Molecular weight variations in the diversity of phospholipase A2 forms in reptile venoms

Daniel J. DuBourdieu, Hideaki Kawaguchi, W. T. Shier

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

Reptile venoms exhibit a wide diversity of phospholipase A2 forms when examined by electrophoretic and chromatographic techniques which separate on the basis of net charge. In principle, diversity in charge among the enzyme forms could result from two type of structural modifications: (i) pretranslational modifications, such as differences in amino acid sequences; (ii) post-translational modifications, such as partial proteolysis or hydrolysis of amide functions of asparagine and glutamine residues. Some types of modifications alter both charge and molecular weigth. A radiolabeling technique has been developed which allows detection of phospholipase A2 enzymes after electrophoretic separation on the basis of molecular weight in polyacrylamide gels containing sodium dodecylsulfate. Venom preparations were radiolabeled by incubation with p-dodecylphenacylbromide, which was prepared radiolabeled to high specific activity with tritium. The labeling agent was dispersed in phosphatidylcholine liposomes and incubated with venom preparations under conditions of optimal enzyme activity. The preparations were then extracted with ether and hexane to remove substrate and unbound label and subjected to polyacrylamide gel electrophoresis under standard conditions. Protein-bound radiolabel was detected by fluorography and autoradiography. Patterns of labeled species obtained by gel electrophoresis of selected venoms using separation on the basis of charge were similar to the patterns of phospholipase A2 variants obtained under the same electrophoretic conditions using enzymatic detection in phosphatidylcholine-containing gels. Examination of a variety of snake and lizard venoms using this technique revealed the presence of multiple molecular weight forms of labeled enzymes, but the extent of diversity was less than that observed with the same venoms using separation of phospholipase electrophoretic variants on the basis of charge. The results are consistent with diversity in reptile venom phospholipase A2 forms being derived from differences in both molecular weight and charge.

Original languageEnglish (US)
Pages (from-to)333-343
Number of pages11
JournalToxicon
Volume25
Issue number3
DOIs
StatePublished - 1987

Fingerprint Dive into the research topics of 'Molecular weight variations in the diversity of phospholipase A<sub>2</sub> forms in reptile venoms'. Together they form a unique fingerprint.

Cite this