Characteristics of an adenosine triphosphatase in erythrocyte membranes stimulated by 2,4-dinitrophenol

Studies were made of the stimulation by 2,4-dinitrophenol (DNP) of an adenosine triphosphatase (ATPase) in stromata of human erythrocytes. Activation by 2,4-dinitrophenol occurs in the range 10^?5 to 10^?3 M and was seen in whole cells, ghosts reconstituted with Mg and ATP, and in osmotic ghosts prepared at a low ratio of cells to water. Phloretin and phloridzin also activated the DNP sensitive system but inhibited it at higher concentrations. DNP increased the K_m and V_max values of the enzyme equally. The DNP sensitive and Na⁺ + K⁺ sensitive enzymes of the stromata were compared. The activities of the two ATPases are additive, require the presence of Mg⁺⁺ and require that the substrate be located at the inner surface of the membrane. The two enzymes differ in their substrate specificity, in their sensitivity to inhibition by ouabain and phloretin and in their sensitivity to some factor in hemolysates. The possible roles of this system in the erythrocyte were discussed.     

Isolation and properties of a T-kininogenase from bovine erythrocyte membranes

A kininogenase from bovine erythrocyte membranes has been purified 140-fold by affinity chromatography on pepstatin A-Agarose followed by ion exchange chromatography on CM Cellulose. The purified enzyme showed an apparent molecular weight of 31,000 daltons as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ItspH optimum is 7.5, and it was totally inhibited by soybean trypsin inhibitor, phenylmethylsulfonylfluoride, aprotinin, pepstatin, and dithiotreitol, suggesting the presence of a disulfide bond(s) whose integrity is(are) essential for maintaining the native three-dimensional structure. The referred enzyme was able to release kinin from a substrate partially purified from rat plasma. The kininogenase was activated by Zn²⁺, Ca²⁺, and cysteine-HCl.     

Calcium ion-dependent p-nitrophenyl phosphate phosphatase activity and calcium ion-dependent adenosine triphosphatase activity from human erythrocyte membranes

In the presence of ATP and of Mg(2+), human erythrocyte membranes show a phosphatase activity towards p-nitrophenyl phosphate which is activated by low concentrations of Ca(2+). The effect of Ca(2+) is strongly enhanced if either K(+) or Na(+) is also present. Activation of the p-nitrophenyl phosphate phosphatase by Ca(2+) reaches a half-maximum at about 8mum-Ca(2+) and is apparent only when the ion has access to the inner surface of the cell membrane. Ca(2+)-dependent phosphatase activity can only be observed if ATP is at the inner surface of the cell membrane, and the presence of ATP seems to be absolutely necessary, since either its removal or its replacement by other nucleoside triphosphates abolishes the activating effect of Ca(2+). The properties of the (ATP+Ca(2+))-dependent phosphatase are very similar to those of the Ca(2+)-dependent ATPase (adenosine triphosphatase), also present in erythrocyte membranes, which probably is involved in Ca(2+) transport in erythrocytes. The similarities suggest that both activities may be properties of the same molecular system. This view is further supported by the fact that p-nitrophenyl phosphate inhibits to a similar extent Ca(2+)-dependent ATPase activity and ATP-dependent Ca(2+) extrusion from erythrocytes.     

The partial reactions in the catalytic cycle of the calcium-dependent adenosine triphosphatase purified from erythrocyte membranes

A preparation of purified erythrocyte membrane ATPase whose activation by Ca2+ is or is not dependent on calmodulin depending on the enzyme dilution was used in the low dilution state for these studies. In appropriate conditions, the purified ATPase in the absence of calmodulin exhibited a Ca2+ concentration dependence identical to that of the native enzyme in the erythrocyte membrane ghost in the presence of calmodulin. Accordingly, an apparent Kd approximately equal to 1 X 10(-7) M was derived for cooperative calcium binding to the activating and transport sites of the nonphosphorylated enzyme. The kinetics of enzyme phosphorylation in the transient state following addition of ATP to enzyme activated with calcium were then resolved by rapid kinetic methods, demonstrating directly that phosphoenzyme formation precedes Pi production, consistent with the phosphoenzyme role as an intermediate in the catalytic cycle. Titration of a low affinity site (Kd approximately equal to 2 X 10(-3) M) with calcium produced inhibition of phosphoenzyme cleavage and favored reversal of the catalytic cycle, indicating that calcium dissociation from the transport sites precedes hydrolytic cleavage of the phosphoenzyme. The two different calcium dissociation constants of the nonphosphorylated and phosphorylated enzyme demonstrate that a phosphorylation-induced reduction of calcium affinity is the basic coupling mechanism of catalysis and active transport, with an energy expenditure of approximately 6 kcal/mol of calcium in standard conditions. From the kinetic point of view, a rate-limiting step is identified with the slow dissociation of calcium from the phosphoenzyme; another relatively slow step following hydrolytic cleavage and preceding recycling of the enzyme is suggested by the occurrence of a presteady state phosphoenzyme overshoot.     

Isolation and properties of a natural inhibitor of the chloroplast adenosine triphosphatase

The complete sequence of protein L17 which is a component of the large subunit of the E. coli ribosome has been determined. Peptides deriving from enzymatic hydrolysis with trypsin, thermolysin, chymotrypsin and S. aureus and A. mellea protease were isolated and sequenced by the DABITC/PITC double coupling method. Some overlapping peptides were obtained after mild acid cleavage of the protein. According to the amino acid sequence protein L17 contains 127 residues and has a molecular mass of 14 365. The primary structure of protein L17 agrees well with the amino acid analysis of the intact protein and its N-terminal sequence as derived from automatic sequencing in an improved Beckman sequencer. Secondary predictions and a search for homologous sequence stretches to other ribosomal proteins were made.     

Guanosine triphosphatase activity in human erythrocyte membranes

Human red cell membranes have the capacity to hydrolyze enzymatically GTD to GDP. The reaction requires magnesium, is not appreciably affected by sodium, potassium or calcium, and is not inhibited by ouabain. Kinetic analysis suggests that there are two separate enzymes in membranes which cleave GTP, a 'high Km' GTPase and a 'low Km' GTPase. Both enzymes are also ATPases, with an approximately equal affinity for GTP and ATP. GTPase activity did not extract from the membrane with spectrin and was not inactivated by antispectrin antibody. Activity was partially destroyed by 0.5% Triton X-100. It seems probable that the low Km GTPase is the sodium- and potassium-independent ATPase of red cell membranes. The identity of the high Km enzyme is not clear.     

Mitochondrial adenosine triphosphatase from human placenta--purification and catalytic properties

1. The purification of ATPase (EC 3.6.1.3) from human placental mitochondria is described. The yield based on mitochondrial enzyme activity was about 70% and the purification was 380-fold. 2. The rate of Mg-ATP hydrolysis was 85 mumole per min per mg of protein under optimum conditions. 3. Nucleoside triphosphates were hydrolyzed by the purified enzyme at decreasing rates in the following order: GTP greater than ITP greater than ATP greater than epsilon-ATP greater than UTP greater than CTP in Tris-HCl buffer (pH 8.0), and in the order: ATP greater than GTP greater than or equal to ITP greater than epsilon-ATP greater than UTP greater than CTP in Tris-bicarbonate buffer at pH 8.0. 4. The values of kinetic parameters are reported. The ATPase reaction deviated from typical Michaelis-Menten kinetics in Tris-HCl buffer but not in Tris-bicarbonate. Eadie-Hofstee plots for Mg-ATP hydrolysis were biphasic in Tris-HCl (Km = 0.2 mM, 0.09 mM) and monophastic in Tris-becarbonate medium (Km = 0.16 mM). 5. In the presence of Mg-ITP or Mg-GTP as substrates no curvature of the reciprocal plots was observed. 6. The results presented reflect the fact that multiple conformations of the enzyme molecule do exist and are probably involved in its regulatory functions. 7. The existence of two kinetically distinct classes of catalytic sites and of an anion-binding site on the placental ATPase is proposed.     

Effect of ozone on erythrocyte membrane adenosine triphosphatase.

Human erythrocyte membrane fragments were exposed to O3 over varying lengths of time. Ozone was found to have a deleterious effect on the ouabainsensitive ATPase (EC 3.6.1.3) in the membrane fragments. After 1 min of exposure to O3, which was generated at a rate of 4.0 mumol/min, ouabain-sensitive ATPase activity decreased to 26% of the control. Ouabain-insensitive ATPase was found to be unaffected by O3 exposure under the test conditions. Additions of ascorbic acid or cysteine, prior to O3 exposure, partially protected the enzyme from inactivation. However, the inactivating effect of O3 could not be reversed by addition of either ascorbic acid or cysteine after exposure. Superoxide dismutase or catalase did not afford significant protection. The enzyme could not be protected by Ellman's reagent. The inactivating effect of O3 on the ouabain-sensitive ATPase was also demonstrated in exposure of intact erythrocytes. No detectable change was observed in glycolytic activity in the hemolysate prepared from O3-treated erythrocytes, however. It was postulated that inactivation of the membrane ATPase by O3 may be responsible for the destructive effect of O3 on the red cell.     

Anion-sensitive adenosine triphosphatase from membranes of rat red blood cells]

Anion-sensitive ATPase was solubilized from membranes to rat red blood cells. The effect of bicarhonate, sulfite and perchlorate on the activity of ATPase was studied. Close resemblance of the properties of ATPase of rat red blood cells and of mitochondrial ATPase was observed.