Purification to homogeneity of human placental acid sphingomyelinase.
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.
Zou
L
L
Institute of Applied Biochemistry, Yagi Memorial Park, Gifu, Japan.
Kojima
N
N
Kito
M
M
Yagi
K
K
D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification*; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D013108:Sphingomyelin Phosphodiesterase / Q000032:analysis / Q000302:isolation & purification*
D010727:Phosphoric Diester Hydrolases; D013108:Sphingomyelin Phosphodiesterase