Dithiothreitol activation of the insulin receptor/kinase does not involve subunit dissociation of the native alpha 2 beta 2 insulin receptor subunit complex

The subunit composition of the dithiothreitol- (DTT) activated insulin receptor/kinase was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and gel filtration chromatography under denaturing (0.1% SDS) or nondenaturing (0.1% Triton X-100) conditions. Pretreatment of 32P-labeled insulin receptors with 50 mM DTT followed by gel filtration chromatography in 0.1% SDS demonstrated the dissociation of the alpha 2 beta 2 insulin receptor complex (Mr 400,000) into the monomeric 95,000 beta subunit. In contrast, pretreatment of the insulin receptors with 1-50 mM DTT followed by gel filtration chromatography in 0.1% Triton X-100 resulted in no apparent alteration in mobility compared to the untreated insulin receptors. Resolution of this complex by nonreducing SDS-polyacrylamide gel electrophoresis and autoradiography demonstrated the existence of the alpha 2 beta 2 heterotetrameric complex with essentially no alpha beta heterodimeric or free monomeric beta subunit species present. This suggests that the insulin receptor can reoxidize into the Mr 400,000 complex after the removal of DTT by gel filtration chromatography. Surprisingly, these apparently reoxidized insulin receptors were also observed to be functional with respect to insulin binding, albeit with a 50% decrease in affinity for insulin and insulin stimulation of the beta subunit autophosphorylation. To prevent reoxidation, the insulin receptors were pretreated with 50 mM DTT followed by incubation with excess N-ethylmaleimide prior to gel filtration chromatography in 0.1% Triton X-100. Under these conditions the insulin receptors migrated as the Mr 400,000 alpha 2 beta 2 complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subunit structure of the purified human placental insulin receptor. Intramolecular subunit dissociation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Insulin receptors purified from human placental membranes by gel-filtration and insulin-agarose affinity chromatography were found to be composed of eight different high molecular weight complexes as identified by nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The subunit stoichiometry of these different high molecular weight forms of the insulin receptor were determined by comparisons of silver-stained gel profiles with the autoradiograms of 125I-insulin specifically cross-linked to the alpha subunit and [gamma-32P]ATP specifically autophosphorylated beta subunit gel profiles. Two-dimensional SDS-polyacrylamide gel electrophoresis in the absence and presence of reductant confirmed the subunit stoichiometries as alpha 2 beta 2, alpha 2 beta beta 1, alpha 2 (beta 1)2, alpha 2 beta, alpha 2 beta 1, alpha 2, alpha beta, and beta, where alpha is the Mr = 130,000 subunit, beta is the Mr = 95,000 subunit, and beta 1 is the Mr = 45,000 subunit. Treatment of the insulin receptor preparations with oxidized glutathione or N-ethylmaleimide prior to SDS-polyacrylamide gel electrophoresis increased the relative amount of the alpha 2 beta 2 complex concomitant with a total disappearance of the alpha 2 beta, alpha 2 beta 1, alpha 2, and free beta forms. The effects of oxidized glutathione were found to be completely reversible upon extensive washing of the treated insulin receptors. In contrast, the effects of N-ethylmaleimide were totally irreversible by washing, consistent with known sulfhydryl alkylating properties of this reagent. The formation of these lower molecular weight insulin receptor subunit complexes was further demonstrated to be due to SDS/heat-dependent intramolecular sulfhydryl-disulfide exchange occurring within the alpha 2 beta 2 complex. These studies demonstrate that the largest disulfide-linked complex (alpha 2 beta 2) is the predominant insulin receptor form purified from the human placenta with the other complexes being generated by proteolysis and by internal subunit dissociation.     

The porcine ovarian luteinizing hormone/human chorionic gonadotropin receptor II. Is the purified receptor an oligomer of identical subunits?

Purified porcine luteinizing hormone/human chorionic gonadotropin receptors were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis following reduction and thermal denaturation and stained with Coomassie Brilliant Blue. A major protein of Mr = 77 +/- 4 X 10(3) and a minor protein of Mr = 66 +/- 4 X 10(3) were observed. Iodoreceptor proteins were resolved into a major component of Mr = 77 +/- 3 X 10(3) and a minor component of Mr = 62 +/- 5 X 10(3) after reduction and thermal denaturation. In the absence of reduction, the iodoreceptor had a major component of Mr 63 +/- 3 X 10(3). Purified human chorionic gonadotropin specifically transferred part of the iodoreceptor from the Mr = 63 X 10(3) species to an Mr = 110-120 X 10(3) species. Purified receptors were analyzed by nondenaturing polyacrylamide gel electrophoresis and identified by specific binding of iodo-human chorionic gonadotropin. Three binding species with approximate Mr = 60 X 10(3), 130 X 10(3), and 260 X 10(3) were identified. Iodoreceptors co-migrated with the Mr = 60 X 10(3) species under the same conditions. Similar results were obtained following renaturation of receptors separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis without reduction and thermal denaturation. These results suggest for the first time that the porcine corpus luteum luteinizing hormone/human chorionic gonadotropin receptor may be a hormone binding monomer of Mr = 60-65 X 10(3), and that the monomer may associate to form hormone binding polymeric receptor complexes.     

Separation and characterization of three insulin receptor species that differ in subunit composition

Partially purified human placental insulin receptor preparations give rise to three distinct insulin-binding peaks when eluted from a Mono Q high-performance liquid chromatography anion-exchange column. We analyzed the basis for this phenomenon by affinity cross-linking of insulin to each peak, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We find that the three insulin-binding peaks represent different molecular weight complexes with the following subunit composition: (alpha beta)2, (alpha beta)(alpha beta'), and (alpha beta')2, where beta' represents a proteolytically derived fragment of the beta subunit. This analysis of subunit composition was confirmed by silver staining of affinity-purified insulin receptor following resolution of the forms on a Mono Q column as described previously. We have characterized the three isolated insulin receptor forms with regard to ligand binding by LIGAND and Scatchard analysis. We also measured insulin-stimulatable autophosphorylation and exogenous kinase activity directed toward poly(Glu/Tyr) (4:1). The three forms of the insulin receptor exhibit similar KD's for insulin binding to the high- and low-affinity sites. The (alpha beta)2 and (alpha beta)(alpha beta') forms of the insulin receptor display superimposable curvilinear Scatchard plots. In contrast, only the intact holoreceptor (alpha beta)2 form demonstrates insulin-stimulatable autophosphorylation and exogenous kinase activity. The (alpha beta)(alpha beta') form has reduced basal kinase activity which was not increased by prior incubation with insulin. The (alpha beta')2 form lacks a kinase domain and consequently demonstrated no kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resolution of two subunits from the molybdenum-iron protein of Azotobacter vinelandii nitrogenase

The Mo-Fe protein of Azotobacter vinelandii nitrogenase was fractionated on 9.5 M urea isoelectric focusing gels and gave three distinct bands (alpha', alpha", beta'). Protein focused on nondenaturing gels gave a single brown band, which when excised and refocused on a denaturing gel gave the three-band pattern. Partial trypsin digestion of the subunits and fractionation of the peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the alpha' and alpha" polypeptide moieties were the same. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the alpha' and beta' proteins with appropriate molecular weight standards indicated Mr = 61,000 and 57,000, respectively. This is consistent with an overall alpha 2 beta 2 mass of 236,000 daltons.     

Multiple forms of rat-liver dihydropteridine reductase identified by their differing isoelectric points

Purified rat-liver dihydropteridine reductase is homogeneous by gel filtration (Mr approximately 51,000), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately 25,500), and native polyacrylamide gel electrophoresis, suggesting that the enzyme is composed of two identical subunits. However, analysis by isoelectric focusing has revealed three enzyme forms with approximate isoelectric points of 6.5, 5.9, and 5.7 (designated forms, I, II, and III, respectively). The three forms, isolated in 65% yield by preparative chromatofocusing, are stable in 0.05 M phosphate buffer, pH 6.8, containing 1 mM beta-mercaptoethanol and exhibit similar kinetic constants when the catalytic activities of the isolated forms are compared with quinonoid dihydrobiopterin as substrate. All forms generate complexes with the enzymatic cofactor NADH which are also detectable by IEF. When examined further by IEF under denaturing conditions in 6 M urea the enzyme demonstrates a differing subunit composition for its three forms. Two distinct subunits, designated alpha and beta, can be identified, and additional evidence suggests that the native enzyme forms I, II, and III represent the three differing dimeric combinations alpha alpha (form I), alpha beta (form II), and beta beta (form III).     

The saxitoxin receptor of the sodium channel from rat brain. Evidence for two nonidentical beta subunits

The saxitoxin receptor of the sodium channel purified from rat bran contains three types of subunits: alpha with Mr approximately 270,000, beta 1 with Mr approximately 39,000, and beta 2 with Mr approximately 37,000. These are the only polypeptides which quantitatively co-migrate with the purified saxitoxin receptor during velocity sedimentation through sucrose gradients. beta 1 and beta 2 are often poorly resolved by gel electrophoresis in sodium dodecyl sulfate (SDS), but analysis of the effect of beta-mercaptoethanol on the migration is covalently attached to the alpha subunit by disulfide bonds while the beta 1 subunit is not. The alpha and beta subunits of the sodium channel were covalently labeled in situ in synaptosomes using a photoreactive derivative of scorpion toxin. Treatment of SDS-solubilized synaptosomes with beta-mercaptoethanol decreases the apparent molecular weight of the alpha subunit band without change in the amount of 125I-labeled scorpion toxin associated with either the alpha or beta subunit bands. These results indicate that the alpha and beta 1 subunits are labeled by scorpion toxin whereas beta 1 is not and that the beta 2 subunit is covalently attached to alpha by disulfide bonds in situ as well as in purified preparations.     

Insulin-dependent covalent reassociation of isolated alpha beta heterodimeric insulin receptors into an alpha 2 beta 2 heterotetrameric disulfide-linked complex

The purified human placental alpha 2 beta 2 heterotetrameric insulin receptor complex was reduced and dissociated into functional alpha beta heterodimers by a combination of alkaline pH and dithiothreitol treatment. Insulin treatment of the isolated alpha beta heterodimeric complex was observed to induce the complete reassociation to an alpha 2 beta 2 heterotetrameric state when analyzed by nondenaturing Bio-Gel A-1.5m gel filtration chromatography. Nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I-insulin affinity cross-linked and 32P-autophosphorylated alpha beta heterodimers demonstrated that the insulin-dependent reassociation to the alpha 2 beta 2 heterotetrameric state occurred both covalently and noncovalently under these conditions. Comparison by reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the insulin-dependent covalent reassociation to an alpha 2 beta 2 heterotetrameric complex was due to the formation of a disulfide linkage(s) between the alpha beta heterodimers. beta subunit autophosphorylation of the control alpha 2 beta 2 heterotetrameric insulin receptor preparation was maximally stimulated within 5 min of insulin preincubation and occurred exclusively in the Mr = 400,000 alpha 2 beta 2 complex. Similarly, maximal insulin-stimulated beta subunit autophosphorylation of the alpha beta heterodimeric preparation occurred within 5 min of insulin pretreatment in the Mr = 210,000 alpha beta complex. However, 4 h of insulin pretreatment of the alpha beta heterodimer preparation induced the formation (6-fold) of a covalent 32P-labeled alpha 2 beta 2 heterotetrameric complex. Maximal stimulation of substrate phosphorylation for the alpha 2 beta 2 heterotetrameric complex was also observed to occur within 5 min of insulin treatment, whereas maximal insulin-stimulated substrate phosphorylation of the alpha beta heterodimeric complex required greater than 4 h. These data demonstrate that (i) insulin treatment can induce the reassociation of the alpha beta heterodimeric complex into a covalent alpha 2 beta 2 heterotetrameric state, and (ii) insulin-dependent protein kinase activation of the alpha beta heterodimeric insulin receptor correlates with the covalent reassociation into a disulfide-linked alpha 2 beta 2 heterotetrameric complex.     

Identification of alpha 2-macroglobulin as a cytokine binding plasma protein. Binding of interleukin-1 beta to "F" alpha 2-macroglobulin

Treatment of normal human plasma with methylamine resulted in the discovery of an interleukin-1 beta(IL-1 beta) binding protein. The protein was labeled with 125I-IL-1 beta and the relative molecular mass (Mr) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein-IL-1 beta complex had a Mr of approximately 400,000 in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis but became dissociated when exposed to beta-mercaptoethanol. The 125I-IL-1 beta labeled protein complex could be immunoprecipitated from plasma by using an anti-alpha 2-macroglobulin (alpha 2M) antiserum. Similarly, a monoclonal antibody (mAb) specific for electrophoretically fast ("F")alpha 2M was able to adsorb the 125I-IL-1 beta labeled complex from plasma. The mAb was also capable of adsorbing "F" alpha 2M-125I-IL-1 beta complexes from binary reaction mixtures, but failed to adsorb free 125I-IL-1 beta. Experiments carried out with purified plasma alpha 2M established that IL-1 beta became bound to alpha 2M only upon reaction with trypsin or methylamine, which results in the appearance of free thiol groups in alpha 2M ("F" alpha 2M). There was no binding of IL-1 beta to the native form of alpha 2M (electrophoretically slow or "S" alpha 2M), which lacks free thiol groups. Pretreatment of "F" alpha 2M with N-ethylmaleimide or [ethylenebis(oxyethylenenitrilo)] tetraacetic acid prevented complex formation between "F" alpha 2M and IL-1 beta. In contrast, the yield of "F" alpha 2M IL-1 beta complex formation was increased severalfold in the presence of 2.5 mM Zn2+. These findings indicate that "F" alpha 2M interacts with IL-1 beta through a thiol-disulfide exchange reaction. Zn2+ may play a major role in bringing together the reactive domains of the adjoining peptide backbones into proper orientation. The ready complex formation between "F" alpha 2M and the pleiotropic cytokine IL-1 beta suggests a novel biological role for "F" alpha 2M, since "F" alpha 2M-IL-1 beta complexes, but not "F" alpha 2M alone, retained IL-1-like activity in the thymocyte costimulator bioassay.     

The isolation of human beta-interferon receptor by wheat germ lectin affinity and immunosorbent column chromatographies

Radioiodinated human beta-interferon-Ser 17 (Betaseron) was reversibly cross-linked to Daudi cells by dithiobis(succinimidylpropionate). The radioactive ligand was cross-linked to three macromolecules forming labeled complexes of apparent Mr values of 130,000, 220,000, and 320,000. Betaseron, human alpha-interferon, human interleukin 2 but not recombinant human gamma-interferon competed with the labeled ligand for binding to these putative receptor(s). Human leukocyte-produced gamma-interferon competed weakly with 125I-Betaseron for binding to Daudi cells. The Betaseron-receptor complex(es) was purified by passage through a wheat germ lectin column followed by chromatography on an anti-interferon immunosorbent column and semipreparative gel electrophoresis. The cross-linked ligand-receptor complex was shown to be highly purified by sodium dodecyl sulfate and acetic acid:urea:Triton X-100 polyacrylamide gel electrophoresis. It can be dissociated into the labeled Betaseron (Mr = 17,000) ligand and a receptor moiety which has an apparent molecular weight of 110,000. The chromatographic behavior of the ligand-receptor complex on wheat germ lectin column suggests that the receptor is a glycoprotein. The described procedure yielded about 1 microgram of Betaseron receptor from 10(10) Daudi cells, estimated to contain a maximum of about 15 micrograms of the receptor.     

Hypoxanthine phosphoribosyltransferase from human brain: purification and partial characterization

A facile and rapid purification procedure, based upon the heat denaturation of extraneous proteins and GMP-Sepharose affinity chromatography, has been used to purify hypoxanthine phosphoribosyltransferase from human brain. A homogeneous enzyme preparation, as judged by sodium dodecyl sulfate and gradient polyacrylamide gel electrophoresis, was obtained. The subunit molecular weight of the enzyme was estimated as 24,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native molecular weight, determined by gradient gel electrophoresis, was approximately 100,000. These results suggest human brain hypoxanthine phosphoribosyltransferase is a tetramer, consistent with recent results reported for the human erythrocyte enzyme. At least three charge variant forms of the human brain enzyme were distinguished by nondenaturing polyacrylamide gel electrophoresis, electrofocusing, and chromatofocusing. Acidic pI values of approximately 5.7, 5.5, and 5.0 were estimated for the three major species.     

Oligomeric structure and catalytic activity of G type casein kinase. Isolation of the two subunits and renaturation experiments

Casein kinase G purified from bovine tissue is an oligomeric cyclic nucleotide-independent protein kinase made of two different monomers, namely an alpha (Mr = 38 kilodaltons) and a self-phosphorylatable beta (Mr = 27 kilodaltons) subunit. Treatment of the native enzyme under denaturing conditions (0.5 M NaCl, 4 M LiCl, and 20 to 35% formamide) resulted in a progressive selective removal of the beta subunit following gel filtration and a correlated loss of activity of the corresponding renatured enzyme. Mild digestion with papain resulted in a proteolytic alteration limited to the beta monomer with a concomitant partial loss of the enzyme activity. Isolation of the alpha and beta casein kinase G subunits was achieved by preparative reversed polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Renaturation of the proteins following sodium dodecyl sulfate removal by acetone and/or Triton X-100 treatment allowed reconstitution of a functional casein kinase G. Whereas the isolated alpha subunit was found to exhibit a weak catalytic activity, addition of the beta subunit was required for recovery of a maximal casein kinase activity. The process was dose-dependent and reached a plateau for an alpha:beta subunit molar ratio of approximately 1 to 1. These data suggest that while the casein kinase G alpha subunit bears the catalytic site, stoichiometric combination with the beta subunit is required for optimal enzymatic activity. A possible role of the beta subunit as a regulatory component of casein kinase G activity in the intact cell remains to be examined.     

Immunological comparison of purified DNA polymerase alpha from embryos of Drosophila melanogaster with forms of the enzyme present in vivo

Specific antisera to purified DNA polymerase alpha from embryos of Drosophila melanogaster and to two of the four constituent subunits (alpha, beta, gamma, and delta) were prepared. These antibodies have revealed the following features of the enzyme. (i) The Mr = 148,000 alpha subunit is very likely derived by in vitro proteolysis from polypeptides with molecular weights of 185,000 and 166,000 that are present in vivo. (ii) The Mr = 60,000 beta subunit occurs in rapidly replicating embryos as both an 85,000- and a 60,000-dalton form, but predominantly as a 60,000-dalton form in more slowly replicating cultured cells. (iii) There is no detectable immunologic cross-reactivity between the four subunits. (iv) There is an abundance of antigenic material in embryos that co-migrates with the delta subunit of the purified enzyme during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.     

Analysis of native forms and isoform compositions of the mouse 90-kDa heat shock protein, HSP90

The 90-kDa heat shock protein, HSP90, of the mouse has two isoforms, alpha and beta, which are electrophoretically separable. We have investigated the native forms of HSP90 molecules under physiological conditions and determined their isoform compositions. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that HSP90 purified from mouse lymphoma L5178Y cells consists of approximately 40% alpha and 60% beta isoforms. Analysis by nondenaturing polyacrylamide gel electrophoresis showed that the purified HSP90 exists predominantly as a dimer, but a considerable amount of monomer was also detected. Western blotting using polyclonal anti-mouse HSP90 antibodies revealed that the native forms of HSP90 in the crude L5178Y cell lysates are also dimer and monomer. The nondenaturing polyacrylamide gel electrophoresis resolved the dimeric forms into two separate bands that were identified as alpha/alpha and beta/beta homodimers by two methods: sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mapping. In addition, the results showed that the monomeric form consists mainly of the beta isoform. Both the alpha and beta isoforms were shown to bind equally to actin filaments.     

The alpha 1 beta 1 heterodimer of ATP synthase

The alpha 3 beta 3 hexamer was reconstituted from the alpha and beta subunits of TF1 portion of ATP synthase of thermophilic bacterium (Kagawa et al. (1989) FEBS Lett. 249, 67). The alpha 1 beta 1 heterodimer of ATP synthase was isolated by high performance liquid chromatography (HPLC) of the alpha 3 beta 3 hexamer in the presence of AT(D)P-Mg. On polyacrylamide gel electrophoresis, both bands corresponding to the dimer and hexamer showed ATPase activity. The alpha 1 beta 1 dimer was dissociated into the equal amounts of the alpha and beta monomers by sodium dodecyl sulfate. The alpha and beta monomers were practically inactive. The alpha 2 and beta 2 homodimers were not detected by electrophoresis and HPLC.     

Lipid transfer protein from Manduca sexta hemolymph

A hemolymph lipid transfer protein (LTP) was isolated from the tobacco hornworm, Manduca sexta. LTP catalyzes net lipid transfer between isolated hemolymph lipoproteins in vitro. An isolation procedure employing density gradient ultracentrifugation and gel permeation chromatography produced a purified protein. LTP is a very high density lipoprotein with a particle Mr greater than 500,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that LTP is comprised of two apoproteins: apoLTP-I (Mr approximately 320,000) and apoLTP-II (Mr approximately 85,000). LTP may have a physiological role in altering the lipid content and composition of the major hemolymph lipoprotein, lipophorin.     

Tac antigen forms disulfide-linked homodimers

Interleukin 2 (IL-2) is responsible for stimulating T-cell proliferation via interaction with specific, high-affinity membrane receptors. Although an IL-2-binding protein has been identified by virtue of its reactivity with a monoclonal antibody that competes with IL-2 for binding (anti-Tac), the complete and precise structure of functional IL-2 receptors is still unknown. To define further the composition of IL-2 receptors, both IL-2 itself and anti-Tac were used as ligands to adsorb membrane proteins for analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (NaDodSO4/PAGE). A variety of experimental approaches yield results indicating that the Tac antigen, which migrates as a single protein on NaDod-SO4/PAGE under reducing conditions, is also expressed as disulfide-linked homodimers and oligomers. Examined under nonreducing conditions, both activated normal human T cells and cell lines from patients with adult T cell leukemia (HUT-102; MT-1) express Tac antigen homodimers (Mr 105,000) in addition to monomers (Mr 54,000). Formation of the disulfide bond is not a consequence of the experimental procedures used to isolate the proteins for analysis, inasmuch as identical results are obtained when the receptor proteins are iodinated and extracted in the presence of N-ethylmaleimide or prepared for electrophoresis in the absence of heat denaturation. Accordingly, these findings point to Tac antigen associating with itself preferentially. The physiologic significance of homodimer and oligomer formation, especially as it relates to the formation of high-affinity IL-2 receptors, is presently unknown.     

Muscarinic cholinergic receptor of rat brain. Factors influencing migration in electrophoresis and gel filtration in sodium dodecyl sulphate

The muscarinic cholinergic receptor present in synaptosomal membranes of rat brain was covalently labelled with the alkylating muscarinic antagonist, tritiated propylbenzilylcholine mustard. The labelled receptor was then solubilized in sodium deoxycholate and sodium dodecyl sulphate, and its migration in polyacrylamide gel electrophoresis and gel filtration in the presence of sodium dodecyl sulphate analysed. Provided both proteolysis and inter-chain disulphide bond formation were vigorously prevented, the receptor from rat forebrain (cerebral cortex plus caudate putamen) migrated, in sodium dodecyl sulphate/polyacrylamide gel electrophoresis, as a broad band of apparent Mr 66000-76000. Two dominantly labelled polypeptides, of apparent Mr 68000 and 73000, could be distinguished as the major components of this band. These multiple species seen in electrophoresis may reflect a structural diversity related to the different binding properties, and modes of action, of this receptor. In electrophoresis using discontinuous buffer systems the labelled receptor readily formed intermolecular disulphide bonds and so aggregated. In particular, if solubilized membranes were reduced with 2-mercaptoethanol, and reformation of disulphide bonds during electrophoresis not prevented, then formation of a dimeric species (apparent Mr 119000-128000) occurred. This probably explains previous reports in the literature of larger-Mr species seen in electrophoresis. During gel filtration, the receptor formed intra-chain disulphide bonds which produced conformational heterogeneity, leading to polydisperse migration. In addition, extensive proteolytic degradation of the receptor occurred due to a protease migrating slightly ahead of the receptor. Both effects were eliminated by alkylation of the solubilized membranes with iodoacetamide before gel filtration. Alkylated receptor migrated on Sephacryl S-300 in 0.5% sodium dodecyl sulphate with an equivalent Stokes' radius of 6.1 nm. This is identical to that of reduced ovalbumin, a molecule with an apparent Mr in gel electrophoresis of 43000. On a different gel matrix, TSK HW 55(S), the receptor migrated with a somewhat larger Stokes' radius, eluting just behind reduced bovine serum albumin (Stokes' radius 8.5 nm; apparent Mr in electrophoresis 67000). Thus the receptor appears to adsorb to the Sephacryl matrix, although even on the TSK gel the receptor eluted as a somewhat smaller protein than expected from its behaviour in gel electrophoresis. Solubilized, alkylated receptor, partly purified by gel filtration so that it was not degraded by endogenous proteases, was not cleaved by mild hydroxylamine treatment.(ABSTRACT TRUNCATED AT 400 WORDS)     

An amphiphilic structure of the ninth component of human complement. Evidence from analysis of fragments produced by alpha-thrombin

Purified human C9 was treated separately with three proteolytic enzymes: trypsin, plasmin, and alpha-thrombin, and the digestion products were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Trypsin initially cleaved the Mr = 71,000 C9 to produce a Mr = 47,000 fragment plus numerous smaller fragments and prolonged digestion reduced the molecule to small polypeptides. Plasmin produced a Mr = 37,000 fragment which was stable to further digestion, plus fragments smaller than Mr = 10,000. Human alpha-thrombin cleaved C9 (7.8% carbohydrate) at a single internal site to produce a Mr = 37,000 fragment (11.3% carbohydrate) and a Mr = 34,000 fragment (3.9% carbohydrate). Statistical analysis of the amino acid compositions of the fragments and alkaline polyacrylamide gel electrophoresis showed that C9 is highly amphiphilic; the Mr = 34,000 fragment contains a majority of the acidic amino acids and migrates rapidly on alkaline gels; the Mr = 37,000 fragment is hydrophobic with a slow electrophoretic mobility. The two fragments remain noncovalently associated, but were separated by sodium dodecyl sulfate-hydroxylapatite chromatography. The NH2-terminal sequence analysis of native C9, of alpha-thrombin-cleaved C9, and for the isolated fragments showed that the acidic Mr = 34,000 fragment is the NH2-terminal C9a domain and the more hydrophobic Mr = 37,000 fragment is the carboxyl-terminal C9b domain. Hemolytic activity of C9 was unaffected by alpha-thrombin cleavage.