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Abstract

  1. Top of page
  2. Abstract
  3. REFERENCES

The behaviour of highly purified glucosylceramide β-glucosidase (glucosylceramidase, EC 3.2.1.45) from human placenta [Furbish, F. S., Blair, H. E., Shiloach, J., Pentchev, P. G. & Brady, R. B. (1977) Proc. Natl Acad. Sci. USA 74, 3560–3563] was investigated in the absence of detergents with structurally modified glucosyl-ceramides inserted into unilamellar liposomes. The reaction between the water-soluble enzyme and the liposomal substrates was significantly dependent on the structure of the lipophilic aglycon moiety of glycolipids: glucosyl-N-acetyl-sphingosines (d-erythro and l-threo) were better substrates than the corresponding glucosylceramides. The l-threo derivatives were poorer substrates with higher apparent Km values than the corresponding d-erythro derivatives. For glucosyl-3-keto-ceramide and glucosyl-dihydro-ceramide (d-erythro), higher Km values were found than for glucosylceramide. Sphingosine, glucosylsphingosine and glucosyl-N-acetyl-sphingosine were the most effective inhibitors of the hydrolysis of glucosylceramide. d-erythro-Ceramide and d-galactosyl-N-acetyl-d-erythro-sphingosine inhibited the hydrolyis of amphiphilic glucosylceramide but not that of water-soluble 4-methyl-umbelliferyl-β-glucoside, suggesting a hydrophobic binding site of the enzyme for the aglycon moiety of its membrane-bound substrate.

Dilution experiments suggested that at least a fraction of the enzyme associates with the liposomes and degrades the lipid substrate even in the absence of activator proteins.

Acidic phospholipids incorporated into liposomes caused a powerful stimulation (30–40-fold) of the glucosylceramide β-glucosidase, whereas acidic sphingolipids (sulphatide, gangliosides GM1 and GD1a) incorporated into liposomes stimulated this enzyme only moderately (3–10-fold).

Abbreviations
4-MuGlc

4-methyl-umbelliferyl-β-d-glucopyranoside

PA

dipalmitoylglycerophosphate

PG

diacylglycerophosphoglycerol (egg yolk)

PS

diacylglycerophosphoserine (bovine brain)

SDS

sodium dodecyl sulfate

Enzyme
 

Lysosomal glucosylceramide β-glucosidase, glucosylceramidase (EC 3.2.1.45)

REFERENCES

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  2. Abstract
  3. REFERENCES
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