Factors controlling the resealing of the membrane of human erythrocyte ghosts after hypotonic hemolysis

Summary In accordance with former observations of Hoffman (1962a), ghost populations obtained by hypotonic hemolysis and subsequent restoration of isotonicity by the addition of alkali salts, were found to be composed of 3 types of ghosts. For our purposes it was useful to distinguish between: (1) ghosts which reseal immediately after hemolysis (type I); these ghosts are incapable of incorporating alkali ions which are added after hemolysis; (2) ghosts which reseal after the addition of alkali ions (type II); salt added to the hemolysate becomes trapped inside these ghosts in the course of the resealing process at temperatures above 0°C; and (3) ghosts which remain leaky regardless of the experimental condition (type III). The discrimination between the various types of ghosts was partly achieved by a kinetic method first devised by Hoffman (1962a), and partly by sucrose density gradient centrifugation.The relative sizes of the 3 fractions depend on the temperature at which hemolysis took place and on the time interval which elapsed between hemolysis and the addition of salt. At 37°C the resealing process is fast. Many of the ghosts reseal before salt can be added to the hemolysate. Hence, the fraction of type I ghosts is high after hemolysis at that temperature. At 0°C resealing is extremely slow. Hence, salt which has been added to the hemolysate at that temperature will enter the ghosts and become trapped during subsequent incubation at 37°C. There are no ghosts of type I and many ghosts of type II (about 60%). Regardless of the temperature at hemolysis, there are always ghosts which do not reseal even after prolonged incubation at 37°C. A method has been designed which permits the preparation of homogeneous populations of type II ghosts.Complexing agents (ATP, EDTA, 2,3-DPG) may prevent the resealing of the ghost membrane. However, they exert this effect only at elevated temperatures and when present in the medium at the instant of hemolysis. At 0°C, the presence of complexing agents in the medium at the instant of hemolysis has no effect on the subsequent resealing at 37°C. The recovery of the ghost membrane takes place in spite of the continued presence of the agents and eventually leads to trapping of these agents inside the resealed ghosts.The experiments support the contention that the complexing agents interact with a membrane constituent which is neither accessible from the inner nor from the outer surface of the cell membrane but becomes exposed during the hemolytic event when the complexing agents penetrate across the membrane. Apparently, at low tempertrures membrane ligands are more successful in competing with the added complexing agents for this constituent than at higher temperatures.Extending former observations of Hoffman, we found that not only Mg⁺⁺ but also Ca⁺⁺ facilitates the resealing process. Perhaps one or the other of the two alkaline earth ions is the membrane constituent which normally participates in the maintenance of the integrity of the red blood cell membrane.     

Relationship of hemolysis buffer structure, pH and ionic strength to spontaneous contour smoothing of isolated erythrocyte membranes

Isolated human erythrocyte membranes crenate when suspended in isotonic medium, but can use MgATP to reduce their net positive curvature, yielding smooth discs and cup forms that eventually undergo endocytosis. An earlier report from this laboratory (Patel, V.P. and Fairbanks, G. (1981) J. Cell Biol. 88, 430-440), has described a phenomenon of ATP-independent shape change in which ghosts prepared by hemolysis and washing in synthetic zwitterionic buffers crenated at 0 degree C, but underwent conversion to smooth discs and cups when warmed in the absence of MgATP. We have further explored the effect of the hemolysis condition on the requirement for ATP in ghost shape change. 25 hemolysis buffers were applied at 10 mM (pH 7.4, 0 degree C). Eight anionic buffers with relatively high ionic strength (e.g., phosphate and diethylmalonic acid (DMA] yielded ghosts requiring ATP for shape change, while two cationic buffers (Bistris and imidazole) and ten synthetic zwitterionic buffers (e.g., Tricine and Hepes) with lower ionic strength produced ghosts that smoothed spontaneously at 30 degrees C. Hemolysis at intermediate ionic strength yielded mixed populations in which spontaneous smoothing was expressed in all-or-none fashion. Maximal ATP-independent shape change was induced by hemolysis at pH 7.3-7.7, while ATP was required after hemolysis at pH less than or equal to 7.1 even when the ionic strength at hemolysis was low. Ghosts requiring ATP could be converted to ATP independence by washing at low ionic strength, but ATP independence could not be reversed readily by washing at high ionic strength. Exposure to low ionic strength at pH greater than 7.1 presumably changes membrane organization in a way that alters the temperature dependence of tensions within the bilayer or skeleton of the composite membrane.     

Role of the interaction between puerarin and the erythrocyte membrane in puerarin-induced hemolysis

Adverse drug reactions (ADR), especially intravenous hemolysis, have largely limited the application of puerarin injections in clinics. This study investigated the underlying mechanisms of puerarin-induced hemolysis. Our results show that puerarin induced concentration-dependent and time-dependent hemolysis when human erythrocytes were incubated in saline solution with more than 2 mM puerarin for over 2 h. However, incubation in PBS or addition of 1 mM of lidocaine to the saline solution completely abolished the hemolysis. Providing materials that could start ATP synthesis did not reverse the hemolysis, and puerarin did not affect Na⁺–K⁺–ATPase activity. In addition, puerarin (0.1–2 mM) did not cause calcium influx or exhibited pro-oxidant activity in erythrocytes. Puerarin exhibited different influences on the membrane microviscosity of erythrocytes in saline and PBS. Moreover, 1 mM lidocaine inhibited 8 mM puerarin-induced reduction of membrane microviscosity in saline solution. SDS–PAGE analysis of membrane proteins revealed that 2 mM puerarin treatment induced the appearance of several new protein bands but attenuated the expression of protein bands 2.1, 3, 4.1, 4.2 and 5. These results suggest that high concentrations of puerarin-induced hemolysis were associated with the changes of membrane lipids and of the composition of erythrocytes membrane proteins but not with ATP depletion, pro-oxidation and calcium influx. These changes could be related to the intercalation of amphiphilic puerarin at high concentration into the erythrocyte membrane in certain media, resulting in membrane disorganization and, eventually, cytolysis. Hence, in clinics, determining the optimal dose of puerarin is critical to avoid overdosing and ADR.     

The sodium and potassium activated ATPase. II. Comparative study of intestinal epithelium and red cells

The Na-K ATPase found in sedimentable fractions of intestinal epithelium of rats hydrolyzed cytidine triphosphate nearly as well as ATP (25% to 50%); was active only in presence of divalent cations, with specificity for Mg (100%), Mn (50%) and Ca (10%); showed a plateau of activation when Mg concentrations were in excess of substrate; and was inhibited by a second divalent cation (Zn > Mn > Ca), and by 3 × 10^?4 M ouabain (50%). Parallel assays of rat red cell ghosts showed differences in substrate specificity (CTP was not utilized), in activation kinetics (activation peak with Mg) and in greater specificity to Mg (Mn was a weaker activator and Zn was a weaker inhibitor). Stabilities also differed in the two preparations: Na? K ATPase of intestinal epithelium was activated by sucrose extraction and denatured during cytolysis at room temperature, while that of red cell fragments was denatured during sucrose extraction and preserved by hemolysis at room temperature. Other properties of Na? K ATPase studied in the two tissues included activation by monovalent cations (optimum at 160 mM Na, 15 mM K), specificity to monovalent cations, and sensitivity to lipid solvents and to some drugs. The data were discussed in terms of comparative properties of Na? K ATPases of various cells. Residual ATPase activities of intestinal epithelium and red cell ghosts were shown to differ in substrate specificity, inhibition and activation. “Residual ATPase” from intestinal epithelium was a zinc-activated nucleoside polyphosphate phosphohydrolase, while ghosts contained Mg? ATPase. Only the latter enzyme was specific to ATP and Mg, activated by Ca in presence of Mg, and sensitive to inhibition by PCMB and Zn.     

The effects of 3-methylindole on hemolysis, transport of Na+, and ATPase activities of bovine erythrocytes.

Biochemical effects of 3MI on cellular membranes were investigated. This study was conducted to examine the effects of 3MI on the hemolysis of erythrocytes, the transport of 22Na+ in resealed erythrocyte ghosts, and on the ATPase activities of erythrocyte membranes. The percent of hemolysis as a function of 3MI incubation time was sigmoidal. Seventy-five percent of the hemoglobin was released with the second 2 hr of incubation during which the concentration of 3MI in the cells reached a plateau of 2500 mug/ml of packed RBC. The effect of 3MI at a subhemolytic concentration on passive and active 22Na+ transport were not significant. The total and Mg2+-dependent ATPase activities in the membranes were significantly increased after 1 hr of incubation with 3MI at concentrations of 100, 200, 300, 400 and 500 mug/ml (P less than or equal to 0/ml (P smaller than or equal to 0.02).     

Characteristics of Ca2+ ion effect on the activity of Mg2+-dependent ATPase system in ghosts and reconstituted erythrocytes]

A good conformity if demonstrated of the kinetics of calcium ions effect on ATPase activity of human and rat erythrocyte ghosts. The increase of calcium concentration in the rat errythrocytes hemolysis medium (above 50-100 micrometer) results in a considerable aggregation of reconstructed vesicles. An activation of ouabaine-sensitive component of Mg2+-dependent ATPase under the increase of intracellular Ca2+ in reconstructed human erythrocytes is observed.     

The inhibition of ATP-dependent shape change of human erythrocyte ghosts correlates with an inhibition of Mg(2+)-ATPase activity by fluoride and aluminofluoride complexes.

The vanadate-sensitive Mg(2+)-dependent ATPase activity of the human erythrocyte ghost is believed to be involved in the shape change events that convert echinocytic ghosts to smoothed forms (biconcave discs and stomatocytes). At physiological salt concentration, pH 7.4, 2 mM ATP, 5 mM Mg2+ and 1 mM EGTA, the Mg(2+)-ATPase activity of ghosts was inhibited strongly by millimolar concentrations of sodium fluoride: I50 = 1.31 +/- 0.23 mM (mean +/- S.D.; n = 12). The addition of aluminium chloride to 15 microM reduced the concentration of NaF required for 50% inhibition to 0.76 +/- 0.21 mM (n = 10). Aluminium alone had only a small inhibitory effect on the ATPase activity (13 +/- 9%; n = 10). Desferrioxamine, a strong chelator of tervalent aluminium ion, failed to reverse the inhibition by fluoride and reversed the inhibition in the presence of aluminium and fluoride back to those values obtained with fluoride alone. Of several metal salts tested only beryllium sulfate was able to replace aluminium as an effective inhibitor in the presence of fluoride. Inhibition of the Mg(2+)-ATPase activity by fluoride and the aluminofluoride complexes correlated with an inhibition of the rate of MgATP-dependent change in red cell ghost shape from echinocytes to smoothed forms. All gross morphological changes of the smoothing process were affected, including the production of discocytes, stomatocytes and endocyctic vesicles.     

Freeze-thaw damage in isolated lobster sarcoplasmic reticulum membranes: A model system for membrane damage

Sarcoplasmic reticulum membrane vesicles (SRV), isolated from the abdominal muscle of Maine lobsters, were put through a freeze-thaw cycle in order to study membrane freezing damage on a molecular basis, The major membrane protein in SRV is a (Ca²⁺ − Mg²⁺) ATPase capable of accumulating Ca²⁺ with the concomitant hydrolysis of ATP, After being frozen and thawed in the presence of NaCl, the SRV showed an increased ATPase activity and a decreased ability to accumulate Ca²⁺. The degree of increased ATPase activity and decreased Ca²⁺ accumulation was dependent upon the NaCl concentration (damage increased with increased NaCl concentration) and cooling rate (damage was only observed at slow cooling rates, i.e., less than 10 °C/min). Slow thawing rates also increased the amount of damage.The freeze-thaw damage of the SRV membranes is probably not due to osmotic shock, since the vesicles are quite resistant to osmotic stress and are highly permeable to small molecules and monovalent ions. Incubation of the SRV in 2 NaCl at 22 °C has no effect on Ca²⁺ accumulation whereas freezing in 0.25 NaCl totally abolishes their ability to take up Ca²⁺. Thus, a combination of salt and low temperature is necessary for damage. The freeze-thaw damage can be largely prevented by the addition of DMSO, glycerol, or PVP. The factors above have implications for the storage of tissue or membranes for subsequent analysis of membrane-bound enzymes. The SRV mimic the behavior of cells in their response to cooling and thawing rates, salts and cryoprotectants.     

Calcium transport in human erythrocytes. Separation and reconstitution of high and low Ca affinity (Mg mca)-AT Pase activities in membranes prepared at low ionic strength.

Human erythrocyte membranes obtained by freeze-thawing of ghosts prepared in the absence or presence of EDTA, by washing with a 12 mosm medium at pH 7.7 or a 2 mosm medium at pH 6.5 contain both high and low Ca affinity (Mg + Ca)-ATPase activities. Incubation of ghosts in a less than 2 mosm medium at pH 7.5 or in 0.1 mm EDTA + 1 Him Tris-maleate (pH 8.0) results in removal of the high affinity (Mg + Ca)-ATPase activity from the membrane in a time dependent manner. Under similar conditions up to 25% of membrane proteins are removed. The soluble protein fraction extracted, although devoid of ATPase activity, reconstitutes with the remaining membrane residue with restoration of original (Mg + Ca)-ATPase activity. Addition of the soluble protein fraction to heat-treated membranes devoid of low affinity (Mg + Ca)-ATPase activity allows reconstitution of more than 33% of the original high affinity (Mg + Ca)-ATPase activity which has a Ca dissociation constant of approximately 1.6μm. Temperature and phospholipase A₂ studies indicate that low affinity (Mg + Ca)-ATPase activity is phospholipid dependent in contrast to high affinity (Mg + Ca)-ATPase activity. Ruthenium red and LaCl₃ inhibit both high and low affinity (Mg + Ca)-ATPase activities with similar potencies. The ease of removal of high affinity (Mg + Ca)-ATPase activity from the membrane by relatively mild conditions suggests that an activator protein or the high affinity (Mg + Ca)-ATPase itself is only loosely attached to the membrane. These studies show that low affinity (Mg + Ca)-ATPase activity is not an artifact and is distinct from high affinity (Mg + Ca)-ATPase activity. The low affinity (Mg + Ca)-ATPase activity is sensitive to Ca²⁺ in the concentration range from below 0.3 μm to 300 μm compatible with an association of this enzyme with Ca transport.     

The expression of optimum ATPase activities in human erythrocytes. A comparison of different lytic procedures.

The exposure of the Na⁺/K⁺/Mg²⁺- and Ca²⁺/Mg²⁺-stimulated ATPase activities in human erythrocytes through the use of several different lytic procedures revealed significant variations in the level of activity. Density (age)-separated as well as mixed-age human erythrocytes were subjected to hemolysis in isotonic buffer using saponin or ethylene glycol, to hemolysis in hypotonie buffer using low osmolarity buffers, or to freeze-thaw to allow potential accessibility to the ATPases. The results ranged from maximum exposure of both types of ATPases in saponin-treated cells, to little or no exposure of activity in ethylene glycol-treated cells, to variable responses in membranes derived by hypotonie hemolysis. The inability to elicit maximum exposure of ATPases in young cells by the freeze-thaw treatment was reversed by the use of saponin lysis in isotonic medium. These results illustrate the importance of the lytic conditions of membrane preparations on the recovery of as well as exposure to ATPase activities. It is concluded that saponin lysis in isotonic buffer medium is the preferred lytic technique for preparation of membranes retaining significant levels of the Na⁺/K⁺/Mg²⁺- and Ca²⁺/Mg²⁺-stimulated ATPases. These data are also discussed in reference to the degree of retention of the activator protein for the $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase system.     

Resealing to small solutes of white erythrocyte membranes after incubation with EDTA, Ca2+, salt, sucrose, phospholipase C

White, stable erythrocyte ghosts can recover their impermeability to small solutes after storage for several days in low-ionic-strength phosphate buffers at 0 °C. The accessibility, to their substrates, of the inner surface enzymes, glyceraldehyde-3-phosphate dehydrogenase, (G3PD), and NADH cytochrome c oxidoreductase, was used to assess resealing. The data from the two enzymes were confirmatory. None of the conditions used to investigate resealing altered the activity of the outer surface enzyme, acetylcholinesterase. Using G3PD activity, ghosts (freshly prepared by gentle stepwise hemolysis in hypotonic phosphate buffers and stored in 11 mm phosphate buffer, pH 7.4) were shown to be slightly sealed (33%). Incubation at 37 °C in the storage buffer with or without EDTA did not alter their permeability. Ionic strength rather than osmotic pressure appears to influence the sealing process since salt (286 mosm) elicited 91% sealing whereas sucrose (278 mosm) had little effect. Calcium in trace amounts caused resealing to 80%. Phospholipase C (C. welchii) completely abolished Ca²⁺-induced resealing. The data were highly reproducible although these ghosts were found to contain only 10 to 20% of the G3PD activity of the leaky ghosts prepared by shock hemolysis in 5 mm phosphate buffer, pH 8.0. The response to the resealing agents was similar regardless of the level of G3PD present. Neither calcium nor ETDA altered the chemical composition (sialic acid, cholesterol, phospholipid) of the membranes. The small amount (5%) of nonspecific loosely bound protein lost during incubation, could not be attributed to any of the test agents. The results suggest that calcium induced the recovery of impermeability by altering the association, distribution, and/or conformation of the proteins and phospholipids within the membrane.     

THE LOCALIZATION OF Mg-Na-K-ACTIVATED ADENOSINE TRIPHOSPHATASE ON RED CELL GHOST MEMBRANES

The lead salt method introduced by Wachstein and Meisel (12) for the cytochemical demonstration of ATPase activity was modified and used to determine sites of activity on red cell ghost membranes. Preliminary studies showed that aldehyde fixation and standard concentrations of the capture reagent Pb(NO₃)₂ resulted in marked inhibition of the ATPase activity of these membranes. By lowering the concentration of Pb²⁺ and incubating unfixed red cell ghosts, over 50% of the total ATPase activity, which included an ouabain-sensitive, Na-K-activated component, could be demonstrated by quantitative biochemical assay. Cytochemical tests, carried out under the same conditions, gave a reaction product localized exclusively along the inner surfaces of the ghost membranes for both Mg-ATPase and Na-K-ATPase. These findings indicate that the ATPase activity of red cell ghosts results in the release of P_i on the inside of the ghost membrane at sites scattered over its inner aspect. There were no deposits of reaction product on the outer surface of the ghost membrane, hence no indication that upon ATP hydrolysis P_i is released outside the ghosts. Nor was there any clear difference in the localization of reaction product of Mg-ATPase as opposed to that of Na-K-ATPase.     

Regulation by calmodulin of the calcium affinity of the calcium-transport ATPase in human erythrocytes

The Ca2+ affinity of (Mg2+ + Ca2+)-ATPase in human red blood cells is regulated by a number of intracellular factors, including the association of the enzyme with the cytosolic Ca2+ binding protein, calmodulin. Ghosts prepared by hypotonic lysis in the presence of 0.1 mM CaCl2, or by a gradual stepwise hemolysis procedure, contain an EDTA-extractable protein whose effects are mimicked by calmodulin, whereas ghosts prepared by extensive washes in the absence of added CaCl2 lack calmodulin and contain only a high molecular weight heat stable activator. Purified calmodulin from human red cells or bovine brain shifts the apparent Ca2+ affinity of (Mg2+ + Ca2+)-ATPase activity in extensively washed ghosts to a high Ca2+ affinity state. The shift was most apparent in ghosts in which the Ca2+ affinity was decreased by EDTA treatment. Calmodulin increased the velocity of (Mg2+ + Ca2+)-ATPase in the EDTA-treated ghosts about 36-fold at a low (1.4 microM) Ca2+ concentration, compared with 6-fold before EDTA treatment. The maximum shift in apparent Ca2+ affinity occurred only in the presence of saturating concentrations of calmodulin. It is concluded that red cell calmodulin confers to the Ca2+ transport ATPase the ability to increase its apparent Ca2+ affinity, as well as its maximum velocity, in response to increases in intracellular Ca2+.     

The isolation of plasma membrane and characterisation of the plasma membrane ATPase from the yeast Candida albicans

Plasma membrane ghosts were isolated from Candida albicans ATCC 10261 yeast cells following stabilisation of spheroplasts with concanavalin A, osmotic lysis and Percoll density gradient centrifugation. Removal of extrinsic proteins with NaCl and methyl alpha-mannoside gave increased ATPase and chitin synthase specific activities in the resultant plasma membrane fraction. Sonication of this fraction yielded unilamellar plasma membrane vesicles which exhibited ATPase and chitin synthase specific activities of 4.5-fold and 3.0-fold, respectively, over those of the plasma membrane ghosts. ATPase activity in the membrane ghosts was optimal at pH 6.4, showed high substrate specificity (for Mg X ATP) and was inhibited 80% by sodium vanadate but less than 4% by oligomycin and azide. The effects of a range of other inhibitors were also characterised. Temperature effects of ATPase activity were marked, with a maximum at 35 degrees C. Breaks in the Arrhenius plot, at 12.2 degrees C and 28.9 degrees C, coincided with endothermic heat flow peaks detected by differential scanning calorimetry. ATPase was solubilised from the plasma membranes with Zwittergent in the presence of glycerol and phenylmethylsulphonyl fluoride and partially purified by glycerol density gradient centrifugation. The solubilised enzyme hydrolysed Mg X ATP at Vmax = 20 mumol X min-1 X mg-1 in the presence of phospholipids, with optimal activity at pH 6.0--6.5.     

Electrophoretic Separation of Different Phophosproteins Associated with Ca-ATPase and Na,K-ATPase in Human Red Cell Ghosts

Ca has been found to increase the quantity of ³²P incorporated into red cell ghosts from [γ-³²P]ATP over the levels obtained by incubation with Mg alone or with Mg + Na, in correlation with the effect of Ca on the associated ATPase activities. When the ³²P-labeled ghosts were solubilized in sodium dodecyl sulfate (SDS) and electrophoresed on acrylamide gels only two bands could be detected either by autoradiography or by counting the sliced gels. The faster moving band (P-2) had the same mobility and the same molecular weight (103,000) as the phosphoprotein found either with Mg alone or with Mg + Na. The slower moving band (P-1) was not found in extensively washed ghosts labeled in the absence of Ca. The molecular weight of P-1 is approximately 150,000. P-1 like P-2 was not affected by pretreatment of intact cells with Pronase before labeling indicating that neither the phosphorylating mechanism nor the phosphoprotein are accessible to externally applied Pronase. The demonstration that a Ca-phosphoprotein is separable from the Na-stimulated phosphoprotein suggests that the Ca-ATPase is distinct from and independent of the Na,K-ATPase. The fact that Ca blocks the dephosphorylation by K of the Na-phosphoprotein indicates that caution is required in interpreting results when the activities of the different phosphoproteins have not been separately determined.     

Isolation and characterization of Neurospora crassa plasma membranes.

The isolation and characterization of plasma membranes from a cell wall-less mutant of Neurospora crassa are described. The plasma membranes are stabilized against fragmentation and vesiculation by treatment of intact cells with concanavalin A just prior to lysis. After lysis, the concanavalin A-stabilized plasma membrane ghosts are isolated by low speed centrifugation techniques and the purified ghosts subsequently converted to vesicles by removal of the bulk of the concanavalin A. The yield of ghosts is about 50% whereas the yield of vesicles is about 20%. The isolated plasma membrane vesicles have a characteristically high sterol to phospholipid ratio, Mg2+-dependent ATPase activity and (Na+ plus K+)-stimulated Mg2+ATPase activity. Only traces of succinate dehydrogenase and 5'-nucleotidase are present in the plasma membrane preparations.     

Effects of calmodulin on the (Ca2+ + Mg2+)ATPase partially purified from erythrocyte membranes.

The stimulation of the (Ca²⁺ + Mg²⁺)ATPase of erythrocyte ghosts by calmodulin was observed not only in intact ghosts, but also in the solubilized (Triton X-100) and partially purified, reconstituted (phosphatidylserine liposomes) forms. Since the solubilized form of the enzyme migrated on Sepharose 6B at a position corresponding to a molecular weight of about 150,000, these results show that calmodulin stimulates by direct interaction with the ATPase complex. Additionally, the effects of calmodulin on erythrocyte ghosts prepared by the Dodge-EDTA method (hypotonic ghosts) and by the method of Ronner et al. (involving lysis followed by an isotonic wash repeated several times) were compared (P. Ronner, P. Gazzotti, and E. Carafoli, 1977, Arch. Biochem. Biophys. 179, 578–583). The (Ca²⁺ + Mg²⁺)ATPase of the hypotonic ghosts was low and was stimulated by added calmodulin while that of the isotonic ghosts was high and changed only slightly upon calmodulin addition; this difference in response to calmodulin persisted in the solubilized and reconstituted forms. Hypotonic ghosts bound ¹²⁵I-labeled calmodulin, while isotonic ghosts did not. This comparison of two types of ghosts showed that isotonic ghosts possess an intact calmodulin-(Ca²⁺ + Mg²⁺)ATPase complex, and that the calmodulin remained with the ATPase during solubilization and reconstitution. The isotonic preparation is a particularly useful method of preparing ghosts with an intact calmodulin-ATPase complex, since it requires no special equipment and produces an enzyme activity which is stable to freezing.     

NaCl Effects on Root Plasma Membrane ATPase of Salt Tolerant Wheat

Wheat seedlings of a salt tolerant cultivar were grown hydroponically in presence and absence of 100 mM NaCl. Roots were harvested, and the plasma membrane (PM) fraction was purified. PM ATPase required a divalent cations for activity (Mg > Mn > Ca > Co > Zn > Ni > Cu), and it was further stimulated by monovalent cations (K > Rb > NH₄ > Li > choline > Cs). The pH optima were 6.0 and 5.6 in absence and presence of 25 mM KCl, respectively. The enzyme was sensitive to vanadate and DCCD but insensitive to azide, oligomycine and nitrate. The enzyme displayed a high preference for ATP but was also able to hydrolyze other nucleotide tri- and diphosphates. The enzyme activity showed a simple Michaelis-Menten kinetics for the substrate Mg²⁺-ATP in both control and salt exposed roots. The polypeptide patterns of control and salt stressed PM fractions, detected by SDS-PAGE, were very similar. NaCl substantially reduced the PM ATPase specific activity, whereas it had little effect on the apparent K_m for Mg²⁺-ATP. Since the root PM ATPase of salt sensitive and resistant genotypes responded similarly to salinity stress, it seems unlikely that the mechanism of salt tolerance in wheat is primarily based on differences in PM ATPase characteristics.     

Disappearance of calcium-induced phase separation in phosphatidylserine-phosphatidylcholine membranes caused by protonation and by electric current

Disappearance of Ca²⁺-induced phase separation in phosphatidylserine-phosphatidylcholine membranes has been studied under several conditions by monitoring electron spin resonance spectrum of spin-labeled phosphatidylcholine. The membranes were prepared in Millipore filters. Electron micrographs of the preparations showed formation of multilayered structures lined on the pore surface. The phase separation was disappeared when the membrane was soaked in non-buffered salt solution (100 ml KCl, pH 5.5). It was markedly contrasting that when the bathing salt solution was buffered no disappearance was observed. Disappearance of the phase separation was also observed when the Ca²⁺-treated membrane was transferred to acidic salt solutions ($\text{?}\text{pH 2.5}$) or to low ionic strength media ($\text{? 10}\text{mM}$) buffered at pH 5.5, and then to the buffered salt solution (100 mM KCl, pH 5.5). These are due to replacement of Ca²⁺ by proton, proton-induced separation, followed by disappearance of the phase separation inthe buffered salt solution. Biological significance of the competition between Ca²⁺ and proton for the phase separation or domain formation in the membranes was emphasized.     

Effect of carbonylcyanide m-chlorophenylhydrazone on the calcium-stimulated ATPase activity of erythrocyte ghosts

Incubation of erythrocyte ghosts with carbonylcyanide m-chlorophenylhydrazone (CCCP) plus Ca²⁺ resulted in inactivation of the Ca²⁺-stimulated ATPase activity. Omission of Ca²⁺ or lowering of the temperature below 25 °C eliminated the inhibitory effect, as also did the presence of ATP during the incubation. On the other hand, the addition of β-mercaptoethanol did not influence the Ca²⁺-dependent inhibition by CCCP. Compared with the level of CCCP which uncouples oxidative phosphorylation, a rather high level (0.5 mM) of CCCP was required to inhibit the ATPase activity in ghosts. However, once the inhibition had been accomplished, almost all of the CCCP could be removed from the ghost membrane by washing with a Ca²⁺-containing solution, without affecting the inhibition of ATPase. If ethylene-glycol-bis(β-aminoethyl acid was included in the washing medium, the inhibition of ATPase was nearly completely reversed by washing. The results indicate that only the Ca²⁺-stimulated, Mg²⁺-ATPase was inhibited by 0.5 mM CCCP, while the remaining components of the ATPase activity were slightly inhibited by higher levels of the uncoupler. Low levels of CCCP (0.1 mM) stimulated the Mg²⁺-ATPase slightly. CCCP was much more specific for the Ca²⁺-stimulated ATPases than N-(1-naphthyl)maleimide, an unusually effective sulfhydryl reagent, and the requirement of Ca²⁺ for inactivation was also quite specific.