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Studies on the nature of adenosine diphosphatase activity from rat liver mitochondria
Abstract:
Adenosine diphosphatase (ADPase) activity was studied in rat liver with [beta-32P]ADP as a substrate. Mitochondria and outer mitochondrial membrane fractions were isolated and assayed for ADPase and various marker enzymes. ADPase activity was strikingly reduced when the outer membranes were removed from the mitochondria whether by digitonin treatment or osmotic shock. Addition of the inter-membrane space subfraction to the purified outer membranes resulted in enhanced ADPase activity. Addition of the inter-mitochondrial membrane enzyme adenylate kinase to outer membranes also produced a large stimulation of activity. The ADPase activity could also be reconstituted in vitro with adenylate kinase and either mitoplast ATPase or ouabain-sensitive (Na+ + K+ + Mg2+)-ATPase. Chloroform-released ATPase, however, was not capable of producing an ADPase activity when combined with adenylate kinase. Gel permeation chromatography of Triton-solubilised outer mitochondrial membranes was unable to resolve ADPase activity from contaminating ATPase. These results suggest that the majority of ADPase activity in rat liver mitochondria consists of the coupled activity of adenylate kinase and ATPase.
Insights
Rat liver mitochondrial adenosine diphosphatase (ADPase) activity relies on outer membrane enzymes. Reconstituting ADPase activity required adenylate kinase and specific ATPases, suggesting coupled enzyme function.
Area of Science:
- Biochemistry
- Cell Biology
- Mitochondrial Function
Background:
- Adenosine diphosphatase (ADPase) plays a role in cellular signaling and metabolism.
- Understanding ADPase localization and function within mitochondria is crucial for comprehending cellular energy regulation.
Purpose of the Study:
- To investigate the localization and enzymatic basis of ADPase activity in rat liver mitochondria.
- To identify the specific enzymes responsible for ADPase activity and their interactions.
Main Methods:
- Isolation of rat liver mitochondria and outer mitochondrial membrane fractions.
- Assay of ADPase activity using [beta-32P]ADP as a substrate.
- Enzyme reconstitution assays with isolated components and marker enzyme analysis.
Main Results:
- ADPase activity was significantly reduced upon removal of mitochondrial outer membranes.
- Reconstitution experiments showed that adenylate kinase combined with specific ATPases (mitoplast or ouabain-sensitive) reconstituted ADPase activity.
- Chloroform-released ATPase failed to reconstitute ADPase activity, and ADPase activity could not be separated from ATPase by chromatography.
Conclusions:
- The majority of rat liver mitochondrial ADPase activity is attributed to the coupled action of adenylate kinase and ATPase.
- Outer mitochondrial membranes are essential for significant ADPase activity.
- Specific ATPase isoforms are involved in the ADPase enzymatic complex.