Al~stract Human placental acid sphingomyelinase (ASM) was purified by sequential chromatography on Con A-Sepharose, octyI-Sepharose and Matrex gel red A. Final purification to apparent homogeneity was achieved by immunoaff'mity chromatography employing polyclonal anti-ASM antibodies. The antibodies also allowed specific detection of ASM by Western blotting at various stages of purification. The ASM activity was enriched about ll0000-fold over that of the crude extract, yielding an enzyme preparation with a specific activity of about 1 mmol/h per mg protein in a detergent-containing assay system. Analysis of the final preparation by SDS-PAGE resulted in a single protein band with a molecular mass of ~ 75 kDa, which was reduced to ,-~60 kDa after complete deglycosylation. Microsequencing of the purified ASM revealed the N-terminal amino acid sequence of the mature placental enzyme.
The processing, intracellular transport, and endocytosis of the G M2 activator protein (G M2 AP), an essential cofactor of -hexosaminidase A for the degradation of ganglioside G M2 , was investigated in human epidermal keratinocytes. The G M2 AP precursor is synthesized as an 18-kDa peptide, which is singly glycosylated, resulting in 22-kDa high mannose and 24 -27-kDa complex glycoforms. A small portion of the 22-kDa form bears phosphomannosyl residues. About 30% of the G M2 AP precursor is secreted during 12 h after synthesis, consisting almost exclusively of complex glycoforms. In a post-Golgi compartment, the intracellular remainder is converted to a 20-kDa mature form within 24 h, bearing a heavily trimmed N-glycan on a 17-kDa backbone. Interestingly, even nonglycosylated G M2 AP is delivered to the lysosome, as shown by tunicamycin treatment and subcellular fractionation. Also, its endocytosis is independent of carbohydrate-linked signals and is even more effective for nonglycosylated G M2 AP. We conclude that a mannose-6-phosphate-independent pathway for the lysosomal delivery of G M2 AP exists in cultured human keratinocytes.
In order to determine the biological function of the three different SAP-B isoforms, SAP-precursor-deficient human fibroblasts were loaded with recombinant SAPprecursor proteins with or without 2-and 3-amino acid insertions, respectively, purified from the medium of the baby hamster kidney cells. They were found to stimulate at nanomolar concentrations the turnover of biosynthetically labeled ceramide, glucosylceramide, and lactosylceramide. Since the physiological function of SAP-B is to stimulate the degradation of sulfatide by arylsulfatase A (EC 3.1.6.1) and globotriaosylceramide by -galactosidase (EC 3.2.1.23) loading studies with the respective exogenously labeled lipids on SAP-precursordeficient fibroblasts were performed. Addition of different purified SAP-precursors to the medium of the lipidloaded fibroblasts showed positive stimulation of the lipid degradation by all three SAP-B isoforms derived from the SAP-precursors. These findings establish that all three forms of the SAP-B can function as sulfatide/ globotriaosylceramide activator.
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