Phospholipase D [phosphatidylcholine cholinehydrolase, EC 3·1·4·4] excreted from Streptomyces chromofuscus was purified from the culture supernatant by precipitation with acetone and column chromatographies on palmitoylated gauze (Pal-G), DEAE-cellulose, and Sephadex G-150 with an overall recovery of 46% and 1000-fold increase in specific activity. The purified enzyme preparation showed a single band on sodium dodecyl sulfate (SDS)polyacrylamide disc gel electrophoresis.The enzyme had a molecular weight of about 50,000 by gel filtration on Sephadex G-150 or about 57,000 by SDS-polyacrylamide disc gel electrophoresis and an isoelectric point (pI) of pH 5·1 on isoelectric focusing. The enzyme hydrolyses lecithin, lysolecithin, sphingomyelin, and cephalin; the relative reaction velocities and Km's for choline-phospholipids were 87% and 1·43 m for lecithin, 100% and 1·67 m for lysolecithin, and 22% and 0·56 nms for sphingomyelin. The enzymatic reaction was optimal at pH 8, and its velocity was appreciably increased by either detergent (Triton X-100, deoxycholate), Ca2+ or both detergent and Ca2+ Diethyl ether stimulated the enzymatic activity by 30%; SDS and EDTA inhibited the activity.Bovine serum albumin, Triton X-100, and lipids (lecithin, lysolecithin, phosphatidic acid, lysophosphatidic acid, palmitic acid, and oleic acid) inhibited adsorption of the purified enzyme onto palmitoyl cellulose (Pal-C) and affected both the enzyme activity and stability:albumin and Triton X-100 increased the activity and enhanced the heat-stability; lysophospholipids decreased the activity but other lipids increased the activity; alt the lipids lowered the heat-stability.The enzyme adsorbed on Pal-C was active, although its activity was about one-ninth of that of free enzyme, and was protected from heat-inactivation.Thus this enzyme appears to possess a hydrophobic site distinct from its catalytic site and to be adsorbed onto Pal-C through the hydrophobic site. Albumin, Triton X-100, and lipids seem to bind to the hydrophobic site and to have an appreciable effect on the enzyme activity and stability. © 1979 By The Journal Of Biochemistry.
