scholarly journals [3H]Ouabain binding and Na+, K+-ATPase in resealed human red cell ghosts.

1983 ◽  
Vol 81 (3) ◽  
pp. 401-420 ◽  
Author(s):  
D G Shoemaker ◽  
P K Lauf
Keyword(s):  
Adenylate Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Adenosine Diphosphate ◽  
Red Cell ◽  
Ouabain Binding ◽  
Intracellular Magnesium ◽  
High Affinity ◽  
Binding Rate ◽  
High Affinity Binding Site

The interaction of the cardiac glycoside [3H]ouabain with the Na+, K+ pump of resealed human erythrocyte ghosts was investigated. Binding of [3H]ouabain to high intracellular Na+ ghosts was studied in high extracellular Na+ media, a condition determined to produce maximal ouabain binding rates. Simultaneous examination of both the number of ouabain molecules bound per ghost and the corresponding inhibition of the Na+, K+-ATPase revealed that one molecule of [3H]ouabain inhibited one Na+, K+-ATPase complex. Intracellular magnesium or magnesium plus inorganic phosphate produced the lowest ouabain binding rate. Support of ouabain binding by adenosine diphosphate (ADP) was negligible, provided synthesis of adenosine triphosphate (ATP) through the residual adenylate kinase activity was prevented by the adenylate kinase inhibitor Ap5A. Uridine 5'-triphosphate (UTP) alone did not support ouabain binding after inhibition of the endogenous nucleoside diphosphokinase by trypan blue and depletion of residual ATP by the incorporation of hexokinase and glucose. ATP acting solely at the high-affinity binding site of the Na+, K+ pump (Km approximately 1 microM) promoted maximal [3H]ouabain binding rates. Failure of 5'-adenylyl-beta-gamma-imidophosphate (AMP-PNP) to stimulate significantly the rate of ouabain binding suggests that phosphorylation of the pump was required to expose the ouabain receptor.

Red cell adenylate kinase activity in AK1 and AK 2-1 phenotypes

1970 ◽  
Vol 33 (4) ◽  
pp. 361-364 ◽  
Author(s):  
SANDRA RAPLEY ◽  
HARRY HARRIS
Keyword(s):  
Adenylate Kinase ◽  
Kinase Activity ◽  
Red Cell

Creatine kinase in serum: 2. Interference of adenylate kinase with the assay.

1976 ◽  
Vol 22 (11) ◽  
pp. 1806-1811 ◽  
Author(s):  
G Szasz ◽  
W Gerhardt ◽  
W Gruber ◽  
E Bernt
Keyword(s):  
Creatine Kinase ◽  
Adenylate Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Hepatic Damage ◽  
Complete Inhibition ◽  
Kinase Assay ◽  
Competitive Inhibitors ◽  
Diadenosine Pentaphosphate ◽  
Adenylate Kinases

Abstract Interference of adenylate kinase with Oliver's method [Biochem. J. 61, 116 (1955)] for creatine kinase is usually suppressed by including an adenylate kinase inhibitor, AMP. We studied the kinetics and compared the inhibition capacities of AMP and diadenosine pentaphosphate. Both are competitive inhibitors, AMP being markedly weaker, with a Ki of about 300 mumol/liter for adenylate kinase from erythrocyte, muscle, and liver. AMP also weakly inhibitis creatine kinase. Diadenosine pentaphosphate inhibits erythrocyte and muscle adenylate kinase strongly (Ki about 0.03 mumol/liter), the liver isoenzyme less strongly (Ki about 3 mumol/liter), and has no effect on creatine kinase up to 100 mumol/liter. All three adenylate kinases may be present in a patinet's serum, causing sample blanks to be high in a creatine kinase assay that lacks inhibitors. In acute hepatic damage, liver adenylate kinase activity in serum can be grossly increased. Use of sufficient diadenosine pentaphosphate alone for complete inhibition is relatively expensive. Consequently, we recommend a combination of both inhibitors. Diadenosine pentaphosphate, 10 mumol, combined with 5 mmol of AMP per liter inhibits adenylate kinase from erythrocytes and muscle by 97% and from liver by 95%.

scholarly journals Decreased pyrimidine nucleoside monophosphate kinase activity in sickle erythrocytes

Blood ◽  
1992 ◽  
Vol 80 (2) ◽  
pp. 512-516 ◽  
Author(s):  
CR Zerez ◽  
NA Lachant ◽  
KM Lent ◽  
KR Tanaka
Keyword(s):  
Blood Cell ◽  
Adenylate Kinase ◽  
Kinase Activity ◽  
The Other ◽  
Pyrimidine Nucleoside ◽  
Phosphogluconate Dehydrogenase ◽  
High Affinity ◽  
Marked Inhibition ◽  
Cell Age ◽  
Sickle Erythrocytes

Abstract We have previously shown that physiologic concentrations of hemin cause marked inhibition of several red blood cell (RBC) enzymes. Because endogenous heme content is elevated in sickle RBCs, we have examined the activity of hemin-sensitive enzymes in these RBCs. One of the hemin- sensitive enzymes, pyrimidine nucleoside monophosphate kinase (PNMK), was shown to have decreased activity in sickle RBCs relative to RBCs of equivalent cell age. The other hemin-sensitive enzymes, including adenylate kinase (AK), pyrimidine 5′-nucleotidase (P5N), 6- phosphogluconate dehydrogenase (6PGD), and aldolase, had activities that were appropriate for cell age. We have also examined the affinity of the hemin-sensitive enzymes to hemin. Using two different methods, PNMK was shown to have the highest binding affinity to hemin. The exquisite sensitivity of PNMK to inhibition by hemin, coupled with the enzyme's high affinity to hemin, may account for the decrease in PNMK activity and the lack of significant decrease in the other hemin- sensitive enzymes in sickle RBCs. These results suggest that the increased endogenous heme content in sickle RBCs may be responsible for the decrease in PNMK activity. Whether the increased endogenous heme content of sickle RBCs can cause hemolysis indirectly by inhibiting RBC enzymes remains to be determined.

scholarly journals Conversion of the Low Affinity Ouabain-binding Site of Non-gastric H,K-ATPase into a High Affinity Binding Site by Substitution of Only Five Amino Acids

2006 ◽  
Vol 281 (19) ◽  
pp. 13533-13539 ◽  
Author(s):  
Li Yan Qiu ◽  
Herman G. P. Swarts ◽  
Elisa C. M. Tonk ◽  
Peter H. G. M. Willems ◽  
Jan B. Koenderink ◽  
...  
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Affinity Binding ◽  
Ouabain Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site

scholarly journals Decreased pyrimidine nucleoside monophosphate kinase activity in sickle erythrocytes

Blood ◽  
1992 ◽  
Vol 80 (2) ◽  
pp. 512-516
Author(s):  
CR Zerez ◽  
NA Lachant ◽  
KM Lent ◽  
KR Tanaka
Keyword(s):  
Blood Cell ◽  
Adenylate Kinase ◽  
Kinase Activity ◽  
The Other ◽  
Pyrimidine Nucleoside ◽  
Phosphogluconate Dehydrogenase ◽  
High Affinity ◽  
Marked Inhibition ◽  
Cell Age ◽  
Sickle Erythrocytes

We have previously shown that physiologic concentrations of hemin cause marked inhibition of several red blood cell (RBC) enzymes. Because endogenous heme content is elevated in sickle RBCs, we have examined the activity of hemin-sensitive enzymes in these RBCs. One of the hemin- sensitive enzymes, pyrimidine nucleoside monophosphate kinase (PNMK), was shown to have decreased activity in sickle RBCs relative to RBCs of equivalent cell age. The other hemin-sensitive enzymes, including adenylate kinase (AK), pyrimidine 5′-nucleotidase (P5N), 6- phosphogluconate dehydrogenase (6PGD), and aldolase, had activities that were appropriate for cell age. We have also examined the affinity of the hemin-sensitive enzymes to hemin. Using two different methods, PNMK was shown to have the highest binding affinity to hemin. The exquisite sensitivity of PNMK to inhibition by hemin, coupled with the enzyme's high affinity to hemin, may account for the decrease in PNMK activity and the lack of significant decrease in the other hemin- sensitive enzymes in sickle RBCs. These results suggest that the increased endogenous heme content in sickle RBCs may be responsible for the decrease in PNMK activity. Whether the increased endogenous heme content of sickle RBCs can cause hemolysis indirectly by inhibiting RBC enzymes remains to be determined.

scholarly journals Side-dependent effects of internal versus external Na and K on ouabain binding to reconstituted human red blood cell ghosts.

1976 ◽  
Vol 67 (5) ◽  
pp. 497-525 ◽  
Author(s):  
H H Bodemann ◽  
J F Hoffman
Keyword(s):  
Blood Cell ◽  
Binding Site ◽  
Red Blood Cell ◽  
Red Cell ◽  
Ouabain Binding ◽  
Binding Properties ◽  
Conformational States ◽  
Binding Rate ◽  
Lack Of Control

The side-dependent effects of internal and external Na and K on the ouabain binding rate, as promoted by inside MgATP, has been evaluated utilizing reconstituted human red blood cell ghosts. Such ghost systems provide the situation where [Na]i, [K]i, [Na]o, and [K]o can each be varied under conditions in which the others are either absent or fixed at constant concentrations. It was found that, in the presence of Ko, increasing either [Na]i or [K]i resulted in decreasing the rate at which ouabain was bound. Changes in [Na]i or [K]i in the absence of Ko were without effect on the ouabain binding rate. Thus, the ouabain binding rate was found to vary inversely with the rate of Na:K and K:K exchange but was independent of the rate of Na:Na exchange. The effect of Ko in antagonizing ouabain binding, as well as the influence of Nao on this interaction, were found to require the presence of either Nai or Ki. The results are interpreted in terms of a model relating the availability of the ouabain binding site to different conformational states of the pump complex. Differences were observed in the ouabain binding properties of red cell ghosts compared to microsomal preparations but it is not known whether the basis for the differences resides in the different preparations studied or in the lack of control of sidedness in the microsomal systems.

P1, P5-Bis-(5′-adenosyl)pentaphosphate: Is this Adenylate Kinase Inhibitor Substrate for Mitochondrial Processes?

1977 ◽  
Vol 32 (9-10) ◽  
pp. 786-791 ◽  
Author(s):  
Josef Köhrle ◽  
Joachim Lüstorff ◽  
Eckhard Schlimme
Keyword(s):  
Adenylate Kinase ◽  
Kinase Inhibitor ◽  
Adenine Nucleotide ◽  
Nucleoside Diphosphate Kinase ◽  
Liver Mitochondria ◽  
Inhibitory Effect ◽  
Rat Liver Mitochondria ◽  
Intermembrane Space ◽  
Rabbit Muscle ◽  
Inner Mitochondrial Membrane

Abstract 1. P1, P5-Bis-(5′-adenosyl)pentaphosphate (Ap5A) inhibits “soluble” adenylate kinase even when this enzyme is an integral part of the complete mitochondrion. The Ki is 10-5м , i. e. about two orders of magnitude higher than the inhibitor constants determined for the purified adenylate kinase of rabbit muscle and an enzyme preparation separated from the mitochondrial intermembrane space. The weaker inhibitory effect is due to a lower accessibility of the enzyme.2. As to be expected Ap5A which is of the “multisubstrate analogue”-type does not affect mito­ chondrial nucleoside diphosphate kinase.3. Though Ap5A owns the structural elements of both ATP and ADP it is not a substrate of the adenine nucleotide carrier, i.e. neither it is exchanged across the inner mitochondrial membrane nor specifically bound.4. Ap5A is not metabolized by rat liver mitochondria.

scholarly journals APPARENT ADENYLATE KINASE ACTIVITY IN VIVO

1953 ◽  
Vol 204 (1) ◽  
pp. 283-288
Author(s):  
Florapearl A. Cobey ◽  
Philip Handler
Keyword(s):  
Adenylate Kinase ◽  
Kinase Activity
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