Adenosine-5 -phosphatase

The changes in a-ketoglutarate decarboxylase and pyruvic acid decarboxylase activities are not the only biochemical alterations observed in thiamine deficiency. The activities of some other enzymes were found to be altered, for example, erythrocyte transke-tolase and adenosine-5 -phosphatase activities are decreased in chicken brain. 

Beriberi is associated with low transketolase activity in the blood, and determination of transketolase activity may be a helpful diagnostic tool in detecting mild forms of thiamine deficiencies. The fact that adding thiamine pyrophosphate to the system restores transketolase activity confers specificity to this procedure. Although the decrease in transketolase activity is easy to explain in view of the role that thiamine plays in that reaction, the mechanism of the alteration in adenosine-5 -phosphatase is not so obvious. The total activity is decreased despite a slight increase of adenosine phosphatase in the nuclei, and the decrease in total activity results from a marked decrease of adenosine phosphatase in the fibers. Increased thiamine pyrophosphate and alkaline phosphatase have also been observed in the optic tectum of the deficient chicken, but these changes remain unexplained. 

TPN differs from DPN in having one more phosphate group, which has been assigned to carbon 2 of the adenylic acid moiety. This is based on several lines of evidence. By splitting the pyrophosphate bond of TPN with potato nucleotide pyrophosphatase it was possible to separate a diphosphoadenosine fragment (compound XI) as the lead salt. Phosphate esterified to carbon 5 was hydrolyzed by an adenosine-5-phosphatase from potato, and the product was found to be identical with adenylic acid a, presumably adenosine-2 -phosphate. Confirmatory evidence has been provided by finding a deaminase active with adenosine-3 -phosphate, adenosine-5 -phosphate, ATP, and DPN but inert with adenosine-2 -phosphate and TPN. ... 

Adenyl cyclase systems isolated from mammals contain as a third component an additional guanosine triphosphate specific subunit and in all probability even more components whose function and structure are not known as yet. It is interesting to note that adenyl cyclases, Na-K-actlvated adenosine phosphatases (ATPases) have been located in the membrane of olfactory neurons and cAMP was found to have the highest concentration in man in the olfactory mucosa. 

Adenosine is formed from ATP via a phosphatase cascade that sequentially involves the diphosphate, ADP, and the monophosphate, AMP. The actions of adenosine are terminated by uptake and rephosphorylation via adenosine kinase to AMP or by cataboHsm via adenosine deaminase to inosine and hypoxanthine. 

If MLCK activates contraction by increasing myosin phosphorylation, then an increase in the activity of myosin light chain phosphatase, MLCP, by decreasing the fraction of myosin which is phosphorylated, should lead to relaxation from the active (contractile) state. Cyclic adenosine monophosphate (AMP) is a strong inhibitor of smooth muscle contraction and it has been suggested that activation of MLCP could result from its phosphorylation via cAMP activated protein kinase (see Figure 5). 

AP = alkaline phosphatase ATPase = adenosine triphosphatase Cardio = cardiovascular d = day(s) Endocr = endocrine F = female Gastro = gastrointestinal Gn pig = guinea pig GOT = glutamic-oxaloacetic transaminase GPT = glutamic-pyruvic transaminase Hemato = hematological hr = hour(s) LDH = lactate dehydrogenase LOAEL = lowest-observable-adverse-effect level M = male Musc/skel = musculoskeletal NOAEL = no-observable-adverse-effect level ... 

Figure 25-8. Control of adipose tissue lipolysis. (TSH, thyroid-stimulating hormone FFA, free fatty acids.) Note the cascade sequence of reactions affording amplification at each step. The lipolytic stimulus is "switched off" by removal of the stimulating hormone the action of lipase phosphatase the inhibition of the lipase and adenylyl cyclase by high concentrations of FFA the inhibition of adenylyl cyclase by adenosine and the removal of cAMP by the action of phosphodiesterase. ACTFI,TSFI, and glucagon may not activate adenylyl cyclase in vivo, since the concentration of each hormone required in vitro is much higher than is found in the circulation. Positive ( ) and negative ( ) regulatory effects are represented by broken lines and substrate flow by solid lines.

Dl-iike receptors activate the Gs transduction pathway, stimulating the production of adenylyl cyclase, which increases the formation of cyclic adenosine monophosphate (cAMP) and ultimately increases the activity of cAMP-dependent protein kinase (PKA). PKA activates DARPP-32 (dopamine and cyclic adenosine 3, 5 -monophosphate-regulated phosphoprotein, 32 kDa) via phosphorylation, permitting phospho-DARPP-32 to then inhibit protein phosphatase-1 (PP-1). The downstream effect of decreased PP-1 activity is an increase in the phosphorylation states of assorted downstream effector proteins regulating neurotransmitter... 

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... 

Aminophthalate anion Atmospheric pressure active nitrogen Analyte pulse perturbation-chemiluminescence spectroscopy Arthromyces rasomus peroxidase Ascorbic acid Adenosine triphosphate Avalanche photodiode 5-Bromo-4-chloro-3-indolyl 2,6-Di-t< r/-bu(yl-4-mclhyl phenol Bioluminescence Polyoxyethylene (23) dodecanol Bovine serum albumin Critical micelle concentration Calf alkaline phosphatase Continuous-addition-of-reagent Continuous-addition-of-reagent chemiluminescence spectroscopy Catecholamines Catechol... 

Brydon and Roberts- added hemolyzed blood to unhemolyzed plasma, analyzed the specimens for a variety of constituents and then compared the values with those in the unhemolyzed plasma (B28). The following procedures were considered unaffected by hemolysis (up to 1 g/100 ml hemoglobin) urea (diacetyl monoxime) carbon dioxide content (phe-nolphthalein complex) iron binding capacity cholesterol (ferric chloride) creatinine (alkaline picrate) uric acid (phosphotungstate reduction) alkaline phosphatase (4-nitrophenyl phosphate) 5 -nucleotidase (adenosine monophosphate-nickel) and tartrate-labile acid phosphatase (phenyl phosphate). In Table 2 are shown those assays where increases were observed. The hemolysis used in these studies was equivalent to that produced by the breakdown of about 15 X 10 erythrocytes. In the bromocresol green albumin method it has been reported that for every 100 mg of hemoglobin/100 ml serum, the apparent albumin concentration is increased by 100 mg/100 ml (D12). Hemolysis releases some amino acids, such as histidine, into the plasma (Alb). 

Creatine phosphokinase activity has been reported to be minimally inhibited by hemolysis. Hemoglobin concentrations of 1.25 g/100 ml inhibit 5% and 2.5 g/100 ml, 12% (N5). However, in methods utilizing adenosine diphosphate in the reaction mixture, hemolysates containing 100 mg of hemoglobin per 100 ml may have apparent activities of 5-100 units/liter. The activity is presumably related to adenylate kinase in the erythrocyte (S33). In methods utilizing adenosine diphosphate in a coupled enzyme reaction with hexokinase and glucose-6-phosphatase, the inhibitory effect can be eliminated by adding sufficient adenosine mono-... 

Phenol, 5-methoxy-3-[2-(3-hydroxy-4-methoxy-phenyl)-ethylj Lf 1.9 3209 Phenylalanine 1 3172 Phloriglucinol, P-D-glucoside St ° Phosphatase, adenosine-5 1 3172 Phosphoric acid 1 3172 Phthalate, N-butyl 0 ... 

Male Fischer 344 rats were fed diets containing 200 ppm AAF for seven weeks to induce hepatocellular altered foci, and were then fed diets containing either 0 or 12 000 ppm di(2-ethylhexyl) phthalate (purity, 98%) for 24 weeks. In foci that were induced by AAF, di(2-ethylhexyl) phthalate reduced the activity of y-GT, as detected histo-chemically, but did not increase the number, mean volume or volume percentage of foci detected by deficiencies in iron storage, glucose-6-phosphatase, adenosine triphosphatase or fibronectin. Although the numbers of haematoxylin/eosin-stained foci were increased in di(2-ethylhexyl) phthalate-treated rats, the volume percentage was not... 

Testicular effects were also investigated after oral administration of 2000 mg/kg bw di(2-ethylhexyl) phthalate for seven consecutive days to 13-week-old male Wistar rats (Saxena et al., 1985). Degeneration was observed in about 40% of the seminiferous tubules. Loss of succinic dehydrogenase, NADH-diaphorase and acid phosphatase activity and increases in adenosine triphosphatase, glucose-6-phosphate dehydrogenase and alkaline phosphatase activity were observed in treated rats. 

Phosphorylation and dephosphorylation Phosphorylation reactions are catalyzed by a family of enzymes called protein kinases that use adenosine triphosphate (ATP) as a phosphate donor. Phosphate groups are cleaved from phosphorylated enzymes by the action of phosphoprotein phosphatases (Figure 5.18). 

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). 

The enzyme obtained by this purification procedure 11), when tested for contaminants under very stringent conditions, was found to be completely free from phosphatase, DNase, ribonuclease, and adenosine deaminase activities. 

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