Temperature transitions of spectrin in solution and in erythrocyte membranes

Temperature transitions of spectrin in solution and in human erythrocyte membranes are recorded in the region t greater than 40 degrees C by irreversible changes in protein fluorescence spectra. Structural changes are completed 20 min after the sample incubation at an increased temperature. Both for isolated spectrin and for erythrocyte ghosts the temperature of half-transition is 46 +/- 1 degree C. There is no transition in the membranes after the removal of spectrin. Transitions in erythrocyte ghosts and in spectrin solution disappear at pH 5 when spectrin is in an aggregated state. Spectrin is suggested to be responsible for the transitions at 50 degrees C; its state in the cells areas more thermostable than in isolated membranes.     

Cross-linkings between spectrin and band 3 in human erythrocyte membranes

A specific structural association between spectrin component 1 and band 3 in human erythrocyte membrane has been demonstrated by covalent cross-linkings, specific labeling, and the technique of two-dimensional gel electrophoresis. A complex of 330,000 daltons, representing 1 + 3, was produced in mildly oxidized membranes at physiologic pH and isotonic conditions but not at hypotonic conditions (< 10 mM KCl or NaCl). The yield of this complex decreased dramatically as the monovalent cation concentration decreased from 90 mM to 30 mM. The presence of Mg⁺⁺ or Ca⁺⁺ (2 mM) at low ionic strength promoted 1 + 3 cross-linking in an amount similar to that produced at isotonic conditions. The specific segment of band 3 involved in the cross-linking was also investigated by means of chymotrypsin digestion of band 3 in the intact red cells. The results showed the cross-links between spectrin component 1 and the 55,000-dalton fragment of band 3 at physiologic pH and isotonic conditions. This is consistent with the idea that band 3 is anchored on or contacted with the submembrane meshwork at the cytoplasmic membrane surface.     

The role of spectrin in erythrocyte ghost endocytosis.

Endocytosis in white ghosts prepared from human erythrocytes was induced by three methods: incubation with Mg-ATP, incubation with 0.1 mM EDTA, and digestion with 20 nanograms (ng.) per ml. of trypsin. In each case the endocytic vacuoles that were produced when separated and analyzed on SDS-polyacrylamide gel electrophoresis were found to be depleted of spectrin. This observation suggested that a requirement for endocytosis is the establishment of spectrin-free domains in the membrane. This hypothesis was tested by pre-incubating ghosts with anti-spectrin antibodies. Pre-incubation with anti-spectrin antibody blocked white ghost endocytosis produced either by Mg-ATP, EDTA, or trypsin. Therefore, it is proposed that spectrin has a key role in the endocytosis process.     

Calcium-induced proteolysis of spectrin and band 3 protein in rat erythrocyte membranes

Calcium-dependent protease activity capable of degrading a number of endogenous proteins was found in rat red blood cell membranes. This protease activity, like that found in human red blood cells, was activated by low concentrations of calcium, but in the rat red blood cells, unlike the human red blood cells, calcium-activated protease activity was membrane-bound. A number of endogenous membrane-bound proteins were degraded after the addition of calcium to the membranes. These included spectrin bands 1 and 2 as well as bands 3, 2.1, and 2.2. No calcium-induced aggregation (transglutaminase activity) was noted in the rat red blood cell membranes.     

Salt and temperature-dependent conformation changes in spectrin from human erythrocyte membranes.

ORD and CD measurements of spectrin, in both the dimer and tetramer association state, indicate a high proportion of alpha-helix in this protein. At temperatures below 27 degrees C and in 0.1 M NaCl, the tetramer has an apparent helix content of 73% and the dimer, 68%. The conformation of both states is dependent on salt concentration and temperature. Low ionic strength solutions of spectrin display lowered sedimentation coefficients and a decreased apparent helix content, indicating perhaps a slight refolding and expansion of the molecule. In addition, spectrin in low ionic strength solutions undergoes a broad temperature-dependent transition spread from 20 to 50 degrees C, while in the presence of salt the transition is sharp and centered on 49 degrees C. The temperature-dependent changes in low ionic strength solutions appear to parallel the dissociation of tetramer to dimer.     

On the mechanism of ATP-induced shape changes in human erythrocyte membranes. I. The role of the spectrin complex

Human erythrocyte ghosts have been shown, by scanning electron microscopy, to undergo ATP-dependent shape changes. Under appropriate conditions the ghosts prepared from normal disk-shaped intact cells adopt a highly crenated shape, which in the presence of Mg-ATP at 37 degrees C is slowly converted to the disk shape and eventually to the cup shape. These changes are not observed with other nucleotides or with 5'-adenylyl imidodiphosphate. Anti-spectrin antibodies, incorporated along with the Mg-ATP into the ghosts in amounts less than equivalent to the spectrin, markedly accelerate the shape changes observed with the Mg-ATP alone. The Fab fragments of these antibodies, however, have no effect. The conclusion is that the structural effect produced by the ATP is promoted by the cross-linking of spectrin by its antibodies, and may therefore itself be some kind of polymerization or network formation involving the spectrin complex on the cytoplasmic face of the membrane. The factors that contribute to the shape of the ghost and of the intact erythrocyte are discussed in the light of these findings.     

The isolation of aggregates of spectrin from bovine erythrocyte membranes.

Aggregated states of spectrin from bovine erythrocyte membranes can be detected in sedimentation velocity experiments. These aggregates have been isolated by means of gel filtration on columns of 4% agarose. They appear to be stable over a wide range of pH and ionic strength, although they are dissociated by sodium dodecyl sulphate. Sedimentation equilibrium measurements yielded values of 960 000 and 480 000 for the molecular weights of the major aggregates, corresponding to a tetramer and dimer, respectively. The presence of different aggregated states in spectrin preparations may explain the wide variation in the reported physical properties of spectrin.     

Isolation of human erythrocyte membranes in glucose solution

A method is described for the preparation or removal of erythrocyte membranes from hemolysates by a glucose solution. The procedure is simple and rapid, requiring centrifugation at 8000g for 2 min. The preparation has microscopic shape and two-dimensional peptide patterns similar to those of the membrane isolated by conventional procedures (10,000g for 20 min). The present procedure is suitable for dealing with a bulky preparation or for removal of erythrocyte membranes from large volumes of hemolysates to purify enzymes and proteins of soluble or membrane fractions.     

Oligomeric states of spectrin in normal erythrocyte membranes: Biochemical and electron microscopic studies

We estimated the relative amounts of oligomeric species of spectrin in 0°C red-cell-membrane extracts, including those released from spectrin-actinpolypeptide 4.1 complexes after mild urea treatment. Spectrin dimers, tetramers, and medium-size oligomers were the prominent species, accounting for 5%–10%, 45%–55%, and 25%–35% of spectrin, respectively. When examined by low-angle rotaryshadowing electron microscopy, these medium-size spectrin oligomers (e.g., hexamers, octamers, decamers, dodecamers, and quadecamers) appeared as polyskelions formed by head-to-head association of three to seven dimers. They were stable species capable of binding to, and subsequent release from, inside-out vesicles without degradation to tetramers or dimers. The data suggest that spectrin tetramers and medium-size oligomers coexist in the normal erythrocyte membrane as the primary native spectrin species.     

Loss of resealing ability in erythrocyte membranes. Effect of divalent cations and spectrin release

Washed human erythrocyte membranes can recover impermeability to macromolecules upon warming in solutions of sufficient ionic strength. This ability is rapidly lost from most ghost preparations in dilute salt solution at temperatures of 15°C or higher. Divalent cations both reseal ghosts in the absence of high ionic strength and prevent loss of resealing ability. The effective concentrations are 40 μM for Ca²⁺ and 200 μM for Mg²⁺. The loss of resealing ability is associated with the release of spectrin polypeptides from the inner surface of the membrane. In ghost preparations that have not become irreversibly leaky, or in the presence of Ca²⁺, loss of spectrin does not occur. These results suggest that an intact spectrin network is required for resealing to macromolecules, and divalent cations stabilize this network. In light of this information, the effect of temperature on resealing kinetics is described.     

Transient dichroism studies of spectrin rotational diffusion in solution and bound to erythrocyte membranes.

Spectrin was purified from human erythrocytes and labeled with the triplet probe eosin-5-maleimide. Rotational diffusion of spectrin was investigated by observing transient dichroism following flash excitation of the probe. Measurements were performed at 4 degrees C in solutions of varying viscosity and with spectrin rebound to spectrin/actin-depleted erythrocyte membranes. In solution, complex anisotropy decays were observed which could not be satisfactorily fitted by the equations for a rod-shaped molecule of appropriate dimensions. When spectrin was rebound to the erythrocyte membrane, a decay in the anisotropy was still present but was markedly less sensitive to solution viscosity and flatter at longer times. In order to overcome the objection that the cytoskeleton is only partially reconstituted when spectrin is rebound, a method was developed for labeling spectrin with eosin-5-maleimide in situ. Anisotropy decays for these labeled membranes exhibited features similar to those obtained for spectrin labeled in solution and subsequently rebound. Taken together, the results provide good evidence for segmental motion of spectrin when incorporated into the erythrocyte cytoskeleton. Upon increasing the temperature, the initial anisotropy ro for both rebound and in situ labeled spectrin decreases, and above 30 degrees C the measured anisotropies are small. Thus, at physiological temperature the probe is almost completely randomized by motions with correlation times less than 10 microseconds.     

Hydrophobic photolabeling in membranes: the human erythrocyte glucose transporter

Human erythrocyte membranes were labeled with a hydrophobic photoactivable reagent, 2-[3H]Diazofluorene. Electrophoretic analysis of the protein fraction showed that several membrane spanning proteins like Band 3 (the anion transporter), Band 4.5 (the glucose transporter), and the sialoglycoproteins PAS 1, 2, and 3 have been labeled. To isolate the diazofluorene-labeled glucose transporter, the membrane preparation was solubilized with Triton X-100 and passed through a DEAE-cellulose column. The flow-through fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioactive analysis of the gel indicated that besides the Band 4.5, two more proteins corresponding to the Band 3 and Band 6 regions also coelute with the glucose transporter in the flow-through fraction. On the other hand, use of n-octyl glucoside gave a relatively better preparation. The 2-[3H]DAF-labeled glucose transporter isolated by the latter method on tryptic digestion indicated that the Mr 18,000 fragment corresponding to the C-terminal transmembrane fragment is labeled.