Tellurite effect on ATPase activity of contractile membrane protein.

The effect of tellurite on ATPase activity of the contractile membrane protein in human erythrocytes was studied. Tellurite, even at a concentration of 0.01 mM, inhibited 25 per cent of the saponin-stimulated ATPase activity of the contractile membrane protein; the inhibition increased with increasing tellurite concentration, and was reversible. On the other hand, in erythrocytes preincubated with tellurite, the ATPase activity of the membrane contractile protein, non-stimulated by saponin, increased, and the increase was further enhanced by washing the erythrocytes. The behaviour is analogous to the tellurite effect on erythrocyte morphology: incubation of erythrocytes with tellurite caused morphological changes which were more pronounced after removing the tellurite by washing. The complex effect of tellurite is discussed.

Red blood cells under mechanical stress.

The effect of mechanical stress on erythrocytes suspended in various media was studied. The ability of the cells to increase their glucose consumption was found to be the major criterion allowing to divide the media into two groups. In plasma, serum or in Ringer's solution supplemented with albumin and glucose the energy consumption by mechanically stressed erythrocytes increased 20 to 50%; no morphological changes of the cells were observed either in suspension or on Giemsa smears. The cells behaved in the same way in Mg2(+)-free medium. The other group included protein-free medium (Ringer's solution supplemented with glucose) and Ca2(+)-free Ringer's solution supplemented with albumin and glucose; under these conditions erythrocytes were unable to raise their energy consumption in response to mechanical stress, and after some period structural impairment of the membrane could be observed on Giemsa smears. No differences in metabolism-associated nucleotide concentrations (ATP, ADP, NAD, NADP) were observed between the samples. Resealed red cell ghosts with high concentrations of intracellular components were prepared as a model of cells with damaged membrane. In these ghosts (with low ATP concentration) mechanical stress produced increased proportions of echinocytes, even in the "native" suspension. These results have confirmed the vital role of the energy-consuming contractile apparatus in the erythrocyte membrane, and supplied a clue to the role of Ca2+ in its activation and to the influence of extracellular proteins on the maintenance of in red cell shape.

Dual effect of calcium on erythrocyte membrane contraction.

Effect of different calcium concentrations on erythrocyte ghost shape and size has been studied. Erythrocyte ghosts are smallest at approximately 10 microM of total calcium content and in the absence of EDTA; both decrease and increase of free Ca2+ results in expansion of ghosts. Changes in erythrocyte ghost size occur at equal Ca2+ concentrations which, as it has been already found, alter ATPase activity of contractile membrane protein. The possibility is discussed that under physiologic conditions contractile membrane protein causes weak membrane contraction which differs from aphysiologic membrane shrinkage brought about particularly by cross-bridges at high Ca2+ concentrations.

Is Ca2+ effect on passive K+ transport mediated by spectrin--dependent ATPase?

The aim of this report was to find which part of the membrane is responsible for the Ca2+ dependence of membrane permeability to K+. We found that the enzyme activity of large contractile complex of membrane proteins, the so called spectrin-dependent ATPase (sp-ATPase) increases at certain Ca2+ concentrations when K+ permeability decreases and vice versa. Ca2+ apparent dissociation constant for sp-ATPase is 6 X 10(-7) M which is the value corresponding to findings of Porzig and Stoffel (1978) for Ca2+ binding to membrane with low K+ permeability. Moreover, 20 chemically quite different substances which inhibit sp-ATPase activity simultaneously increase in the same concentrations K+ permeability and vice versa. No exception was found. The results show that low membrane permeability to K+ occurs at such conformation of sp-ATPase at which its enzyme activity may fully be manifested whereas at other conformations permeability to K+ increases. The conformation of sp-ATPase seems to affect gating mechanism of a respective channel for passive K+ transport through membrane.

Energy requirements of erythrocytes under mechanical stress.

Factors influencing an increase of glucose consumption in erythrocytes under mechanical stress were studied. Under the shear stress of 2000-5000 s-1 the glucose consumption goes up 20-50%. This effect disappears in protein-free media, in Ca2+-free media and in resealed red ghosts; in all these cases certain morphological deviations were observed in the stressed cells. These results confirm the vital role of energy-consuming contractile apparatus of erythrocyte membrane in restoration of the smooth biconcave shape after deformation.

Is flexoelectricity the coupling factor between chemical energy and osmotic work in the pump? A model of pump.

The following pump model is proposed. A gate is responsible for pump specificity. The actual driving force of the transport of ions against the electrochemical potential gradient is the electric field originating from an altered curvature of the phospholipid bilayer around the pump. The physical origin of this curvature-induced electric field arises from a basic liquid crystal property of lipid bilayers called flexoelectricity. Alterations occurring in phospholipid bilayer arrangement are due to changed conformation of protein; the main energy source of this change is ATP. Consequently, the energy of ATP is transformed, in our pump model, into osmotic work in following steps: ATP + protein (conformation I)----protein (conformation II)----alterations in phospholipid bilayer arrangement----electric field----active transport of ions. This model is the most simple one. In Na, K-pump there is a bidirectional ion transport. In our model of Na, K-pump three conformational states of pump proteins and two different electric fields formed sequentially in opposite directions are supposed.

Inhibition of phagocytosis of polymorphonuclear leucocytes by adenosine and HoCl3 in vitro.

Phagocytosis of polymorphonuclear leucocytes treated with NaF, HoCl3 and adenosine were studied. The highest concentration used was 25 mM of NaF, 25 mM of adenosine and 5 mM of HoCl3. It was ascertained that these substances, inhibitors of erythrocyte contractile protein, inhibit both phagocytosis and ability of polymorphonuclear leucocytes to change their shape. These unfavourable effects may be induced by the chemicals interfering with polymorphonuclear leucocytes contractile protein. NaF, HoCl3 and adenosine are also responsible for morphological changes in the cell nucleus.

Is there any connection between heat inactivation of spectrin-dependent ATPase and loss of smooth biconcave shape of red cells?

Spectrin-dependent ATPase activity was measured in membranes from native human erythrocytes and erythrocytes heated for 20 min at different temperatures. This activity was found to decline when the erythrocytes were heated at 48 degrees C and higher. The break in ATPase activity corresponds to morphological changes in erythrocytes found by Crome and Mollison [Brit. J. Haematol. (1964) 10, 137]. The role of spectrin-dependent ATPase in erythrocyte shape maintenance is discussed.

The role of spectrin-dependent ATPase in erythrocyte shape maintenance.

The paper reports on the relationship between spectrin-dependent ATPase and erythrocyte shape. It can be seen from various experiments that different treatment of erythrocyte membrane which brings about alteration in activity of spectrin-dependent ATPase, also changes erythrocyte shape. We suggest that spectrin-dependent ATPase is a large actomyosin-like protein complex with some characteristics of contractile protein which, under suitable conditions, causes a physiological tension (contraction) of the membrane. Energetically rich state of spectrin-dependent ATPase seems to be responsible for this phenomenon.

Changes in fluidity of erythrocyte membranes after storage of erythrocytes and regeneration of cellular ATP level.

The membrane fluidity of freshly collected human erythrocytes, of erythrocytes stored for 3-4 weeks and of stored erythrocytes rejuvenated with glucose and inosine was investigated by measuring polarization of fluorescence emission of 1,6-diphenyl-1,3,5-hexatriene and N-phenyl-1-naphthylamine. The fluidity of membranes prepared from stored erythrocytes was higher than that of fresh erythrocytes. After rejuvenation of erythrocytes with glucose and with or without inosine the membrane fluidity decreased. These changes were probably due to variations of ATP levels in the erythrocytes.

Does the conformation of Mg-ATPase (spectrin-dependent ATPase) influence the passive permeability to K+?

The processing of human erythrocytes disclosed changes in Mg-ATPase activity following action of Pb2+ and Nile blue, and changes of permeability of K+ after treatment with Nile blue. The obtained results and those from previous papers can be summarized as follows : Substances decreasing the activity of stimulated membrane Mg-ATPase (spectrin-dependent ATPase) in red blood cells increase the passive permeability to K+, and substances increasing the stimulated Mg-ATPase activity decrease the passive permeability to K+. A hypothesis is proposed that the conformation of Mg-ATPase is secondarily reflected in the state of the proper path for K+ transport through the membrane; thus the rate of passive permeability to K+ is influenced.

Activation of Mg-ATPase (spectrin-dependent ATPase) by Ca2+.

The dependence of saponin-stimulated Mg-ATPase activity in the erythrocyte membrane on Ca2+ concentration was studied. In the membrane of freshly sampled human erythrocytes we found for this enzyme and Ca2+ an apparent dissociation constant of 0.611 mumol/l (SE +/- 0.106 mumol/l) and Hill coefficient of 0.93 (SE +/- 0.05). The enzyme is in most probability identical with Ca,Mg-ATPase of high affinity to Ca2+ described also as spectrin-dependent Ca,Mg-ATPase.

[Effect of vitamin E on the mechanical fragility and the Mg++-ATPase of rabbit erythrocytes]. / Der Einfluss des Vitamin E auf die mechanische Fragilität und die Aktivität der Mg++-ATPase von Kaninchenerythrozyten.

We have established that Mg++ activated ATPase in red cells of rabbits is not changed by vitamin E injection, even if the mechanical resistance of these red cells is increased. Enzyme activity, however, cannot be correlated with this increased mechanical resistance. Probably, the effect of vitamin E on membrane lipids may be supposed to exist, though its effect on membrane proteins cannot be fully excluded.

The role of Mg++-ATPase (actomyosin-like protein) in maintaining the biconcave shape of erythrocytes.

Chemically different substances known to change the Mg++-ATPase activity in the red cell membrane, likewise alter the red cell shape. Normal human red cells retain their biconcave shape only when the activity of this enzyme remains unchanged. The present work deals with the possibility that Mg++-ATPase may cause certain tension in the membrane responsible for the biconcave shape of the erythrocyte.