红细胞膜带4.2蛋白的结构与功能

带4.2蛋白是一种重要的红细胞膜蛋白,与红细胞的形态、可变形性及携氧功能有至关重要的联系。它通过与带3蛋白(阴离子通道蛋白)、锚蛋白结合,稳定的连接在细胞膜的内表面,连接着膜骨架网架结构与细胞膜,是膜骨架与脂质双分子层连接的重要纽带。带4.2蛋白的缺失会引起球形或椭圆形红细胞增多症及不同程度的溶血性贫血,严重的情况需要摘除脾脏来进行治疗。近年来研究认为,带4.2蛋白在维持细胞膜骨架的完整性和稳定性方面扮演了重要角色。现对带4.2蛋白结构及功能的研究状况进行综述。     

Aberrant protein pattern in red cell membranes of a patient with mild hemolytic anemia

A 17-year-old patient with mild hemolytic anemia known since early childhood displayed an aberrant protein pattern of his red cell membranes when analyzed in SDS-polyacrylamide gel electrophoresis (SDS-PAGE). A double protein band in the low molecular weight region (molecular weight of about 33,000) was distinctly reduced or missing in the membranes that were investigated concomitantly with those of controls on two occasions at an interval of 6 months'. The phosphorylation pattern of membrane phosphoproteins, on the other hand, did not seem to differ from that of controls. It is suggested that a causal relationship existed between the observed membrane abnormality and the mild hemolytic anemia.     

Assessment of the red cell proteome of young patients with unexplained hemolytic anemia by two-dimensional differential in-gel electrophoresis (DIGE)

Erythrocyte cytosolic protein expression profiles of children with unexplained hemolytic anemia were compared with profiles of close relatives and controls by two-dimensional differential in-gel electrophoresis (2D-DIGE). The severity of anemia in the patients varied from compensated (i.e., no medical intervention required) to chronic transfusion dependence. Common characteristics of all patients included chronic elevation of reticulocyte count and a negative workup for anemia focusing on hemoglobinopathies, morphologic abnormalities that would suggest a membrane defect, immune-mediated red cell destruction, and evaluation of the most common red cell enzyme defects, glucose-6-phosphate dehydrogenase and pyruvate kinase deficiency. Based upon this initial workup and presentation during infancy or early childhood, four patients classified as hereditary nonspherocytic hemolytic anemia (HNSHA) of unknown etiology were selected for proteomic analysis. DIGE analysis of red cell cytosolic proteins clearly discriminated each anemic patient from both familial and unrelated controls, revealing both patient-specific and shared patterns of differential protein expression. Changes in expression pattern shared among the four patients were identified in several protein classes including chaperons, cytoskeletal and proteasome proteins. Elevated expression in patient samples of some proteins correlated with high reticulocyte count, likely identifying a subset of proteins that are normally lost during erythroid maturation, including proteins involved in mitochondrial metabolism and protein synthesis. Proteins identified with patient-specific decreased expression included components of the glutathione synthetic pathway, antioxidant pathways, and proteins involved in signal transduction and nucleotide metabolism. Among the more than 200 proteins identified in this study are 21 proteins not previously described as part of the erythrocyte proteome. These results demonstrate the feasibility of applying a global proteomic approach to aid characterization of red cells from patients with hereditary anemia of unknown cause, including the identification of differentially expressed proteins as potential candidates with a role in disease pathogenesis.     

Hemolysis of human erythrocytes induced by tamoxifen is related to disruption of membrane structure

Tamoxifen (TAM), the antiestrogenic drug most widely prescribed in the chemotherapy of breast cancer, induces changes in normal discoid shape of erythrocytes and hemolytic anemia. This work evaluates the effects of TAM on isolated human erythrocytes, attempting to identify the underlying mechanisms on TAM-induced hemolytic anemia and the involvement of biomembranes in its cytostatic action mechanisms. TAM induces hemolysis of erythrocytes as a function of concentration. The extension of hemolysis is variable with erythrocyte samples, but 12.5 microM TAM induces total hemolysis of all tested suspensions. Despite inducing extensive erythrocyte lysis, TAM does not shift the osmotic fragility curves of erythrocytes. The hemolytic effect of TAM is prevented by low concentrations of alpha-tocopherol (alpha-T) and alpha-tocopherol acetate (alpha-TAc) (inactivated functional hydroxyl) indicating that TAM-induced hemolysis is not related to oxidative membrane damage. This was further evidenced by absence of oxygen consumption and hemoglobin oxidation both determined in parallel with TAM-induced hemolysis. Furthermore, it was observed that TAM inhibits the peroxidation of human erythrocytes induced by AAPH, thus ruling out TAM-induced cell oxidative stress. Hemolysis caused by TAM was not preceded by the leakage of K(+) from the cells, also excluding a colloid-osmotic type mechanism of hemolysis, according to the effects on osmotic fragility curves. However, TAM induces release of peripheral proteins of membrane-cytoskeleton and cytosol proteins essentially bound to band 3. Either alpha-T or alpha-TAc increases membrane packing and prevents TAM partition into model membranes. These effects suggest that the protection from hemolysis by tocopherols is related to a decreased TAM incorporation in condensed membranes and the structural damage of the erythrocyte membrane is consequently avoided. Therefore, TAM-induced hemolysis results from a structural perturbation of red cell membrane, leading to changes in the framework of the erythrocyte membrane and its cytoskeleton caused by its high partition in the membrane. These defects explain the abnormal erythrocyte shape and decreased mechanical stability promoted by TAM, resulting in hemolytic anemia. Additionally, since membrane leakage is a final stage of cytotoxicity, the disruption of the structural characteristics of biomembranes by TAM may contribute to the multiple mechanisms of its anticancer action.     

Phosphorylation of a 25 kDa protein is induced by thermostable direct hemolysin of Vibrio parahaemolyticus

Thermostable direct hemolysin (TDH) is a possible virulence factor produced by Vibrio parahaemolyticus. Although TDH has a variety of biological activities, including hemolytic activity, the biochemical mechanism of action remains uncertain. Here we analysed biochemical events, especially phosphorylation, caused by TDH in erythrocytes, and found that TDH caused significant phosphorylations of proteins on erythrocyte membrane. Phosphorylation of proteins was studied using γ-³²P ATP and SDS-PAGE. A number of protein kinase inhibitors were tested, to determine which types of kinases were involved in the phosphorylation events. TDH induced the phosphorylation of two proteins on membranes of human erythrocyte that are sensitive to TDH. The estimated molecular weight of these proteins was 25 and 22.5 kDa. Interestingly, the 22.5 kDa, but not the 25 kDa protein, was phosphorylated on the membrane of TDH-insensitive (resistant) horse erythrocytes. Moreover, a mutant TDH (R7), which retained binding ability but lost hemolytic activity, also phosphorylated only the 22.5 kDa protein on human erythrocyte membranes. Among the protein kinase inhibitors used the protein kinase C inhibitors, (staurosporine and calphostin C) showed marked inhibition of phosphorylation of 25 kDa protein. In addition to phosphorylation, these protein kinase C inhibitors suppresssed hemolysis by TDH. These results indicate that the phosphorylation of the 25 kDa protein seems to be essential for the hemolysis by TDH after it binds to erythrocyte membranes.     

Proteomic identification of erythrocyte membrane protein deficiency in hereditary spherocytosis

Hereditary spherocytosis (HS) is the most common congenital hemolytic anemia in Caucasians, with an estimated prevalence ranging from 1:2000 to 1:5000. The molecular defect in one of the erythrocytes (RBC) membrane proteins underlying HS like; spectrin-α, spectrin-β, ankyrin, band 3 and protein 4.2 that lead to membrane destabilization and vesiculation, may change the RBCs into denser and more rigid cells (spherocytes), which are removed by the spleen, leading to the development of hemolytic anemia. It is classified as mild, moderate and severe, according to the degree of the hemolytic anemia and the associated symptoms. Two-dimensional gel electrophoresis (2-DE) is potentially valuable method for studying heritable disorders as HS that involve membrane proteins. This separation technique of proteins based upon two biophysically unrelated parameters; molecular weight and charge, is a good option in clinical proteomics in terms of ability to separate complex mixtures, display post-translational modifications and changes after phosphorylation. In this study, we have used contemporary methods with some modifications for the solubilisation, separation and identification of erythrocyte membrane proteins in normal and in HS RBCs. Spectrin alpha and beta chain, ankyrin and band 3 proteins expression differences were found with PDQuest software 8.0.1. and peptide mass fingerprinting (PMF) analysis performed for identification of proteins in this study.     

Contact-dependent hemolytic activity distinct from deforming activity of Bartonella bacilliformis

Although Bartonella bacilliformis causes a severe anemia in humans, this study presents the first report of hemolytic activity by B. bacilliformis. The activity was not apparent in culture supernatants but was reliably detected when B. bacilliformis cells were centrifuged onto erythrocytes prior to incubation. Abrogation of hemolytic activity by proteinase K treatment suggested the hemolysin was a Bartonella protein. Even though hemolysis required relatively long incubation times, de novo protein synthesis was not required to produce the protein. A preparation containing factors released by B. bacilliformis, including deformin, a B. bacilliformis protein able to induce pits and invaginations in erythrocyte membranes, had some ability to lyse erythrocytes. However, pre-deformed erythrocytes did not lyse faster or to a greater extent than control erythrocytes after the addition of B. bacilliformis cells. Inhibition of deformation caused by B. bacilliformis cells with the erythrocyte ATPase inhibitor, vanadate, did not affect hemolytic activity. This study suggests hemolytic activity and deforming activity are attributable to different B. bacilliformis proteins.     

Role ofN-Myristylation in Targeting of Band 4.2 (Pallidin) in Nonerythroid Cells

Band 4.2 (pallidin) is a major erythrocyte membrane protein which has been detected in a number of nonerythroid cell types. Increasing evidence suggests that band 4.2 is involved in maintaining membrane stability in the erythrocyte. For example, band 4.2 binds to the integral membrane protein band 3 and to cytoskeletal proteins in the erythrocyte membrane, and band 4.2 deficiency results in varying degrees of hemolytic anemia. We have previously shown that human erythrocyte band 4.2 is myristylated at its penultimate glycine. Here we report that when expressed in both Sf9 and COS cells, myristylated forms of band 4.2 are detected at different intracellular locations than nonmyristylated forms. We also show that the unspliced form of human erythrocyte band 4.2 (a minor form in reticulocytes which contains an additional 30 amino acids after the first three N-terminal amino acids compared to the major erythroid form) is myristylated only at a barely detectable level, while mouse erythrocyte band 4.2 (homologous to the major erythroid form of human band 4.2) is myristylated at a level comparable to that of human band 4.2. These results suggest that myristylation plays a key role in the targeting of band 4.2 to specific intracellular locations and is likely to have a role in the function of this protein.     

Isolation, partial chemical and biological characterization of thymone B

A new approach to the study of the molecular arrangements of proteins in membranes is described. Irradiation with visible light of native erythrocytes or washed erythrocyte membranes suspended in buffers containing a) riboflavin, fluorescein or fluorescein coupled to dextran and b) ³H-labelled tryptophan resulted in incorporation of radioactivity into the membrane proteins. Polyacrylamide gel electrophoresis of solubilized membranes followed by radioactivity measurements of the separated membrane proteins revealed that in native erythrocytes the protein components known to be located at the exterior cell surface, Band 3 and the major sialoglycoproteins became specifically labelled, whereas in washed lysed cells all of the major membrane proteins were labelled.     

Expression Levels of the ABCG2 Multidrug Transporter in Human Erythrocytes Correspond to Pharmacologically Relevant Genetic Variations

We have developed a rapid, simple and reliable, antibody-based flow cytometry assay for the quantitative determination of membrane proteins in human erythrocytes. Our method reveals significant differences between the expression levels of the wild-type ABCG2 protein and the heterozygous Q141K polymorphic variant. Moreover, we find that nonsense mutations on one allele result in a 50% reduction in the erythrocyte expression of this protein. Since ABCG2 polymorphisms are known to modify essential pharmacokinetic parameters, uric acid metabolism and cancer drug resistance, a direct determination of the erythrocyte membrane ABCG2 protein expression may provide valuable information for assessing these conditions or for devising drug treatments. Our findings suggest that erythrocyte membrane protein levels may reflect genotype-dependent tissue expression patterns. Extension of this methodology to other disease-related or pharmacologically important membrane proteins may yield new protein biomarkers for personalized diagnostics.     

Regulation of erythrocyte ghost membrane mechanical stability by chlorpromazine

Chlorpromazine (CPZ), a widely used tranquilizer, is known to induce stomatocytic shape changes in human erythrocytes. However, the effect of CPZ on membrane mechanical properties of erythrocyte membranes has not been documented. In the present study we show that CPZ induces a dose-dependent increase in mechanical stability of erythrocyte ghost membrane. Furthermore, we document that spectrin specifically binds to CPZ intercalated into inside-out vesicles depleted of all peripheral proteins. These findings imply that CPZ-induced mechanical stabilization of the erythrocyte ghost membranes may be mediated by direct binding of spectrin to the bilayer. Membrane active drugs that partition into lipid bilayer can thus induce cytoskeletal protein interactions with the membrane and modulate membrane material properties.     

Combined deletion of mouse dematin-headpiece and beta-adducin exerts a novel effect on the spectrin-actin junctions leading to erythrocyte fragility and hemolytic anemia

Dematin and adducin are actin-binding proteins of the erythrocyte "junctional complex." Individually, they exert modest effects on erythrocyte shape and membrane stability, and their homologues are expressed widely in non-erythroid cells. Here we report generation and characterization of double knock-out mice lacking beta-adducin and the headpiece domain of dematin. The combined mutations result in altered erythrocyte morphology, increased membrane instability, and severe hemolysis. Peripheral blood analysis shows evidence of severe hemolytic anemia with reduced number of erythrocytes/hematocrit/hemoglobin and an approximately 12-fold increase in the number of circulating reticulocytes. The presence of a variety of misshapen and fragmented erythrocytes correlates with increased osmotic fragility and reduced in vivo life span. Despite the apparently normal protein composition of the mutant erythrocyte membrane, the retention of the spectrin-actin complex in the membrane under low ionic strength conditions is significantly reduced by the double mutation. Atomic force microscopy reveals an increase in grain size and a decrease in filament number of the mutant membrane cytoskeleton, although the volume parameter is similar to wild type erythrocytes. Aggregated, disassembled, and irregular features are visualized in the mutant membrane, consistent with the presence of large protein aggregates. Importantly, purified dematin binds to the stripped inside-out vesicles in a saturable manner, and dematin-membrane binding is abolished upon pretreatment of membrane vesicles with trypsin. Together, these results reveal an essential role of dematin and adducin in the maintenance of erythrocyte shape and membrane stability, and they suggest that the dematin-membrane interaction could link the junctional complex to the plasma membrane in erythroid cells.     

Calreticulin, a component of the endoplasmic reticulum and of cytotoxic lymphocyte granules, regulates perforin-mediated lysis in the hemolytic model system.

Cytotoxic lymphocytes kill virally infected cells with specialized cytotoxic granules containing perforin, a protein that forms toxic pores in the target cell membrane. These specialized cytotoxic granules also contain calreticulin, an endoplasmic reticulum chaperone protein. The calcium-independent association of perforin and calreticulin prompted our evaluation of calreticulin's potential to function as a regulatory molecule that protects cytotoxic lymphocytes from their own perforin. We report here that 10(-7) M calreticulin blocked perforin-mediated lysis in the hemolytic model system using erythrocytes as targets. Previously, we found that millimolar levels of calcium in the hemolytic assays dissociate high-affinity perforin-calreticulin complexes, which makes it unlikely that perforin associates with calreticulin in solution when hemolysis is blocked. Calreticulin may affect perforin at the erythrocyte membrane. We observed calcium-dependent binding of calreticulin to erythrocyte membranes with a Kd of 2.7 x 10(-7) M and a saturation average of 10(5) molecules calreticulin per erythrocyte. At concentrations that blocked hemolysis, calreticulin occupied many of the calreticulin membrane-binding sites and was in molar excess of perforin. These observations open the possibilities that membrane-bound calreticulin prevents hydrophobic entry of perforin into membranes and (or) prevents perforin from assembling into polyperforin pores.     

Three Cases of Hemolytic Anemia with Erythrocyte Pyruvate Kinase Deficiency in Alberta

Erythrocyte pyruvate kinase (PK) activity was determined by the lactate dehydrogenase-coupled spectrophotometric assay. The effects of modifications in the buffer and in substrate concentrations were studied. Three patients with congenital non-spherocytic hemolytic anemia were deficient in erythrocyte PK, and were evidently homozygous for this deficiency. The daughter of one patient and the parents of another had intermediate PK levels and were probably heterozygous. The erythrocyte adenosine triphosphate (ATP) level was low in one patient, high in another. Adenosine triphosphatase activity of the erythrocyte membranes of one patient was normal.     

Challenges for red blood cell biomarker discovery through proteomics

Red blood cells are rather unique body cells, since they have lost all organelles when mature, which results in lack of potential to replace proteins that have lost their function. They maintain only a few pathways for obtaining energy and reducing power for the key functions they need to fulfill. This makes RBCs highly sensitive to any aberration. If so, these RBCs are quickly removed from circulation, but if the RBC levels reduce extremely fast, this results in hemolytic anemia. Several causes of HA exist, and proteome analysis is the most straightforward way to obtain deeper insight into RBC functioning under the stress of disease. This should result in discovery of biomarkers, typical for each source of anemia. In this review, several challenges to generate in-depth RBC proteomes are described, like to obtain pure RBCs, to overcome the wide dynamic range in protein expression, and to establish which of the identified/quantified proteins are active in RBCs. The final challenge is to acquire and validate suited biomarkers unique for the changes that occur for each of the clinical questions; in red blood cell aging (also important for transfusion medicine), for thalassemias or sickle cell disease. Biomarkers for other hemolytic anemias that are caused by dysfunction of RBC membrane proteins (the RBC membrane defects) or RBC cytosolic proteins (the enzymopathies) are sometimes even harder to discover, in particular for the patients with RBC rare diseases with unknown cause. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Molecular characterization of the hemolysin determinant of Serratia marcescens.

The nucleotide sequence of a 7.3-kilobase-pair fragment of DNA encoding a hemolytic activity from Serratia marcescens was determined. Two large open reading frames were identified, designated shlA (Serratia hemolysin) and shlB, capable of encoding polypeptides of 165, 056 and 61,897 molecular weight, respectively. Both reading frames were expressed in vivo. The shlB gene product was localized to the outer membrane of Escherichia coli cells harboring the S. marcescens hemolysin determinant. Consistent with this location, a signallike sequence was identified at the N terminus of the polypeptide predicted from the nucleotide sequence of the shlB gene. Hyperexpression of the shlB locus permitted the identification of two shlB-encoded polypeptides of 65,000 and 62,000 molecular weight, respectively. Determination of the N-terminal amino acid sequence of the purified 62,000-molecular-weight protein confirmed that it was the mature form of the ShlB protein initially synthesized as a precursor (65,000-molecular-weight protein). By using polyclonal antisera raised against the purified proteins, ShlA and ShlB were identified in the outer membrane of S. marcescens. The shlA gene product was shown to interact with erythrocyte membranes, confirming it as the hemolysin proper. Both hemolysis and the interaction of ShlA with erythrocyte membranes did, however, require the ShlB function. Progressive deletion of the C terminus of the ShlA protein gradually reduced hemolytic activity until 37% of the amino acids had been removed. Elimination of 54% of the amino acids produced a nonhemolytic protein which, however, was still capable of associating with erythrocyte membranes.     

Protein and lipid components of the pigeon erythrocyte membrane.

The plasma membrane of the nucleated pigeon erythrocyte was isolated by a method that is simple, reproducible and minimally disruptive, the final preparation consisting of whole cell 'ghosts', recovered at over 40% yield. Alternative methods, which yield membrane fragments, were also tested and some of their possible disadvantages demonstrated. Analysis of the protein components of the isolated membranes by gel elctrophoresis in the presence of sodium dodecyl sulphate revealed that their composition is very similar to that of the proteins of human erythrocyte membranes. However, two major proteins are unique to the nucleated cell membrane; these have apparent mol.wts. of 97000 and 57000. Also, the bands designated 4.2 (74500 mol.wt.) and 6 (35000 mol wt.) by Steck [(1974) J. Cell Biol. 62, 1-19] for the human cell membrane are absent from pigon cell membrane. Glycosylated membrane proteins could not be detected in gels stained with the periodate-Schiff-base procedure. Analysis of membrane phospholipids revealed the same components known to be present in mammalian erythrocytes, though in different proportions. These findings are discussed in the light of known physiological and biochemical differences between avian and mature mammalian erythrocytes.     

Identification of Re lipopolysaccharide-binding protein on murine erythrocyte membrane

Our recent studies have suggested that bacterial lipopolysaccharide (LPS) attaches to Pronase-sensitive proteins on the murine erythrocyte membrane. In the present study, in order to identify the LPS-binding protein on the murine erythrocyte membrane, a unique method to detect LPS-binding protein on a nitrocellulose membrane was developed. Murine erythrocyte membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred electrophoretically onto a nitrocellulose membrane. The membrane was incubated with LPS of Salmonella minnesota R595 (Re LPS) in phosphate-buffered saline (PBS), after the remaining sites were blocked with gelatin in PBS. We were able to obtain a non-background stain by adding the nonionic detergent octylglucoside at the low concentration of 0.1% to the Re LPS solution. The Re LPS bound to the protein on the nitrocellulose membrane was exposed to affinity purified anti-Re LPS antibodies (IgG) and then to alkaline phosphatase-conjugated anti-IgG. The alkaline phosphatase was detected on the membrane by an enzymatic reaction. This method demonstrated that Re LPS was bound to an erythrocyte protein of 96 kDa. Treatment of erythrocytes with Pronase led to disappearance of the Re LPS-binding protein on the erythrocyte membrane. There was no difference between LPS-responder and LPS-nonresponder murine erythrocyte membranes in amount and molecular weight of the Re LPS-binding protein.     

The activity of membranes reconstituted from HVJ envelope proteins and lipids to induce hemolysis and fusion between liposomes and erythrocytes

A simple method for preparation of lipid-free envelope proteins (HN protein and F protein) of HVJ (Sendai virus) was developed. Reconstituted 'envelopes' were then prepared from envelope proteins and various lipids by the detergent dialysis method, and the activity to induce hemolysis and fusion between liposome and erythrocyte was studied. Lipid-free envelope protein aggregates could induce hemolysis and liposome-erythrocyte fusion. The activity was however greatly augmented by incorporation of envelope proteins into membrane of viral total lipids. Hemolytic and fusogenic activity was somewhat augmented by incorporation of envelope proteins into dipalmitoylphosphatidylcholine/cholesterol (1:1, molar ratio) and dimyristoylphosphatidylcholine/cholesterol (1:1), though the augmentation was lower than that observed with viral total lipids. When 'envelopes' were reconstituted with the proteins and viral total lipids supplemented with phosphatidylethanolamine, two kinds of 'envelopes' were prepared; one was permeable to Dextran (Mr 75000) and hemolytic, and the other was impermeable to Dextran and nonhemolytic. The latter acquired hemolytic activity after subjection to freezing and thawing, and its barrier function was lost concomitantly. The study suggests that envelope proteins (HN protein and F protein) could function without lipids but their activity was greatly influenced by not only the composition of additional lipids but also mode of arrangement of components on the reconstituted membranes.     

Immunological identification of G protein alpha- and beta-subunits in tail membranes of bovine spermatozoa.

Heterotrimeric G proteins are believed to play important roles as signal transducing components in various mammalian sperm functions. To assess the distribution of G proteins in bovine sperm tails, we purified membranes by hypoosmotic swelling of bovine spermatozoa followed by disruption of plasma membranes in a homogenizer and various centrifugation steps. Electron microscopy revealed highly purified membranes of bovine sperm tails. Subsequently, antisera against synthetic peptides were used to identify G proteins in immunoblots. An antiserum directed against the C-terminal decapeptide of Gi3 and detecting all known pertussis toxin-sensitive alpha-subunits, reacted specifically with a 40-kDa protein. In contrast, various other specific peptide antisera against alpha-subunits did not detect any G protein in enriched tail membranes. An antiserum recognizing the beta 2-subunit of G proteins and an antiserum reacting with both beta 1- and beta 2-subunits identified a 35-kDa protein in sperm tail membranes. In contrast, antisera against the 36-kDa beta 1-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein alpha-subunits nor G protein beta-subunits were found in the cytosol. Our results suggest that G proteins in membranes of tails of bovine spermatozoa most likely belong to a novel subtype of G protein alpha-subunits, whereas the putative beta-subunit could be identified as a beta 2-subunit.