Cobra Venom Cytotoxin Free of Phospholipase A 2 and Its Effect on Model Membranes and T Leukemia Cells

Abstract

Membrane-active toxins from snake venom have been used previously to study protein-lipid interactions and to probe the physical and biochemical states of biomembranes. To extend these studies, we have isolated from Naja naja kaowthia (cobra) venom a cytotoxin free of detectable phospholipase A2 (PLA2). The amino acid composition, pI (10.2), and net charge of the cytotoxin compares well with membrane-active toxins isolated from venoms of other cobras. The cytotoxin, shown by a spin label method, associates with PLA2 in buffers at pH values between 7.0 and 5.0, but not at pH 4.0. It is suggested that cytotoxin and PLA2 (pI close to 4.8) associate electrostatically in the native venom. The effect of the cytotoxin on model phospholipid membranes was studied by EPR of spin probes in oriented lipid multilayers and 1H-NMR of sonicated liposomes. The cytotoxin did not significantly affect the packing of lipids in pure phosphatidylcholine (PC) membranes and in PC membranes containing 10 mol% phosphatidic acid (PA) or cardiolipin (CL). However, the cytotoxin induced an increase in membrane permeability and formation of nonbilayer structures in PC membranes containing 40 mol% of PA or CL. The purified cytotoxin was cytocidal to Jurkat cells, but had little effect on normal human lymphocytes. However, both Jurkat cells and normal lymphocytes were killed equivalently when treated with 10(-9) m PLA2 and 10(-5) m cytotoxin in combination. From its effect on model membranes and Jurkat cells, it is suggested that purified cytotoxin preferentially targets and disrupts membranes that are rich in acidic phospholipids on the extracellular side of the plasma membrane.

The possible role of nonbilayer structures in regulating ATP synthase activity in mitochondrial membranes

“…1). These two effects are most likely related to the interactions of CTII with acidic phospholipids that are in direct contact with F 0 group proteins [14]. Such effects were not observed when DCCD-BP was added to mitochondria, possibly because hydrophobic DCCD-BP is incapable of interacting with mitochondrial membranes in an aqueous solution.…”

Snake Venom Cytotoxins, Phospholipase A2s, and Zn2+-dependent Metalloproteinases: Mechanisms of Action and Pharmacological Relevance

“…These cytotoxins kill cells by non-selectively disrupting cell membranes [12,14]. Cytotoxins exhibit various physiological effects which are mainly regulated by modifying cell membrane structure and function [2,12,14,28,29].…”

“…These cytotoxins kill cells by non-selectively disrupting cell membranes [12,14]. Cytotoxins exhibit various physiological effects which are mainly regulated by modifying cell membrane structure and function [2,12,14,28,29]. Various pharmacological applications have been suggested for cytotoxins such as their ability to destroy various types of cancer cells [2,12,14,30].…”

“…PLA 2 s are commonly used as enzyme models to study mechanisms that modulate phospholipid hydrolysis [15,37,38]. Given that overactive lipid hydrolysis by endogenous phospholipases stimulates chronic inflammation, there is considerable interest by the medical community for finding ways to decrease PLA 2 activity in chronic inflammatory conditions like rheumatoid arthritis, asthma, and autoimmune disorders.…”

Toxin synergism in snake venoms

“…An example of such synergistic enhancement includes cytotoxins from different cobra species. Cobra cytotoxins have been shown to enhance PLA2 activity through complex formation and deformation of cell membranes, causing cellular lysis due to hydrolysis of phospholipids (Gasanov et al, 1997, Gasanov et al, 2014. On a more toxin-specific level, Chaim-Matyas et al (1995) showed that cytotoxin P4 from Naja nigricollis (black-necked spitting cobra) venom acted synergistically with various PLA2s when cancer cell lines were subjected to these toxins in vitro, causing cell lysis.…”