Structural-functional relationships in pig heart AMP-deaminase in the presence of ATP, orthophosphate, and phosphatidate bilayers.

The secondary structure of pig heart AMP-deaminase (AMP-d) in the absence and in the presence of orthophosphate or dioleoyl phosphatidic acid (DOPA) or ATP was investigated by FT-IR spectroscopy. While the latter substance activates the enzyme, orthophosphate is a well-known negative allosteric effector and DOPA exerts a noncompetitive inhibition on AMP-deaminase. Small changes in the secondary structure of AMP-d were induced by the above mentioned substances. Only DOPA reduced the thermal stability of AMP-d and avoided protein intermolecular interactions suggesting structural-functional relationships in AMP-d in the presence of the above substances and a possible role of phosphatidic acid in the subtle regulation of AMP-d activity by temporary binding of the enzyme to cellular membranes.

Structural properties and thermal stability of human liver and heart fatty acid binding proteins: a Fourier transform IR spectroscopy study.

The secondary structure and the thermal stability of human liver (L-FABP) and heart (H-FABP) fatty acid-binding proteins were analyzed, in the absence and in the presence of oleic acid, by Fourier transform ir spectroscopy. The study was done in order to gain information on the secondary as well three-dimensional structure of L-FABP and to check the possible H-FABP self-association that has been found to occur in rat and pig H-FABP. Comparison of human L-FABP and H-FABP ir spectra reveals that, in spite of the low sequence homology, the two proteins have similar secondary and probably tertiary structures. The ir data indicates that a larger amount of beta-strands are exposed to the solvent in H-FABP as compared to L-FABP, suggesting minor differences in the three-dimensional structures of these proteins. The binding of oleic acid to L-FABP and H-FABP stabilizes their structures and does not modify their secondary structure. The ir spectra neither confirm nor exclude self-association of human H-FABP.

A comparison of the secondary structure of human brain mitochondrial and cytosolic 'malic' enzyme investigated by Fourier-transform infrared spectroscopy.

The secondary structure of human brain cytosolic and mitochondrial 'malic' enzymes purified to homogeneity has been investigated by Fourier-transform IR spectroscopy. The absorbance IR spectra of these two isoenzymes were slightly different, but calculated secondary-structure compositions were essentially similar (38% alpha-helix, 38-39% beta-sheet, 14% beta-turn and 9-10% random structure). These proportions were not affected by succinate, a positive effector of mitochondrial 'malic' enzyme activity. IR spectra indicate that the tertiary structures of human brain cytosolic and mitochondrial 'malic' enzymes are slightly different, and addition of succinate does not cause conformational changes to the tertiary structure of the mitochondrial enzyme. Thermal-denaturation patterns of the cytosolic and mitochondrial enzymes, obtained from spectra recorded at different temperatures in the absence or presence of Mg2+, suggest that the tertiary structure of both isoenzymes is stabilized by bivalent cations and that the cytosolic enzyme possesses a more compact tertiary structure.

Acetate-induced changes of adenine nucleotide levels in rat liver.

The changes in adenine nucleotide concentration induced by acetate were investigated in rat liver in situ and in isolated rat hepatocytes. Adenosine monophosphate (AMP) concentration increased approximately threefold within 15 minutes after intraperitoneal injection of sodium acetate. A small but significant decrease in adenosine triphosphate (ATP) concentration also occurred. Consequently, the ATP/AMP ratio decreased from approximately 14 (the value found in control or sodium chloride-injected rats) to approximately 3 (the value found in sodium acetate-injected rats). Adenosine diphosphate (ADP) concentration increased slightly, but this was statistically nonsignificant. Total adenine nucleotide concentrations after acetate injection remained essentially the same as those in control rats. Adenylate energy charge decreased after acetate administration. No significant changes in nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) concentrations were found after sodium acetate injection. Similar patterns of changes in adenine nucleotide concentrations were found in isolated rat hepatocytes incubated in the presence of acetate. These data indicate that acetate, which appears in human blood either during hemodialysis with acetate-containing solution or after ethanol consumption, may alter energy equilibrium of adenine nucleotides in the liver. This is due to the conversion of ATP to AMP in the course of acetate to acetyl-coenzyme A (CoA) activation. It is therefore possible that accelerated ATP turnover in the liver may contribute both to the "intolerance to acetate" in patients subjected to dialysis with the sodium acetate-containing solution and to the pathogenesis of gout associated with excessive ethanol consumption.

The interaction of phospholipid bilayers with pig heart AMP deaminase: Fourier-transform infrared spectroscopic and kinetic studies.

The interaction of pig heart AMP deaminase with different chemical species of phosphatidylcholine and with natural plasma membranes has been investigated. Phospholipids added to the system either as natural biological membranes (plasma membrane vesicles) or in the form of liposomes containing unsaturated phosphatidylcholine considerably enhanced AMP deaminase activity. The secondary structure of pig heart AMP deaminase in the absence and in the presence of dioleoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine liposomes was investigated by Fourier-transform infrared spectroscopy. Quantitative analysis of the amide I band showed that the enzyme contains 45% beta-sheets, 28% alpha-helix, 16% turns and 11% non-ordered structure. In the presence of dioleoyl phosphatidylcholine liposomes, the beta/alpha content ratio decreased; this decrease was dependent on the amount of lipid added. This phenomenon was not observed in the case of dipalmitoyl phosphatidylcholine liposomes. These data suggest a possible role for membrane phospholipids in the regulation of AMP deaminase activity.

Changes of nucleotide content in human and rat heart during cardiac surgery and ischemia.

The influence of ischemia on purine nucleotide and their catabolite concentration in human myocardium was investigated during surgery of acquired and congenital heart defects. This was compared with the influence of ischemia on rat heart. Concentrations of adenine and guanine nucleotides and their catabolites were measured in the extracts of heart biopsies taken at the onset of ischemia and at the time of reperfusion. The content of myocardial ATP in human heart decreased from the initial value of 22.3 +/- 1.1 to 14.6 +/- 1.5 nmol/mg protein and total adenine nucleotide pool decreased from 34.2 +/- 1.8 to 27.6 +/- 1.5 nmol/mg protein during the operation. Significant increases in myocardial concentrations of purine catabolites were also observed with the most prominent rise in inosine from below 0.5 at the onset of the ischemia to 3.0 +/- 0.5 nmol/mg protein at the time of reperfusion. A positive correlation was demonstrated between the concentration of purine catabolites in the heart at the end of ischemia with the decrease of both ATP and the total nucleotide pool. An interesting metabolic specificity of the ischemic human heart appeared to be only a small accumulation of inosine monophosphate (IMP). The increase of IMP in the rat heart after ischemia was several-fold higher. Thus, cardiac surgery of congenital and acquired heart defects was associated with a significant decrease in myocardial adenylate pool and a single biopsy collected at the end of ischemia seems to be sufficient to evaluate the extent of this metabolic and possibly functional impairment of the heart.

Formation and breakdown of uridine in ischemic hearts of rats and humans.

In contrast to cardiac purine metabolism, little is known about pyrimidine catabolism in heart. We therefore investigated uridine and uracil formation in ischemic rat and human hearts. Human donor hearts accumulated uridine 3 x (P < 0.05) before implantation. Hearts released this pyrimidine during implantation or correction of cardiac defects. During the former systemic blood uridine rose 38% (P < 0.05). In explanted human hearts, uridine was the only pyrimidine released during reperfusion; isolated, perfused rat hearts produced initially 3 x more uracil than uridine. Uridine phosphorylase activity in human heart homogenate was 3.4 mU/g wet weight, i.e. 60 x lower than that in rat myocardium (198 mU/g, P < 0.02); its purine counterpart, nucleoside phosphorylase, differed much less in activity (0.32 and 1.12 U/g, respectively; P < 0.001). Thus human heart is virtually devoid of uridine phosphorylase, contrasting rat heart. Consequently uridine accumulates in ischemic human heart while uracil production predominates in rat heart.

Purines, lactate and phosphate release from child and adult heart during cardioplegic arrest.

The release of lactate, phosphate and purine catabolites from the heart in adult and children undergoing cardiac surgery was recorded. The compounds were determined in the coronary effluent collected during subsequent infusions of cardioplegic solution into the coronary root. As compared to the infusion just after onset of ischemia, both in adults and children manifold increase of the release was observed during subsequent infusions. The rates of release of lactate, phosphate and purines (adenosine + inosine + hypoxanthine) were 1.5 to 2.5 times higher in children than in adult hearts during the second cardioplegic infusion and 3 to 7 times higher during the third cardioplegic infusion in spite of a more frequent infusion of cardioplegic solution in children. A much greater increase of the release of lactate, phosphate and purines provides evidence for more severe metabolic injury during cardioplegic arrest to the heart in children than in adults.

Adenylate degradation products release from the human myocardium during open heart surgery.

Purine degradation products were determined in the human heart coronary sinus effluent collected from patients undergoing cardiac surgery, during infusion of a cardioplegic solution. At the onset of cardiopulmonary bypass the mean concentrations of adenosine, inosine and hypoxanthine were 0.1, 0.5 and 0.3 mumol/l, respectively. Ischemic arrest leads to a progressive increase of the respective levels to 1.4 17.8 and 9.6 mumol/l after 60-80 min of ischemia. Xanthine concentration was undetectable (less than 0.2 mumol/l) throughout. A substantial urate release (20 mumol/l) was observed which decreased with the duration of ischemia. Xanthine oxidoreductase activity in human myocardium was found to be below the detection limit (0.1 mU/g wet weight). Thus, urate release represented wash out of urate which had accumulated in the tissue.

Comparison of kinetic and regulatory properties of high S0.5 form of AMP deaminase from chicken and pigeon liver with AMP deaminase from rat and ox liver.

1. The high S0.5 form of AMP deaminase from avian liver was shown to display a two times lower S0.5 value than the single mammalian enzyme form. 2. Avian enzymes showed several fold higher affinity to the activator (ATP) but lower affinity to inhibitors (GTP and Pi) than the mammalian AMP deaminases. 3. GTP was shown to exert a biphasic: activating and inhibitory effect on all the enzymes tested, the chicken and pigeon enzymes being activated within a much broader range of effector concentration. 4. In the presence of 3 mM ATP the activity of avian enzymes was not affected by high GTP and Pi concentrations, in contrast to AMP diaminase from rat liver which was strongly inhibited by GTP under the same experimental conditions. 5. The differences of the regulatory properties described are discussed in terms of adjustment of avian liver AMP deaminase to a faster adenylates' catabolism and thus urate synthesis.

The influence of phosphatidate bilayers on pig heart AMP deaminase. Crucial role of pH-dependent lipid-phase transition.

Phosphatidate bilayers composed of dilauroylphosphatidate, dimyristoylphosphatidate, dipalmitoylphosphatidate and dioleoylphosphatidate were prepared. Their interaction with AMP deaminase isolated from pig heart was investigated. Dioleoylphosphatidate bilayers were found to exert non-competitive inhibition on the AMP deaminase with a Ki of 15 x 10(-6) M. This inhibition is three orders of magnitude stronger than that exerted by orthophosphate. The phosphatidate species containing saturated fatty acids were either non-inhibitory or inhibited enzyme activity rather poorly. However, alkalinization of the medium from pH 6.5 to pH 7.9 led to the inhibition of pig heart AMP deaminase by dilauroylphosphatidate bilayers. This was accompanied by the fluidization of the saturated phosphatidate species, i.e. the lowering of their phase transition temperature in alkaline pH, as measured by light-scattering and fluorescence scans. The possible significance of these findings for the regulation of AMP deaminase activity in vivo by natural membranes is discussed.

Two forms of AMP deaminase in bovine heart.

Two forms of bovine heart AMP deaminase were separated by phosphocellulose column chromatography. Form A with lesser affinity to phosphocellulose exhibited a hyperbolic type of substrate curve and was relatively insensitive to ATP. Form B was strongly activated by 1 mM ATP and its substrate saturation kinetics (without ATP) indicated a cooperative effect. The alteration of adenylate energy charge affected forms A and B differently but within the physiological range, the activity of both forms decreased with the increase in energy charge. The Mr of the two forms was identical as estimated by gel filtration. These activities are compared to those of AMP deaminases from the hearts of other species.