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Purification and characterization of developmentally regulated AMP deaminase from Dictyostelium discoideum
D P Malliaros1, D L Kozwich, E G Jahngen
1Department of Chemistry, University of Lowell, MA 01854.
Abstract:
AMP deaminase, the enzyme that catalyzes the conversion of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia, was purified from the cellular slime mold, Dictyostelium discoideum in the nutrient-deprived state. The native enzyme had an apparent molecular weight of 199,000 daltons. Its apparent Km was 1.6 mM and its Vmax was 1.0 mumol min-1 mg-1, as measured by the release of IMP From AMP. The enzyme, like other AMP deaminases, was found to be activated by ATP, and inhibited either by GTP or inorganic phosphate. It was also specific for the deamination of AMP. Deaminase activity was increased either when vegetative cells were placed in a nutrient-deprived medium (for up to 6 h) or when vegetative cells were treated with the drug hadacidin. In cells actively growing in complete media, enzyme activity was more non-specific, hydrolyzing adenosine as well as AMP. AMP deaminase in D. discoideum appears to be stage-specific and developmentally regulated, possibly serving to regulate the adenylated nucleotide pool and the interconversion to guanylated nucleotides during early morphodifferentiation.
Insights
This study purified AMP deaminase from Dictyostelium discoideum, revealing its role in regulating nucleotide pools during development. The enzyme
Area of Science:
- Biochemistry
- Molecular Biology
- Developmental Biology
Background:
- Cellular slime molds like Dictyostelium discoideum undergo significant developmental changes.
- Adenosine monophosphate (AMP) deaminase is a key enzyme in nucleotide metabolism.
- Understanding enzyme regulation is crucial for deciphering developmental processes.
Purpose of the Study:
- To purify and characterize AMP deaminase from Dictyostelium discoideum.
- To investigate the enzyme's kinetic properties and regulatory mechanisms.
- To explore the enzyme's potential role in developmental regulation.
Main Methods:
- Purification of AMP deaminase from nutrient-deprived Dictyostelium discoideum.
- Determination of enzyme kinetics (Km, Vmax) using AMP as substrate.
- Analysis of enzyme activation and inhibition by various nucleotides and phosphate.
Main Results:
- Purified native AMP deaminase with an apparent molecular weight of 199,000 daltons.
- Enzyme exhibited Michaelis constant (Km) of 1.6 mM and Vmax of 1.0 μmol min-1 mg-1.
- Activity was enhanced by ATP and inhibited by GTP and inorganic phosphate; specific for AMP deamination.
Conclusions:
- AMP deaminase in Dictyostelium discoideum is stage-specific and developmentally regulated.
- The enzyme likely regulates the adenylated nucleotide pool and interconversion to guanylated nucleotides.
- Its activity is modulated by nutrient availability and specific chemical treatments, impacting early morphodifferentiation.