A novel approach for the identification of isozymes using inhibitors.

Carboxylesterase isozymes of a termite and a fungus, have been identified by exploiting their differential sensitivity towards organophosphate inhibitors. The inhibition curves obtained by plotting pI (negative logarithm of inhibitor concentration) versus per cent inhibition are used to distinguish the isozymes. The termite and its associated fungus possess 2 and 4 isozymes respectively. Each of the purified isozyme gave a single sigmoidal inhibition curve with its characteristic I50 value. The pattern of the inhibition curve of the crude extract is found to mimic that of the mixture of the purified isozymes in both termite and fungus. This method provides not only identification of isozymes but also serves as a criterion of homogeneity.

Purification and characterisation of a carboxylesterase from the latex of Synadenium grantii Hook,'f'.

The latex of Synadenium grantii was found to contain esterolytic activity. Polyacrylamide gel electrophoretic study coupled with substrate and inhibitor specificity studies revealed the presence of multiple forms of carboxylesterases and cholinesterases in the latex. One of the carboxylesterases of the latex was purified by acetone fractionation, carboxymethyl- Sephadex chromatography and Sepharose-6B gel filtration. The homogeneity of the enzyme was established by polyacrylamide gel electrophoresis, isoelectric focussing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme consists of a single polypeptide chain with a molecular weight of 14,000. The amino acid analysis of the purified enzyme revealed that it contained a greater number of neutral and acidic, compared to basic amino acid residues. The isoelectric pH of the enzyme was found to be 4·0. The enzyme was a glycoprotein as revealed by periodic acid Schiff-staining technique. Studies with different organophosphate and carbamate inhibitors showed that this enzyme was sensitive to organophosphates. The product inhibition studies with this enzyme showed linear competitive inhibition with acetate and linear non-competitive inhibition with 1- naphthol.

Tissue esterases of Exoristina sorbillans (Uzi fly).

Polyacrylamide gel disc electrophoretic technique was used to examine the esterase pattern of nead, haemolymph, alimentary canal., ovary and testis of the Uzi fly. The zymograms revealed the varied pattern of esterases both in number and type. This varied pattern suggested several roles for these enzymes present in different tissues.