The role of valence on the high-affinity binding of Griffonia simplicifolia isolectins to type A human erythrocytes

The Griffonia simplicifolia-I (GS-I) isolectins have been used to probe the effect of lectin valence on their high-affinity binding to human erythrocytes. These tetrameric lectins are composed of A and B subunits and constitute a series of five isolectins (A4, A3B, A2B2, AB3, B4). The A subunit is specific for alpha-D-GalNAc end groups and binds to the blood type A determinant GalNAcalpha1, as well as to terminal alpha-D-Gal groups found on type B cells. The B subunit is specific for alpha-D-Gal end groups, and binds very specifically to type B erythrocytes. This series of isolectins is tetravalent (A4), trivalent (A3B), divalent (A2B2), and monovalent (AB3) for type A erythrocytes; thus, this system provides the opportunity to examine the effect of lectin valency on the association constants of these GS-I isolectins binding to cells. Cell binding experiments carried out using 125I-labeled GS-I isolectins and type A human erythrocytes allowed us to demonstrate that (1) the association constant of the isolectin monovalent for alpha-D-GalNAc (AB3) is virtually identical to its association constant for the haptenic sugar methyl-N-acetyl-alpha-D-galactosaminide, reported previously, and (2) the association constant of the GS-I isolectins for human type A erythrocytes increases with increasing valency of the isolectin. These results indicate that the increased affinity displayed by the GS-I isolectins for human type A erythrocytes is dependent on their multivalency, and not on an extended binding site nor on nonspecific, or noncarbohydrate, interactions of the lectin with the cell surface. These findings should be of general relevance to understanding the high-affinity interactions observed between other multivalent proteins and multivalent ligands (e.g., cell surfaces).     

Role of the prenyl group on the G protein gamma subunit in coupling trimeric G proteins to A1 adenosine receptors

The coupling of receptors to heterotrimeric G proteins is determined by interactions between the receptor and the G protein alpha subunits and by the composition of the betagamma dimers. To determine the role of the gamma subunit prenyl modification in this interaction, the CaaX motifs in the gamma1 and gamma2 subunits were altered to direct modification with different prenyl groups, recombinant betagamma dimers expressed in the baculovirus/Sf9 insect cell system, and the dimers purified. The activity of the betagamma dimers was compared in two assays: formation of the high affinity agonist binding conformation of the A1 adenosine receptor and receptor-catalyzed exchange of GDP for GTP on the alpha subunit. The beta1gamma1 dimer (modified with farnesyl) was significantly less effective than beta1gamma2 (modified with geranylgeranyl) in either assay. The beta1gamma1-S74L dimer (modified with geranylgeranyl) was nearly as effective as beta1gamma2 in either assay. The beta1gamma2-L71S dimer (modified with farnesyl) was significantly less active than beta1gamma2. Using 125I-labeled betagamma subunits, it was determined that native and altered betagamma dimers reconstituted equally well into Sf9 membranes containing A1 adenosine receptors. These data suggest that the prenyl group on the gamma subunit is an important determinant of the interaction between receptors and G protein gamma subunits.     

Quaternary structure regulates hemin dissociation from human hemoglobin

Rate constants for hemin dissociation from the alpha and beta subunits of native and recombinant human hemoglobins were measured as a function of protein concentration at pH 7.0, 37 degrees C, using H64Y/V68F apomyoglobin as a hemin acceptor reagent. Hemin dissociation rates were also measured for native isolated alpha and beta chains and for recombinant hemoglobin tetramers stabilized by alpha subunit fusion. The rate constant for hemin dissociation from beta subunits in native hemoglobin increases from 1.5 h-1 in tetramers at high protein concentration to 15 h-1 in dimers at low concentrations. The rate of hemin dissociation from alpha subunits in native hemoglobin is significantly smaller (0.3-0.6 h-1) and shows little dependence on protein concentration. Recombinant hemoglobins containing a fused di-alpha subunit remain tetrameric under all concentrations and show rates of hemin loss similar to those observed for wild-type and native hemoglobin at high protein concentration. Rates of hemin dissociation from monomeric alpha and beta chains are much greater, 12 and 40 h-1, respectively, at pH 7, 37 degrees C. Aggregation of monomers to form alpha1beta1 dimers greatly stabilizes bound hemin in alpha chains, decreasing its rate of hemin loss approximately 20-fold. In contrast, dimer formation has little stabilizing effect on hemin binding to beta subunits. A significant reduction in the rate of hemin loss from beta subunits does occur after formation of the alpha1beta2 interface in tetrameric hemoglobin. These results suggest that native human hemoglobin may have evolved to lose heme rapidly after red cell lysis, allowing the prosthetic group to be removed by serum albumin and apohemopexin.     

Expression, purification, and properties of recombinant barley (Hordeum sp.) hemoglobin. Optical spectra and reactions with gaseous ligands

A cDNA encoding barley hemoglobin (Hb) has been cloned into pUC 19 and expressed in Escherichia coli. The resulting fusion protein has five extra amino acids at the N terminus compared with the native protein, resulting in a protein of 168 amino acids (18.5 kDa). The recombinant Hb is expressed constitutively. Extracts made from the bacteria containing the recombinant fusion construct contain a protein with a subunit molecular mass of approximately 18.5 kDa comprising approximately 5% total soluble protein. Recombinant Hb was purified to homogeneity according to SDS-polyacrylamide gel electrophoresis by sequential polyethylene glycol precipitation and fast protein liquid chromatography. Its native molecular mass as assessed by fast protein liquid chromatography-size exclusion was 40 kDa suggesting that it is a dimer. Ligand binding experiments demonstrate that 1) barley Hb has a very slow oxygen dissociation rate constant (0.0272 s-1) relative to other Hbs, and 2) the heme of ferrous and ferric forms of the barley Hb is low spin six-coordinate. The subunit structure, optical spectrum, and oxygen dissociation rate of native barley hemoglobin are indistinguishable from those obtained for the recombinant protein. The implications of these kinetic data on the in vivo function of barley Hb are discussed.     

Novel aspects of tetramer assembly and N-terminal domain structure and function are revealed by recombinant expression of human AMP deaminase isoforms

AMP deaminase isoforms purified from endogenous sources display smaller than predicted subunit molecular masses, whereas baculoviral expression of human AMPD1 (isoform M) and AMPD3 (isoform E) cDNAs produces full-sized recombinant enzymes. However, nearly 100 N-terminal amino acid residues are cleaved from each recombinant polypeptide during storage at 4 degreesC. Expression of N-truncated cDNAs (DeltaL96AMPD1 and DeltaM90AMPD3) produces stable recombinant enzymes exhibiting subunit molecular masses and kinetic properties that are similar to those reported for purified isoforms M and E. Conversely, wild type recombinant isoforms display significantly higher Km(app) values in the absence of ATP. Gel filtration analysis demonstrates native tetrameric structures for all recombinant proteins, except the wild type AMPD1 enzyme, which forms aggregates of tetramers that disperse upon cleavage of N-terminal residues at 4 degreesC. These data: 1) confirm that available literature on AMP deaminase is likely derived from N-truncated enzymes and 2) are inconsistent with a new model proposing native trimeric structure of an N-truncated rabbit skeletal muscle AMP deaminase (Ranieri-Raggi, M., Montali, U., Ronca, F., Sabbatini, A., Brown, P. E., Moir, A. J. G., and Raggi, A. (1997) Biochem. J. 326, 641-648). N-terminal residues also influence actomyosin-binding properties of the enzyme, which are enhanced and suppressed by AMPD1 and AMPD3 sequences, respectively. Finally, co-expression of AMPD1 and AMPD3 recombinant polypeptides produces tetrameric enzymes with either isoform-specific or mixed subunits, and also reveals that tetramer assembly is driven by relative polypeptide abundance with no apparent preference for like subunits.     

Studies on the disulfide bonds of glycoprotein hormones. Complete reduction and reoxidation of the disulfide bonds of the alpha subunit of bovine luteinizing hormone

Reoxidation of the disulfide bonds of the alpha subunit of bovine luteinizing hormone (LH) after their complete reduction both in the presence and absence of denaturing agent yields a product which is indistinguishable from the native subunit in its electrophoretic pattern on polyacrylamide gels and in its ability to recombine with the beta subunits of both luteinizing hormone and thyrotropin. The circular dichroism spectrum of the reoxidized alpha subunit is essentially identical to that of native alpha subunit except that its maximum at 233 nm is smaller than observed with native LHalpha. The intact hormone preparations obtained by recombination of reoxidized alpha subunit with native LH-beta exhibit electrophoretic patterns in polyacrylamide gels, elution profiles on gel filtration, binding activities to a membrane fraction from rat testes, and circular dichroism spectra identical to those of native LH and recombinants of native LH-alpha with the beta subunit. Recombinants of native or reoxidized LH-alpha with the beta subunit of thyrotropin are also indistinguishable in their electrophoretic patterns on polyacrylamide gels and in their in vivo activities of stimulating 32P uptake in thyroids of day-old chicks. While this study does not preclude that the alpha subunit may be biosynthesized as part of a larger precursor protein, the data demonstrate that sufficient information is present in the linear sequence of the alpha subunit to allow folding and formation of disulfide bonds to yield a functional alpha subunit.     

PDGF-alpha receptor subunit expression down-regulated by IL-1beta in human periodontal ligament cells

The responses of cells to the distinct PDGF isoforms have been correlated directly to the relative numbers of specific PDGF receptor subunits on the cell surface. The modulation of PDGF-alpha receptor subunits, the major subunit expressed in human periodontal ligament (PDL) cells, by cytokines present in the periodontal wound site, such as interleukin-1 (IL-1), may be an important factor influencing regenerative outcomes. The purpose of the present study was to examine the effects of IL-1 beta on PDGF-alpha receptor subunit expression in human PDL cells. Primary cultures of human PDL cells were treated with IL-1 beta over a range of concentrations. We assessed PDGF-alpha receptor subunits by examining the mitogenic responses of cells to PDGF-AA, specific binding of 125I-labeled PDGF-AA, immunofluorescent analysis of PDGF-alpha receptor subunits, and PDGF-alpha receptor subunit mRNA levels using Northern blot analysis. The results demonstrate a significant concentration-dependent decrease in 3H-thymidine incorporation in response to PDGF-AA following IL-1 beta treatment (p < 0.001). This decreased response correlated directly with IL-1-induced decreases in 125I-labeled PDGF-AA binding (p < 0.01), the numbers of immunolabeled PDGF-alpha receptor subunits, and in PDGF-alpha receptor subunit mRNA levels. However, when combined with TGF-beta, IL-1 beta did not show additional down-regulation in proliferative response to PDGF-AA or PDGF-alpha receptor subunits beyond that achieved with these factors individually. These experiments identify IL-1 beta, along with TGF-beta, as significant inhibitors of PDGF stimulation in human PDL cells, acting through the down-regulation of PDGF-alpha receptor subunit expression.     

Kinetic properties of a single nucleotide binding site on chloroplast coupling factor 1 (CF1)

The kinetics of nucleotide binding to spinach chloroplast coupling factor CF1 in a fully inhibited state were investigated by stopped-flow experiments using the fluorescent trinitrophenyl analogue (NO2)3Ph-ADP. The CF1 was in a state in which two of the three binding sites on the beta subunits were irreversibly blocked with ADP, Mg2+ and fluoroaluminate, while the three binding sites on the alpha subunits were occupied by nucleotides [Garin, J., Vincon, M., Gagnon, J. & Vignais, P. V. (1994) Biochemistry 33, 3772-3777)]. Thus, it was possible to characterise a single nucleotide-binding site without superimposed nucleotide exchange or binding to an additional site. (NO2)3Ph-ADP binding to the remaining site on the third beta subunit was characterised by a high dissociation rate of 15 s(-1), leading to a very low affinity (dissociation constant higher than 150 microM). Subsequent to isolation, CF1 preparations contained two endogenously bound nucleotides. Pre-loading with ATP yielded CF1 with five tightly bound nucleotides and one free nucleotide-binding site on a beta subunit. Pre-loading with ADP, however, resulted in a CF1 preparation containing four tightly bound nucleotides and two free nucleotide binding sites. One of the two free binding sites was located on a beta subunit, while the other was probably located on an alpha subunit.     

His64(E7)-->Tyr apomyoglobin as a reagent for measuring rates of hemin dissociation

To develop an assay for hemin dissociation, His64(E7) was replaced by Tyr in sperm whale myoglobin producing a holoprotein with a distinct green color due to an intense absorption band at 600 nm. Val68(E11) was replaced by Phe in the same protein to increase its stability. When excess Tyr64-Val68 apoglobin is mixed with either metmyoglobin or methemoglobin, the solution turns from brown to green, and the absorbance changes can be used to measure complete time courses for hemin dissociation from either holoprotein. This assay has been used to measure rates of hemin dissociation from native metmyoglobin, four myoglobin mutants (Ala64(E7), Ala68(E11), Phe68(E11), and Glu45(CD3)), native methemoglobin, valence hybrid hemoglobins, and two mutant hemoglobins ((alpha(Gly-E7)beta(native))2, and (alpha(native)beta(Gly-E7))2). Two kinetic phases were observed for hemin dissociation from native human hemoglobin at pH 7.0 and 37 degrees C. Valence and mutant hybrid hemoglobins were used to assign the faster phase (k = 7.8 +/- 2.0 h-1) to hemin dissociation from ferric beta subunits and the slower (k = 0.6 +/- 0.15 h-1) to dissociation from alpha subunits. The corresponding rate for wild-type metmyoglobin is 0.007 +/- 0.004 h-1.     

The Ost1p subunit of yeast oligosaccharyl transferase recognizes the peptide glycosylation site sequence, -Asn-X-Ser/Thr-

Other laboratories have established that oligosaccharyl transferase (OST) from Saccharomyces cerevisiae can be purified as a protein complex containing eight different subunits. To identify the OST subunit that recognizes the peptide sites that can be glycosylated, we developed photoaffinity probes containing a photoreactive benzophenone derivative, p-benzoylphenylalanine (Bpa), as part of an 125I-labeled peptide that could be expected to be glycosylated. We found that Asn-Bpa-Thr peptides served as substrates for OST and that photoactivation of these probes in the presence of microsomes abolished the OST activity. Photoactivation of 125I-labeled Asn-Bpa-Thr in the presence of microsomes resulted in specific covalent labeling of a protein doublet of molecular mass 62 and 64 kDa. By carrying out the photoactivation of the probe using microsomes containing epitope-tagged Ost1p, we demonstrated that the 125I-labeled protein was Ost1p. Radiolabeling of this protein was dependent on irradiation at 350 nm. No labeling was detected using a probe containing Ala instead of Thr as the third amino acid residue. We conclude that Ost1p is the subunit of the OST complex that recognizes the peptide sites in the nascent chains that are destined to be glycosylated.     

Mechanism of auxiliary subunit modulation of neuronal alpha1E calcium channels

Voltage-gated calcium channels are composed of a main pore-forming alpha1 moiety, and one or more auxiliary subunits (beta, alpha2 delta) that modulate channel properties. Because modulatory properties may vary greatly with different channels, expression systems, and protocols, it is advantageous to study subunit regulation with a uniform experimental strategy. Here, in HEK 293 cells, we examine the expression and activation gating of alpha1E calcium channels in combination with a beta (beta1-beta4) and/or the alpha2 delta subunit, exploiting both ionic- and gating-current measurements. Furthermore, to explore whether more than one auxiliary subunit can concomitantly specify gating properties, we investigate the effects of cotransfecting alpha2delta with beta subunits, of transfecting two different beta subunits simultaneously, and of COOH-terminal truncation of alpha1E to remove a second beta binding site. The main results are as follows. (a) The alpha2delta and beta subunits modulate alpha1E in fundamentally different ways. The sole effect of alpha2 delta is to increase current density by elevating channel density. By contrast, though beta subunits also increase functional channel number, they also enhance maximum open probability (Gmax/Qmax) and hyperpolarize the voltage dependence of ionic-current activation and gating-charge movement, all without discernible effect on activation kinetics. Different beta isoforms produce nearly indistinguishable effects on activation. However, beta subunits produced clear, isoform-specific effects on inactivation properties. (b) All the beta subunit effects can be explained by a gating model in which subunits act only on weakly voltage-dependent steps near the open state. (c) We find no clear evidence for simultaneous modulation by two different beta subunits. (d) The modulatory features found here for alpha1E do not generalize uniformly to other alpha1 channel types, as alpha1C activation gating shows marked beta isoform dependence that is absent for alpha1E. Together, these results help to establish a more comprehensive picture of auxiliary-subunit regulation of alpha1E calcium channels.     

Characterization of active recombinant his-tagged oxygenase component of Comamonas testosteroni B-356 biphenyl dioxygenase

Biphenyl (BPH) dioxygenase oxidizes BPH to 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356. The enzyme comprises a two-subunit iron-sulfur protein (ISPBPH), a ferredoxin FERBPH, and a ferredoxin reductase REDBPH. REDBPH and FERBPH transfer electrons from NADH to an Fe-S active center of ISPBPH which activates molecular oxygen for insertion into the substrate. In this work B-356 ISPBPH complex and its alpha and beta subunits were purified from recombinant Escherichia coli strains using the His-bind QIAGEN system. His-tagged B-356 ISPBPH construction carrying a single His tail on the N-terminal portion of the alpha subunit was active. Its major features were compared to the untagged enzyme. In both cases, the native form is an alpha3beta3 heteromer, with each alphabeta unit containing a [2Fe-2S] Rieske center (epsilon455 = 8,300 M-1 cm-1) and a mononuclear Fe2+. Although purified His-tagged alpha subunit showed the characteristic absorption spectra of Rieske-type protein, reassociation of this enzyme component and His-tagged beta subunit to reconstitute active ISPBPH was weak. However, when His-tagged alpha and beta subunits were reassembled in vitro in crude cell extracts from E. coli recombinants, active ISPBPH could be purified on Ni-nitrilotriacetic acid resin.     

The expression of voltage-dependent calcium channel beta subunits in human cerebellum

The beta subunits of voltage-dependent calcium channels, exert marked regulatory effects on the biophysical and pharmacological properties of this diverse group of ion channels. However, little is known about the comparative neuronal expression of the four classes of beta genes in the CNS. In the current investigation we have closely mapped the distribution of beta1, beta2, beta3 and beta4 subunits in the human cerebellum by both in situ messenger RNA hybridization and protein immunohistochemistry. To our knowledge, these studies represent the first experiments in any species in which the detailed localization of each beta protein has been comparatively mapped in a neuroanatomically-based investigation. The data indicate that all four classes of beta subunits are found in the cerebellum and suggest that in certain neuronal populations they may each be expressed within the same cell. Novel immunohistochemical results further exemplify that the beta voltage-dependent calcium channel subunits are regionally distributed in a highly specific manner and studies of Purkinje cells indicate that this may occur at the subcellular level. Preliminary indication of the subunit composition of certain native voltage-dependent calcium channels is suggested by the observation that the distribution of the beta3 subunit in the cerebellar cortex is identical to that of alpha(1E). Our cumulative data are consistent with the emerging view that different native alpha1/beta subunit associations occur in the CNS.     

Restoration of 125I-alpha-bungarotoxin binding activity to the alpha subunit of Torpedo acetylcholine receptor isolated by gel electrophoresis in sodium dodecyl sulfate

The four subunits (alpha, beta, gamma, delta) of the acetylcholine receptor from Torpedo californica have been isolated by preparative gel electrophoresis in sodium dodecyl sulfate. After removal of the sodium dodecyl sulfate by dialysis of the polypeptides against a cholate-containing buffer, the alpha subunit, but not the other chains, binds 125I-alpha-bungarotoxin in a saturable manner. The binding affinity, 0.1-0.2 microM, is approximately 10(4)-fold lower than that observed for native acetylcholine receptor. For three preparations of alpha subunit, 1 mol of subunit bound 0.87, 0.38, and 0.33 mol of 125I-alpha-bungarotoxin at saturation. The binding was inhibited by cholinergic ligands, although the apparent affinities of these ligands for alpha were 50-100-fold lower than that found for the native receptor. These results indicate that at least part of the alpha-bungarotoxin binding site resides on the alpha subunit.     

Electrospray mass spectrometry studies of non-heme iron-containing proteins

The oligomeric state and the metal atom stoichiometry of a series of non-heme iron-containing, multimeric proteins have been measured using electrospray ionization (ESI) in a time-of-flight (TOF) mass spectrometer. The proteins were obtained both from natural sources and by overexpression of recombinant DNA in Escherichia coli. ESI-TOF mass spectra of the metalloproteins present in nondenaturing solutions exhibit peaks corresponding to the multimeric forms of the holoproteins containing the expected number of metal atoms. Capillary-skimmer dissociation of the holoproteins produces a series of ions, which allows an exact count of the number of metal atoms present in each subunit, and also provides an indication of the oxidation state of the metal atoms. Two recombinant proteins, Phascolopsis gouldii hemerythrin (Pg-Hr) and Desulfovibrio vulgaris rubrerythrin (Dv-Rr), have been examined as well as hemerythrin isolated from Lingula reevii (Lr-Hr). ESI-TOF measurements of the aqueous solution of Pg-Hr at pH 6 yields ions of mass 108,783 Da, in close agreement with the calculated average molecular mass of an intact octameric holoprotein. Capillary-skimmer dissociation of the ions of the holoprotein produces a mass spectrum that contains peaks corresponding to a low m/z monomer and a high m/z heptamer. The masses of the monomer ions produced in this manner are assigned to the aposubunit, [subunit + Fe - 3H]+, and [subunit + 2Fe - 6 H]+. Naturally occurring Lr-Hr is composed of two subunits with average molecular masses measured under denaturing conditions by ESI-TOF to be 13,877.0 Da for the alpha-subunit and 13,517.5 Da for the beta-subunit. Under nondenaturing conditions, a multimeric species with a molecular weight of 110,663 Da is measured by ESI-TOF, corresponding to an alpha 4 beta 4 octamer. Capillary-skimmer dissociation of the alpha 4 beta 4 oligomer produces ions corresponding to both types of monomers (alpha and beta) and the corresponding heptamers (alpha 3 beta 4 and alpha 4 beta 3). In ESI-TOF measurements of recombinant rubrerythrin Dv-Rr using nondenaturing conditions, the principal ion observed corresponds to a homotetramer with an average molecular mass of 86,844 Da. Capillary-skimmer dissociation of the rubrerythrin tetramer leads to formation of a series of peaks corresponding to the subunit of the apoprotein and to subunits containing from one to three specifically bound iron atoms.     

Cerebrolysin ameliorates performance deficits, and neuronal damage in apolipoprotein E-deficient mice

We report a method for the purification and radioactive labeling of human TSH receptor (TSHR). The method is based on the construction of a fusion TSHR (TSHR-Xa-BIO) which consists of the N-terminal 725 amino acids of human TSHR linked to the 4-amino acid Xa protease cleavage site and the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase (the C-terminal domain directs the efficient posttranslational biotinylation of the protein). TSHR-Xa-BIO was produced in HeLa cells using recombinant vaccinia virus. The expressed protein was fully functional and was biotinylated with an efficiency of about 90%. Streptavidin-agarose-immobilized TSHR-Xa-BIO was labeled with 125I using the chloramine T oxidation procedure and specifically eluted from the solid phase after cleavage with protease Xa. Isolated native radiochemically pure 125I-labeled TSHR specifically interacted with pathological autoantibodies in the sera of patients with Graves' disease, and thus could be useful for the detection of these autoantibodies by immunoprecipitation analysis.     

Left ventricular diastolic function in young adults: the Coronary Artery Risk Development in Young Adults Study

Shiga-like toxin type I (SLT-I) is a cytotoxin produced by certain strains of Escherichia coli. SLT-I is a bipartite molecule comprised of A (SLT-IA) and B (SLT-IB) subunits. In holotoxin, five B subunits are arranged in a pentameric ring and bind to globotriaosylceramide receptors on the surface of susceptible eukaryotic cells. The SLT-IB pentamer is noncovalently associated with one A subunit that has N-glycosidase activity and ultimately causes the death of targeted cells. Using a previously described overexpression vector, plasmid SBC32, we developed a two-step procedure for the purification of biologically active recombinant SLT-IB. Periplasmic proteins were extracted from E. coli JM105(pSBC32), fractionated by ammonium sulfate precipitation, and separated by isoelectric focusing in a pH 3-10 gradient. SLT-IB was present in fractions with pH values between 5.0 and 6.0, consistent with its reported pI of 5.8. SLT-IB was purified to homogeneity in a second step by native polyacrylamide gel electrophoresis. Purified SLT-IB was characterized for biological and biochemical activity. When analyzed by native polyacrylamide gel electrophoresis, the majority of SLT-IB had an apparent molecular weight of 38,900, consistent with a pentameric subunit association. Chemical cross-linking of SLT-IB with disuccinimidyl suberate resulted in species with molecular weights corresponding to dimeric, trimeric, tetrameric, and pentameric forms of B subunit. SLT-IB was not cytotoxic to Vero cells at concentrations as high as 10 micrograms/ml and protected Vero cells from native SLT-I. Purified SLT-IB maintained its ability to associate with SLT-IA to form holotoxin that exhibited toxicity similar to native toxin.     

A hierarchy of disulfide-bonded subunits: the quaternary structure of Eudistylia chlorocruorin

The quaternary structure of the cysteine-rich, approximately 3500-kDa chlorocruorin (Chl) from the marine polychaete Eudistylia vancouverii was investigated using maximum entropy deconvolution of the electrospray ionization mass spectra (ESIMS). The native Chl provided two groups of peaks, at approximately 25 and approximately 33 kDa, and one peak at approximately 66 kDa. ESIMS of the reduced and reduced and carbamidomethylated Chl and of its subunits obtained by HPLC provided the complete subunit composition of the Chl. Two groups of nonglobin linker chains were observed: L1a-f (25 000.4, 25 017.9, 25 039.6, 25 057.0, 25 074.4 and 25 096.8 Da) and L2a-d (25 402.7, 25 446.0, 25 461.6 and 25 478.3Da) (+/-2.5 Da), with relative intensities L1:L2 = 5:2. Six globin chains were found, a1, a2, and b1-4, with reduced masses of 16 051.5, 16 172.4, 16 853.5, 17 088.9, 17 161.2 and 17 103.6 (+/-1.0 Da) and relative intensities of 8:4:1:4:2:1, respectively. Disulfide-bonded dimers and a tetramer of globin chains were identified: D1 = a1 + b3 at 33 207.1; D2 at 33 374.1, which had a cysteinylated Cys (a2 + b2 + Cys); and D3 = a1 + b4 at 33 149.4 Da (+/-3.0 Da), with relative intensities D1:D2:D3 = 5:4:1 and T = a1 + a2 + b1 + b2 at 66 154.8 +/- 4.0 Da. A 206-kDa dodecamer subunit obtained by dissociation of the Chl in 4 M urea [Qabar, A. N., et al. (1991) J. Mol. Biol. 222, 1109-1129], was found to consist only of tetramers T. A model was proposed for the Chl, based on a dimer:tetramer ratio of 2:1: four 206-kDa dodecamers (trimer of tetramers) and 48 dimers tethered to a framework of 30 L1 and 12 L2 linker chains. The 144 globin chains (2480 kDa) and 42 linker chains (1059 kDa) provide a total mass of 3539 kDa, in good agreement with the 3480 +/- 225 kDa determined previously by STEM mass mapping. The hierarchy of disulfide-bonded globin subunits observed for Eudistylia Chl provides a built-in heterogeneity of hexagonal bilayer structures.     

Formation of native hepatitis C virus glycoprotein complexes

The hepatitis C virus (HCV) glycoproteins (E1 and E2) interact to form a heterodimeric complex, which has been proposed as a functional subunit of the HCV virion envelope. As examined in cell culture transient-expression assays, the formation of properly folded, noncovalently associated E1E2 complexes is a slow and inefficient process. Due to lack of appropriate immunological reagents, it has been difficult to distinguish between glycoprotein molecules that undergo productive folding and assembly from those which follow a nonproductive pathway leading to misfolding and aggregation. Here we report the isolation and characterization of a conformation-sensitive E2-reactive monoclonal antibody (H2). The H2 monoclonal antibody selectively recognizes slowly maturing E1E2 heterodimers which are noncovalently linked, protease resistant, and no longer associated with the endoplasmic reticulum chaperone calnexin. This complex probably represents the native prebudding form of the HCV glycoprotein heterodimer. Besides providing a novel reagent for basic studies on HCV virion assembly and entry, this monoclonal antibody should be useful for optimizing production and isolation of native HCV glycoprotein complexes for serodiagnostic and vaccine applications.