Identification and subcellular localization of sphinganine-phosphatases in rat liver.
One of the primary products of [4,5-3H]sphinganine phosphate, added to fibroblast cultures, is sphinganine [Van Veldhoven and Mannaerts (1994) Biochem. J. 299, 597-601], implicating the physiological action of (a) hitherto unknown phosphatase(s). We have now further characterized this activity in rat liver. In homogenates, the dephosphorylation appeared to be catalysed by multiple enzymes. A low-affinity system was active at acidic pH, whereas at physiological pH values hydrolysis was carried out by a high-affinity enzyme. The latter was sensitive to Zn2+ and detergents and possessed a pH optimum of 7.5. Upon cell fractionation the major portion of the high-affinity activity was recovered in the nuclear and microsomal fractions. Further separation of the microsomal fraction showed an association predominantly with vesicles derived from the plasma membrane. Likewise, when plasma membranes were prepared from the nuclear fraction, the high-affinity phosphatase co-purified with the plasma membrane markers. From the differential effects of bivalent cations, chelators, water-soluble and amphiphilic phosphate esters, detergents and other compounds, it could be concluded that the plasma membrane-associated sphinganine-phosphatase activity is not due to alkaline phosphatase, dolichol-phosphatase, the N-ethylmaleimide-insensitive phosphatidate phosphatase or ceramide-phosphatase. The dephosphorylation observed at acidic pH in homogenates appeared also to be enriched in purified plasma membranes and might represent a side-activity of ceramide-phosphatase. We speculate that the high-affinity phosphatase, which is especially active in neuronal tissues, plays a role in the attenuation of bioactive phosphorylated sphingoid bases such as sphingenine phosphate, and propose to name it sphingosine-phosphatase.
De Ceuster
P
P
Katholieke Universiteit Leuven, Afdeling Farmakologie, Belgium.
Mannaerts
G P
GP
Van Veldhoven
P P
PP
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002518:Ceramides / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000648:ultrastructure*; D008297:Male; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis*; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology*; D014018:Tissue Distribution; D015032:Zinc / Q000494:pharmacology
D002518:Ceramides; D004492:Edetic Acid; D010744:Phosphoric Monoester Hydrolases; D015032:Zinc; D013110:Sphingosine; C005682:safingol