Purification and properties of a phospholipase C that has high activity toward sphingomyelin from Aspergillus saitoi.
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).
Matsuoka
S
S
Research Laboratories, Tokyo Tanabe Co., Ltd., Akabane, Japan.
Kimura
H
H
Kiuchi
A
A
Ohkawa
H
H
Yagi
K
K
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
D013109:Sphingomyelins; D010767:Phosphorylcholine; C010733:2-hexadecanoylamino-4-nitrophenylphosphorylcholine; D010738:Type C Phospholipases