Abstract
Glucocerebrosidase from normal human spleen, and spleen from cases of neurologic (types 2 and 3) and nonneurologic (type 1) Gaucher's disease, was delipidated and inactivated by extraction from membranes with sodium cholate and ice-cold 1-butanol. Control glucocerebrosidase was stimulated markedly by large quantities (20-30 micrograms/assay) of phosphatidylserine (PS), or by a combination of smaller amounts (1-2 micrograms) of PS and 3 micrograms of a heat-stable factor (HSF) derived from the spleen of a patient with Gaucher's disease. The residual glucocerebrosidase from a nonneurologic case, but not a neurologic case, was also responsive to PS and HSF. The combination of HSF and PS decreased the Km of the normal enzyme for 4-methylumbelliferyl-beta-D-glucopyranoside from 8.0 to 1.6 mM. These effectors also increased the reactivity of glucocerebrosidase to the inhibitor conduritol B epoxide; HSF alone had no effect (t1/2 = 19 +/- 0.5 min) whereas the maximum rate of inactivation (t1/2 = 4.0 min) by conduritol B epoxide was achieved in the presence of a mixture of PS (1 microgram) and HSF (3 micrograms). Phosphatidylglycerol (PG) and phosphatidic acid, also acidic phospholipids, were effective activators of glucocerebrosidase. Varying the fatty acid composition of PG had little effect on its ability to stimulate glucocerebrosidase activity. However, in the case of phosphatidylcholine (PC), a weaker activator than PG or PS, fatty acid composition had a significant impact on the ability of this neutral lipid to activate glucocerebrosidase; dilinoleoyl-PC and dicaproyl-PC were moderately effective activators, but distearoyl-PC and dioleoyl-PC were almost totally inactive. The mono-, and di-, and trisialogangliosides (GM1, GD1, and GT1 were less than half as effective as PS as activators of glucocerebrosidase. These results indicate that acidic phospholipids and the heat-stable factor may both play a role in explaining the genetic heterogeneity of Gaucher's disease.
Highlights
HSF alone had no effect whereas markedlyby acidic phospholipids, the maximum rate of inactivation ( t l l z= 4.0 min) by few comparative studiesdescribing the activatorlipid requireconduritol B epoxide was achieved in the presence of a mixture of PS (1 pg) and HSF (3 pg)
The first evidence of abiochemical distinction between neurologic and nonneurologic cases of Gaucher’s disease was di, and trisialogangliosides (GMl,Gol, and GT1 were providedrecentlybyWenger and Roth [15]. They demonless than half as effective as PS as activators of gluco- strated tha0t -glucosidase assays supplemented wiathmixture cerebrosidase. These results indicate that acidic phos- of PS,’ Triton X-100, and partially purified HSF [16] actipholipids and the heat-stable factor may both play a vated 0-glucosidase in crude extractsof leukocytes and fibrorole in explaining the genetic heterogeneityof Gauch- blasts from controls and a case of type 1 Gaucher’s disease, er’s disease
The authors did not address therelative importance of the HSF and Gaucher’sdisease presents mostoften in a benign formthat involves visceral organs, but spares the central nervous system. This autosomal recessive glycosphingolipidosis appears asa devastating neurologic diseasewith extensive central nervous system involvement acidic phospholipid to thesuccessful use of the 0-glucosidase assay for the biochemical differentiation of neurologic and nonneurologic Gaucher’s disease cases
Summary
Acts primarily by increasing the sensitivityof the enzyme to exogenous lipid activators, particularlyPS; and HSF aloneis. The results of the present studyof the residual glucocere- the active sitoef glucocerehrosidase to CBE It seems that the brosidase in neurologic and nonneurologic Gaucher’s disease HSF acts as a co-activator; Berent and Radin’s use of support our earlier findings[6];that is, residual enzyme in a the term “coglucosidase” [31,32] may be appropriate. One reason for considering it to Members of another class of acidic lipids, namely ganglio- be physiologically irrelevant was that large amounts of HSF sides, were only half as effective as PS in restoring activity towere needed to cause moderate stimulation of the glucocereglucocerebrosidase from control spleen This observation in- brosidase preparations inuse at that tim(e33).
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