Oxidative damage to sarcoplasmic reticulum Ca2+-pump induced by Fe2+/H2O2/ascorbate is not mediated by lipid peroxidation or thiol oxidation and leads to protein fragmentation

The major protein in the sarcoplasmic reticulum (SR) membrane is the Ca²⁺ transporting ATPase which carries out active Ca²⁺ pumping at the expense of ATP hydrolysis. The aim of this work was to elucidate the mechanisms by which oxidative stress induced by Fenton's reaction (Fe²⁺ + H₂O₂ HO· + OH^–+ Fe³⁺) alters the function of SR. ATP hydrolysis by both SR vesicles (SRV) and purified ATPase was inhibited in a dose-dependent manner in the presence of 0–1.5 MM H₂O₂ plus 50 M Fe²⁺ and 6 mM ascorbate. Ca²⁺ uptake carried out by the Ca²⁺-ATPase in SRV was also inhibited in parallel. The inhibition of hydrolysis and Ca²⁺ uptake was not prevented by butylhydroxytoluene (BHT) at concentrations which significantly blocked formation of thiobarbituric acid-reactive substances (TBARS), suggesting that inhibition of the ATPase was not due to lipid peroxidation of the SR membrane. In addition, dithiothreitol (DTT) did not prevent inhibition of either ATPase activity or Ca²⁺ uptake, suggesting that inhibition was not related to oxidation of ATPase thiols. The passive efflux of ⁴⁵Ca²⁺ from pre-loaded SR vesicles was greatly increased by oxidative stress and this effect could be only partially prevented (ca 20%) by addition of BHT or DTT. Trifluoperazine (which specifically binds to the Ca²⁺-ATPase, causing conformational changes in the enzyme) fully protected the ATPase activity against oxidative damage. These results suggest that the alterations in function observed upon oxidation of SRV are mainly due to direct effects on the Ca²⁺-ATPase. Electrophoretic analysis of oxidized Ca²⁺-ATPase revealed a decrease in intensity of the silver-stained 110 kDa Ca²⁺-ATPase band and the appearance of low molecular weight peptides (MW < 100 kDa) and high molecular weight protein aggregates. Presence of DTT during oxidation prevented the appearance of protein aggregates and caused a simultaneous increase in the amount of low molecular weight peptides. We propose that impairment of function of the Ca²⁺-pump may be related to aminoacid oxidation and fragmentation of the protein.Abbreviations AcP acetylphosphate - BHT butylhydroxytoluene - DTT dithiothreitol - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - SDS sodium dodecyl sulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - SR sarcoplasmic reticulum - SRV sarcoplasmic reticulum vesicles - TBA thiobarbituric acid - TBARS thiobarbituric acid-reactive substances - TFP trifluoperazine     

Ca2+-Mg2+-activated adenosine triphosphatase in plasma and granule membranes in non-secreting and secreting mast cells

An adenosine triphosphatase (ATP) activated by Ca²⁺ or Mg²⁺ is shown morphologically on the outer surface of non-secreting and secreting rat peritoneal mast cells. ATPase having the same properties is also seen on the external surface of the other peritoneal cells, i.e. macrophages, mononuclear cells and lymphocytes. When histamine release from the mast cells was induced by exposing them to antigen (anaphylactic reaction) or compound 48/80, ATPase activated by Ca²⁺ or Mg²⁺ could in addition be demonstrated in the granule membranes. Granule membrane ATPase is also shown in non-secreting mast cells after freezing and thawing. ATPase on the outer surface of the plasma membrane is seen in the secreting mast cells as in the non-secreting cells except in the areas where the plasma membrane fuses with the granule membrane. The role of ATPase in granule secretion process has been discussed.     

Differential susceptibility of murine T and B lymphocytes to freeze-thaw and hypotonic shock

Experiments were carried out to determine if thymic-derived lymphocytes (T-cells) could be differentially damaged by hypotonic and/or freeze-thaw stress. The uptake of ³H-thymidine after stimulation of murine spleen cells with phytohemagglutinin-P (PHA-P) or bacterial endotoxin (LPS) was used as an indicator of recovery. Optimal freezing and thawing techniques showed that 100% of LPS-responsive cells could be recovered, compared to 65% of PHA-responsive cells. These differences could be increased by treatment of spleen cells with 0.17 m NH₄Cl prior to freezing and thawing. This represented a recovery of 50% of LPS-responsive cells and less than 10% of PHA-responsive cells. A similar effect could be obtained by treating NH₄Cl-treated spleen cells with distilled water prior to culture. It is hypothesized that T-cells are more susceptible to osmotic damage than B-cells due to their differences in membrane characteristics.     

Factors affecting the repair of sublethal freeze-thaw damage in mammalian cells: II. The effect of ouabain

Mammalian cells were able to repair sublethal damage sustained during exposure to freeze-thaw conditions if they were incubated at 37 °C during the repair period. Repair was also observed when the cells were incubated at 37 °C in medium containing 10^?4m ouabain but this was not the case with 10^?3m ouabain. Cells exposed to either 10^?3 or 10^?4m ouabain before freezing and thawing showed reduced survival indicating the requirement for the prior operation of the (Na⁺ ? K⁺) — ATPase system to avoid additional lethal damage.     

Proteins from frost-hardy leaves protect thylakoids against mechanical freeze-thaw damage in vitro

We have isolated protein fractions from cold-acclimated, frost-hardy cabbage (Brassica oleracea L.) and spinach (Spinacia oleracea L.) leaves which protect isolated thylakoids from non-hardy spinach against mechanical membrane rupture during an in-vitro freeze-thaw cycle. No protective activity was found in similar preparations from non-hardy leaves. The proteins protected the membranes from damage by reducing their solute permeability during freezing and by increasing their expandability during thawing. The proteins act by increasing the resistance of the membranes against the osmotic stress to which they are exposed during a freeze-thaw cycle. In the absence of cryoprotectants this stress results in membrane rupture.This investigation was supported by the Deutsche Forschungsge-meinschaft.     

Optimal conditions for the preservation of mouse lymph node cells in liquid nitrogen using cooling rate techniques

The factors that affect the survival of mouse lymphocytes throughout a procedure for storage at ?196 °C have been studied both for the improvement of recovery and the possible extension to the mouse system of cell selection by freezing. After thawing, the survival of cells cooled at different rates in dimethyl sulphoxide (DMSO, 5 or 10%, $\text{v}\text{v}$) was assessed from the [³H]thymidine incorporation in response to phytohaemagglutinin and concanavalin A. Before freezing the protection against freezing damage increased with time (up to 20 min) in DMSO (5%, $\text{v}\text{v}$) at 0 °C. Superimposed upon this effect was toxicity due to the DMSO. During freezing and thawing the cooling rate giving optimal survival was 8 to 15 °C/min for cells in DMSO (5%) and 1 to 3 °C/min for DMSO (10%). Omission of foetal calf serum was detrimental. Rapid thawing (>2.5 °C/min) was superior to slow thawing. After thawing dilution at 25 or 37 °C greatly improved cell survival compared with 0 °C; at 25 °C survival was optimal (75%) at a moderate dilution rate of 2.5 min for a 10-fold dilution in FCS (10%, $\text{v}\text{v}$) followed by gentle centrifugation (50g).Dilution damage during both thawing and post-thaw dilution may be due to osmotic swelling as DMSO and normally excluded solutes leave the cell. The susceptibility of the cell membrane to dilution damage may also be increased during freezing. The need to thaw rapidly and dilute at 25 °C after thawing is probably due to a decrease in dilution stress at higher temperatures. Optimisation of dilution procedures both maximised recovery and also widened the range of cooling rates over which the cells were recovered. These conditions increase the possibility of obtaining good recovery of a mixed cell population using a single cooling procedure. Alternatively, if cell types have different optimal cooling rates, stressful dilution may allow their selection from mixed cell populations.     

Assessment of cellular damage during cooling to −196 °C using radiochemical markers

The release of ten radiochemical markers from MRC-5 and CHO cells after cooling at various rates and thawing from temperatures in the range of 0 to ?196 °C was measured. Many of these radiochemicals had specific sites of attachment on or within the cell and the aim was to determine the effect of freeze-thaw stresses on various parts of the cell. Cell death during cooling and thawing was, in most instances, accompanied by osmotic damage and loss of cytoplasmic constituents. Significant damage to the cell membrane occurred only after the cell was already dead and was related to the disruption of cells killed at higher temperatures and to osmotic stress during rewarming. The release of cations and other cytoplasmic markers was correlated to cell shrinkage and dehydration. The data were used to assess the relative effects of some of the proposed damaging factors in freeze-thaw injury (thermal shock, ice damage, dilution shock, etc.). CHO cells showed a much higher survival rate and release of cations after fast cooling than MRC-5 cells. This, and additional circumstantial information, indicated that CHO cells survived freeze-thaw cycles better than MRC-5 cells because they are able to dehydrate more readily, even at fast cooling rates.     

ANALYSIS OF THE EXPRESSION OF GENES FOR P-TYPE ATPASE IN THE HALOTOLERANT ALGA DUNALIELLA SALINA (CHLOROPHYCEAE)1

A partial complementary DNA (cDNA) (DSA8) for a P-type ATPase was obtained from the halotolerant alga Dunaliella salina (Dunal) Teod. (Chlorophyceae). The cDNA exhibited greater than 90% homology to the cDNA for a H⁺-ATPase in D. bioculata Butcher. The expression of the gene that corresponded to DSA8 was decreased strongly by increases in NaCl concentration. The expression of a gene that corresponded to another ATPase (DSA1; possibly for a Ca²⁺-ATPase) from D. salina did not show the same decrease as did the DSA8. However, increased osmotic pressure due to glycerol resulted in the same decrease in the DSA8 gene. Under salt or osmotic stress, the activity of a H⁺-ATPase from microsomes of this alga also decreased. We suggest that expression of the gene for the plasma membrane H⁺-ATPase of D. salina is regulated by osmotic pressure rather than by the concentration of NaCl.     

Parameters of biological freezing damage in simple solutions: Catalase: II. Demonstration of an optimum-recovery cooling-rate curve in a membraneless system

Seeded solutions of catalase in neutral 10 mM potassium phosphate buffer exhibited characteristic rate dependencies for freeze-thaw damage: Damage increased as the cooling rate was increased, and as the warming rate was decreased. The pattern of warming-rate dependence was independent of the prior cooling rate and also of the addition of KCl or of NaCl to the buffer. In contrast, the cooling-rate curve became almost flat upon addition of 0.1 M KCl, suggesting increased damage from concentrating solute at low cooling rates. In the presence of added NaCl, frank optimum-recovery cooling-rate curves were generated. At low NaCl levels (less than 10 mM) the optimum occurred at 0.5 °C/ min; at 27 and 81 mM NaCl, the optimum shifted to 5 and 20 °C/min, respectively. By comparison with KCl, it appears that the major factor causing damage at low cooling rates in NaCl is acidification. The factor causing damage at high cooling rates remains obscure. The argument that it is due to the trapping of the enzyme molecules at interfaces at high dilution, to be subsequently damaged by shearing stress or dehydration during the recrystallization attending slow warming, is mitigated by the finding that inactivation remains a function of the initial enzyme concentration at all cooling rates. The possibility that a particular conformational state is trapped in an unfavorable temperature zone was also considered: Three simple models were formulated, and the relative order of recovery was deduced for the possible sequences of fast and slow cooling and warming. The permutation observed for catalase was inconsistent with any of these three mechanisms, although they may be pertinent for the red cell and other systems. A final possibility, not yet explored, is that rapid cooling causes damage by producing nonequilibrium freezing, with large deviations of pH and/or solute concentration from those expected at equilibrium.     

Effects of freezing on the release and inactivation of chloroplast coupling factor 1

The three high-molecular-weight subunits of chloroplast coupling factor (CF₁) are the primary proteins released from pyrophosphate-washed thylakoids exposed to freezing. Identical subunit profiles are found in the supernatant proteins of thylakoids exposed to different intensities of freezing stress by the inclusion of sugars with varying degrees of cryoprotective efficiency. Isolated CF₁ is inactivated by freezing in the presence of NaCl, glucose, and sucrose but raffinose can protect against loss of enzymatic activity during freezing. The low specific activity of the supernatant proteins released from the thylakoid and the inability to recover the Ca²⁺-dependent ATPase activity lost from the membrane suggest that inactivation accompanies release of CF₁ during freezing.     

In Vivo Perturbation of Membrane-Associated Calcium by Freeze-Thaw Stress in Onion Bulb Cells : Simulation of This Perturbation in Extracellular KCl and Alleviation by Calcium

Incipient freeze-thaw stress in onion bulb scale tissue is known to cause enhanced efflux of K⁺, along with small but significant loss of cellular Ca²⁺. During the post-thaw period, irreversibly injured cells undergo a cytological aberration, namely, `protoplasmic swelling.' This cellular symptom is thought to be caused by replacement of Ca²⁺ from membrane by extracellular K⁺ and subsequent perturbation of K⁺ transport properties of plasma membrane. In the present study, onion (Allium cepa L. cv Sweet Sandwich) bulbs were slowly frozen to either −8.5°C or −11.5°C and thawed over ice. Inner epidermal peels from bulb scales were treated with fluorescein diacetate for assessing viability. In these cells, membrane-associated calcium was determined using chlorotetracycline fluorescence microscopy combined with image analysis. Increased freezing stress and tissue infiltration (visual water-soaking) were paralleled by increased ion leakage. Freezing injury (−11.5°C; irreversible) caused a specific and substantial loss of membrane-associated Ca²⁺ compared to control. Loss of membrane-associated Ca²⁺ caused by moderate stress (−8.5°C; reversible) was much less relative to −11.5°C treatment. Ion efflux and Ca²⁺-chlorotetracycline fluorescence showed a negative relationship. Extracellular KCl treatment simulated freeze-thaw stress by causing a similar loss of membrane-associated calcium. This loss was dramatically reduced by presence of extracellular CaCl₂. Our results suggest that the loss of membrane-associated Ca²⁺, in part, plays a role in initiation and progression of freezing injury.     

Manifestations of cell damage after freezing and thawing

The nature of the primary lesions suffered by cells during freezing and thawing is unclear, although the plasma membrane is often considered the primary site for freezing injury. This study was designed to investigate the nature of damage immediately after thawing, by monitoring several functional tests of the cell and the plasma membrane. Hamster fibroblasts, human lymphocytes, and human granulocytes were subjected to a graded freeze-thaw stress in the absence of cryoprotective compound by cooling at -1 degree C/min to a temperature between -10 and -40 degrees C, and then were either warmed directly in water at 37 degrees C or cooled rapidly to -196 degrees C before rapid warming. Mitochondrial function in the cells was then assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), fluorescein diacetate (FDA), colony growth, and osmometric response in a hypertonic solution. Cells behaved as osmometers after cooling at -1 degree C/min to low temperatures at which there were no responses measured by other assays, indicating that the plasma membrane is not a primary site for injury sustained during slow cooling. These results also indicate that the FDA test does not measure membrane integrity, but reflects the permeability of the channels through which fluorescein leaves the cells. Fewer cells could respond osmotically after cooling under conditions where intracellular freezing was likely, implying that the plasma membrane is directly damaged by the conditions leading to intracellular freezing. A general model of freezing injury to nucleated mammalian cells is proposed in which disruption of the lysosomes constitutes the primary lesion in cells cooled under conditions where the cells are dehydrated at low temperatures.     

Freezing damage and frost tolerance of the photosynthetic apparatus studied with isolated mesophyll protoplasts of Valerianella locusta L.

Mesophyll protoplasts were isolated from unhardened and cold-acclimated leaves of Valerianella locusta L. and subjected to freeze-thaw treatment. To evaluate the extent and course of freezing injury, photosynthetic reactions of whole protoplasts and of free thylakoid membranes, liberated from protoplasts by osmotic lysis, were measured. In addition, the integrity of the protoplasts was determined by microscopy. The results reveal an increased frost tolerance of protoplasts isolated from acclimated leaves with respect to all parameters measured. CO₂-dependent O₂ evolution (representing net photosynthetic CO₂ fixation of protoplasts) was the most freezing-sensitive reaction; its inhibition due to freeze-thaw treatment of protoplasts was neither correlated with disintegration of the plasma membrane, nor was it initiated by inactivation of the thylakoid membranes. The frost-induced decline of protoplast integrity was not closely correlated to thylakoid damage either. Freezing injury of the thylakoid membranes was manifested by inhibition of photosynthetic electron transport and photophosphorylation. Both photosystems were affected by freezing and thawing with strongest inhibition occurring in the water-oxidation system or at the oxidizing site of photosystem II. Photophosphorylation responded more sensitively to freezing stress than electron transport, although uncoupling (increased permeability of the thylakoid membranes to protons) was not a conspicuous effect. The data are discussed in relation to freezing injury in leaves and seem to indicate that frost damage in vivo is initiated at multiple sites.Abbreviations Chl chlorphyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbazide - Hepes 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid - MES 2-(N-morpholino)-ethanesulfonic acid - PS I photosystem I - PS II photosystem II     

The changes in erythrocyte Ca<Superscript>2+</Superscript>-ATPase activity induced by PEG-1500 and low temperatures

Various organic compounds are applied upon cryopreservation and their adding into cell suspension causes modification of subcellular systems, providing cell survival during freeze–thawing. The aim of the study was to assess the modifying effect of cryoprotectant PEG-1500 and low temperatures on Ca²⁺-ATPase activity in saponin-permeabilized erythrocytes. PEG-1500 was revealed to inhibit erythrocyte Ca²⁺-ATPase activity despite the presence of endogenous effectors able to stimulate the enzyme function. Presumably, the Ca²⁺-ATPase modification was determined by the physicochemical properties of the polymer solution, since the removal of PEG-1500 out of the medium recovered the enzyme activity. Reversibility of Ca²⁺-ATPase inhibition was characteristic of erythrocytes both exposed to cryoprotectant without freezing and frozen–thawed in the PEG-1500 presence. The cell freeze–thawing without cryoprotectant had no effect on Ca²⁺-ATPase, suggesting that membrane form of enzyme is cryoresistent. Although the efficiency of erythrocyte cryopreservation with PEG-1500 depends on the incubation temperature before freezing stage, the functional indices of Ca²⁺-ATPase in erythrocytes exposed to PEG-1500 at 37 and 5–7°C had no significant distinctions if the subsequent ATP hydrolysis was conducted at 37°C. However, the enzyme activity was additionally slowed down when the temperature of enzymatic reaction was decreased to 5–7°C after erythrocyte preincubation with PEG-1500 under the same conditions. The identified changes in Ca²⁺-ATPase activity in erythrocytes in the PEG-1500 presence were most likely determined by a modifying effect of the cryoprotectant on the membrane structure; as a result, the Ca²⁺-ATPase endogenous effectors present in the medium could not overcome the restrictions imposed on the enzyme function by a modified membrane macroenvironment.     

Exogenous Ca2+ alleviates waterlogging-caused damages to pepper

Ca²⁺ has been considered as a necessary ion for alleviation of stress-induced damages in plants. We investigated effects of exogenous Ca²⁺ on waterlogging-induced damage to pepper and its underlying mechanisms. Pepper seedlings under stress were treated by spraying of 10 mM CaCl₂. Applying exogenous Ca²⁺ increased the biomass of pepper leaves and roots, improved photosynthetic characteristics, membrane permeability, root activity, osmotic substance contents, antioxidant enzyme and alcohol dehydrogenase activities, while it reduced lactate dehydrogenase activity. It maintained hydroxide radical contents and activities of malate dehydrogenase and succinate dehydrogenase relatively high. Our results suggested that applying exogenous Ca²⁺ could regulate osmotic substance contents, antioxidant system activity, root respiration, and metabolism, and subsequently alleviate waterlogging-induced damages to pepper plants.     

Hormonal control of uterine contraction: Characterization of cyclic AMP-dependent membrane properties in the myometrium

A mitochondria-free membrane fraction prepared from rat myometrium accumulated ⁴⁵Ca²⁺ in the presence of oxalic acid and ATP. The rate of transport of Ca²⁺ into the membranous vesicles was increased by greater than 50% in the presence of 3′,5′-cyclic AMP, but not by 2′,3′-cyclic AMP or 5′-AMP. Membrane ATPase activity was stimulated by cyclic AMP in a manner similar to Ca²⁺-transport. ATPase activity was stimulated by Mg²⁺; slight additional stimulation was obtained in the presence of Na⁺ and K⁺ but not in the presence of Ca²⁺. Despite the cyclic AMP sensitivity of membrane ATPase activity, the absence of any effect of inhibitors of Ca²⁺-transport suggest it has little to do with Ca²⁺ accumulation by the membranes.Cyclic AMP-induced increase in Ca²⁺-transport and membrane ATPase activity was duplicated in vivo by incubating uteri in 10⁻⁴ M isoproterenol prior to membrane isolation. Isoproterenol has been previously shown to increase myometrial cyclic AMP levels, and changes in Ca²⁺-transport by cell membranes in relation to intracellular cyclic AMP levels may be the mechanism through which hormones modulate uterine contractility.     

Role of matrix metalloprotease-2 in oxidant activation of Ca<Superscript>2+</Superscript>ATPase by hydrogen peroxide in pulmonary vascular smooth muscle plasma membrane

Exposure of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant H₂O₂ (1 mM) stimulated Ca²⁺ATPase activity. We sought to determine the role of matrix metalloprotease-2 (MMP-2) in stimulating Ca²⁺ATPase activity by H₂O₂ in the smooth muscle plasma membrane. The smooth muscle membrane possesses a Ca²⁺-dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease activity was found to be inhibited by EGTA, 1: 10-phenanthroline, a2-macroglobulin and tissue inhibitor of metalloprotease-2 (TIMP-2) indicating that the Ca²⁺-dependent 72 kDa protease is the MMP-2. Western immunoblot studies of the membrane suspension with polyclonal antibodies of MMP-2 and TIMP-2 revealed that MMP-2 and TIMP-2, respectively, are the ambient matrix metalloprotease and the corresponding tissue inhibitor of metalloprotease in the membrane. In addition to increasing the Ca²⁺ATPase activity, H₂O₂ also enhanced the activity of the smooth muscle plasma membrane associated protease activity as evidenced by its ability to degrade¹⁴C-gelatin. The protease activity and the Ca²⁺ATPase activity were prevented by the antioxidant, vitamin E, indicating that the effect produced by H₂O₂ was due to reactive oxidant species(es). Both basal and H₂O₂ stimulated MMP-2 activity and Ca²⁺ATPase activity were inhibited by the general inhibitors of matrix metalloproteases: EGTA, 1: 10-phenanthroline, α₂-macroglobulin and also by TIMP-2 (the specific inhibitor of MMP-2) indicating that H₂O₂ increased MMP-2 activity and that subsequently stimulated Ca²⁺ATPase activity in the plasma membrane. This was further confirmed by the following observations: (i) adding low doses of MMP-2 or H₂O₂ to the smooth muscle membrane suspension caused submaximal increase in Ca²⁺ATPase activity, and pretreatment with TIMP-2 prevents the increase in Ca²⁺ATPase activity; (ii) combined treatment of the membrane with low doses of MMP-2 and H₂O₂ augments further the Ca²⁺ATPase activity caused by the respective low doses of either H₂O₂ or MMP-2; and (iii) pretreatment with TIMP-2 prevents the increase in Ca²⁺ATPase activity in the membrane caused by the combined treatment of MMP-2 and H₂O₂.     

Calpain I activates Ca2+ transport by the reconstituted erythrocyte Ca2+ pump

Summary Calpain I purified from human erythrocyte cytosol activates both the ATP hydrolytic activity and the ATP-dependent Ca²⁺ transport function of the Ca²⁺-translocating ATPase solubilized and purified from the plasma membrane of human erythrocytes and reconstituted into phosphatidylcholine vesicles. Following partial proteolysis of the enzyme by calpain I, both the initial rates of calcium ion uptake and ATP hydrolysis were increased to near maximal levels similar to those obtained upon addition of calmodulin. The proteolytic activation resulted in the loss of further stimulation of the rates of Ca²⁺ translocation or ATP hydrolysis by calmodulin as well as an increase of the affinity of the enzyme for calcium ion. However, the mechanistic Ca²⁺/ATP stoichiometric ratio was not affected by the proteolytic treatment of the reconstituted Ca²⁺-translocating ATPase. The proteolytic activation of the ATP hydrolytic activity of the reconstituted enzyme could be largely prevented by calmodulin. Different patterns of proteolysis were obtained in the absence or in the presence of calmodulin during calpain treatment: the 136-kDa enzyme was transformed mainly into a 124-kDa active ATPase fragment in the absence of calmodulin, whereas a 127-kDa active ATPase fragment was formed in the presence of calmodulin. This study shows that calpain I irreversibly activates the Ca²⁺ translocation function of the Ca²⁺-ATPase in reconstituted proteoliposomes by producing a calmodulin-independent active enzyme fragment, while calmodulin antagonizes this activating effect by protecting the calmodulin-binding domain against proteolytic cleavage by calpain.     

Thermodynamic modeling and cryomicroscopy of cell-size, unilamellar, and paucilamellar liposomes

Cryomicroscope studies of large unilamellar liposomes indicate that liposomes are an excellent model for studying membrane response to freezing and thawing. Liposomes are attractive for such use because they can be custom-manufactured for a particular investigation. In addition, liposome responses to freezing and thawing mimic real cell behavior in a number of significant ways. Analogous behavior includes osmotic shrinkage at slow cooling rates, internal ice formation at fast cooling rates, comparable nucleation temperatures, and a variety of comparable thawing responses. Experimental determination has been made of the equilibrium osmotic properties and the nonequilibrium water transport properties of the egg lecithin liposomes used in the freezing studies. These properties have been used in a computer model to simulate volume changes resulting from water transport during freezing and thawing. Comparison between computer model predictions and experimental data for the liposome volume response during freezing indicates reasonable agreement whereas computer simulations of volume response during thawing do not match experimental data well.     

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.