Role of the carbohydrate part of yeast acid phosphatase.
Acid phosphatase, purified from the yeast Saccharomyces cerevisiae, was completely deglycosylated by endo-beta-N-acetylglucosaminidase H or by HF treatment. Three protein bands were obtained on sodium dodecyl sulfate (SDS)-electrophoresis, with molecular weights of 73,000, 71,000 and 61,500. The released carbohydrate chains varied in size from 12 to 142 mannose units. To study the role of carbohydrate chains in the structure and function of acid phosphatase, a comparison of the properties of the partially deglycosylated enzyme with the native one was performed. The 60% deglycosylated enzyme retained the original activity, and CD and fluorescence spectra showed that the native conformation of the enzyme was preserved. The 90% deglycosylated enzyme showed a pronounced loss of enzyme activity, accompanied by the disruption of the three-dimensional structure. The partially deglycosylated enzyme was less soluble and more susceptible to denaturing effects of heat, pH, urea, and guanidine hydrochloride. Under conditions of electrophoresis, the partially deglycosylated enzyme dissociated, indicating a possible role of carbohydrate chains in maintaining the dimeric structure of the enzyme. Susceptibility of acid phosphatase toward proteolysis was drastically increased by deglycosylation.
D000118:Acetylglucosaminidase / Q000494:pharmacology; D000135:Acid Phosphatase / Q000378:metabolism*; D002241:Carbohydrates / Q000502:physiology*; D002942:Circular Dichroism; D004355:Drug Stability; D006858:Hydrofluoric Acid / Q000494:pharmacology; D046911:Macromolecular Substances; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight; D011487:Protein Conformation; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D002241:Carbohydrates; D046911:Macromolecular Substances; D000135:Acid Phosphatase; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D006858:Hydrofluoric Acid