THE PURIFICATION AND PARTIAL CHARACTERIZATION OF PHOSPHOLIPASE C FROM CLOSTRIDIUM PERFRINGENS
Abstract
A method for the purification of phospholipase C from Clostridium perfringens (PLC-Cp) was developed that resulted in an essentially pure preparation of enzyme that was highly active and was obtained in good yield. This enzyme was subsequently analyzed for its physical and enzymatic characteristics, as well as its effects on muscle cell cultures. PLC-Cp appears to be a monomer of about 42,500 molecular weight; however, aggregated forms were observed under the conditions of both sodium dodecylsulfate and basic pH polyacrylamide gel electrophoresis. The higher order aggregates were enzymatically inactive and their appearance was sensitive to chelating and reducing agents. PLC-Cp was found to be a metalloenzyme, containing 2 moles zinc per mole protein. Three isoenzymic forms were observed upon isoelectric focusing with isoelectric points of 4.6, 4.8, and 5.1. The amino acid composition was determined and found to contain 55% hydrophilic residues. No carbohydrate could be detected. To facilitate the enzymatic characterization of this enzyme two assay methods were developed which utilized phosphatidylcholine (PC) dispersed in either ethanol or sodium deoxycholate (DOC). PLC-Cp was found to hydrolyze dispersions of PC in DOC about 4.9-times better than when PC was dispersed in ethanol. Several different divalent cations were found to affect PLC-Cp activity, but Ca('2+) and Co('2+) appeared to be the most efficient. Examination of the substrate specificity of PLC-Cp showed that it preferred PC but could also hydrolyze sphingomyelin. Various analogs of PC were examined as inhibitors of PC hydrolysis, but none were completely effective. PLC-Cp was shown to be the causative agent in the inhibition of fusion and the stimulation of phospholipid synthesis in muscle cell cultures. These two effects of PLC-Cp treatment may be unrelated as phospholipase A(,2) treatment inhibited fusion but did not increase phospholipid synthesis.
Degree
Ph.D.
Subject Area
Biochemistry
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