Role of sulfhydryl groups in the function of glucosidase I from mammary gland

• Published in: The Journal of biological chemistry 1993-03, Vol.268 (9), p.6445-6452
• Main Authors: PUKAZHENTHI, B. S, NAGARAJA MUNIAPPA, VIJAY, I. K
• Format: Article
• Language: English
• Subjects: alpha -glucosidase; alpha-Glucosidases - chemistry; alpha-Glucosidases - physiology; Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Biological and medical sciences; Carbohydrate Sequence; Cattle; disulfide bonds; Disulfides - metabolism; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Female; Fundamental and applied biological sciences. Psychology; mammary glands; Mammary Glands, Animal - enzymology; Molecular Sequence Data; Rats; Rats, Sprague-Dawley; role; structure-activity relationships; Sulfhydryl Compounds - physiology; Transferases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)53272-5

Glucosidase I initiates the processing of asparagine-linked glycoproteins by excising the distal alpha 1,2-linked glucosyl residue from the Glc3Man9GlcNAc2 oligosaccharide, soon after its en bloc transfer from the lipid-linked donor to the nascent polypeptide. 1-Deoxynojirimycin, an analog of D-glucose, is a potent competitive inhibitor of the enzyme. Sulfhydryl-seeking reagents also strongly inhibit the enzyme, implying the involvement of an -SH group in its activity. To test this hypothesis, glucosidase I was purified from the rat mammary gland and its active site was loaded with 1-deoxynojirimycin, to protect such a group(s), while -SH groups on the remaining surface of the enzyme were blocked with N-ethylmaleimide or para-chloromercuriphenylsulfonic acid. Deoxynojirimycin was removed by dialysis to expose the active site -SH group(s). This group(s) was then tagged with 3-(N-maleimidopropionyl)biocytin (MPB) and detected with 125I-streptavidin on Western blots. A series of experiments is presented to show that indeed a critical -SH group(s) is located within the catalytic site of the enzyme. Additionally, the enzyme also possesses one or more sulfhydryls and disulfide bonds in its primary structure. The experimental approach outlined here should apply to identify reactive sulfhydryl groups in other catalytically active proteins.