Apparent Subunit Molecular Weight Research Articles

Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis.

Elementary bodies (EB) of Chlamydia trachomatis serotypes C, E, and L2 were extrinsically radioiodinated, and whole-cell lysates of these serotypes were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Autoradiography of the polypeptide profiles identified a major surface protein with an apparent subunit molecular weight of 39,500 that was common to each C. trachomatis serotype. The abilities of nonionic (Triton X-100), dipolar ionic (Zwittergent TM-314), mild (sodium deoxycholate and sodium N-lauroyl sarcosine), and strongly anionic (SDS) detergents to extract this protein from intact EB of the L2 serotype were investigated by SDS-PAGE analysis of the soluble and insoluble fractions obtained after each detergent treatment. Only SDS readily extracted this protein from intact EB. Sarkosyl treatment selectively solubilized the majority of other EB proteins, leaving the 39,500-dalton protein associated with the Sarkosyl-insoluble fraction. Ultrastructural studies of the Sarkosyl-insoluble EB pellet showed it to consist of empty EB particles possessing an apparently intact outer membrane. No structural evidence for a peptidoglycan-like cell wall was found. Morphologically these chlamydial outer membrane complexes (COMC) resembled intact chlamydial EB outer membranes. The 39,500-dalton outer membrane protein was quantitatively extracted from COMC by treating them with 2% SDS at 60 degrees C. This protein accounted for 61% of the total COMC-associated protein, and its extraction resulted in a concomitant loss of the COMC membrane structure and morphology. The soluble extract obtained from SDS-treated COMC was adsorbed to a hydroxylapatite column and eluted with a linear sodium phosphate gradient. The 39,500-dalton protein was eluted from the column as a single peak at a phosphate concentration of approximately 0.3 M. The eluted protein was nearly homogeneous by SDS-PAGE and appeared free of contaminating carbohydrate, glycolipid, and nucleic acid. Hyperimmune mouse antiserum prepared against the 39,500-dalton protein from serotype L2 reacted with C. trachomatis serotypes Ba, E, D, K, L1, L2, and L3 by indirect immunofluorescence with EB but failed to react with serotypes A, B, C, F, G, H, I, and J, with the C. trachomatis mouse pneumonitis strain, or with the C. psittaci feline pneumonitis, guinea pig inclusion conjunctivitis, or 6BC strains. Thus, the 39,500-dalton major outer membrane protein is a serogroup antigen of C. trachomatis organisms.

Intra-strain heterogeneity of gonococcal pili is related to opacity colony variance.

We have purified pili from isogenic opacity colony variants that were derived from 14 gonococcal strains. Pili purified from opaque colonies of one strain usually differed from pili purified from transparent colonies of the same strain. In 10 of the 14 strains examined, the apparent subunit molecular weight of pilin isolated from the opaque variants was larger than that seen with pilin obtained from transparent variants. In addition there were demonstrable intra-strain differences in the isoelectric point and buoyant density of pili derived from the opacity variants. Because gonococci express differing opacity phenotypes during the menstrual cycle, it is possible that the pili of these organisms may also alter in vivo.

A two-subunit cytochrome c oxidase (cytochrome aa3) from Paracoccus dentrificans.

Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, EC 1.9.3.1) was purified from the cytoplasmic membrane of the bacterium Paracoccus denitrificans. The enzyme contains two heme groups (a and a3) and two copper atoms per minimal unit, oxidizes mammalian cytochrome c at a high rate, and, when incorporated into liposomes, generates an electrochemical proton gradient during cytochrome c oxidation. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis reveals only two subunits of apparent molecular weights 45,000 and 28,000; they appear to correspond to the two largest mitochondrially made subunits of the seven-subunit cytochrome c oxidase isolated from yeast mitochondria. Because of its structural simplicity. Paracoccus cytochrome c oxidase offers new possibilities for exploring the mechanism of cytochrome c oxidase function.

Secretion of alpha 1-antitrypsin by an established human hepatoma cell line and by human/mouse hybrids.

The human hepatoma cell line SK-HEP-1 has been shown by radioimmunoelectrophoresis to synthesize and secrete a protein which coprecipitates with human alpha 1-antitrypsin. This protein was indistinguishable from serum alpha 1-antitrypsin in terms of electrophoretic mobility, apparent subunit molecular weight (47,000), and binding to concanavalin-A. The protein identified as alpha 1-antitrypsin (alpha 1 AT) was secreted by seven clones derived from SK-HEP-1 and by twelve out of eighteen hybrid clones derived from the fusion of SK-HEP-1 with mouse RAG cells. There was no correlation between the expression of alpha 1 AT and that of human enzymes assigned to sixteen different autosomes. There was an imperfect correlation between the expression of alpha 1 AT and of the two chromosome 9 marker enzymes AK1 and AK3 (two discordant clones).

Membrane association and orientation of rat renal activities capable of degrading glutathione

1. 1. γ-Glutamyltranspeptidase and a peptidase which cleaves cysteinylglycine and its S-derivatives are localized within the brush border membrane of the proximal straight tubule. 2. 2. The transpeptidase has previously been shown to be an amphipathic protein in which the membrane binding and catalytic domains are separated by a papain-sensitive sequence of amino acids. 3. 3. The Triton-purified peptidase exhibits a higher apparent subunit molecular weight (150,000) than the papain-purified peptidase (135,000). 4. 4. The presence of the papain-sensitive sequence of amino acids was required for the incorporation of the Triton-purified peptidase into lecithin vesicles. 5. 5. Papain treatment of isolated brush border membrane vesicles results in 90–95% solubilization of transpeptidase and peptidase activities without altering the internal volume of the vesicles. 6. 6. At least 92% of both activities associated with vesicles can be inactivated with specific Fab antibodies or precipitated by indirect immunoprecipitation. 7. 7. The non-accessible γ-glutamyltranspeptidase and peptidase activities were shown to be associated with the internal surface of a unique population of inverted vesicles. 8. 8. Therefore, the two activities are orientated exclusively on the outside of the brush border membrane, consistent with their proposed participation in the extracellular degradation of glutathione and mercapturic acid precursors.

Ca2+-dependent protein phosphorylation of purely cholinergic Torpedo synaptosomes.

Preincubation of intact, purely cholinergic Torpedo synaptosomes with [32P]Pi results in the incorporation of 32P into about 10 specific proteins. Depolarizing the Torpedo synaptosomes by a high K+ buffer or treatment with the Ca2+ ionophore A23187 result in Ca2+ uptake, in acetylcholine (ACh) release, and in a marked increase of 32P incorporation into a specific protein band with an apparent subunit molecular weight of 100,000 (band alpha). The kinetics of synaptosomal 45Ca2+ uptake, of 32P incorporation into band alpha, and of ACh release is similar and reach maximal values about 45 s after the synaptosomes have been treated. Sr2+ and Ba2+ can replace Ca2+ in evoking both K+ depolarization-dependent ACh release and 32P incorporation into band alpha. The effectiveness of these ions (Ca2+ greater than Sr2+ greater than Ba2+) is similar in both cases. The data presented suggest that Ca2+ accumulation by Torpedo synaptosomes leads to an increase in the phosphorylation of a specific protein and to ACh release. This phosphoprotein may be involved in the regulation of presynaptic processes which underly ACh release.

Polypeptide composition of acetylcholine receptor purified from teleost and Elasmobranch electroplax membranes

Acetylcholine receptor (AcChR) was solubilized and purified from membranes derived from electric organs of the marine fish Torpedo marmorata, Torpedo nobiliana, Narcine brasliensis, and of the freshwater eel, Electrophorus electricus, using techniques originally developed for Torpedo californica ( J. Schmidt and M. Raftery, 1972, Biochem. Biophys. Res. Commun. 49, 572–578; 1973, Biochemistry 12, 852–856. The conditions used were identical in each case and the goal was to determine the degree of similarity between receptors from each source since conflicting reports have appeared with regard to polypeptide composition. The Torpedo and Narcine preparations were of high specific activity and exhibited four polypeptide components of apparent molecular weights 64, 59, 50, and 40 × 10 3 upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Two components were observed upon gel electrophoresis in sodium cholate or upon sucrose density gradient centrifugation, representing monomeric and dimeric forms. Eel acetylcholine receptor exhibited three major subunits of apparent molecular weights 57, 49, and 40 × 10 3. The amino acid and neutral sugar composition of the purified receptor preparations have been determined. The results support the contention that the receptor is composed of several types of polypeptide.

Purification and properties of phosphorylated isocitrate dehydrogenase of Escherichia coli.

Phosphorylated NADP+-isocitrate dehydrogenase (EC 1.1.1.42) has been purified to electrophoretic homogeneity from in vivo 32P-labeled Escherichia coli. The cells used as the source of phosphorylated enzyme were harvested 1 h after the addition of 5 mCi of [32P]orthophosphoric acid and 25 mM sodium acetate to cultures grown to early stationary phase on a low phosphate medium with limiting glucose. Double immunodiffusion and autoradiography demonstrated immunological identity between the 32P-labeled NADP+-isocitrate dehydrogenase and the enzyme isolated from glucose-grown E. coli. The phosphoenzyme had an apparent subunit molecular weight of 51,000 as determined by denaturing acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and the radioactivity co-electrophoresed with NADP+-isocitrate dehydrogenase activity when purified enzyme was subjected to nondenaturing gel electrophoresis. [32P]Phosphoserine was identified following partial acid hydrolysis of the purified phosphoenzyme.

Studies of the composition of purified Torpedo californica acetylcholine receptor and of its subunits

Under conditions that limit proteolytic degradation, the detergent-solubilized purified receptor protein from Torpedo californica exists in monomeric and dimeric forms. The purified receptor complex is composed of four different polypeptide subunits of apparent molecular weights 40 000, 50 000, 60 000, and 65 000. The individual polypeptides have been purified and their amino acid compositions have shown them to be relatively hydrophobic. In addition, the carbohydrate composition of the intact receptor complex and of the individual polypeptides has been determined. Amino acid analysis provided evidence for the occurrence of a component with chromatographic properties similar to those of phosphoserine. Treatment of receptor with CH3NH2 in base, a condition which provided quantitative modification of O-phosphoserine residues in beta-casein, completely eliminated the peak corresponding to phosphoserine following mild acid hydrolysis. We conclude that the receptor contains O-phosphoserine residues to the extent of approximately seven residues per molecule and these residues occur in all constituent polypeptides. Other forms of O-substituted serine and threonine were also shown to occur, most likely as glycosylated residues.

An analysis of the protein, glycoprotein and monosaccharide composition of dictyostelium discoideum plasma membranes during development

Qualitative and quantitative changes in the protein and glycoprotein components of the plasma membrane of the cellular slime mould Dictyostelium discoideum have been detected by analysis of sodium dodecyl sulphate-polyacrylamide gel electrophoretic patterns. The amounts of proteins of subunit molecular weight 220 000, 91 000, 63 000, 59 000, 56 000 increased during the acquisition of aggregation competence, while proteins of subunit molecular weight 82 000 and 22 000 decreased. The amounts of glycoproteins with apparent subunit molecular weights 285 000, 150 000, 137 000, 100 000, 53 000, 50 500 and 30 500 increased during differentiation while a 125 000 dalton component decreased dramatically in amount. The neutral and amino sugar composition of the plasma membrane was also analyzed and found to remain essentially unchanged during the first 12 h of differentiation. The major sugars were mannose, fucose, and glucosamine; galactose and galactosamine were also present, but in lower amounts.

Fibroblast cellular and plasma fibronectins are similar but not identical

Fibronectins are multimeric, adhesive glycoproteins present on cell surfaces and circulating in blood. Cellular fibronectin produced by fibroblasts in vitro and fibronectin isolated from plasma are known to be very similar immunologically and biochemically. We investigated whether or not they are identifical. Purified chicken and human cell-surface fibronectins are 150-fold more active in hemagglutination of fixed erythrocytes than plasma fibronectins. Cell-surface fibronectin is also 50-fold more active in restoring a more normal morphology to transformed cells originally missing the protein. However, in two other assays that measure cell attachment to collagen and cell spreading, cell-surface and plasma fibronectins have identical specific activities. In sodium dodecyl sulfate polyacrylamide gels, the subunits of human and chicken plasma fibronectins have significantly smaller apparent subunit molecular weights than cellular fibronectins present on cell surfaces or secreted into culture media. These differences are also present in a characteristic large subfragment of both forms of fibronectin after limited proteolysis by trypsin. We conclude that by both biological and biochemical criteria, cellular and plasma fibronectins are similar but not identical.

Evidence for role of glycoprotein carbohydrates in membrane transport: specific inhibition by tunicamycin.

Using tunicamycin, we have investigated the role of glycoproteins in membrane transport. Tunicamycin is a glucosamine-containing antibiotic that specifically inhibits dolichol pyrophosphate-mediated glycosylation of asparaginyl residues of glycoproteins. Inhibition of protein glycosylation in chick embryo fibroblasts by tunicamycin or other inhibitors of glycosylation resulted in defective transport of glucose, uridine, and amino acid analogs (alpha-aminoisobutyrate and cycloleucine). The defect in glucose transport is accompanied by decreased glucose metabolism, as determined by rates of CO2 and lactate production. In contrast, tunicamycin treatment did not affect other membrane-associated processes, such as secretion of fibronectin and procollagen, uptake of glucose by passive diffusion, Na+/K+ ATPase and adenylate cyclase activities, or stimulation of adenylate cyclase by prostaglandin and cholera toxin. Two glucose/glycosylation-regulated membrane proteins with apparent subunit molecular weights of 95,000 and 75,000 were induced by tunicamycin treatment. Our results indicate that glycoprotein glycosylation is required for membrane transport.

The hemoglobin ofpseudodoras, a South American catfish: Isolation, characterization and ligand binding studies

1. The hemoglobin of the Amazonian catfishPseudodoras sp. was isolated and characterized; it comprises a single component.2. The hemoglobin's subunit composition is similar to that of other teleost hemoglobins. The apparent native molecular weight as determined by gel filtration is 66,000. The apparent subunit molecular weight is 14,300 by sodium dodecyl sulfate electrophoresis. The hemoglobin does not polymerize after oxidation by potassium ferricyanide.3. The hemoglobin lacks a Root effect. A small Bohr effect is evident in the phosphate-free hemoglobin:Δlog p1/2/ΔpH is no more than about −0.1 to −0.2 and increases toΔlog p1/2/ΔpH = −0.4 in the presence of 1 mM ATP. The cooperativity, as determined byn of the Hill equation, is low, varying from 0.8 to 1.7 between pH 6.1 and 8.6.4. Thep1/2 values of stripped hemoglobin solutions are extremely low, less than 0.5 mm Hg at all pH values examined between pH 6.1 and 9.0. The high oxygen affinity is reflected primarily in the CO combination rate which resembles that found in myoglobins and isolated subunits of human hemoglobin.5. Both the CO combination rate and the O2 dissociation rate determined by stopped-flow spectrophotometry are pH and phosphate sensitive. Between pH 6.2 and 8.1 the COon rate increases about 5-fold in the phosphate-free hemoglobin. Addition of 1 mM ATP causes a depression in the rate at all pH values examined. The O2off rate decreases 7-fold going from pH 6.0 to 8.2 in stripped hemoglobin solutions. Addition of 1 mM ATP induces a 10-fold decrease over the same range. At pH values below 6.0 a depression in the O2off rate occurs in the stripped hemoglobin, indicative of an acid Bohr effect.

Purification and properties of four species of lysyl oxidase from bovine aorta.

Lysyl oxidase of bovine aorta was resolved into four enzymically active species by elution from DEAE-cellulose with a salt gradient in 6m-urea, consistent with purification results obtained with enzyme of other tissues [Stassen (1976) Biochim. Biophys. Acta438, 49-60]. In the present study, each of the four peaks of activity was purified to apparent homogeneity by subsequent chromatography on gel-filtration media in 6m-urea. Each enzyme is eluted as a species with mol.wt. approx. 30000 under these conditions, although lysyl oxidase polymerizes to a series of multimers with molecular weights ranging up to 1000000 in the absence of urea. The apparent subunit molecular weight of each enzyme species determined by electrophoresis in sodium dodecyl sulphate and 8m-urea is approx. 32000-33000. The amino acid compositions of the purified forms of lysyl oxidase are similar to each other, although sufficient differences exist to conclude that each is a unique molecular species. Incorporation of alpha-toluenesulphonyl fluoride into the purification scheme does not alter the resolution of enzyme into four species, suggesting that proteolysis during isolation is not the basis of the heterogeneity. The similar sensitivities of each form of enzyme to chelating agents and to semicarbazide and isoniazid indicate that each requires the participation of a metal ion, presumably Cu(2+), and of a carbonyl compound for enzyme function. The present study describes a method for the purification of multiple species of lysyl oxidase and reveals that significant chemical differences exist between the different enzyme forms.

Phylogenetic origins of immune recognition: Naturally occurring DNP-binding molecules in chordate sera and hemolymph

A method is described which allows the isolation of small quantities of DNP-binding antibodies of both high- and low-molecular-weight classes. The only detectable DNP-binding protein in the serum of placoderm-derived vertebrates was immunoglobulin in nature, and of the order of ≤ 1% total serum immunoglobulin. Proteins in the serum of the urochordate Pyura stolonifera, which bind to both DNP and erythrocyte antigens, showed charge heterogeneity and an apparent subunit molecular weight of approximately 65,000 to 70,000 daltons.

Isolation and partial characterization of human erythrocyte membrane NADH: (acceptor) oxidoreductase.

The NADH: (acceptor) oxidoreductase (EC 1.6.99.3) was isolated from human erythrocyte ghosts by a procedure including Triton X-100 solubilization, affinity chromatography on an NAD+-Sepharose 4B column, ammonium sulfate precipitation, and isoelectric focusing. This enzyme preparation was characterized by a single band on the urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by a single precipitin line with its corresponding antiserum on double diffusion and immunoelectrophoresis. A 103-fold purification indicates that the oxidoreductase represents approximately 1% of the ghost protein mass. The specific activity of the purified enzyme was 112 units/mg protein. The pH optimum was 6.8 and the isoelectric point, pI, was 6.6 The oxidoreductase has a specificity for NADH as a cofactor. The NADPH was ineffective as a reducing agent. The enzyme activity was strongly temperature-dependent, displaying maximal activity between 35 and 40 degrees C. The energy of activation was 4.9 kcal. The enzyme activity was inhibited by sulfhydryl reagents, anionic detergents, and divalent ions. The amino acid composition of the purified enzyme is characterized by the presence of all common amino acids including half-cystine and tryptophan. The results of carbohydrate and lipid analyses indicated that the oxidoreductase is a glycolipoprotein with fucose, galactose, mannose, and glucosamine as the sugar components and cholesterol and sphingomyelin as the lipid constituents. The apparent subunit molecular weight estimated by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of 2-mercaptoethanol was 40,000. The antiserum completely inhibited the enzymic activity at the equivalence point. We suggest that the membrane-bound NADH: (acceptor) oxidoreductase might be a transmembrane protein.

Studies on the characterization of human erythrocyte acetylcholinesterase and its interaction with antibodies

Human erythrocyte membranes were solubilized in 5% Triton X-100 and the acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was isolated by affinity chromatography utilizing a specific inhibitor, trimethyl-p- aminophenyl ammonium chloride, bound to Sepharose 4B. After a repeated chromatography acetylcholinesterase was found to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunization of rabbits with acetylcholinesterase elicited the formation of an antiserum which gave single precipitin lines with the enzyme on immunodiffusion and rocket, crossed and immuno-electrophoreses. The purified enzyme had a specific activity of 418 units/mg protein. The K m value of acetylcholinesterase with acetylthiocholine as substrate was 1.5 × 10 −4 M . Isoelectric focusing of acetylcholinesterase in the presence of Triton X-100 and within the pH ranges of 3–10 and 3–6 exhibited a single peak of enzyme activity with a PI of 4.8. The results of amino acid and carbohydrate analyses showed that acetylcholinesterase is a glycoprotein with a carbohydrate/protein weight ratio of 0.16 and glucose, galactose, mannose, glucosamine, galactosamine and sialic acid as the sugar components. The N-terminal amino acid was blocked. Lipid, phosphorus and fatty acid analyses indicated phosphatidylserine and cholesterol as the major lipid components of acetylcholinesterase. The apparent subunit molecular weight estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol was 160 000 and in its presence, 80 000. The kinetic studies showed a competitive inhibition of acetylcholinesterase by its antibodies. Agglutination of human red cells by monospecific antiserum to acetylcholinesterase confirmed that the antigenic site(s) of the enzyme is localized on the outer surface of the erythrocyte membrane.

Developmentally regulated lectin from embryonic chick pectoral muscle. Purification by affinity chromatography.

A lectin, whose specific activity in soluble extracts of embryonic chick pectoral muscle increases strikingly between 8 and 16 days of development, has been purified by affinity chromatography on derivatized Sepharose 4B coupled to p-aminophenyl-beta-D-lactoside. After affinity chromatography the lectin is pure except for minor contamination with another protein possibly representing a second muscle lectin. The latter can be completely removed by preparative isoelectric focusing. The purified lectin has an apparent molecular weight of 30,000 and an apparent subunit molecular weight of 15,000. Its isoelectric point is 4.0. The most potent saccharide inhibitors tested were thiodigalactoside and lactose. An antibody has been raised to the pure lectin. Studies with this antibody indicate that the lectin is present both on the surface of and within myoblasts.

Purification of porcine and human ristocetin-Willebrand factor.

A plasmatic component required for the ristocetin-induced aggregation of platelets has been purified from normal human and porcine plasma by gel filtration (4% agarose) and anion-exchange chromatography (DEAE cellulose). No factor VIII coagulant activity was found associated with the purified human or porcine component. Urea sodium dodecylsulfate electrophoretic analysis of the purified component of both species indicated that the apparent molecular weight with intact disulfides is in excess of 500,000; after disulfide reduction with 2-mercaptoethanol, single components with an apparent subunit molecular weight of 230,000 were observed. Purified porcine ristocetin-Willebrand factor (RWF) co-sedimented in sucrose gradients with the factor present in normal plasma. Amino acid analysis of both human and porcine RWF indicated that all normal amino acids are present, whereas amino sugars were undetected. However, lipid analysis indicated 1% to 2% lipids present, including monoglycerides, di- and tri-glycerides, cholesterol, cholesterol esters, some free fatty acids, and a trace of phospholipid. A single line of identity was observed between normal human plasma and purified human RWF when immunodiffusion plates were run with purified rabbit anti-human RWF immunoglobulins. Antisera raised against human and porcine RWF's do not inhibit the factor VIII coagulant activity of the homologous plasma, nor is "spontaneously occurring" human factor VIII inhibitor neutralized by the isolated material of either species.

Biological and biochemical properties of Phaseolus vulgaris isolectins.

Affinity-purified phytohemagglutinin from red kidney bean resolves into five isolectins by SP-Sephadex ion exchange chromatography. Recoveries ranging from 30 to 130 mg of protein for each isolectin are easily achieved. The isolectins have similar amino acid compositions which differ only in threonine, lysine, and arginine. A distinguishing feature of the amino acid composition is the total lack of sulfur-containing amino acids. Each isolectin contains about 4% mannose and 2.2% N-acetyl-D-glucosamine. All isolectins on electrophoresis form single protein bands under denaturing and nondenaturing conditions in polyacrylamide gels, and all have apparent subunit molecular weights of 33,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isolectins are also homogeneous by ultracentrifugation and have apparent native molecular weights of 115,000 +/- 4,130, suggesting tetrameric quaternary structures. Whereas 80% of the starting erythroagglutinin activity is recovered, one of the five isolectins possesses 50% of that original activity. As sequentially eluted from the ion exchange column, each isolectin displays progressively higher erythroagglutinin and lower lymphocyte mitogenic activities. Based on their relative biological activities, the isolectins are assigned the structures L4, L3E1, L2E2, L1E3, and E4, where L and E represent lymphocyte- and erythrocyte-reactive subunits, respectively, and the subscripts represent the proposed subunit composition.