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Bee venom is known to contain high amounts of phosphatidase A and to cause degranulation and histamine release in rat mast cells. In the present study bee venom was fractionated on Sephadex G 50, whereby the phosphatidase A (Fraction F I) was separated from the histamine releasing activity (Fratcion F II). Various aspects of the F II-induced histamine release from rat peritoneal mast cells were studied, namely the dose-response curve, the time course of the reaction, the pH and temperature dependency, and the influence of inhibitors. It is concluded that this bee venom fraction acts by a mechanism similar to that of compound 48/80. This is in contrast to findings reported for melittin, a histamine releasing bee venom fraction described by other authors.

Summary The part played in the mechanism of haemolysis by the lysophosphatides and the fatty acids is reviewed. The following hypothesis is put forward: the haemolysis might be due under certain physiological and pathological circumstances to an increase of the lysophosphatide/phosphatide ratio within the erythrocyte membrane. Three main mechanisms may produce haemolysis: 1. an alteration of the erythrocyte metabolism, 2. the activation of the plasmatic phosphatidase A and 3. the activation of complement at the cell surface. Several arguments in favour of the role of the above mechanisms in haemolytic conditions are presented. Resume Le role joue par les lysophosphatides et les acides gras dans les mecanismes de I'hemolyse est etudie a la lumiere des donnees de la litterature. L'hypothese suivante est proposee et discutee: dans diverses conditions physiologiques et pathologiques, I'hemolyse est conditionnee par une augmentation du rapport lysophosphatides-phosphatides dans la membrane erythrocytaire; celle-ci peut etre le resultat de trois mecanismes principaux: 1. une alteration intrinseque du metabolisme erythrocytaire, 2. une activation de la phosphatidase A plasmatique et 3. une activation du complement a la surface erythrocytaire. Les arguments plaidant pour l'intervention de ces mecanismes dans les etats hemolytiques sont passes en revue. Zusammenfassung An Hand der Literatur wird die Bedeutung der Lysophosphatide und der Fettsauren bei der Hamolyse untersucht. Folgende Hypothese wird zur Diskussion gestellt: Wesentlich fur das Zustandekommen der Hamolyse unter physiologischen und pathologischen Bedingungen ist eine Zunahme des Lysophosphatid Phosphatidverhaltnisses in der Erythrozytenmembran. Die Verschiebung dieses Verhalt-nisses ist die Folge von drei Mechanismen: 1. einer Veranderung des Erythrozytenstoffwechsels, 2. einer Aktivierung der plasmatischen Phosphatidase A und 3. einer Komplementaktivierung an der Erythrozytenoberflache. Die Argumente, die fur und wider diese Hypothese sprechen werden eingehend dargelegt.

A study was made of the action of phosphatidase D (phospholipase D) from Clostridium perfringens (C. welchii) on five constituents of the yolk of hens' eggs (α- and β-lipovitellin, the low-density lipoprotein fraction, granules, and lecithin) in aqueous solution, in the absence and presence of chloroform. Although the untreated lipovitellins were little affected by the enzyme, after absorption of chloroform, a large proportion of their phospholipids was hydrolyzed. In this respect they resembled lecithin but differed from the low-density yolk lipoproteins, whose phospholipids were rapidly hydrolyzed by the enzyme in the presence or absence of chloroform. The mechanism of this type of chloroform activation is discussed.

Bee and snake venoms containing phosphatidase A are known to cause degranulation of mast cells and histamine release. This effect has been ascribed to their phosphatidase content. Recent studies have shown that a factor in bee venom, not related to phosphatidase A, releases histamine from rat peritoneal mast cells. In the present study semipurified bee venom phosphatidase A was subjected to separation by gel filtration and the fractions tested for degranulating effect on rat mesentery mast cells. The results show that this effect was due to a factor which was not phosphatidase A and not identical with the earlier demonstrated histamine releasing principle in bee venom.

Intravenous injection of snake venom phosphatidase A into rabbits produced rapid and marked changes in the plasma phospholipids. Five to fifteen minutes after injection there was a marked decrease in plasma lecithin and an increase in plasma lysolecithin. At the same time red blood cell sphering had occurred and lysolecithin was found attached to the red blood cells. Within 2 hr after injection the plasma lysolecithin dropped toward normal, whereas plasma lecithin remained low. The decrease in plasma lysolecithin was paralleled by reversion of the spheric red cell shape to normal bidiscoid. Evidence was obtained for disappearance of plasma phospholipids from the blood stream after phosphatidase injection.

Two bee venom fractions, F I and F II, obtained by gel filtration, and compound 48/80, were shown to release histamine from skin and lung tissue of the rat. F I is the phosphatidase A containing fraction, and F II contains a basic polypeptide. Dose-response relationships, the time course of the histamine release, and the influence of enzyme inhibitors were studied. The results with F II and compound 48/80 were similar, suggesting that the two substances activate the same kind of release mechanism. It is concluded that F II is not related to the earlier described bee venom polypeptides melittin and aparnin. The mode of action of phosphatidase A was distinctly different from that of F II or compound 48/80. The results are consistent with the assumption that it acts in a “non-specific.” way by hydrolysing tissue phosphatides to lyso compounds, which in turn damage the tissue.

Summary In a controlled (but not blind) trial anti-snake-venom serum alleviated asthma in 45 out of 60 cases. In 34 cases success was obtained within 3 to 8 days and in the remaining 11 cases in 2 to 6 weeks. The period during which the patients remained free from asthmatic attacks varied from 1 to 12 months. During the remainder of the expected asthmatic period the attacks were less frequent, and, as judged by the incidence of status asthmaticus and need for adrenaline and cortisone, milder than in the pre-treatment year. Only 3 of 15 unsuccessful cases benefited from prednisolone. Mild serum reactions were confined to 7 partially successful or unsuccessful cases. The good effect of anti-snake-venom serum is ascribed mainly to its antiphosphatidase A activity. This suggests the possibility of treating bronchial asthma with antiphosphatidase A or the specific inhibitor of phosphatidase A.

Mast cell granules, obtained from sonically disintegrated rat peritioneal cells and suspended in sucrose, 0.34 M, were found to release histamine in the presence of bee venom. Three venom fractions were obtained by gel filtration; two of these had strong histamine releasing capacity, namely “F I” containing phosphatidase A, and “F III” with direct hemolytic properties. The third fraction, “F II”, which releases histamine from intact mast cells, had little effect on granules. Various possibilities for the release mechanism, namely phosphatidase activity, surface activity, and cation exchange, are discussed.

Release of histamine and formation of smooth-muscle stimulating principles (spasmogenic lipids) occurred when minced guinea-pig lung tissue was exposed to antigen or to bee venom phosphatidase A. In agreement with earlier reports the anaphylactic histamine release was a rapid process, and it could be inhibited by anoxia in the absence of glucose, and by potassium cyanide, ninhydrin and A’-ethyl-maleimide. The histamine release induced by phosphatidase A was not influenced by these inhibitors or by anoxia, and the release was slower than in anaphylaxis. The spasmogenic lipids formed had similar chromatographic and biological properties to those reported for the corresponding principles formed in anaphylaxis, and the process of formation was inhibited by oxygen lack and by the above enzyme inhibitors. One noteworthy feature of the spasmogenic lipid formation was that the inhibition caused by anoxia could not be counteracted by glucose. The results support earlier hypotheses regarding the mode of action of phosphatidase A on guinea-pig lung tissue. It is suggested that mast cells are not necessarily involved in the formation of spasmogenic lipids occurring in connection with histamine release induced by phosphatidase A in the guinea-pig lung. The bee venom fraction F II, which liberates histamine from rat tissues by a mechanism resembling that of compound 48/80, did not cause release of histamine or formation of spasmogenic lipids from the guinea-pig lung tissue in concentrations up to 100 mug/ml.

Received February 1, 1961 After the finding of MacFarlane and Knight (1941) that the hemolytio factor of C.welchii a-toxin exhibits phosphatidase activity, various specificity characteristics of this enzyme (phosphatidase D) have been described (MacFarlane, 1948; Zamecnik et al., 1947). As regards the ability of phosphatidase D to liberate phosphoryl choline from saturated lecithins, opinions appear to differ. Hanahan and Veroamer (1954) observed that in an etherethanol medium both a fully saturated and an unsaturated lecithin were completely hydrolysed, which fact was also found by Meduski et al. (1956) in an aqueous medium. On the other hand, Long and Maguire (1954) reported that in the latter medium saturated lecithins resisted phosphatidase D action. In the course of an investigation into the susoeptibility of red cells from different animal species against hemolytic agents, it appeared necessary to obtain additional information on the substrate specificity of phosphatidase D. Beside variations in the phosphatide composition we found the fatty acid pattern of glycero-phosphatides from red cell membranes to vary greatly between various species of mammals (Kagl et al. 1960a). For this reason the action of phosphatidase D was studied on a series of synthetic lecithins carrying very different fatty

Determination of enzyme activities and starch-gel electrophoretic fractionation at pH 8·6 were carried out on fresh venom from 116 fer-de-lances, Bothrops atrox , captured in 15 localities of all ecological zones of Costa Rica and kept in captivity for several months. Fractions containing chymotryptic and thrombic activities were the most toxic, whereas L-amino acid dehydrogenase, phosphatidase A and various phosphatases were much less toxic. Geographic variations, probably due to topographic barriers, were exemplified by electrophoretically polymorphic L-amino acid dehydrogenase and a trypsin-like enzyme with multiple action on blood clotting. Although the variable proteins were present in venoms of all geographic areas, fer-de-lance populations from the Atlantic and Pacific zones could be distinguished from each other by the frequency of occurrence of certain forms of these proteins and by the degree of activity of several venom enzymes. Biochemical individuality was shown by extensive individual differences within single populations. These intraspecific variations are of genetic origin since they are not correlated with size of the animals, duration of their captivity, fasting or venom storage before the analysis. Biochemical properties are more sensitive than morphological characteristics to show differences at the species level.

WE reported recently that a fraction of venom of the Australian tiger snake (Notechis scutatus scutatus), rich in haemolysin, protects mice against both the dermonecrotic and lethal action of staphylococcal alpha toxin. Such an effect is observed when this fraction of the venom is injected either together with the toxin or separately1.