Resolution of erythrocyte membrane proteins by two-dimensional electrophoresis.

A two-dimensional electrophoresis method has been developed which solubilizes erythrocyte membrane proteins, and which resolves the components of the band that migrates in detergent gels as if its molecular mass were 95,000 daltons. This method uses gel electrophoresis with sodium dodecyl sulfate in the first dimension and phenol, aqueous urea, and acetic acid in the second dimension. The 95,000 dalton band is known to contain several different membrane proteins, including those associated with anion transport, glucose transport, and (Na+,K+) transport. Two-dimensional electrophoresis resolved this band into one major spot and several minor ones. Pronase digestion of whole erythrocytes, followed by preparation of ghosts and two-dimensional electrophoresis, showed that only the major component of this band was digested by pronase.     

High resolution two-dimensional gel electrophoresis of human erythrocyte membrane proteins.

Three different two-dimensional (2-D) gel electrophoretic techniques have been modified to provide high resolution of human erythrocyte membrane proteins. The resulting gels were referenced to the established one-dimensional (1-D) sodium dodecylsulfate (SDS) gel electrophoretic profile, and the effects of endogenous proteolysis and cytosolic contamination were studied. It is concluded that in vitro proteolysis and cytosolic contamination do not contribute significantly to the patterns observed on the 2-D gels, under the conditions used for erythrocyte ghost preparation. The procedures require only small quantities of blood; as many as twenty 2-D gel profiles can be obtained from 5 ml of blood. The combination of nonequilibrium isoelectric focusing (IEF) in the first dimension, SDS electrophoresis in the second dimension, and very sensitive silver staining techniques resolves more than 250 individual protein spots. This appears to be the most useful single procedure for the analysis of red cell membrane proteins. Membrane protein profiles from patients with Duchenne muscular dystrophy, Wernicke-Korsakoff syndrome, and acanthocytosis with degeneration of the basal ganglia were compared with normal controls. The patterns for Duchenne muscular dystrophy and Wernicke-Korsakoff syndrome were not different from normal patterns. The pattern for the patient with acanthocytosis and degeneration of the basal ganglia consistently showed a high level for one protein in the 100,000 mol. wt. range.     

Comparison of human and pigeon erythrocyte membrane proteins by one- and two-dimensional gel electrophoresis

Pigeon and human bands 1 and 2 (spectrin) and 5 (actin) are conserved. Band 3 anion porters have similar SDS positions, but the pigeon porter has a higher isoelectric point. Both anion porters are inhibited by similar doses of pyridoxal phosphate. Many differences are apparent in minor bands.     

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.     

Study of the aggregation of membrane proteins of erythrocyte ghosts using SDS-PAAG electrophoresis

Using the method of electrophoresis in SDS-PAAG the authors showed a diminution of proteins of bands I + II (spectrins) and III (major integral protein) after irradiation of erythrocyte ghosts with doses of 50 to 1000 Gy. We failed to ascertain that radiation-induced lipid peroxidation is involved into membrane protein aggregation. Among the radiolysis products, OH-radicals were shown to contribute markedly to the radiation effect observed.     

Two-dimensional polyacrylamide-gel electrophoresis of the proteins and glycoproteins of the human erythrocyte membrane.

A two-dimensional polyacrylamide gel electrophoresis technique has been developed, improving the analytical separation of some proteins and glycoproteins of the human erythrocyte membrane. Freshly prepared membranes are totally solubilized, subjected to dodecylsulfate--polyacrylamide gel electrophoresis in the first dimension, followed by electrophoresis in the second dimension, using a detergent-free polyacrylamide gradient gel. By this method the proteins of the human erythrocyte membrane could be resolved into a two-dimensional pattern, which has been shown to be highly reproducible with respect to various blood-groups and within one blood-group from specimen to specimen. The method enables especially the investigation of the hydrophobic and very likely integrated membrane proteins and glycoproteins. Thus, band III[Fairbanks, G., Steck, Th. & Wallach, D. F. H., Biochemistry, 10, 2606--2617 (1971)] could be shown to consist of five proteins, one of them being the major glycoprotein of the human erythrocyte membrand. The two spectrin bands differed considerably in their two-dimensional patterns. The value of the given method for the investigation of membrane defects, which may be linked with various diseases of human erythrocytes, could be demonstrated in the case of two patients suffering from congenital dyserythropoetic anaemia.     

Capillary gel electrophoresis: separation of major erythrocyte membrane proteins

A new separation method of human erythrocyte membrane proteins by sodium dodecyl sulfate capillary gel electrophoresis (SDS–CGE) is described. In this method, a replaceable gel matrix was used. Seven major erythrocyte membrane proteins, α-and β-spectrin, ankyrin 2.1, band 3 (anion-exchanger), 4.1a and b, and 4.2 (pallidin), were separated and identified by SDS–CGE method. High reproducible migration times of these proteins (inter-assay coefficients of variation less than 2%), as well as quantification (inter-assay coefficients of variation less than 11%) were obtained. This new SDS–CGE method may provide important diagnostic evidence for hereditary spherocytosis. It can be a powerful diagnostic tool in place of SDS polyacrylamide gel electrophoresis for erythrocyte membrane protein analysis.     

Calcium effects on human erythrocyte membrane proteins

The effects of Ca²⁺ on human erythrocyte membrane proteins were examined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Ca²⁺ had several effects on normal human erythrocyte membrane proteins. It affected the binding of cytoplasmic proteins to the membrane, produced a non-reversible aggregation of several membrane proteins and activated apparent proteolysis of membrane proteins. The Ca²⁺ effect could be obtained with isolated, washed membranes when the erythrocyte cytoplasm was added. These studies indicate that the Ca²⁺-induced membrane proteolysis and aggregation effects are not due simply to its presence at the time of hemolysis as previously suggested (Carraway, K.L., Triplett, R.B. and Anderson, D.R. (1975) Biochim. Biophys. Acta 379, 571–581), but are the result of more complex interactions between the erythrocyte membrane and cytoplasmic factors.     

Interaction of molybdenum with human erythrocyte membrane proteins

This study describes the interaction of molybdenum with blood components. Molybdenum-99 was added to blood, and after four washings, 3% of the total radioactivity was found in red cells. More specifically, the radioactivity was determined to be associated with the cell membrane. Molybdenum-99 in the +VI form did not interact with the human erythrocyte membrane; however, Mo(V) forms did interact. Of five different compounds, the highes uptake was observed with a brown Mo(V)-ascorbate complex generated from Mo(VI) and ascorbic acid in the molar ratio 1∶20. A membrane suspension of Mo-ascorbate-treated human erythrocytes was prepared and the solubilized proteins were separated on a polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS). Molybdenum-99 binding to spectrin was demonstrated, as well as some minor interactions with membrane hemoglobin and bands 6 and 8.     

Differential solubilization of human erythrocyte cell membrane proteins by maleic anhydride.

A method is described to differentially solubilize the proteins of erythrocyte membranes using maleic anhydride. Based on this solubilization, the two groups of proteins appear to be distinct in their molecular organization within the membrane structure. From the operational criteria of peripheral and integral proteins of biological membranes, it is suggested that maleic anhydride by itself solubilizes mostly the peripheral proteins, while, in the presence of urea, it solubilizes almost all the proteins. It is suggested that maleic anhydride could be used to solubilize, fractionate and characterize the membrane proteins. Since maleyl groups can be removed from proteins, reconstitution of membrane structure from solubilized components could be studied.     

Massspectrometric analyses of transmembrane proteins in human erythrocyte membrane

It is difficult to understand the functional mechanisms of integral membrane proteins without having protein chemical information on these proteins. Although there have been many attempts to identify functionally important amino acids in membrane proteins, chemically and enzymatically cleaved peptides of integral membrane proteins have been difficult to handle because of their hydrophobic properties. In the present study, we have applied an analytical method to transmembrane proteins combining amino acid sequencing, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, and liquid chromatography with electrospray ionization (LC/ESI) mass spectrometry. We could analyze most (97%) of the tryptic fragments of the transmembrane domains of band 3 as well as other minor membrane proteins. The peptide mapping of the transmembrane domain of band 3 was completed and the peptide mapping information allowed us to identify the fragments containing lysine residues susceptible to 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and to 2,4-dinitrofluorobenzene (DNFB). This method should be applicable to membrane proteins not only in erythrocyte membranes but also in other membranes.     

Thermotropic properties of human erythrocyte membrane proteins as affected by hydroxychloroaromatic compounds

The thermal stability of the anion transport protein (band 3) and other proteins of the human erythrocyte membrane, as influenced by hydroxychloroaromatic (HO-Cl2-Ar) compounds, was studied by differential scanning calorimetry. Various hydroxychlorodiphenyl ethers (HO-Clx-DPEs) and hexachlorophene, but not pentachlorophenol, caused a marked decrease in the thermal stability of band 3. Most of the other calorimetric transitions of the erythrocyte membrane were only slightly affected. The activity of HO-Clx-DPEs toward lowering the transition temperature of band 3 generally increased with the degree of chlorination, and was somewhat dependent on the position of hydroxyl substitution. At higher concentrations of HO-Clx-DPEs, there was a decrease in the enthalpy change and a broadening of the endothermic transition of band 3. The order of effectiveness of these compounds, as determined from band 3 denaturation temperatures, was similar to the order of potency previously observed for hemolysis of human erythrocytes.