Properties of acid ceramidase from human spleen.
We have characterised ceramidase activity in extracts of human spleen from control subjects and from patients with Gaucher disease. In Triton X-100 extracts of control spleens, a broad pH optimum of pH 3.5-5.0 was found; no ceramidase activity was detectable at neutral or alkaline pH. About 45-60% of acid ceramidase could be extracted from spleen without detergents, but for complete extraction, Triton X-100 was required. For the radiolabelled substrate oleoylsphingosine, a Km of 0.22 +/- 0.09 mM and a Vmax of 57 +/- 11 nmol/h per mg protein was calculated in spleen from a control subject. Flat-bed isoelectric focussing in the presence of Triton X-100 revealed a pI of 6.0-7.0 for acid ceramidase; similar values were found for sphingomyelinase and glucerebrosidase. HPLC-gel filtration indicated that in the presence of Triton X-100, acid ceramidase has an Mr of about 100 kDa. In the absence of detergents, the enzyme forms high-molecular-weight aggregates. Similar aggregation behaviour was observed for sphingomyelinase, while the elution of beta-hexosaminidase was not affected by detergents. The elution profile of glucocerebrosidase was only slightly altered by Triton X-100. There was no difference in the properties of acid ceramidase present in spleen from control subjects and from patients with type I Gaucher disease.
Al
B J
BJ
Department of Biochemistry, Faculty of Medicine, University of Amsterdam, The Netherlands.
Tiffany
C W
CW
Gomes de Mesquita
D S
DS
Moser
H W
HW
Tager
J M
JM
Schram
A W
AW
D055573:Acid Ceramidase; D000581:Amidohydrolases / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D055572:Ceramidases; D002851:Chromatography, High Pressure Liquid; D005776:Gaucher Disease / Q000201:enzymology*; D005962:Glucosylceramidase / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols; D013154:Spleen / Q000201:enzymology*; D001617:beta-Glucosidase / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D011092:Polyethylene Glycols; D017830:Octoxynol; D001617:beta-Glucosidase; D005962:Glucosylceramidase; D001619:beta-N-Acetylhexosaminidases; D000581:Amidohydrolases; C526693:ASAH1 protein, human; D055573:Acid Ceramidase; D055572:Ceramidases