Normal Ionic Strength Research Articles

Orientational disorder and motion of weakly attached cross-bridges

In a relaxed muscle fiber at low ionic strength, the cross-bridges may well be in states comparable to the one that precedes the cross-bridge power stroke (Schoenberg, M. 1988. Adv. Exp. Med. Biol. 226:189-202). Using electron paramagnetic resonance (EPR) and (saturation transfer) electron paramagnetic resonance (ST-EPR) techniques on fibers labeled with maleimide spin label, under low ionic strength conditions designed to produce a majority of weakly-attached heads, we have established that (a) relaxed labeled fibers show a speed dependence of chord stiffness identical to that of unlabeled, relaxed fibers, suggesting similar rapid dissociation and reassociation of cross-bridges; (b) the attached relaxed heads at low ionic strength are nearly as disordered as in relaxation at physiological ionic strength where most of the heads are detached from actin; and (c) the microsecond rotational mobility of the relaxed heads was only slightly restricted compared to normal ionic strength, implying great motional freedom despite attachment. The differences in head mobility between low and normal ionic strength scale with filament overlap and are thus due to acto-myosin interactions. The spectra can be modeled in terms of two populations: one identical to relaxed heads at normal ionic strength (83%), the other representing a more oriented population of heads (17%). The spectrum of the latter is centered at approximately the same angle as the spectrum in rigor but exhibits larger (40 degrees) axial probe disorder with respect to the fiber axis. Alternatively, assuming that the chord stiffness is proportional to the fraction of attached crossbridges, the attached fraction must be even more disordered than 400, with rotational mobility nearly as great as for detached cross-bridges.

The pH, conductivity and osmolality of low ionic strength solutions used within the U.K. for the antiglobulin test.

Low ionic strength solutions (LISS) for use in the antiglobulin test were obtained from 356 U.K. laboratories. Of the 324 laboratories using LISS to suspend the test red cells and who returned details of their LISS technique, 15 used unequal proportions of red cell suspension and serum despite the LISS being formulated for use with equal proportions. Of the 22 laboratories mixing LISS with serum and red cells suspended in a normal ionic strength medium, four used a LISS preparation formulated for a LISS-suspension technique and three used a commercially available LISS-addition preparation using proportions of red cells, serum and LISS not recommended by the manufacturer. The mean (standard deviation) pH, conductivity and osmolality of the 334 LISS preparations for LISS-suspension was 6.9 (0.2), 4.1 (0.4) mS/cm and 298 (15) mmole/kg, respectively. It is suggested that attention should be paid to the osmolality and, particularly, conductivity during the preparation of LISS because values were observed that were clearly outside the acceptable range cited in the Guidelines for the Blood Transfusion Services in the United Kingdom, i.e. pH 6.7 +/- 0.2, conductivity 3.7 +/- 0.3 mS/cm and osmolality 295 +/- 5 mmole/kg.

Rotational dynamics of spin-labeled F-actin during activation of myosin S1 ATPase using caged ATP

The most probable source of force generation in muscle fibers in the rotation of the myosin head when bound to actin. This laboratory has demonstrated that ATP induces microsecond rotational motions of spin-labeled myosin heads bound to actin (Berger, C. L. E. C. Svensson, and D. D. Thomas. 1989. Proc. Natl. Acad. Sci. USA. 86:8753-8757). Our goal is to determine whether the observed ATP-induced rotational motions of actin-bound heads are accompanied by changes in actin rotational motions. We have used saturation transfer electron paramagnetic resonance (ST-EPR) and laser-induced photolysis of caged ATP to monitor changes in the microsecond rotational dynamics of spin-labeled F-actin in the presence of myosin subfragment-1 (S1). A maleimide spin label was attached selectively to cys-374 on actin. In the absence of ATP (with or without caged ATP), the ST-EPR spectrum (corresponding to an effective rotational time of approximately 150 microseconds) was essentially the same as observed for the same spin label bound to cys-707 (SH1) on S1, indicating that S1 is rigidly bound to actin in rigor. At normal ionic strength (micro = 186 mM), a decrease in ST-EPR intensity (increase in microsecond F-actin mobility) was clearly indicated upon photolysis of 1 mM caged ATP with a 50-ms, 351-nm laser pulse. This increase in mobility is due to the complete dissociation of Si from the actin filament. At low ionic strength (micro, = 36 mM), when about half the Si heads remain bound during ATP hydrolysis, no change in the actin mobility was detected, despite much faster motions of labeled S1 bound to actin. Therefore, we conclude that the active interaction of Si, actin,and ATP induces rotation of myosin heads relative to actin, but does not affect the microsecond rotational motion of actin itself, as detected at cys-374 of actin.

Modifications of single acetylcholine-activated channels in BC3H-1 cells. Effects of trimethyloxonium and pH.

We have examined the effects of chemical modification with trimethyloxonium (TMO) and changes in external pH on the properties of acetylcholine (ACh)-activated channels in BC3H-1 cells, a clonal muscle cell line. TMO reacts covalently and specifically with carboxylic acid moieties in proteins to convert them to neutral methyl esters. In BC3H-1 cells TMO modification reduces the whole-cell response to ACh measured at negative membrane potentials by approximately 60%. G omega seal patch-clamp recordings of single ACh channel currents showed that the reduction in ACh sensitivity is due to alterations in both the current-carrying and the kinetic properties of the channels. Under all our experimental conditions, i.e., in external solutions of normal or low ionic strength, with or without external divalent cations, and at external pHs between 5.5 and 8.1, TMO treatment reduced ACh single-channel conductance to 70-90% of normal. The effects of TMO on channel kinetics were dependent on the ionic conditions. In normal ionic strength solutions containing both calcium and magnesium ions TMO modification reduced the channel average open time by approximately 25%. A similar reduction in open time was seen in calcium-free solution, but was not present when both calcium and magnesium ions were absent from the external solution. Lowering the ionic strength of the solution increased the mean open time in normal channels by about threefold, but did not affect the kinetics of modified channels. In low ionic strength solutions normal ACh channel open times were maximal at approximately pH 6.7 and decreased by three- to fourfold at both acid and alkaline pH. TMO modification removed the pH dependence of channel kinetics, and average open times were short at all pHs between 5.5 and 8.1. We suggest that TMO modifies normally titratable groups on the external surface of ACh channels that help to determine both the gating and permeability properties of ACh channels.

Some Properties of Two Erythromycin-dependent Strains of Escherichia coli

Strains of Escherichia coli can be isolated that require erythromycin for growth. With one strain, AM, a range of antibiotics, including chloramphenicol, tetracycline, spectinomycin, kasugamycin and rifampicin, will substitute for erythromycin on solid and in liquid media; nalidixic acid supports growth in liquid but not on solid media. With a second strain, 103, chloramphenicol, tetracycline and spectinomycin support growth in liquid media but on solid medium only chloramphenicol substitutes for erythromycin. In media of higher than normal ionic strength, strain AM, but not strain 103, can grow in the absence of antibiotics. Possible reasons for these complex phenotypes are discussed.

Studies of stallion sperm survival: preservation of progressive motility of stallion spermatozoa by low ionic strength media.

Preservation of stallion sperm forward motility was studied using a video recording system in semen diluted with media of different ionic strength and sodium content. After 8 hr of incubation at room temperature, semen diluted in a low ionic strength media containing sucrose displayed 65 +/- 9% motility with 68 +/- 3% of the motile sperm showing forward motility (diameter of head trajectory greater than or equal to 80 microns). In contrast, sperm populations diluted and incubated with a normal ionic strength media containing sodium had 56 +/- 7% motile sperm of which only 36 +/- 7% displayed forward progression. Replacement of the sodium ion in the normal ionic strength media by a different cation (choline) produced results similar to those of the media containing high sodium concentration. These results indicate that the preservation of stallion sperm progressive motility is highly dependent on the ionic strength of the media. Membrane depolarization caused by the replacement of ions by noncharged particles in the sperm's external medium might be responsible for the sustained coordination of the flagellar movements.

Pulmonary surfactant apoprotein A structure and modulation of surfactant secretion by rat alveolar type II cells.

The pulmonary surfactant apoprotein with a reduced denatured molecular mass of 26-38 kDa (PSP-A) has recently been identified as an inhibitor of surfactant phospholipid secretion by isolated rat alveolar type II cells. We have investigated some of the structural determinants of PSP-A that are relevant to the inhibitory process. The PSP-A was isolated from rats given an intratracheal instillation of silica. The yield of PSP-A from silica-treated animals was 20-40-fold higher than that obtained from untreated animals. Reduction of PSP-A with 2-mercaptoethanol caused a reversible loss of biological activity that was restored by mild oxidation. Alkylation of the protein with excess iodoacetamide also led to inactivation, although titration with 5,5'-dithiobis-(2-nitrobenzoic acid) indicated that the protein initially contained no free sulfhydryl moieties. Neither alkylation nor reduction plus alkylation completely prevented the formation of oligomers as determined by gel permeation analysis. The apparent molecular mass of PSP-A at 4 degrees C in low ionic strength buffers was 1.6 megadaltons, and at 37 degrees C in normal ionic strength buffers was greater than 1.5 megadaltons. Removal of the oligosaccharide moiety with endoglycosidase F also had no effect upon biological activity. Five distinct monoclonal antibodies recognizing peptides epitopes on PSP-A were produced. All monoclonal antibodies exhibited similar affinity for PSP-A and recognized the delipidated and deglycosylated form. Four monoclonal antibodies reacted with epitopes on PSP-A that altered its function as an inhibitor. One monoclonal antibody was clearly ineffective at altering the activity of PSP-A. These results demonstrate that: 1) disulfide bonds are required for the activity of PSP-A, 2) disruption of disulfides does not prevent the formation of oligomeric forms of PSP-A, 3) the oligosaccharide moiety is not essential for biological activity, and 4) monoclonal antibodies can be used to map the epitopes responsible for biological activity.

Optical ellipsometry on the diffraction order of skinned fibers. pH-induced rigor effects

The polarization properties of light diffracted from single-skinned fibers of skeletal muscles have been examined under conditions in which the bathing solution pH and the ionic strength are changed. For fibers in the relaxed state, we observe large decreases in both the total depolarization signal, r, and the total diffraction birefringence signal, delta nT, upon pH change from 7.0 to 8.0 at normal ionic strength. However, if the ionic strength is raised, then the r-value change as the pH changes from pH 7.0 to pH 8.0 is much smaller. If the rigor state is achieved at pH 8.0, and 0 mM ATP under either of the ionic strength conditions, the fiber can still be stretched. Rigor stiffness for this state is only approximately 20% that of the value of the stiffness at pH 7.0 rigor. Electron micrographs obtained under this pH 8.0 rigor state show that the overlap region can be decreased upon stretching the fiber, signifying a different kind of weaker-binding rigor state. Optically, the weaker-binding rigor state has a lower depolarization signal and larger form birefringence than the strong-binding rigor state. To convert from one type of rigor state (pH 7.0) to the other rigor state (pH 8.0), or vice versa, the fiber must first be relaxed. Apparently, either of the rigor states can block the full impact of the pH effect.

Surface components involved in bacterial pathogen-plant host recognition.

During their initial association with plant hosts, pathogenic bacteria interact with plant cell walls. The results of this interaction appear to determine whether bacterial multiplication will take place. With one group of bacterial plant pathogens (e.g. Agrobacterium tumefaciens), attachment to the host surface appears essential for pathogenesis. With another group (e.g. Pseudomonas solanacearum), only those strains that do not attach to the host cell wall are able to multiply in the intercellular spaces. Attachment of many incompatible strains to tobacco mesophyll cell walls leads to a rapid hypersensitive response (HR) and a drastic reduction in bacterial multiplication. Our working hypothesis is that these differences in host response to strains of P. solanacearum are the result of a recognition response in which surface components of both host and pathogen play important roles. Our approach is based on the use of spontaneous or transposon (Tn5)-generated mutants of strains K60 (virulent) and B1 (avirulent) that differ in surface components and in their ability to attach to host cells and to induce the HR. A study of the surface components of bacterial and tobacco cell walls has led to the tentative conclusion that bacterial lipopolysaccharide (LPS) and plant hydroxyproline-rich glycoproteins mediate initial attachment, apparently as a result of charge-charge interaction. This initial attachment is reversed by high salt concentrations during the first 15 min, but not thereafter. Firm attachment appears to depend on hydrophobic interactions mediated by bacterial pili. At the normal ionic strength of intercellular fluids, extracellular polysaccharide (EPS) appears to inhibit only the pili-mediated attachment. Several HR- mutants of strain B1 have been obtained by Tn5 insertion, but they remain avirulent on tobacco. We are examining the EPS, LPS and pili production, and the attachment characteristics of these strains.

Photoelectric signals generated by bovine rod outer segment disk membranes attached to a lecithin bilayer

Purified bovine rod outer segment disk membranes were attached to a lecithin bilayer membrane. After photoexcitation with a 500-nm flash delivered by a dye laser, a negative photovoltage was observed on the bilayer under normal ionic strengths (100 mM KCl), which had a rise phase of 1-3 ms at 20 degrees C. The photoresponse was obviously due to bleaching of rhodopsin as it decreased for successive flashes of light. It originated most probably during the metarhodopsin-I metarhodopsin-II (meta-I-II) transition of rhodopsin because it was pH dependent at 2 degrees C but not at 20 degrees C. At 10 mM KCl, i.e., under hypotonic conditions, a positive photovoltage with slower kinetics than at high salt was observed. As the disk membranes were merely attached to the bilayer membrane, the photovoltage was apparently due to a light-induced transmembrane potential change in the disk membranes. Possible electrogenic mechanisms underlying the photosignal will be discussed.

Characterization of the C1q receptor on a human macrophage cell line, U937.

The binding of C1q to the human macrophage cell line U937 has been studied. Fluorescence microscopy with fluorescein-conjugated F(ab')2 anti-C1q antibody showed that 100% of the cell population is able to bind exogenous C1q. Monomeric C1q binding to U937 cells is very weak at normal ionic strength (I0.15) and was therefore investigated at I0.07, conditions which stabilize the binding. However, aggregation of C1q on dextran sulphate or a lipid A-rich lipopolysaccharide allowed a firm, binding at I0.15. Quantitative binding studies with monomeric 125I-C1q showed a concentration-dependent, saturable, specific and reversible binding involving specific membrane receptors. Scatchard plots of C1q binding indicated [1.6 +/- 0.7 (1 S.D.)] X 10(6) sites per cell with an equilibrium constant of (2.9 +/- 1.8) X 10(7) M-1 at I0.07. The location of the molecule region mediating C1q binding was established with collagen-like fragments prepared by partial pepsin digestion, confirming earlier results obtained by inhibition studies.

A role of ionic strength on the inotropic effects of osmolarity change in frog atrium.

The influence of varying the ionic strength of the bathing solution on the contraction of chemically skinned frog atrial muscle fibers was studied. The rate of tension development activated by calcium slows as the ionic strength is elevated. The size of caffeine contracture, however, was larger in the fiber preloaded with calcium at a higher ionic strength. There was a decrease in the maintained tension at 10(-6) M Ca when a fiber was bathed in a high ionic strength solution. Returning to a normal ionic strength solution caused a transient tension increase. When the fiber was bathed in a low ionic strength solution, the maintained tension increased transiently to a high value and then declined to reach a plateau. The response was also observed in a solution of pCa 8. In the caffeine-treated fiber or in the fiber bathed in ATP free solution, although the maintained tension level was changed corresponding to the altered ionic strength, the transient responses were blocked. These responses were not much influenced by the kinds of salts used to change the ionic strength. When osmolarity of the medium was altered by sucrose, transient responses were not induced. The results could qualitatively explain the isometric tension change of an intact fiber of frog atrium bathed in a hyper- or hypotonic solution.

A study of optimal reaction conditions for an assay of the human alternative complement pathway.

The optimal conditions for performance of a sensitive functional assay for the human alternative complement pathway were studied. The serum dilution causing 50% lysis of rabbit erythrocytes in magnesium EGTA buffer is designated the APH50 titer. Optimal reaction conditions for the assay were pH 7.2, incubation temperature 37 degrees C, incubation time 60 minutes, and magnesium concentration 0.002 M. Lowering the ionic strength of the buffer from 0.150 M to 0.0125 M increased APH50 titers nearly 2.5-fold, but decreased the reproducibility of titers. Significant fluid-phase conversion of C3 at 37 degrees C in low ionic strength buffer was demonstrated by crossed immunoelectrophoresis. Using the optimal reaction conditions, in normal ionic strength buffer the mean APH50 +/- SD for 45 normal adults was 25.3 +/- 5.7 U/mL. Heparin, an inhibitor of the alternative pathway, decreased APH50 by 50% at a concentration of 100 U heparin/mL serum, and totally abolished alternative pathway hemolytic activity at 1,000 U heparin/mL serum, while lowering CH50 titers to a much lesser degree. When increasing doses of zymosan were used for complement activation in vitro, the per cent APH50 depletion at low doses of zymosan was at least twice the per cent depletion of CH50 or antigenic P, B, and C3. A striking dichotomy between nearly complete APH50 depletion and normal or near normal CH50 and hemolytic C4 levels was documented for a human burn patient and for a baboon infused with a lethal dose of Escherichia coli lipopolysaccharide. Therefore, we documented a substantially greater sensitivity of APH50 than of conventional complement determinations for detecting complement consumption by alternative pathway activators.

Some functional and morphological characteristics of an acutely dispersed purified cell suspension of rat lactotrophs prepared with Percoll.

A cell suspension containing more than 90% lactotrophs can be prepared from enzymically dispersed adenohypophyses obtained from male rats pretreated with estradiol. The lactotrophs are separated from the mixed cell population by centrifugation on a discontinuous density gradient prepared from a commercial preparation of colloidal silica (Percoll, Pharmacia). The method allows isopycnic separation of these delicate cells under very mild conditions; normal ionic strength and normal pH were maintained throughout the gradient, centrifugal acceleration did not exceed 1600 X g, and all procedures were done at room temperature. Histological verification that at least 90% of the cells were lactotrophs was done using specific immunoperoxidase staining. The functional capability of the lactotrophs was established by measuring the dose--response to the dopamine agonist bromocriptine and to thyrotropin-releasing hormone (TRH). Bromocriptine decreased spontaneous release in a dose-related way over the concentration range of 10(-10) to 10(-8) M. TRH, which causes an in vivo release of prolactin (PRL) in estrogen-primed rats, produced a dose-related increase in the release of PRL over the concentration range of 3 X 10(-10) to 3 X 10(-8) M after the high spontaneous release had been previously reduced by bromocriptine (3 X 10(-8) M).

Evidence for cross-bridge attachment in relaxed muscle at low ionic strength.

In vitro at low ionic strength (mu = 0.02 M) and 5 degrees C, myosin subfragment-1 shows significant binding to regulated actin in the presence of ATP, independent of the concentration of free Ca2+. Under the same conditions, single skinned rabbit psoas muscle fibers develop force only in the presence of Ca2+ and are relaxed in its absence. However, the stiffness, measured with very rapid stretches (0.5% of muscle length in 0.1 ms), is high even when the fibers are relaxed. This "rapid stiffness" of the resting muscle is sensitive to ionic strength, becoming small at normal ionic strength (mu = 0.17 M). At low ionic strength, the rapid stiffness is approximately proportional to the overlap between the actin and myosin filaments. At zero overlap (sarcomere length = 3.8 microns), the stiffness is less than 20% of the value measured at full overlap. This remaining 20% is relatively insensitive to ionic strength, like the passive resting tension, and it may in fact be due to the structures responsible for the resting tension. Thus, both in vitro binding and the effect of overlap on rapid stiffness measurements in fibers suggest that cross-bridges are attached to actin in relaxed muscle at low ionic strength.

Mechanism of enhancement of blood group antibody reactions in low ionic strength.

When the ionic strength of blood samples is reduced, blood group antibodies bind to red blood cells that lack the corresponding antigen. Upon normalization of the ionic strength, the antibodies elute into the supernatant fluid. We compared, in semiquantitative terms, this nonspecific and reversible binding of various blood group antibodies. Studied with anti-CD antibodies showed that the nonspecific binding increased when the ion strength was progressively lowered. In 1/10 normal ionic strength, practically all of a high-titer anti-CD antibody bound to Rh(D)-negative RBC (phenotype ce). The majority of the other antibodies studied (anti-C, -c, -Fya, -Fyb, and -S) behaved similarly in 1/10 normal ionic strength, but the binding of some (anti-E, -s, and -Jka) was, at times, less complete. Most examples of anti-K antibodies, however, demonstrated very poor nonspecific binding to K-negative RBC in low ionic strength (LIS). The authors propose that the augmentation of specific blood group antibody reactions in LIS is the direct result of this nonspecific binding, and that the failure of these conditions to support the nonspecific binding of anti-K antibodies might explain the lack of augmentation (and, at times, even the impairment) of the reactions of anti-K with its antigen.

Reduced red cell filtrability due to red cell plasma protein interactions.

The adsorption of protein on red cell surface was studied by the measurement of protein in plasma and incubation of blood with 131I-albumin. The protein concentration of the moderately hypertonic plasma clearly decreased by the addition of red cells. The decay counts of 131I-albumin increased in the centrifuged red cell segment as the ionic strength in the plasma increased. For the interpretation of these results it was assumed that the human red cells suspended in plasma are covered with albumin under the normal ionic strength and adsorb more albumin molecules when the ionic strength in the plasma is elevated by the addition of NaCl. A multilayer adsorption of albumin is suggested at the ionic strength of 450 mOsmol. The excess adsorption of albumin on red cells reduces the filtrability of red cells through the nuclepore filter.

Complement and Ig uptake by erythrocytes in low ionic strength solutions.

We studied the uptake of immunoglobulin (Ig) and complement from plasma by red blood cells (RBC) under low ionic strength (LIS) conditions using Rh-negative RBC and anti-Rh antibodies. The ionic strength of plasma/RBC mixtures was lowered by dialysis against 5% mannitol, pH 6.0. The studies revealed that bromelin-modified RBC failed to bind either Ig or complement in low ionic strength conditions, but that unmodified RBC bound the majority of anti-D. The bound anti-D (consisting of IgG1, IgG3, IgA, and IgM molecules) could be eluted into 0.9% NaCl yielding a higher recovery of antibody than that obtained by specific absorption-elution techniques. Although uptake of Ig by RBC was independent of complement activation, the subsequent elution of bound Ig into normal ionic strength solutions was not. Small amounts of Ig remained on EC43 and EC4, but not on RBC that did not become complement sensitized during low ionic strength exposure. These findings suggest that Ig remained attached to RBC via a complement-RBC bond.

Chloride transport by self-exchange and by KCl salt diffusion in gramicidin-treated red blood cells.

The permeability of gramicidin-treated human red blood cell membranes to K+ and Cl- has been measured at normal ionic strength (1) by tracer exchange at steady-state distribution of salt, and (2) by net transport of salt in the presence of a salt concentration gradient. Under both conditions KCl was the only inorganic salt in cells and medium. In the studies of self-exchanges the electrical driving force on the ions was zero. Calculaton of permeability coefficients from net salt transport was simplified because the experiment was designed as a special case of the Nerst-Planck diffusion regime, i.e. the single salt case. Gramicidin altered the cell membranes from being anion to become cation selective. Gramicidin increased the potassium exchange without affecting the chloride exchange measurably. The chloride exchange showed saturation kinetics as does chloride exchange in normal cells. The net transport of KCl in the presence of a constant concentration gradient increased to a constant value with increasing gramicidin concentration. At high gramicidin concentrations (0 degree C, pH 7.2) the "chloride permeability coefficient" calculated from tracer exchange (1.9 x 10(-6) cm/s) was 290 times the chloride permeability coefficient calculated from net salt transport (0.65 x 10(-8) cm/s). The latter value corresponds to a chloride conductance of 4.2 x 10(-6) ohm-1 cm-2. The chloride permeability coefficient was 2.1 x 10(-6) cm/s at 25 degrees C (pH 6.8) indicating a value of 3 for the Q25. It appears that normal red cells are anion selective in the sense that anion permeability exceeds cation permeability with a factor of more than a hundred between 0 degrees C and body temperature. The anion exchange, i.e. the Hamburger shift, is a tightly coupled transport process which is several orders of magnitude faster than anion transport by salt diffusion.

The effect of cryogenic storage on human erythrocyte membrane proteins as determined by polyacrylamide-gel electrophoresis

A study was conducted to determine the effects of freezing on the major membrane proteins of isolated human erythrocyte membranes. Membranes in low or normal ionic strength medium were frozen at slow or fast freezing rates. The membrane protein composition and elution of proteins from the membranes were studied utilizing polyacrylamide-gel electrophoresis in a sodium dodecyl sulfate or an acetic acid-urea-phenol solvent system. Neither a change in the composition of the membrane proteins nor any elution of membrane protein during freezing and thawing was observed. The data indicate that any human erythrocyte membrane damage during freezing and thawing was not related to a change in major membrane protein composition. Human red cell membranes were stable at −80 or −196 °C in the absence of a cryoprotective agent.