title: Purification and properties of a phospholipase C that has high activity toward sphingomyelin from Aspergillus saitoi.
abstract: An enzyme hydrolyzing sphingomyelin was purified from extracts of solid cultures of Aspergillus saitoi 7041 by fractionation with isopropanol followed by successive column chromatographies on DEAE-Sepharose CL-6B, butyl-Toyopearl 650 M, and phenyl-Sepharose CL-4B. The preparation of purified enzyme was homogeneous and had an activity increased 81-fold over that of the isopropanol fraction. The yield was about 65%. The molecular weight was estimated to be 54,000 by sodium dodecyl sulfate-gel electrophoresis. The enzyme solution had a violet color and contained iron atoms. The enzyme catalyzed the hydrolysis of sphingomyelin to N-acylsphingosine and phosphorylcholine. The optimum pH for hydrolytic activity was around 3.5. The Km values for sphingomyelin and 2-hexadecanoylamino-4-nitrophenylphosphorylcholine were 0.11 and 0.33 mM, respectively. The enzyme also catalyzed the hydrolysis of other phospholipids; the order of its hydrolytic activity at a substrate concentration of 2.5 mM was phosphatidylcholine greater than or equal to sphingomyelin = phosphatidylethanolamine = lysophosphatidylethanolamine greater than phosphatidyl DL-glycerol = phosphatidyl L-serine greater than phosphatidylinositol. From these results, this enzyme appears to be a new type of phospholipase C(phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3).
authors: Research Laboratories, Tokyo Tanabe Co., Ltd., Akabane, Japan.
authors: S
authors: S
authors: Matsuoka
authors: H
authors: H
authors: Kimura
authors: A
authors: A
authors: Kiuchi
authors: H
authors: H
authors: Ohkawa
authors: K
authors: K
authors: Yagi
mesh_terms: D001230:Aspergillus / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008970:Molecular Weight; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism*; D010738:Type C Phospholipases / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D013109:Sphingomyelins; D010767:Phosphorylcholine; C010733:2-hexadecanoylamino-4-nitrophenylphosphorylcholine; D010738:Type C Phospholipases