Complete enzymatic deglycosylation of native sex steroid-binding protein (SBP or SHBG) of human and rabbit plasma: Effect on the steroid-binding activity
| Autor: | W Zhang, Philip H. Petra, Patrick R. Griffin, J R Yates rd, K Moore |
|---|---|
| Rok vydání: | 1992 |
| Předmět: |
Glycosylation
Hydrocortisone medicine.medical_treatment Oligosaccharides Biochemistry Steroid chemistry.chemical_compound Pregnancy Sex Hormone-Binding Globulin medicine Animals Humans Testosterone Molecular Biology Polyacrylamide gel electrophoresis Chromatography High Pressure Liquid Progesterone chemistry.chemical_classification Estradiol biology Dihydrotestosterone Oligosaccharide Molecular Weight Enzyme chemistry biology.protein Electrophoresis Polyacrylamide Gel Female Rabbits Digestion Neuraminidase Research Article medicine.drug |
| Zdroj: | Protein Science. 1:902-909 |
| ISSN: | 1469-896X 0961-8368 9066-1907 |
| DOI: | 10.1002/pro.5560010708 |
| Popis: | An enzymatic procedure for the complete removal of the N-linked and O-linked oligosaccharide side chains of the sex steroid-binding proteins (SBP or SHBG) of human and rabbit plasma under native conditions is described. Deglycosylation was catalyzed by N-glycanase, neuraminidase, and O-glycanase and was monitored by SDS-PAGE, lectin blotting, and molecular weight analyses by electrospray mass spectrometry. Digestion of rabbit SBP with N-glycanase generated a major 39,777-Da protein and two minor ones of 39,389 and 39,545 Da. The molecular weight of the major protein agrees with the molecular weight calculated from the sequence of the sugar-free polypeptide monomer (39,769 Da: Griffin, P.R., Kumar, S., Shabanowitz, J., Charbonneau, H., Namkung, P.C., Walsh, K.A., Hunt, D.F., & Petra, P.H., 1989, J. Biol. Chem. 264, 19066-19075), whereas the other two are deglycosylated proteolytic cleavage products lacking the TQR and TQ sequences at the amino-terminus. The N- and O-linked side chains of human SBP were removed by sequential digestion with N-glycanase and neuraminidase/O-glycanase. A 38,771-Da protein was generated, which agrees well with the molecular weight of the sugar-free polypeptide monomer (Walsh, K.A., Titani, K., Kumar, S., Hayes, R., & Petra, P.H., 1986, Biochemistry 25, 7584-7590). N-deglycosylation of human and rabbit SBP has no effect on the steroid-binding activity, but removal of the O-linked side chains of N-deglycosylated human SBP results in an apparent 50% loss of steroid-binding activity and an increase in the Kd for the binding of 5 alpha-dihydrotestosterone from 0.3 mM to 0.9 nM.(ABSTRACT TRUNCATED AT 250 WORDS) |
| Databáze: | OpenAIRE |
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