Zymoblot, a new microtechnique used to detect enzyme activity in spiroplasmas and bacteria

Abstract

A new microtechnique that detects enzyme activity in prokaryotes is described. The technique, designated zymoblot, is based on the immobilization of negatively charged enzymes from an alkaline extract spotted onto a nitrocellulose membrane. The presence of specific enzyme activity in the extract is selectively assayed with a reaction mixture containing the corresponding substrate. The enzyme–substrate reaction produces an insoluble colored product that accumulates at the site. The zymoblot technique offers the advantages of simplicity, sensitivity, reproducibility, speed, and the use of microquantities of reactants. The protein in the spot can be visualized by a technique termed "proteinblot," in which the protein is stained with Coomassie blue. Esterase and tyrosinase were detected by the zymoblot method in six spiroplasmas including four strains of Spiroplasma citri, one of Spiroplasma kunkelii, and one of Spiroplasma melliferum, and two bacteria, Pseudomonas syringae pv. syringae B301D and Escherichia coli K-12. Acid phosphatase, alkaline phosphatase, alanine dehydrogenase, peroxidase, and 6-phosphogluconate dehydrogenase were not detected in any of the spiroplasmas, but were each detected in one or both of the walled bacteria.Key words: spiroplasma, enzyme, protein, zymoblot.