Inactivation of (Na-++K-+)-stimulated ATPase by a cytotoxic protein from cobra venom in relation to its lytic effects on cells.

The mechanism of action of the cytotoxic protein P6 isolated from cobra venom (Naja naja) which shows preferential cytotoxicity particularly to Yoshida sarcoma cells has been studied by its effects on the membrane-bound enzyme (Na-++K-+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) of a variety of cell systems. Evidence obtained with Yoshida sarcoma cells, dog and human erythrocytes and three tissue culture cell lines KB (human oral carcinoma), Hela (human cervix carcinoma) and L-132 (human lung embryonic) shows that inhibition of (Na-++K-+)-ATPase by the P6 protein can be correlated with its lytic activity. (Na-++k-+)-ATPase of Yoshida sarcoma membrane fragments inactivated by P6 protein could be reconstituted by the addition of phosphatidylserine and phosphatidic acid. It is conceivable that lysis of cells by the P6 protein may be due to an imbalance of K-+ and Na-+ in the cell which leads to swelling and disintegration of the membrane structure. Observations indicate that the P6 protein combines with membrane constituents of susceptible cells. The overall evidence suggests that both the specificity of its protein structure and the highly basic nature of the P6 protein are factors which enable it to compete with the lipid moiety maintaining the (Na-++k-+)-ATPase of the susceptible cells in proper conformation for activity.

Comparative study of three basic polypeptides from snake venoms in relation to their effects on the cell membrane of normal and tumor cells.

Basic polypeptides producing a variety of effects on animals and cells have been isolated from snake venoms. Many possess common structural features and also produce similar pharmacological effects. This has led to doubt as to the specificity of each polypeptide. Study of toxin gamma (cardiotoxin from Naja nigricollis), cytotoxin P6 (from naja naja, preferentially cytotoxic to certain cells) and neurotoxin alpha (Naja nigricollis) under identical conditions shows that they are separate entities though having some common structural properties. The amino acid composition shows certain resemblance between the nontoxic polypeptides, P6 and toxin gamma, as compared to the neurotoxin alpha. Their molecular weights are of the same order. Sulphydryl groups are absent in all but they possess a high proportion of disulphide linkages. The behavior of toxin gamma, cytotoxin P6 and neurotoxin alpha on Yoshida sarcoma cells and human erythrocytes demonstrate that whereas cytotoxin P6 was more active in lysing Yoshida sarcoma cells the order of activity was reversed in the human erythrocytes. Apparently these two cell systems respond differently to the action of the two polypeptides suggesting that they bind to different membrane receptors. The selectively displayed in changing the membrane permeability of different cells is probably dependent not ony on their basic charge but on the specificity of their protein structure.

Influence of charge on the inactivation of membrane bound (Na+ + K+)-ATPase of Yoshida sarcoma cells by inhibitor proteins from cobra venom.

Inactivation of (Na+ + K+)-ATPase of Yoshida sarcoma cells and beef brain microsomes by phospholipase A2 and a cytotoxin P6 from snake venom has been examined in relation to their activity to degrade phospholipids. Cytotoxin P6 which was most basic and devoid of phospholipase activity was most effective in inhibiting the (Na+ + K+)-ATPase of Yoshida sarcoma cells. Phospholipase A2 from Naja naja which was most active in degrading phospholipids was least effective in inhibiting (Na+ + K+)-ATPase in Yoshida sarcoma cells or in beef brain microsomes. Addition of trace amounts of cytotoxin P6 to the phospholipase considerably enhanced the inactivation of (Na+ + K+)-ATPase. The evidence suggests that the charge of the inhibitor protein and its specific structure play an important role in the inactivation of (Na+ + K+)-ATPase.

Inactivation of membrane-bound (Na+ +K+)-ATPase of Yoshida sarcoma cells and cobra venom cytotoxin complex with the glycolipid components of the enzyme system.

The molecular mechanisms involved in the inactivation of (Na+ + K+)-stimulated ATPase of Yoshida sarcoma cells by a cytotoxic protein (P6) from cobra venom have been examined. The overall data obtained using purified (Na+ + K+)-stimulated ATPase of Yoshida sarcoma cells suggest that cytotoxin P6 combines with phosphatidyl serine and a glycolipid which are closely associated with (Na+ + K+)-stimulated ATPase which in turn may lead to the inactivation of the enzyme in this cell system.

Elevation of serum polyamines in malignant lymphomas and acute myeloid leukemia.

In a study of 89 cases of hematological cancers including 55 cases of Hodgkin's disease, 21 of non-Hodgkin lymphoma and 13 of acute myeloid leukemia, the serum total polyamines were considerably elevated (1.2-5.7 nmol/ml) as compared to observations on control values for eight healthy individuals (0.62-0.87 nmol/ml). The assay procedure was based on enzymes oxidizing polyamines isolated from Russell's viper venom. Serial determination of polyamines in the sera of eight cases of Hodgkin's disease done before and after chemotherapy or radiotherapy showed positive correlation with clinical status of the disease. Analysis of individual polyamines in four patients with Hodgkin's disease indicated that putrescine may be a more sensitive marker of the diseased state. By the present method it was undetected in the control sera but rose significantly in the sera of patients with Hodgkin's disease. Overall data suggest that serum polyamines may be useful as indicators of onset of relapse in patients with Hodgkin's disease and could thus be helpful in early start of treatment.