ATPase inhibitor from yeast mitochondria. Purification and properties.

Area of Science:

• Mitochondrial biochemistry
• Enzyme inhibition
• Yeast physiology

Background:

• Mitochondria from Candida utilis and Saccharomyces cerevisiae exhibit ATPase-inhibitor activity.
• This activity is influenced by yeast growth conditions, notably decreasing with high glucose concentrations, suggesting glucose-mediated repression.

Purpose of the Study:

• To isolate and characterize the ATPase inhibitor from Candida utilis mitochondria.
• To compare the properties of the Candida utilis ATPase inhibitor with that of beef heart.
• To investigate the interaction and binding characteristics of the inhibitor with its target ATPase.

Main Methods:

• Isolation and purification of the ATPase inhibitor from Candida utilis mitochondria.
• Determination of molecular weight using amino acid composition and SDS-PAGE.
• Isoelectric focusing and cross-reactivity assays with beef heart ATPase.
• Binding studies using 14C-labeled inhibitor and inhibitor-depleted submitochondrial particles.

Main Results:

• The ATPase inhibitor was purified to homogeneity with a molecular weight of 6000-7500 Da.
• Candida utilis inhibitor has a lower molecular weight, different amino acid composition, and a more acidic isoelectric point compared to beef heart inhibitor.
• Despite differences, it cross-reacts with beef heart ATPase, and its interaction requires Mg2+-ATP. Binding affinity (KD) was found to be approximately 10^-7 M.

Conclusions:

• Candida utilis possesses a unique mitochondrial ATPase inhibitor regulated by glucose.
• The inhibitor shares functional similarities with beef heart ATPase inhibitor, indicating conserved mechanisms.
• The study provides insights into the molecular properties and binding interactions of this yeast mitochondrial ATPase inhibitor.