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Ammonium ions enhance proteolytic activation of adenylate cyclase and decrease its sensitivity to inhibition by
S M Yeung1, I Shoshani, D Stübner
1Department of Physiology and Biophysics, School of Medicine, State University of New York, Stony Brook 11794-8661.
Archives of Biochemistry and Biophysics
|June 1, 1989
Summary
Ammonium ions activate adenylate cyclase by increasing its Vmax and decreasing its sensitivity to proteases. Polyamines counteract this effect, impacting enzyme kinetics and P-site inhibition.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Pharmacology
Background:
- Adenylate cyclase (AC) is a crucial enzyme in cellular signaling pathways.
- Proteolytic activation and modulation by small molecules are key regulatory mechanisms for AC.
- Understanding the interactions of ions and polyamines with AC provides insights into enzyme regulation.
Purpose of the Study:
- To investigate the effects of ammonium salts and polyamines on the proteolytic activation of rat brain adenylate cyclase.
- To examine the influence of these compounds on the enzyme's sensitivity to inhibition at the 'P'-site.
- To elucidate the mechanisms underlying ammonium- and polyamine-mediated modulation of adenylate cyclase activity.
Main Methods:
- Detergent-solubilized adenylate cyclase from rat brain was used.
- Enzyme activation was achieved using a bovine sperm serine protease and guanosine 5 -O-(3-thiotriphosphate (GTPγS).
- Kinetic parameters (Vmax, Km) and inhibition constants (IC50) were determined under various conditions.
Main Results:
- Ammonium bicarbonate (NH4HCO3) activated proteolytically modified adenylate cyclase, increasing Vmax and reducing sensitivity to further proteolysis.
- Ammonium ions (NH4+) enhanced both reversible (Mn2+) and irreversible (GTPγS) AC activation, with optimal effects at specific concentrations.
- Polyamines inhibited the stimulatory effect of NH4+ on Mn2+-activated AC and altered the enzyme's Km and sensitivity to P-site inhibitors like 2 ,5 -dideoxyadenosine (2 ,5 -ddAdo).
Conclusions:
- Ammonium ions modulate adenylate cyclase activity through mechanisms potentially independent of Gs activation, affecting enzyme kinetics and stability.
- Polyamines antagonize ammonium's stimulatory effects and influence AC regulation via interactions affecting substrate binding and allosteric inhibition.
- These findings highlight the complex interplay of ions and polyamines in regulating adenylate cyclase activity and its response to different activators and inhibitors.