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Purification to Homogeneity of Human Placental Acid Sphingomyelinase
Author(s) -
Zou L.,
Kojima N.,
Kito M.,
Yagi K.
Publication year - 1989
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1989.tb00058.x
Subject(s) - chromatofocusing , chromatography , sphingomyelin , agarose , enzyme , size exclusion chromatography , chemistry , enzyme assay , gel electrophoresis , sodium dodecyl sulfate , substrate (aquarium) , sepharose , polyacrylamide gel electrophoresis , biochemistry , biology , membrane , ecology
Acid sphingomyelinase was purified to homogeneity from human placenta in the presence of a dialyzable detergent, n‐octyl‐beta‐D‐glucopyranoside. The major steps in the procedure included column chromatographies with Con A‐Sepharose, sphingosylphosphorylcholine‐Sepharose 4B, hexyl‐agarose, and Mono P. The purified enzyme with pI 7.4 had a specific activity of approx 170,000 units/mg protein with a yield of 3.6%. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis showed a single protein band of Mr 62,000. Gel filtration with a Superose 12 column gave a single peak, and the enzyme in the presence 50 mM n‐octyl‐beta‐D‐glucopyranoside was of Mr 123,000, indicating that the native enzyme occurs in a dimeric form. The optimal pH was 5.5 with both sphingomyelin and an artificial substrate, 2‐N‐hexadecanoylamino‐4‐nitrophenylphosphorylcholine. The Km values were 55 microM with sphingomyelin and 340 microM with the artificial substrate. The enzyme activity was not affected by Mg2+ (1–5 mM), confirming that the enzyme is acid sphingomyelinase. The enzyme was stable at −80 degrees C for more than 4 months. In addition to the enzyme with pI 7.4, the Mono P chromatofocusing gave two peaks (pI 7.0 and 6.7) possessing the enzymatic activity.