Isolation of phospholipase a isoenzymes from Najanaja venom and their action on membrane-bound enzymes

Salach, James I.; Turini, Paola; Hauber, J.; Seng, R.; Tisdale, H.D.; Singer, Thomas P.

doi:10.1016/0006-291x(68)90402-6

Cited by 39 publications

(11 citation statements)

References 11 publications

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“…Several reports in the literature suggest that phospholipase A2 acts toward saturated egg phosphatidylcholine or dipalmitoyl phosphatidylcholine at a much slower rate than toward natural egg phosphatidylcholine -as much as 1/30 the rate for some forms of the enzyme (43,52). On the other hand, we found that phospholipase A?…”

Section: Effect Of Thermotropic Phase Transitions On Enzymatic Activitymentioning

confidence: 48%

Interaction of enzymes with mixed micelles of phospholipid and detergent: Analysis of the phospholipase A₂–dipalmitoyl phosphatidylcholine–triton X 100 system

Dennis

1974

J Supramol Struct

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Physical studies on the formation and structure of mixed micelles of the nonionic surfactant Triton X-100 and phospholipids and enzymatic studies on the action of phospholipase A, toward these mixed micelles are presented. Results of nmr intensity, line width, and T I determinations, as well as gel chromatography and centrifugation experiments on the interaction of Triton X-100 with egg and dipalmitoyl phosphatidylcholine, are presented and discussed. The structure of mixed micelles is discussed in terms of a working schematic model which is consistent with the experimental results. Kinetic studies on phospholipase A, (Naja naja) action are then analyzed in terms of this model. The temperature dependence of phospholipase A, action toward dipalmitoyl phosphatidylcholine is considered in terms of the effect of thermotropic phase transitions on mixed micelle formation. The phospholipase A, -dipalmitoyl phosphatidylcholine-Triton X-I00 system is then considered as an artificial model system for studying the effect of lipid phase separations on biological activity.For the last few years, our laboratory has been studying the action of phospholipase A, toward phosphatidylcholine as substrate in mixed micelles with the surfactant Triton X-100, employing largely kinetic techniques to characterize the enzyme (1, 2 ) and nuclear magnetic resonance (nmr) techniques t o characterize the substrate (3-5). Our orientation has been that of attempting t o develop a suitable in vitro system for studying the detailed mechanism of action of enzymes of lipid metabolism; these enzymes normally act on substrates that are part of complex structures rather than ones which are molecularly dispersed (6-8). In addition, these studies provide: a) information on the use of and possible effect of Triton and other surfactants (often referred t o as "detergents") in the assay of enzymes that act on or require lipids (9), b) information about the process of protein solubilization, since mixed micelles are formed when surfactants are employed 682 0 1974 Alan R. Liss, Inc.,

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Section: Effect Of Thermotropic Phase Transitions On Enzymatic Activitymentioning

confidence: 48%

Interaction of enzymes with mixed micelles of phospholipid and detergent: Analysis of the phospholipase A₂–dipalmitoyl phosphatidylcholine–triton X 100 system

Dennis

1974

J Supramol Struct

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“…Increasing the amount of enzyme and/or time of incubation did not result in an appreciably higher degradation ratio. The phospholipase A2 activity found in crude N. naja venom, however, is known not to be due to the presence of one single compound, but rather to a complex mixture of various isoenzymes [12]. Since the molecular weights of those isoenzymes do not differ very much (if at all) from one another, they cannot be separated by ordinary gel filtration.…”

Section: Discussionmentioning

confidence: 99%

The action of cobra venom phospholipase A2 isoenzymes towards intact human erythrocytes

Roelofsen

Trip

Verheij

et al. 1980

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Summary1. Cobra venom phospholipase A2 from three different sources has been fractionated into different isoenzymes by DEAE ion-exchange chromatography. 2. Treatment of intact human erythrocytes with the various isoenzymes revealed significant differences in the degree of phosphatidylcholine hydrolysis in those cells. 3. It is argued that the plateaus observed in dose-response curves for such treatments may be caused by an increase in lateral surface pressure within the outer half of the membrane due to the production of free fatty acids and lyso-compounds.Phospholipases A2 (EC 3.1.1.4) purified from snake venoms (in particular from Naja naja venom) have been widely used as tools to study the disposition of phospholipids in biological membranes (for a recent review see Ref. 1). These enzymes have been particularly useful in elucidating the localization of phospholipids in the erythrocyte membrane [2], and the results thus obtained for the localization of phosphatidylcholine in that mem. brane could be quantitatively confirmed by studies using phosphatidylcholine exchange protein [3,4]. The reason why phospholipases could successfully be applied became apparent only recently, when analyses involving 3'P-NMR showed that even after extensive phospholipase treatments of the membrane, the residual phospholipids and products of hydrolysis remain in a bilayer configuration [5]. Indeed, fatty acids and lysophosphatidylcholine associate to form bilayers [6]. While the reliability of the results and the conclusions drawn from the above studies have been questioned by Martin et

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“…Addition of 50 mM EDTA in the presence of 10 mM Ca++ also abolished activity. Strontium was not able to substitute for (23). Table 3 shows ,B-bungarotoxin to possess one-third the phospholipase activity of V. russellii enzyme but to be twice as potent as N. naja enzyme.…”

Section: Methodsmentioning

confidence: 99%

beta-Bungarotoxin, a pre-synaptic toxin with enzymatic activity.

Strong¹,

Goerke²,

Oberg³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

155

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ABSTRACT,5-Bungarotoxin, a pre-synaptic neurotoxin isolated from the venom of the snake Bungarus multicinctus, has been shown to modify release of neurotransmitter at the neuromuscular junction. In this communication, we demonstrate that jP-bungarotoxin is a potent phospholipase A2 (phosphatide 2-acyl hydrolase, EC 3.1.1.4), comparable in activity with purified phospholipase enzymes from Naja naja and Vipera russeii. The phospholipase activity of fl-bungarotoxin requires calcium and is stimulated by deoxycholate. When strontium replaces calcium no phospholipase activity is detected. Since neuromuscufar transmission is not blocked when calcium is re laced by strontium, it was possible to examine the effects of the toxin on neuromuscular transmission in the presence of strontium. Under these conditions, when the phospholipase activity should be inhibited, the toxin has little or no effect on neuromuscular transmission. If fl-bungarotoxin owes its toxicity in part to its enzymatic activity, then it must be placed in a different class from those toxins which produce their effect by binding passively to an appropriate receptor.

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Isolation of phospholipase a isoenzymes from Najanaja venom and their action on membrane-bound enzymes

Cited by 39 publications

References 11 publications

Interaction of enzymes with mixed micelles of phospholipid and detergent: Analysis of the phospholipase A₂–dipalmitoyl phosphatidylcholine–triton X 100 system

Interaction of enzymes with mixed micelles of phospholipid and detergent: Analysis of the phospholipase A₂–dipalmitoyl phosphatidylcholine–triton X 100 system

The action of cobra venom phospholipase A2 isoenzymes towards intact human erythrocytes

beta-Bungarotoxin, a pre-synaptic toxin with enzymatic activity.

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Isolation of phospholipase a isoenzymes from Najanaja venom and their action on membrane-bound enzymes

Cited by 39 publications

References 11 publications

Interaction of enzymes with mixed micelles of phospholipid and detergent: Analysis of the phospholipase A2–dipalmitoyl phosphatidylcholine–triton X 100 system

Interaction of enzymes with mixed micelles of phospholipid and detergent: Analysis of the phospholipase A2–dipalmitoyl phosphatidylcholine–triton X 100 system

The action of cobra venom phospholipase A2 isoenzymes towards intact human erythrocytes

beta-Bungarotoxin, a pre-synaptic toxin with enzymatic activity.

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Interaction of enzymes with mixed micelles of phospholipid and detergent: Analysis of the phospholipase A₂–dipalmitoyl phosphatidylcholine–triton X 100 system

Interaction of enzymes with mixed micelles of phospholipid and detergent: Analysis of the phospholipase A₂–dipalmitoyl phosphatidylcholine–triton X 100 system