Purification and properties of AMP-deaminase from human uterine smooth muscle. Regulation by adenylate energy charge and activated fatty acids

At pH 7.0 and physiological concentrations of the main regulatory ligands (ATP, ADP, orthophosphate), human uterine muscle AMP-deaminase follows a hyperbolic type of saturation kinetics with S0.5 parameter value about 2 mM. The enzyme is regulated by adenylate energy charge (AEC) variations, being the most active at the AEC value 0.5-0.6 or 0.5-0.7, depending on the size of the total adenine nucleotide pool. Long-chain acyl-CoA strongly inhibit activity of the enzyme, influencing mainly the maximum velocity of the reaction.     

AMP-deaminase from normal and cirrhotic human liver: A comparative study

AMP-deaminase (EC 3.5.4.6) is an enzyme of nucleotide breakdown involved in regulation of adenine nucleotide pool in mammalian cells. Reaction catalysed by AMP-deaminase constitutes a rate-limiting step in adenine nucleotide catabolism in liver. In this study kinetic and regulatory properties of AMP-deaminase purified from normal and cirrhotic human liver were investigated. In comparison to AMP-deaminase extracted from the normal human liver, AMP-deaminase extracted from the cirrhotic liver was less sensitive towards substrate analogues, and only a very limited response towards pH and adenylate energy charge changes tested for enzyme isolated from this tissue source had been observed. At physiological pH 7.0, in the absence and in the presence of important allosteric effectors (ATP, ADP, GTP and orthophosphate), AMP-deaminases from the two sources studied manifested different regulatory profiles, with half-saturation constant (S0.5) values being distinctly higher for the enzyme extracted from the pathological organ. In contrast to AMP-deaminase isolated from the normal, healthy liver, where presence of relatively large (68 kDa) protein fragment was also detected, only smaller protein fragments were identified, while SDS-PAG electrophoresis of AMP-deaminase isolated from the cirrhotic liver was performed. The obtained results indicate clearly that advanced proteolytic processes occurring in the cirrhotic liver may affect structural integrity of AMP-deaminase studied, making enzyme less active and less sensitive to regulatory action of important allosteric effectors.     

Human liver AMP-deaminase – oligomeric forms of the enzyme

AMP-deaminase (EC 3.5.4.6) is a key enzyme of nucleotide breakdown involved in regulation of adenine nucleotide pool in the liver. Mechanisms regulating activity of the enzyme are not completely elucidated, till now. In this paper experimental data indicating on the potential regulatory significance of changes in oligomeric structure of the enzyme are presented. SDS-PAG electrophoresis of human liver AMP-deaminase revealed the presence of three enzyme fragments. Only largest of them (the protein fragments weighing 68 kDa) reacted immunologically with monoclonal anti- (human liver) AMP-deaminase antibodies. At physiological pH 7.0, in the absence of regulatory ligands, reaction catalysed by human liver AMP-deaminase was strongly dependent on enzyme concentration used, with half-saturation constant (S_0.5) values increasing significantly with the degree of enzyme dilution. Preincubation with activated long-chain fatty acids – substances promoting dissociation of oligomeric enzymes, inhibited the activity of AMP-deaminase studied nearly completely. Gel filtration on Sepharose CL-6B column demonstrated existence of at least three active oligomeric forms of human liver AMP-deaminase. We postulate that oligomeric structure of the enzyme is a factor determining regulatory profile of AMP-deaminase studied.     

Purification and properties of AMP-deaminase from human uterine smooth muscle

AMP-deaminase from human uterine smooth muscle has been isolated, and properties of the enzyme were characterized. At pH 7.0, and in the presence of 100 mM potassium chloride the enzyme manifests a distinctly sigmoidal type of kinetics, with S0.5 parameter value about 12 mM. 1 mM ATP strongly activates the enzyme, and diminishes the value of S0.5 to 1.2 mM. In contrast to that 2.5 mM orthophosphate slightly inhibits the activity of AMP-deaminase studied and increases the S0.5 to about 14 mM. Similarly to ATP, orthophosphate does not influence the maximum velocity of the reaction. Electrophoresis in the presence of sodium dodecyl sulphate revealed that the molecular weight of human smooth muscle AMP-deaminase subunit is close to 37 kDa.     

Comparative immunologic and kinetic evaluation of AMP-deaminase isolated from normal human liver and hepatocellular carcinoma (HCC)

In the present paper physico-chemical properties of AMP-deaminase purified from human liver neoplasm-hepatocellular carcinoma (HCC) were investigated and compared with these obtained for the enzyme from normal, unaffected tissue.     

AMP-deaminase from human term placenta

AMP-deaminase from human term placenta was chromatographed on a phosphocellulose column and physico-chemical and immunological properties of the purified enzyme were investigated. At physiological pH7.0, in the absence of regulatory ligands (control conditions) studied AMP-deaminase manifested sigmoid-shaped substrate saturation kinetics, with half-saturation parameter (S_0.5) value of about 7 mM. Addition of important allosteric effectors (ATP, ADP or orthophosphate) modified kinetic properties of studied AMP-deaminase, influencing mainly the value of S_0.5 parameter. Micromolar concentrations of stearylo-CoA inhibited potently the enzyme making it no longer sensitive towards 1 mM ATP-induced activation. SDS-PAGE electrophoresis of the purified enzyme revealed presence of 68 kDa protein fragment, reacting with anti-(human) liver AMP-deaminase antibodies. Experimental results presented indicate that liver type of AMP-deaminase is an enzyme form present in human term placenta.     

Full-size form of human liver AMP-deaminase?

AMP-deaminase from human liver was purified by two-step phosphocellulose chromatography, and SDS-PAG electrophoresis of the most active enzyme fraction eluted has been performed. The largest of the protein fragments revealed had a size (92 kDa) of an apparent full-size enzyme subunit, and reacted positively with antibodies produced against specific human ampd2 gene product. Three-day storage at cold room temperature modified significantly the electrophoretical pattern of the enzyme, evidencing continuous and progressive degradation of its structure. This is a first report evidencing the presence of apparent full-size form of human liver AMP-deaminase in preparation obtained from endogenous source.