERVE

Abstract— The phospholipase A1 (EC 3.1.1.32) and A2 (EC 3.1.1.4) activities of rat sciatic nerve homogenates have been studied. With phosphatidylcholine as substrate normal nerve had significant activity of both types at pH 5.0. Substantial increases occurred in nerve undergoing Wallerian degeneration after transection, beginning as early as 2 days after operation and rising to eight times normal values by the second week.

ON THE SITE OF ACTION OF PHOSPHATIDE ACYL-HYDROLASE ACTIVITY OF RAT BRAIN HOMOGENATES ON LECITHIN

Abstract— ‐The hydrolysis of 2‐ [14C]acyl‐labelled and [Me‐3H]choline‐labelled lecithins by rat brain homogenates has been studied. The acyl‐labelled substrate was hydrolysed with the production of both radioactive lysolecithin and radioactive free fatty acid in the proportions of 60 per cent and 40 per cent; traces of labelled mono‐ and diglyceride were also formed.

THE LIBERATION OF ACTIVE ENZYMES FROM BRAIN TISSUE BY LYSOLECITHIN

IN 1951, ORD and THOMPSON described the ability of a number of surface-active agents to ‘clear’ the opaque supernatants obtained by the centrifugation of water homogenates of whole rat brain. In an attempt to purify the true cholinesterase of brain they further showed that a non-ionic surface-active agent, acetyl alcoholpolyoxyethylene condensate, was able to ‘solubilize’ this enzyme without causing any inactivation. GROSSE and TAUBOCK (1942) had earlier briefly reported that a brain brei suspended in physiological saline can be cleared with lysolecithin. A more detailed description of this action of lysolecithin on nervous tissue has recently been given by WEBSTER (1957), who has shown that when uncentrifuged homogenates of whole brain tissue are incubated at 38”c with lysolecithin, a very rapid and almost complete ‘clearing’ of the opaque homogenate occurs; following a 2 hr incubation with 0.007 M-lysokcithin, the optical transmission, using a neutral grey filter, of a 1 : 20 homogenate of rat brain in 0.025 M-sodium bicarbonate is equal to about 80 per cent of the optical transmission of water, while about 60 per cent optical transmission is reached after only 10 min incubation; the optical transmission of the homogenate at zero time was 5 per cent of that of water. In view of this ‘solubilizing’ effect on the greater part of the components of the neurones, glial cells and myelin sheath lipids present in these homogenates, it seemed of interest to determine next what effects, if any, the lysolecithin, and the lysis produced by this compound, might have on the enzymic activities of the original homogenates. WEIL (1930) had earlier studied the effect of cobra venom, now known to contain a phospholipase A , on segments of spinal cord, and MORRISON and ZAMECNIK (1950), on the basis of similar observations, had suggested that if lysolecithin were to be formed inside the nervous system, localized demyelination might be expected to occur. We have therefore, in the first instance, been primarily interested in those enzymes which, if released into solution, might be expected to exert a further destructive action on cclls or cell membranes, or which might interfere with the normal processes of excitation and transmission in unaffected nervous tissue. We have begun by assaying in lysolecithin-cleared preparations the levels of activity of the cholinesterases and of mono-amine oxidase, enzymes which are or may be concerned with the inactivation of transmitter or excitor substances, and of the proteinases, esterases, phosphatases and transaminases, which might be expected, if working in an unco-ordinated fashion, to exert degradative changes on the normal cells. A preliminary account of part of this work has already been published (MARPLES, THOMPSON and WEBSTER, 1957). From the point of view of any possible pathological significance, it would clearly be desirable to know what effect, if any, lysolecithin

ON THE PHOSPHOLIPASE A2 ACTIVITY OF HUMAN CEREBRAL CORTEX

Abstract— Preparations of phospholipase Az have been obtained from human cerebral cortex. The enzyme was extracted from acetone‐dried tissue and purified by heat‐treatment and gel filtration on Sephadex.

PHOSPHOLIPASE A IN HUMAN BRAIN: A1-TYPE AT ALKALINE pH

Abstract— A novel phospholipase A1, present in human cerebral cortex and active at pH 9.25 upon ultrasonicated phosphatidylethanolamine is described. It has been purified 39‐fold from acetone‐dried powders of cortex by ammonium sulphate fractionation at 25% saturation, followed by Sephadex G150 gel filtration. A molecular weight of approx 500,000 has been found by Sepharose 6B gel filtration. The enzyme was slightly stimulated by 2 mM‐Ca2+ and was inhibited by 1 mM‐Hg2+ and by all detergents tested. The enzyme hydrolysed phosphatidylcholine at 18% of the rate for phosphatidylethanolamine, lysophospholipids to a lesser degree and neutral lipids not at all. There appeared to be a preference for fluid phosphatidylethanolamine substrates. The similarities of the enzyme to other phospholipases A1 are discussed.