Acid sphingomyelinase of human placenta: purification, properties, and 125iodine labeling.
Human placental acid sphingomyelinase was highly purified in the presence of Triton X-100. DEAE-Sephacel chromatography and chromatofocusing were the most effective steps in the purification procedure. Enzyme purification was 380,000 nmol/mg protein/h. Characterization and radioiodination were carried out with the chromatofocusing fraction containing highly purified enzyme. The purified enzyme contained no activity of eleven other lysosomal hydrolases but hydrolyzed bis-p-nitrophenyl phosphate slowly compared with [14C]sphingomyelin and chromogenic substrates. SDS-gel electrophoresis revealed two distinct protein bands with molecular weights of 70,500 and 39,800. This enzyme had a molecular weight of 200,000 as determined by analytical gel filtration. The pH optimum was 5.0 and Km was 52.6 x 10(-5) M for [14C]sphingomyelin. Highly purified sphingomyelinase was labeled with 125iodine by the use of Enzymobeads. Labeled sphingomyelinase preparation was rapidly cleared from blood with t1/2 of 1 min. It was absorbed mostly into the liver and presumably largely excreted from there. This labeled enzyme may be useful in metabolic studies in normal animals and animal models of genetic lysosomal storage disorders.
D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D002850:Chromatography, Gel; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D007525:Isoelectric Focusing / Q000379:methods; D007553:Isotope Labeling; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D013108:Sphingomyelin Phosphodiesterase / Q000302:isolation & purification / Q000378:metabolism*
D007457:Iodine Radioisotopes; D010727:Phosphoric Diester Hydrolases; D013108:Sphingomyelin Phosphodiesterase