The nature of the stimulatory action of the protein 'coglucosidase' on glucocerebrosidase was investigated with the use of highly purified cofactor from bovine spleen, radioactive glucosyl ceramide and methylumbelliferyl-beta-glucoside. A complex between glucosidase and either substrate could not be detected under equilibrium and non-equilibrium binding conditions. Complex formation between stimulating protein and the enzyme could be shown by the binding of the enzyme to an affinity column containing coglucosidase. This binding could be blocked by adding phosphatidylserine to the enzyme. The lipid also stimulated the enzyme. Additional evidence for binding of the enzyme to the two kinds of stimulators was the finding that they protected the enzyme against inactivation by N-ethylmaleimide and chloromercuriphenylsulfonate. A role for lipids in the stimulatory action of coglucosidase was shown by extracting lipids from the enzyme; this resulted in a loss of basal enzyme activity and of sensitivity to activation by the protein. Adding back to the lipids or phosphatidylserine increased the sensitivity of the delipidated enzyme to coglucosidase. Using the crude, unextracted enzyme we could show that low concentrations of phosphatidylserine augmented the effectiveness of coglucosidase but high concentrations of the lipid blocked the effect of the protein. It is proposed that lipids, particularly acidic ones, act on solubilized glucocerebrosidase to produce an enzyme conformation which allows binding and stimulation by coglucosidase. At higher lipid concentrations, the acidic lipids bind, in competition with coglucosidase, to the latter's binding site on the enzyme.
MATERIALS AND METHODS Materials used. MUG,* phosphatidyl serine from bovine brain, Triton X-100, and guanidine-HCl were from Sigma Chemical, and Stains All, from Polysciences (Warrington, Pa). Glucosyl ceramide was isolated from a Gaucher spleen (5) and [6-zH]glucosyl ceramide was prepared from it chemically (6). Coglucosidase from human Gaucher spleen was kindly furnished by Dr. Robert H. Glew and Lydia B. Danz Abbreviation used: MUG, methylumbelliferyl-~-D-glucopyranoside.
ABSTRACSendai virus induces human peripheral blood leukocytes to produce high levels of tumor necrosis factor (TNF) mRNA. TNF mRNA can represent as much as 0.6% of the total mRNA. Kinetic studies indicate that the level of TNF mRNA peaks about 2 hours before that of IFN-a mRNA produced in the same system. Although the peak levels of TNF and IFN-a mRNA were similar, TNF in the culture supernatants was at a 200 fold lower level than IFN--. Cloning and sequence analysis of TNF cDNA isolated from peripheral blood leukocytes RNA showed that normal human cells in response to Sendai virus produce TNF identical to that previously isolated and cloned from tumor-derived cell lines. A bacterial expression system was used to produce the cloned TNF at a maximum level of 2 X 106 units per ml of culture.
A family of beta-glucosidase-stimulating proteins (called cohydrolase SPH-I here) was isolated from bovine, Gaucher human and control human spleens. All preparations exhibited a similar pattern of four major electrophoretic bands in polyacrylamide when stained with the cationic dye, Stains-All. The bovine bands migrated more rapidly, while the two types of human cohydrolase migrated very similarly. The two human preparations differed in several respects: the concentration was much higher in Gaucher spleen; the Gaucher factors eluted a little earlier from gel permeation and decyl agarose columns; much more of the cohydrolase was bound by a concanavalin A column; the control bands stained less intensely in gels than the Gaucher bands. Antibodies raised in rabbits to bovine cohydrolase reacted with all three preparations. All four bands from Gaucher cohydrolase showed similar ability to stimulate glucosidase and to bind the antibodies. It is evident that the cohydrolases from control and Gaucher spleens are similar in many respects, yet differ in some secondary fashion, possibly in carbohydrate content. It is suggested that Gaucher cohydrolase is formed from normal cohydrolase by the nonenzymatic action of cellular glucose over a period of many years, due to slowed catabolism of the cofactor.
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