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Isolation and characterization of AMP deaminase from mammalian (rabbit) myocardium
J K Thakkar1, D R Janero, C Yarwood
1Research Department, CIBA-GEIGY Corporation, Summit, NJ 07901.
Abstract:
AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) is a ubiquitous enzyme in eukaryotes, which may play a role in ATP catabolism during myocardial ischaemia. We report isolation of AMP deaminase from rabbit myocardium with a 19% recovery and a 650-fold enrichment, using a newly devised protocol involving sequential cation-exchange, gel-permeation and affinity chromatographies. The cardiac AMP deaminase preparation described was electrophoretically and chromatographically homogeneous and contained one unique N-terminal residue (leucine). The isolated enzyme was sensitive to various cations (K+, Mg2+, Ca2+). The pH optimum of purified cardiac AMP deaminase was 6.8, its pI was 6.5, and it displayed substrate-specificity toward 5'-AMP. The subunit molecular mass of rabbit heart AMP deaminase on SDS/PAGE (81 kDa) and the holoenzyme molecular mass as estimated by non-denaturing size-exclusion h.p.l.c. (330 kDa) indicated that the native enzyme was a tetramer. Cardiac AMP deaminase displayed a sigmoidal substrate-saturation curve in the presence of 100 mM KCl. Apparent Michaelis constants were a Km of 5.8 mM AMP and a Vmax. of 11.1 mumol/min per mg of protein. ATP and ADP were positive allosteric effectors of cardiac AMP deaminase: the apparent Km was decreased to 1.7 mM by 1.0 mM ATP. The enzyme was inhibited by GTP, coformycin, coformycin 5'-phosphate, palmitoyl-CoA, inorganic phosphate compounds, and the metal chelator o-phenanthroline. No inhibition either by product nucleotide (IMP) or by nicotinamide nucleotides was detected when these agents were examined at concentrations up to 2.5 mM. We conclude that this enzyme preparation offers a means by which the kinetic mechanism and regulation of mammalian cardiac AMP deaminase may be directly investigated.
Insights
Researchers isolated and purified AMP deaminase (AMP aminohydrolase) from rabbit heart, revealing its tetrameric structure and allosteric regulation by ATP and ADP. This work facilitates further study of cardiac AMP deaminase
Area of Science:
- Biochemistry
- Enzymology
- Cardiovascular Research
Background:
- Adenosine monophosphate (AMP) deaminase is crucial in cellular energy metabolism and has potential roles in myocardial ischemia.
- Understanding the enzyme's properties is vital for investigating its function in cardiac conditions.
Purpose of the Study:
- To isolate and characterize AMP deaminase from rabbit myocardium.
- To elucidate the enzyme's kinetic properties, quaternary structure, and regulatory mechanisms.
Main Methods:
- Sequential chromatography (cation-exchange, gel-permeation, affinity) for enzyme purification.
- SDS-PAGE and size-exclusion HPLC for molecular mass determination.
- Enzyme kinetics assays, including substrate saturation and allosteric effector analysis.
Main Results:
- Purified cardiac AMP deaminase is a homogeneous tetramer (330 kDa) with 81 kDa subunits.
- The enzyme exhibits optimal activity at pH 6.8, is substrate-specific for 5'-AMP, and shows sigmoidal kinetics with KCl.
- ATP and ADP act as positive allosteric effectors, decreasing the apparent Km for AMP.
Conclusions:
- A robust method for isolating and purifying rabbit cardiac AMP deaminase was established.
- The characterized enzyme provides a valuable tool for investigating mammalian cardiac AMP deaminase's kinetic mechanism and regulation.