Adenosine-5 -phosphate deaminase

The problem of a poor detection limit was caused by high background ATP and by the low sensitivity of the luciferin-luciferase (L-L) reagent. We have already developed an ATP elimination system using two ATP degrading enzymes (adenosine phosphate deaminase and apyrase) and a surfactant tolerant luciferase that was a mutated Luciola lateralis firefly luciferase. We optimized this elimination system, and investigated its suitability as a detection system. 

Fig. 173. Interconversion of inosine, adenosine and guanosine monophosphates 1 Inosine monophosphate dehydrogenase 2 guanosine monophosphate synthase 3 guanosine monophosphate reductase 4 adenylosuccinate synthetase 5 adenylosuccinate lyase 6 adenosine (phosphate) deaminase...

Attention has been drawn to the potential of phosphoric acid anhydrides of nucleoside 5 -carboxylic acids (14) as specific reagents for investigating the binding sites of enzymes. For example, (14 B = adenosine) inactivates adenylosuccinate lyase from E. coli almost completely, but has little effect on rabbit muscle AMP deaminase. The rate of hydrolysis of (14) is considerably faster than that of acetyl phosphate, suggesting intramolecular assistance by the 3 -hydroxyl group or the 3-nitrogen atom. 

The isomerism existing between the pairs of nucleotides was attributed to the different locations of the phosphoryl residues in the carbohydrate part of the parent nucleoside,49 63 since, for instance, the isomeric adenylic acids are both hydrolyzed by acids to adenine, and by alkalis or kidney phosphatase to adenosine. Neither is identical with adenosine 5-phosphate since they are not deaminated by adenylic-acid deaminase,68 60 and are both more labile to acids than is muscle adenylic acid. An alternative explanation of the isomerism was put forward by Doherty.61 He was able, by a process of transglycosidation, to convert adenylic acids a" and 6 to benzyl D-riboside phosphates which were then hydrogenated to optically inactive ribitol phosphates. He concluded from this that both isomers are 3-phosphates and that the isomerism is due to different configurations at the anomeric position. This evidence is, however, open to the same criticism detailed above in connection with the work of Levene and coworkers. Further work has amply justified the original conclusion regarding the nature of the isomerism, since it has been found that, in all four cases, a and 6 isomers give rise to the same nucleoside on enzymic hydrolysis.62 62 63 It was therefore evident that the isomeric nucleotides are 2- and 3-phosphates, since they are demonstrably different from the known 5-phosphates. The decision as to which of the pair is the 2- and which the 3-phosphate proved to be a difficult one. The problem is complicated by the fact that the a and b" nucleotides are readily interconvertible.64,64... 

Figure 22.17 Summary of mechanisms to maintain the ATP/ADP concentration ratio in hypoxic myocardium. A decrease in the ATP/ADP concentration ratio increases the concentrations of AMP and phosphate, which stimulate conversion of glycogen/ glucose to lactic acid and hence ATP generation from glycolysis. The changes also increase the activity of AMP deaminase, which increases the formation and hence the concentration of adenosine. The latter has two major effects, (i) It relaxes smooth muscle in the arterioles, which results in vasodilation that provides more oxygen for aerobic ATP generation (oxidative phosphorylation). (ii) It results in decreased work by the heart (i.e. decrease in contractile activity), (mechanisms given in the text) which decreases ATP utilisation.

Purine nucleotides are degraded by a pathway in which they lose their phosphate through the action of 5 -nucleotidase (Fig. 22-45). Adenylate yields adenosine, which is deaminated to inosine by adenosine deaminase, and inosine is hydrolyzed to hypoxanthine (its purine base) and D-ribose. Hypoxanthine is oxidized successively to xanthine and then uric acid by xanthine oxidase, a flavoenzyme with an atom of molybdenum and four iron-sulfur centers in its prosthetic group. Molecular oxygen is the electron acceptor in this complex reaction. 

Muscular work is accompanied by the production of ammonia, the immediate source of which is adenosine 5 -phosphate (AMP).301 302 This fact led to the recognition of another substrate cycle (Chapter 11) that functions by virtue of the presence of a biosynthetic pathway and of a degradative enzyme in the same cells (cycle A, Fig. 25-17). This purine nucleotide cycle operates in the brain303 304 as well as in muscle. The key enzyme 5-AMP aminohydrolase (AMP deaminase step a, Fig. 25-17) also occurs in erythrocytes and many other tissues.304 305 Persons having normal erythrocyte levels but an absence of this enzyme in muscles suffer from muscular weakness and cramping after exercise.306... 

Hog spleen acid DNase, as obtained by the above procedure, is completely free of contaminating phosphatase, exonuclease, and adenosine deaminase activities. The enzyme has a weak intrinsic hydrolytic activity on bis(p-nitrophenyl) phosphate and the p-nitrophenyl derivatives of deoxyribonucleoside 3 -phosphates (see Section III,D,3). 

After purine nucleotides have been converted to the corresponding nucleosides by 5 -nucleotidases and by phosphatases, inosine and guanosine are readily cleaved to the nucleobase and ribose-1-phosphate by the widely distributed purine nucleoside phosphorylase. The corresponding deoxynucleosides yield deoxyribose- 1-phosphate and base with the phosphorylase from most sources. Adenosine and deoxyadenosine are not attacked by the phosphorylase of mammalian tissue, but much AMP is converted to IMP by an aminohydrolase (deaminase), which is very active in muscle and other tissues (fig. 23.20). An inherited deficiency of purine nucleoside phosphorylase is associated with a deficiency in the cellular type of immunity. 

Many isoenzymes have been identified from various human tissue sources however, our consideration will deal with six erythrocytic systems that have received routine crime laboratory status. These are phosphoglucomutase (PGM), adenylate kinase (AK), adenosine deaminase (ADA), glucose-6-phosphate dehydrogenase (G-6-PD), 6-phosphogluconate dehydrogenase (6-PGD) and erythrocytic acid phosphatase (EAP). 

Thus, uridine-cytidine kinase converts uridine and cytidine to UMP and CMP, respectively thymidine kinase converts thymidine to dTMP and adenosine kinase converts adenosine to AMP. Specific kinases convert monophospho-nucleotides to dinucleotides using ATP as a phosphate donor. The conversion of diphosphonucleotides to triphosphonucleotides is carried out by a nonspecific nucleoside diphosphate kinase. This includes both the ribo- and deoxy-ribonucleotides. Cytosine and its nucleoside and nucleotide transformations are often associated with the metabolism of uracil and its nucleosides and nucleotides. Note that UTP can give rise to CTP (Figure 10.9), and also that, in the presence of cytidine deaminase, cytidine can be converted to uridine. 

A phosphorylase from Escherichia coli has been purified it is specific for 2-deoxy-D-ribosyl phosphate, but can use uracil, 2-thiouracil, 5-amino-uracil, 5-bromouracil, and 2-thiothymine as a pyrimidine. Deaminases of adenosine, 2-deoxyadenosine, cytidine, and 2-deoxycytidine have been detected in Escherichia coli. 

Figure 4.4 The HPLC analysis of a reaction mixture containing AMP and alkaline phosphatase. Separations were carried out on a reversed-phase column with a mobile phase of potassium phosphate (pH 5.5) and 10% methanol. The column was eluted isocratically, and the detection was at 254 nm. Two sets of tracings were obtained, according to the following schedules. For the original reaction mixture (A) immediately after the addition of enzyme, (B) after 10 minutes, and (C) after 15 minutes. For the reaction mixture to which had been added EHNA (5 /xAf), an inhibitor of adenosine deaminase, the suspected contaminant (D ) after 2 minutes, ( ) after 10 minutes, and (F) after 40 minutes. (From Rossomando et al., 1981.)...

Figure 10.11 AMP can be formed by adenosine kinase (1) in a reaction that uses ATP as the phosphate donor and forms ADP as the second reaction product. Alternatively, AMP can be deaminated to IMP by the enzyme AMP deaminase (2) and converted to inosine (INO) by a 5 -nucleotidase activity (3). Finally, AMP can be phosphorylated to ADP by the enzyme AMP kinase (4).

The salvage pathway does not involve the formation of new heterocyclic bases but permits variation according to demand of the state of the base (B), i.e. whether at the nucleoside (N), or nucleoside mono- (NMP), di- (NDP) or tri- (NTP) phosphate level. The major enzymes and routes available (Scheme 158) all operate with either ribose or 2-deoxyribose derivatives except for the phosphoribosyl transferases. Several enzymes involved in the biosynthesis of purine nucleotides or in interconversion reactions, e.g. adenosine deaminase, have been assayed using a method which is based on the formation of hydrogen peroxide with xanthine oxidase as a coupling enzyme (81CPB426). 

Adenosine deaminase O.IM KCl and O.IM phosphate, pH 7.0 2mM mercaptopurine riboside (substrate analogue)... 

It is of interest that within several years after the observations of Hopkinson et al. (H13), other human erythrocytic enzymes such as phosphoglucomutase, glucose 6-phosphate dehydrogenase, phosphogluco-nate dehydrogenase, adenylate kinase, peptidase, and adenosine deaminase were explored intensively with respect to their polymorphism (H2, HU). However, we shall concern ourselves here only with acid phosphatase. 

---