Cardiac adenylate deaminase: molecular, kinetic and regulatory properties under phosphate-free conditions

Abstract

Adenylate deaminase (EC 3.5.4.6) may help to regulate the adenine nucleotide catabolism characteristic of such disease states as myocardial ischaemia. We report analysis of the molecular, kinetic and allosteric properties of rabbit heart adenylate deaminase when extracted and purified under phosphate-free conditions (i.e., with Hepes/KOH). The enzyme's subunit molecular mass (approximately 81 kDa), pI (6.5), substrate specificity for 5'-AMP, and activation by K+ were identical in the absence or presence of phosphate. At each chromatographic step during isolation without phosphate, cardiac adenylate deaminase showed a lower apparent activity as compared with the enzyme prepared with phosphate present. Kinetic constants for the phosphate-free rabbit heart adenylate deaminase preparation (Km 0.54 mM AMP; Vmax. 1.4 mumol/min per mg of protein) were approximately 10-fold lower than those of the enzyme isolated with phosphate. The same irreversible decrease in kinetic constants could be achieved by dialysing phosphate from the phosphate-containing enzyme preparation. The relationship between enzyme activity and substrate concentration was sigmoidal in the presence of phosphate, but hyperbolic in its absence. Cardiac adenylate deaminase under phosphate-free conditions was no longer allosterically activated by ATP and ADP, yet remained inhibitable by GTP. Enzyme inhibition by the transition-state mimic coformycin was not influenced by phosphate status. The phosphate-free preparation of rabbit heart adenylate deaminase was markedly labile and extremely susceptible to proteolysis by trypsin or chymotrypsin. The inactivation kinetics and fragmentation pattern in response to controlled proteolysis depended on whether the enzyme had been isolated with or without phosphate present, suggesting a conformational difference between the two enzyme preparations. These data constitute direct evidence that the absence of phosphate irreversibly converts cardiac adenylate deaminase into a pseudo-isoenzyme with distinct kinetic, regulatory and stability properties.

Role of the HPRG Component of Striated Muscle AMP Deaminase in the Stability and Cellular Behaviour of the Enzyme

“…The EDTA-eluted fraction showed a higher stability during storage and a hyperbolic kinetics, that may be ascribed to the protection against loss of activity exerted by its higher HPRG content. Since the alteration of stability of rabbit AMPD1 observed when the molar ratio HPRG/AMPD was reduced is similar to that described for rabbit AMPD3 in the absence of phosphate [ 105 ], we can reasonably assume that the stabilizing effects of phosphate on cardiac AMPD3 could be explained by the protection exerted by the ion against loss of the HPRG component of the enzyme.…”

“…Thakkar et al [ 105 ] showed that SDS/PAGE of rabbit heart AMPD3 purified under phosphate-free conditions gave rise to three bands of approx. 80 kDa, 70 kDa, and 50 kDa.…”

“…As effect of the absence of phosphate, rabbit heart AMPD3 was no longer activated by ATP and ADP, and showed a hyperbolic curve of the enzyme activity as function of substrate concentration, whereas that obtained with the enzyme purified in the presence of phosphate was sigmoidal. On the basis of these observations, the authors suggest that the cardiac AMPD isoforms previously described may represent pseudo-isoenzyme species generated by an irreversible molecular alteration of one basic tissue-specific isoform caused by the absence of phosphate [ 105 ]. However, all these data could be now interpreted considering that the preparation of rabbit heart AMPD3 without phosphate yields the catalytic subunits of the enzyme dissociated from the HPRG subunits that are lost during the purification.…”

Metalloproteinase Inhibitor Counters High-Energy Phosphate Depletion and AMP Deaminase Activity Enhancing Ventricular Diastolic Compliance in Subacute Heart Failure

“…Cardiac AMPD may help regulate adenine nucleotide catabolism during myocardial ischemia (Thakkar et al, 1994) under oxidative stress conditions (Tavazzi et al, 2001), increased ADP (Chung and Bridger, 1976), and sustained ␤-stimulation (Hohl, 1999). In skeletal muscle in vivo, AMPD activity increase during muscular work is mediated by ADP, AMP, and pH (Wheeler and Lowenstein, 1979).…”

Gene Expression Profiles of Cultured Rat Cardiomyocytes (H9C2 Cells) in Response to Arsenic Trioxide at Subcytotoxic Level and Oxidative Stress

“…Thus the activation of AMP deaminase may deplete ATP in cardiomyocytes and cause heart failure. 14,15) It is interesting that arsenic increased the mRNA transcript of AMP deaminase. At present, we do not know whether the increase in AMP deaminase is related to oxidative stress, which is specifically induced only by arsenic, and further study is ongoing.…”