Assay for in vivo yeast invertase activity using NaF

Area of Science:

• Biochemistry
• Enzymology
• Microbiology

Background:

• Invertase activity assays typically measure sucrose hydrolysis products like glucose.
• Whole yeast cells can metabolize these sugars, causing inaccuracies in enzyme activity measurements.
• Periplasmic invertase activity is crucial for understanding yeast metabolism.

Purpose of the Study:

• To develop a more accurate method for determining invertase activity in yeast.
• To overcome errors caused by monosaccharide uptake and metabolism by whole yeast cells.
• To provide a reliable assay for samples with low invertase activity.

Main Methods:

• Pre-incubation of yeast cells with 50 mM sodium fluoride, an enolase inhibitor.
• Inhibition of the glycolytic pathway to prevent monosaccharide metabolism.
• In vivo measurement of invertase activity under initial rate conditions.
• Utilizing cell concentrations up to 64 mg/ml.

Main Results:

• The sodium fluoride pre-incubation effectively blocked the glycolytic pathway.
• This method significantly improved the accuracy of invertase activity determination.
• The assay proved particularly effective for yeast strains exhibiting low invertase activity.
• Accurate initial rate measurements were achieved.

Conclusions:

• Blocking the glycolytic pathway with sodium fluoride is a viable strategy for accurate yeast invertase assays.
• This improved method enhances the reliability of enzyme activity measurements, especially in low-activity samples.
• The findings contribute to a better understanding of yeast enzymatic processes and metabolic regulation.