The adenylate kinase of human plasma, erythrocytes and platelets in relation to the degradation of adenosine diphosphate in plasma

1. Adenylate kinase (EC 2.7.4.3) has been shown to be present in human plasma obtained by conventional means and the adenylate-kinase activities of plasma and of lysed and intact human platelets and erythrocytes have been measured at 37 degrees by sensitive spectrophotometric methods. 2. The activities found in plasma ranged from 2.7 to 22.9mumoles of ADP formed/min./l. and in lysed platelets and lysed erythrocytes mean values of 0.79 and 12.0mumoles of ADP formed/min./10(9) cells respectively were found. Intact platelets and erythrocytes showed little or no activity. 3. The apparent K(m) of plasma adenylate kinase for ADP was found to be 1.4-1.6mm. 4. The adenylate-kinase activity of plasma was correlated with the free haemoglobin present and the larger part of the activity could be accounted for by haemolysis occurring either during the withdrawal of the blood or in vivo. 5. Aggregation of platelets by ADP, collagen fibres or thrombin released up to 16% of the platelet adenylate kinase into the suspending medium. 6. Measurement of the rate of breakdown of 1.6mum-ADP in plasma gave values of about 0.1mmu-mole/min./ml. This was not increased by addition of sufficient erythrocyte lysate to increase the activity of plasma adenylate kinase five to ten times. 7. It was concluded that the activity of adenylate kinase found in plasma, even after aggregation of the platelets, is insufficient to account for the rate of breakdown of low concentrations of ADP usually observed, and that another enzyme is responsible for this process.     

An arsenical analogue of adenosine diphosphate.

An analogue of ADP was made in which the terminal phosphono-oxy group, -O-PO(OH)2, has been replaced by the arsonomethyl group, -CH2-AsO(OH)2. This compound cannot form a stable analogue of ATP because anhydrides of arsonic acids are rapidly hydrolysed, so that any enzyme that phosphorylates ADP and accepts this analogue as a substrate should release orthophosphate in its presence. The analogue proves to be a poor substrate for 3-phosphoglycerate kinase (V/Km is diminished by a factor of 10(2)-10(3)) and a very poor substrate for pyruvate kinase (V/Km is diminished by a factor of 10(5)-10(6)). No substrate action was detected with adenyl kinase and creatine kinase.     

Synthesis of simple adenosine diphosphate ribose analogues

[See figures]. The synthesis of analogues of adenosine diphosphate ribose and acetylated adenosine diphosphate ribose, modified at the northern pentose, is reported. The stereochemistry at the acetylated centers was chosen to minimize acetyl migration and dictated the overall synthetic strategy.     

Changes in concentration of adenosine triphosphate and adenosine diphosphate in individual preimplantation sheep embryos.

Concentrations of ATP and ADP were measured in 156 sheep embryos by means of an ultramicrofluorescence assay. Stages of preimplantation development measured included unfertilized oocytes through blastocyst-stage embryos. ATP concentrations remained constant through the 8-cell stage; then ATP decreased significantly (p < 0.025) at the morula stage and remained low through the blastocyst stage. ADP concentrations did not change throughout the embryonic stages measured. Decreased levels of ATP with constant levels of ADP caused the ATP:ADP ratio to decrease significantly (p < 0.025) between the 8-cell and morula stages. We suggest that the increase in glucose uptake by sheep embryos observed at the morula stage of development may be due, in part, to a decrease in the ATP:ADP ratio.     

Regulatory effects of adenosine diphosphate on the activity of the plasma membrane ATPase of corn roots

Plasma membrane enriched microsomal fraction was isolated from corn root cells by sucrose density centrifugation. The ATPase activity as measured by the release rate of inorganic phosphate, was decreased by the presence of modifiers which included diethylstilbestrol, vanadate, N,N'-dicyclohexylcarbodiimide, and miconazole. The presence of ADP also decreased the rate of ATP hydrolysis. Furthermore, a preincubation of the membrane with ADP significantly reduced the inhibitory effects of these membrane ATPase modifiers. Since the modes of interaction of these modifiers with the enzyme are different, the results suggest that the binding of ADP may stabilize the plasma membrane ATPase in a modifier insensitive state.     

Fluorometric determination of plasma adenosine concentrations using high-performance liquid chromatography

Methods are described for the fluorometric determination of plasma adenosine concentrations, using HPLC. Plasma obtained from blood of dogs treated with erythro-(2-hydroxy-3-nonyl)adenine hydrochloride and dipyridamole was deproteinized with perchloric acid and the neutralized sample was put sequentially onto a SepPak C18 and boronic acid affinity column. Subsequently, adenosine in the final elution was converted to 1,N6-ethenoadenosine and was quantitated by HPLC with a fluorescence detector. The percentage recovery of adenosine added to the deproteinized plasma was nearly 100%. In the adenosine deaminase treated plasma, the increase in adenosine concentration of even 4 nM can be accurately determined. The control renal venous plasma concentrations of adenosine in anesthetized dogs were 19.9 +/- 1.9 nM, a significantly higher value than the corresponding arterial concentrations (12.7 +/- 1.1 nM), thereby suggesting the renal release of adenosine. This release was markedly enhanced following the removal of the renal arterial occlusion. Thus, taken together with the in vivo results, the present method is sensitive, hence most useful for the determination of plasma adenosine concentrations.     

Nonenzymic adenosine 5'-diphosphate ribosylation of poly(adenosine diphosphate ribose)

Poly(adenosine 5'-diphosphate ribose) [poly(ADP-ribose]) is spontaneously ADP-ribosylated when it is incubated with nicotinamide adenine dinucleotide, especially in 0.5 M NaCl and at an alkaline pH. The ADP-ribose residues are monomeric and are attached to the middle of polymer chains. The linkage is similar to, and may be identical with, that of the branch points that are created in cells. RNA is also spontaneously ADP-ribosylated, but not DNA.     

Presence and turnover of adenosine diphosphate ribose in human erythrocytes.

ADP-ribose was detected in human red blood cells (RBC) at 0.45 +/- 0.1 microM concentrations. These levels could be estimated after purification of ADP-ribose by means of three sequential HPLC fractionations of RBC extracts. Extraction was performed by sonication of RBC either in trichloroacetic acid, followed by centrifugation, or in carbonate-bicarbonate buffer, pH 10.0, followed by rapid ultrafiltration. Neither procedure of extraction caused artefactual formation of ADP-ribose. Prolonged incubation of intact RBC in isotonic buffer containing labeled orthophosphate resulted in the slow incorporation of radioactivity into ADP-ribose. Identification of the labeled ADP-ribose was confirmed upon incubation of the purified metabolite with nucleotide pyrophosphatase, yielding radioactive 5'-AMP and ribose 5-phosphate, while its exposure to a nonspecific deaminase resulted in the quantitative formation of labeled inosine diphosphate ribose.