Amir Askari

Regulation of AMP deaminase by 2,3-diphosphoglyceric acid: a possible mechanism for the control of adenine nucleotide metabolism in human erythrocytes

Abstract 1. 1.AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) activities of dialyzed and undialyzed hemolyzates of human erythrocytes in the presence of optimal concentrations of K+ were measured. Whereas the activity of the dialyzed hemolyzate was a linear function of the hemolyzate concentration, that of the undialyzed hemolyzate was not. These results suggested the presence of a dialyzable inhibitor of the enzyme in the hemolyzate. 2. 2.Effects of several organic phosphates of erythrocytes on AMP deaminase were studied, 2,3-Diphosphoglyceric acid, at concentrations usually found in erythrocytes, was found to be an inhibitor of the enzyme. 3. 3.The substrate-velocity curve of AMP deaminase is a sigmoid. In the presence of 2,3-diphosphoglyceric acid the curve became more sigmoidal in shape. The effects of varying concentrations of 2,3 diphosphoglyceric acid on AMP deaminase in the presence and absence of ATP were also determined. In the presence of ATP, when the per cent inhibition of velocity was plotted against the concentration of 2,3-diphosphoglyceric acid a sigmoidal curve was obtained. 4. 4.The data suggest that 2,3-diphosphoglyceric acid may control the adenine nucleotide content of the intact erythrocyte through an allosteric regulation of the AMP deaminase activity.

Uptake of Some Quaternary Ammonium Ions by Human Erythrocytes

In many biophysical studies on erythrocytes some quaternary ammonium ions are used as replacements for Na+ and K+ of the physiological solutions. The object of this work was to study the possible uptake of quaternary ammonium ions by erythrocytes. Uptake of C14–choline chloride and C14–tetramethylammonium chloride by human erythrocytes was proved. It was shown that the compounds were neither incorporated into phospholipids of the cell nor converted to any other metabolites. Studies of uptake as a function of time, at several external concentrations of choline and tetramethylammonium, showed that within the first 4 hours uptake was a linear function of time regardless of the external concentration of the quaternary ammonium ions. The effects of various external concentrations of choline and tetramethylammonium ions on the rate of uptake by the cells were studied. The results showed the presence of two distinct mechanisms for the uptake of choline: one, a facilitated uptake mechanism which becomes saturated at low external concentrations of the ion; the other, a simple diffusion mechanism in which the rate of uptake is proportional to concentration. For the facilitated part of the uptake the external choline concentration at which half-maximum rate was obtained was found to be 0.02 mm. Although the kinetic studies with tetramethylammonium ion were not as extensive as those with choline, they did suggest the presence of similar mechanisms for the uptake of both ions. Tetramethylammonium and tetraethylammonium ions were shown to be competitive inhibitors of the facilitated choline uptake.

Studies on the partial reactions catalyzed by the (Na2+ + L+-activated ATPase. I. Effects of simple anions and nucleoside triphosphates on the alkali-cation specificity of the p-nitrophenylphosphatase

Abstract 1. 1. The K+-dependent p-nitrophenylphosphatase activity of the (Na2+ + K+)-activated. ATPase complex is stimulated by the addition of Na+ + ATP. Since oligomycin blocks the (Na+ + ATP)-stimulation, but not the K+-dependent activity, the existence of a “K+-sensitive sit” and a “Na+-sensitive site” is indicated. The object of this work was to learn more about these sites through kinetic studies. 2. 2. The “K+-sensitive site” responded to Li+, Rb+, and Cs+; but the “Na+-sensitive site” showed absolute specificity for Na+. 3. 3. The order of cation specificity of the “K+-sensitive site” (K+ = Rb+ > Cs+ > Li+), and the absolute specificity of the “Na+-sensitive site” remained constant under conditions (e.g., change in the major anion of the assay medium) which had been used to demonstrate the changing specificity of another alkali-cation-activated enzyme (AMP deaminase). 4. 4. The “+-sensitive site” could be demonstrated only in the presence of certain nucleoside triphosphates (ATP, ITP and CTP). Nucleoside diphosphates, nucleoside monophosphates, pyrophosphate and orthophosphate had no activating effects in the presence of Na+. 5. 5. A variety of simple anions were found to have inhibitory effects on the enzyme. Because of this, and due to the impurity of the enzyme, the complex kinetic data were of little value for mechanistic interpretations. 6. 6. Na+ inhibited the enzyme both in the presence and absence of an activator cation. From the kinetic data it was not possible to determine if this inhibition was exerted at the same “Na+-sensitive site” that is involved in the (Na+ + ATP)-activation of the enzyme.

Studies on the partial reactions catalyzed by the (Na+ + K+)-activated ATPase. II. Effects of oligomycin and other inhibitors of the ATPase on the p-nitrophenylphosphatase.

Abstract 1. 1. It is known that the K+-activated p-nitrophenylphosphatase, in contrast to (Na+ + K+)p-activated ATPase, is not inhibited by oligomycin; and that when p-nitrophenylphosphatase is activated by Na+ + K+ + ATP, the activity becomes partially sensitive to oligomycin. The object of this work was the further study of the mechanism of action of oligomycin on the enzyme complex. 2. 2. It was shown that oligomycin blocks the stimulation of the activity that is caused by Na+ + ATP. The K+-dependent portion of the activity remained insensitive to oligomycin. 3. 3. In the presence of Na+ and low concentrations of K+, oligomycin stimulated the p-nitrophenylphosphatase. This effect of oligomycin, like the activating effect of ATP, was accompanied by a decrease in the apparent Km of K+. 4. 4. The curve of activation of enzyme as a function of oligomycin concentration had a sharp peak. Maximum activation obtained with oligomycin never approached that obtained with ATP. It was concluded that Na+-dependent binding of either ATP or oligomycin to a modifying site affects the interaction of K+ with the enzyme, and that higher concentrations of oligomycin block the modifying effects of ATP and lower oligomycin concentrations. 5. 5. Ethacrymic acid, chlorpromazine, N-ethylmaleimide, and Dio-9 inhibited the (Na+ + K+)-activated ATPase and the K+-activated p-nitrophenylphosphatase. In this respect these inhibitors are more similar to ouabain than to oligomycin.

Effects of monovalent cations and ATP on erythrocyte AMP deaminase

Summary 1. Human erythrocytes contain an AMP deaminase (AMP aminolrydrolase, EC 3.5.4.6) which requires the presence of certain alkali cations. Since the activation of this enzyme from other tissues by ATP had been reported, the effects of ATP and alkali cations on the enzyme from human, dog and cat erythrocytes were studied. 2. The human erythrocyte enzyme could be activated by either K+ or NH4+. No activation by Na+, Li+, Rb+ or Cs+ could be shown. ATP alone did not affect the enzyme. However, in the presence of ATP the enzyme could be activated not only by K+ and NH4+, but also by Na+, Li+ and Rb+. Experiments suggested that ATP was not chemically altered. 3. The enzymes from cat and dog erythrocytes were activated by ATP alone. The alkali cations had no effect on these enzymes either in the presence or absence of ATP. 4. The results were interpreted in terms of a previous suggestion that the ATP-induced changes in protein charge could lead to changes in the nature of the counter-ions associated with the protein.

A fluorimetric assay method for the K+-phosphatase associated with the (Na+ + K+)-activated ATPase

1. 1. Data are presented which indicate that umbelliferone phosphate is a substrate for the K+-activated phosphatase which is found in preparations of (Na+ + K+)-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3). 2. 2.|The hydrolysis of umbelliferone phosphate by this enzyme resembles that of p-nitrophenyl phosphate in that it requires Mg2+ and is activated by K+ and this activation is inhibited by ouabain. 3. 3. At low concentrations of K+, it is further activated by ATP in the presence of Na+ and this extra activation is inhibited by oligomycin. 4. 4. The fluorimetric assay method, which has previously been used in the determination of acid and alkaline phosphatases, is simple, convenient and highly sensitive.

Alkali cation-activated AMP deaminase of erythrocytes: some properties of the membrane-bound enzyme.

Abstract 1. 1.Human erythrocyte membranes were prepared by several different methods. All preparations, including hemoglobin-free membranes, contained some alkali cation-activated AMP deaminase (AMP aminohydrolase, EC 3.5.4.6). 2. 2.Various washing procedures and fragmentation of membranes by sonication showed that although part of the activity of some membrane preparations could be released, a portion of the enzyme activity was tightly bound to the membrane. 3. 3.Intact erythrocytes did not deaminate AMP which was added to the incubation medium. This suggested that, unlike the situation in muscle, AMP deaminase was not on the outer surface of the membrane. 4. 4.Substrate-velocity curve of the membrane enzyme was hyperbolic in contrast to the sigmoid curve of the soluble enzyme. 5. 5.The effects of ouabain and ethacrynic acid, which are inhibitors of (Na+-K+)-activated ATPase, on the membrane AMP deaminase were studied. Ouabain did not affect the enzyme. Ethacrynic acid was found to be a potent inhibitor of the enzyme.

Vitamin C—Effects on the Na+, K+ adenosine triphosphate phosphohydrolase complexes of several tissues

Abstract The effects of ascorbate and dehydroascorbate on the Na+, K+-adenosine triphosphatase (ATPase) preparations of several tissues were studied. Both compounds inhibited the ATPase activities of the preparations obtained from the brains of rat, guinea pig and beef; but they did not affect the enzyme activities of the rat kidney, rat heart, beef heart and human red cells. Both ascorbate and dehydroascorbate produced half-maximal inhibition of the Na+, K+-ATPase and the K+-dependent phosphatase activities of the rat brain enzyme at a concentration of 5 × 10−5 M. Maximum inhibition was obtained at 0.5 mM. Comparison of the inhibitory effects of the vitamin with those of ouabain and oligomycin indicated qualitative differences between the nature of the interactions of these inhibitors with the enzyme complex. The inhibited enzyme, obtained by preincubation with the vitamin, could not be reactivated by washing. Presence of histidine and EDTA did not prevent the inhibitory effects of the vitamin. Of the several simple lactones that were tested, only angelicalactone proved to be an inhibitor of the enzyme. These studies suggest a possible role of vitamin C in the control of active transports of Na+ and K+ in certain tissues.

EFFECT OF MONOVALENT CATIONS ON THE ADENOSINETRIPHOSPHATASE OF SONICATED ERYTHROCYTE MEMBRANE.

Abstract 1. 1. Human erythrocyte membrane contains a Mg2+-dependent adenosinetriphosphatase activity. The enhancement of this activity requires the simultaneous presence of Na+ and K+. When the membranes were broken by ultrasonic vibrations, the adenosinetriphosphatase activity was enhanced with either Na+ and K+, and in this case enhancement was no longer affected by ouabain. 2. 2. The effects of Mg2+, Ca2+, pH, and detergents on the various manifestations of the adenosinetriphosphatase activity, namely: (a) the Na+-plus-K+-dependent form, (b) the single-ion-dependent form, and (c) the ion-independent form, were studied. The results did not suggest the presence of more than one enzyme. 3. 3. Since the single-ion-dependent adenosinetriphosphatase activity was revealed merely by a physical change in the method of preparation (sonication), it is reasonable to suspect that it and the Na+-plus-K+-dependent activity are different manifestations of a single enzymic activity.

Red cell Na+, K+-ATPase: A method for estimating the extent of inhibition of an enzyme sample containing an unknown amount of bound cardiac glycoside

Abstract Na + , K + -ATPase activities of the membranes obtained from intact red cells that are exposed to ouabain, digoxin, and digitoxin are inhibited. The extent of inhibition of each enzyme sample can be found by the following two assays: 1) Activity is measured by the addition of enzyme to a buffered solution containing 2 mM ATP, 3 mM Mg 2+ , 1 mM EDTA, 100 mM Na + , and 25 mM K + . Since little regeneration of the inhibited enzyme occurs under these conditions, the measured activity is that of the partially inhibited enzyme. 2) Enzyme is preincubated for ten minutes in the same solution from which Mg 2+ and K + are omitted, and then assayed by the addition of Mg + and K + . Since the inhibited enzyme is completely regenerated during the preincubation period, the activity measured here serves as a control for that determined in the first assay.