Subunit structure of external invertase from Saccharomyces cerevisiae

Area of Science:

• Enzymology
• Glycobiology
• Molecular Biology

Background:

• External invertase from Saccharomyces cerevisiae is heavily glycosylated, comprising 50% carbohydrate by mass.
• High carbohydrate content complicates accurate molecular weight determination using traditional methods like centrifugation or electrophoresis.

Purpose of the Study:

• To accurately determine the molecular weight and subunit composition of Saccharomyces cerevisiae external invertase.
• To investigate the structure of the enzyme after removal of its carbohydrate moieties.

Main Methods:

• Enzymatic deglycosylation using endo-beta-N-acetyl-glucosaminidase H from Streptomyces plicatus.
• Determination of molecular weight of carbohydrate-free invertase.
• Terminal sequence analysis using carboxypeptidases A, B, and Y.

Main Results:

• Removal of oligosaccharide chains enabled accurate molecular weight assessment.
• Carbohydrate-free invertase was found to be composed of two subunits, each weighing 60,000 daltons.
• Sequence analysis indicated that these two subunits are identical.

Conclusions:

• The core protein of Saccharomyces cerevisiae external invertase consists of two identical 60 kDa subunits.
• Enzymatic deglycosylation is an effective strategy for characterizing heavily glycosylated proteins.