Disulphide bridges of the heavy chain of human immunoglobulin G2

Amino acid sequences around the disulphide bridges of the heavy chain of an immunoglobulin of the gamma2 subclass have been studied. The protein was digested with pepsin and the digest fractionated by Sephadex. Screening of the eluate by one-dimensional electrophoresis of oxidized and unoxidized samples was used as an assay and pools of fractions were prepared. Identification by diagonal electrophoresis of several inter- and intra-chain disulphide bridges was done on the pooled fractions. The inter-heavy-chain bridged peptide included four cystine residues. Comparison with proteins of other human subclasses indicated that the intrachain bridges identified are the bridges of the invariable section of gamma2 heavy chains. The amino acid sequence of one cysteic acid peptide that may have been derived from the variable part of the molecule was determined. Partial reduction followed by carboxymethylation with radioactive iodoacetate of two proteins of the gamma2 class showed a number of labelled peptides that could be identified as being related to the inter-chain bonded cystine residues.     

The recombination of dimers of immunoglobulin peptide chains

1. Both the gamma and light peptide chains of human pooled and myeloma immunoglobulin G can be prepared as non-aggregating dimers at pH5.4 in 4mm-sodium acetate buffer. The dimeric state is maintained by non-covalent bonds, since the formation of interchain disulphide bonds was prevented by alkylation of the thiol groups. In the case of the light chains there is some evidence that the dimers are in equilibrium with a small amount of monomer. 2. When such dimers of the gamma and light chains are mixed at pH5.4 in 4mm-sodium acetate buffer they combine rapidly, yielding a product that resembles the original immunoglobulin G in its physicochemical and antigenic properties. However, the original optical rotatory dispersion spectrum was regained only with the homogeneous myeloma protein. The recombined pooled immunoglobulin G had a spectrum slightly different from the original, suggesting that at least some of the recombinant molecules had not regained native conformations. 3. Dimers of gamma chains stabilized by interchain disulphide bonds were able to recombine with light chains. However, light chains stabilized in the dimeric state by interchain disulphide bonds would not combine with gamma chains. 4. The chains of rabbit immunoglobulin G behave similarly to the human chains in this system, apart from the alkylated light chains showing clearer evidence of monomeric components.     

Location of disulfide bonds within the sequence of human serum cholinesterase

Human serum cholinesterase was digested with pepsin under conditions which left disulfide bonds intact. Peptides were isolated by high pressure liquid chromatography, and those containing disulfide bonds were identified by a color assay. Peptides were characterized by amino acid sequencing and composition analysis. Human serum cholinesterase contains 8 half-cystines in each subunit of 574 amino acids. Six of these form three internal disulfide bridges: between Cys65-Cys92, Cys252-Cys263, and Cys400-Cys519. A disulfide bond with Cys65 rather than Cys66 was inferred by homology with Torpedo acetylcholinesterase. Cys571 forms a disulfide bridge with Cys571 of an identical subunit. This interchain disulfide bridge is four amino acids from the carboxyl terminus. A peptide containing the interchain disulfide is readily cleaved from cholinesterase by trypsin (Lockridge, O., and La Du, B. N. (1982) J. Biol. Chem. 257, 12012-12018), suggesting that the carboxyl terminus is near the surface of the globular tetrameric protein. The disulfide bridges in human cholinesterase have exactly the same location as in Torpedo californica acetylcholinesterase. There is one potential free sulfhydryl in human cholinesterase at Cys66, but this sulfhydryl could not be alkylated. Comparison of human cholinesterase, and Torpedo and Drosophila acetylcholinesterases to the serine proteases suggests that the cholinesterases constitute a separate family of serine esterases, distinct from the trypsin family and from subtilisin.     

The disulphide bonds of an Asian influenza virus neuraminidase

The arrangement of the disulphide bonds in the pronase-released neuraminidase heads of the Asian influenza virus A/Tokyo/3/67 have been examined by cyanogen bromide fragmentation, enzymic digestion and diagonal peptide mapping. There are 9 intrachain disulphide bridges and one interchain bridge which links pairs of monomers at the distal end of the stalk region of the neuraminidase tetramer. The disulphide bond arrangements of the remaining 3 half-cystine residues in the membrane-embedded stalk region of the neuraminidase were not examined.     

Interchain disulphide bridges of mouse immunoglobulin M.

Mouse IgM (immunoglobulin M) was selectively and partially reduced and treated with iodo[2-14C]acetate to label the interchain disulphide bridges. The carboxymethylation was studied in some detail. The labelled peptides were purified, sequenced and positioned by homology with human IgM. Only peptides originating from three interchain disulphide bridges were labelled, in contrast with the four labelled bridges obtained in human IgM under the same conditions. These peptides are homologous to human bridge peptides forming the heavy-light bridge and two inter-heavy bridges, one present in the CMU2 region and the other in the C-terminal region. The inter-heavy bridge in the Cmu2 region was alone cleaved and radioactively labelled in selectively reduced IgM held together as a pentamer by non-covalen interactions. The same bridge was the only one to be totally cleaved in subunits released after more extensive, though still selective, reduction. In the light of these results a possible arrangement of the disulphide bridges of the mouse IgM.     

The disulphide bridges of immunoglobulin κ-chains

The arrangement of the disulphide bridges of the major component of the light chains of immunoglobulins (kappa-chains) has been studied in the Bence-Jones proteins. Three disulphide bridges have been found. An interchain bridge at the C-terminus has been shown to occur in the dimers of all the proteins studied and was characterized by symmetrical peptides. In the monomer form, the C-terminal half-cystine of the corresponding peptides was linked to a lone half-cystine residue. A second common disulphide-bridge peptide in which a single amino acid difference could be related to the Inv factors of the individual proteins was found in Bence-Jones proteins and in the kappa-chains of normal and abnormal immunoglobulins. Peptides characteristic of a third disulphide bridge studied in three specimens were found to have differences in some residues, but also striking similarities. A methionine peptide has also been characterized in two specimens as a by-product of the technique employed. It is suggested that a general manner of folding may be a common feature of the heterogeneous population of kappa-chains: one bridge which folds an invariable stretch of the chain, another bridge which folds a stretch that varies from protein to protein, and a bridge at the C-terminus which is the interchain link.     

Isolation and biochemical characterization of the alpha- and beta-subunits of glycoprotein IIb of human platelet plasma membrane.

The alpha- and beta-subunits of glycoprotein IIb (GPIIb) of human platelet plasma membrane were isolated in fully reduced, partially reduced and alkylated, and fully alkylated forms, by size-exclusion chromatography after reduction of pure GPIIb. The sugar moiety of GPIIb alpha accounts for 16.4% of its total weight, whereas that of GPIIb beta accounts for only 10.2%. The molar percentages (per 100 mol of total amino acids) of neuraminic acid and galactose in the alpha-subunit more than double those in the beta-subunit, whereas galactosamine is present only in GPIIb alpha. From the amino acid and sugar compositions the acidic nature of both subunits was confirmed. The Mr values obtained, 114,000 for GPIIb alpha and 22,200 for GPIIb beta, are in very good agreement with those obtained by physical methods. We found by stepwise reduction of pure GPIIb with dithioerythritol that GPIIb alpha and GPIIb beta are joined by a single interchain disulphide bridge, while the remaining half-cystine residues participate in intrachain bonds, six in GPIIb alpha and one in GPIIb beta, the intersubunit disulphide bond being that reduced first. Neither of the two subunits is liberated from isolated plasma membranes when this GPIIb interchain bond is reduced in isolated membranes.     

Fb''2, a new peptic fragment of human immunoglobulin G.

The digestion of a human IgG1 K myeloma protein with pepsin in the presence of 8M-urea was observed to produce a fragment, designated Fb'2, which differed from the products of aqueous peptic digestion and from other characteristic immunoglobulin digestion products. 2. Fragment Fb's was also found when two other IgG1/K proteins were treated similarly. 3. Sedimentation-equilibrium studies showed the mol.wt. of fragment Fb'2 to be 56800. 4. On reduction, two equivalents of each of three peptides were released from fragment Fb's; these were characterized by N- and C-terminal determinations and by amino acid sequencing. 5. Fragment Fb'2 was shown to consist of the constant regions of both light chains, from residue Ile-117 to the C-terminus, and the CH1 domains and hinge region of the heavy chains, from residue Val-113 to residue Met-252, with a gap of five residues within the intrachain disulphide loop, between residues Leu-174 and Tyr-180.     

Protein folding/refolding analysis by mass spectrometry. Scrambling of disulphide bridges in insulin.

In this paper we present a protocol that allows a dynamic analysis of disulphide-bridge formation, based on freezing the intermediates by acid/acetone precipitation, followed by digestion with pepsin and direct fast-atom-bombardment mass-spectrometric analysis. A rapid definition of the exact nature of disulphide bridges formed can be obtained via a definitive assignment of disulphide-linked peptides according to their unique mass values. With the use of an appropriate thiol concentration, scrambling of the native disulphide bonds in bovine insulin occurs, and the process is catalysed by protein disulphide-isomerase (EC 5.3.4.1). The disruption of native and the formation of new disulphide bonds can be monitored as described above, and interestingly B-chain dimers containing Cys-B7-Cys-B7 and Cys-B7-Cys-B19 bonds are detected.     

Large-scale prediction of disulphide bridges using kernel methods, two-dimensional recursive neural networks, and weighted graph matching

The formation of disulphide bridges between cysteines plays an important role in protein folding, structure, function, and evolution. Here, we develop new methods for predicting disulphide bridges in proteins. We first build a large curated data set of proteins containing disulphide bridges to extract relevant statistics. We then use kernel methods to predict whether a given protein chain contains intrachain disulphide bridges or not, and recursive neural networks to predict the bonding probabilities of each pair of cysteines in the chain. These probabilities in turn lead to an accurate estimation of the total number of disulphide bridges and to a weighted graph matching problem that can be addressed efficiently to infer the global disulphide bridge connectivity pattern. This approach can be applied both in situations where the bonded state of each cysteine is known, or in ab initio mode where the state is unknown. Furthermore, it can easily cope with chains containing an arbitrary number of disulphide bridges, overcoming one of the major limitations of previous approaches. It can classify individual cysteine residues as bonded or nonbonded with 87% specificity and 89% sensitivity. The estimate for the total number of bridges in each chain is correct 71% of the times, and within one from the true value over 94% of the times. The prediction of the overall disulphide connectivity pattern is exact in about 51% of the chains. In addition to using profiles in the input to leverage evolutionary information, including true (but not predicted) secondary structure and solvent accessibility information yields small but noticeable improvements. Finally, once the system is trained, predictions can be computed rapidly on a proteomic or protein-engineering scale. The disulphide bridge prediction server (DIpro), software, and datasets are available through www.igb.uci.edu/servers/psss.html.     

Assembly of immunoglobulin M. Blocked thiol groups of intracellular 7S subunits

We have shown previously that immunoglobulin M (IgM) is present within IgM-forming cells mainly in its 7S subunit form (IgMs), whereas only fully assembled IgM pentamers are secreted. There is no spontaneous polymerization of intracellular IgMs in cell lysates, suggesting that the 7S subunits had blocked cysteine residues. This suggestion was explored and confirmed in the present paper. Radioactive IgM (secreted) and IgMs (intracellular) were prepared by sucrose-density-gradient centrifugation after incubation of cells of the IgM-producing mouse myeloma MOPC 104E with [(3)H]leucine. We investigated the susceptibility to reduction of fully assembled mouse IgM and its reconstitution from subunits by analysis by polyacrylamide-gel electrophoresis under dissociating conditions. With increasing concentrations of dithioerythritol, interchain disulphide bonds were cleaved in the following order: inter-IgMs subunit, intra-IgMs subunit H-H, intra-IgMs subunit H-L. Removal of the reducing agent from IgM-reduction mixtures by filtration through Sephadex G-25 caused partial reconstitution of IgM at low protein concentrations (5-100mug/ml) and total reconstitution at higher protein concentrations (300mug/ml or more). Isolated radioactive intracellular IgMs showed no tendency to polymerize unless first treated with a reducing agent; under optimum conditions removal of the reducing agent caused 70% of the subunits to be assembled into IgM. Similar assembly occurred when IgMs was isolated from cells that had been lysed in the presence of an irreversible alkylating reagent (iodoacetamide). The intracellular IgMs cysteine residues responsible for inter-IgMs linkage therefore appear to be reversibly blocked within the cells. Assembly into IgM is thus controlled by removal of this block during secretion.     

Rule of antibody structure. Primary structure of a human monoclonal IgA-immunoglobulin (myeloma protein Tro). VII. Purification and characterization of the disulfide bridges]

Myeloma Protein Tro has been isolated from the plasma of a myeloma patient. Monomeric IgA was separated from its polymer (by chromatography on Sephadex G-200). Both the forms were split with pepsin or cyanogen bromide and, if necessary, with thermolysin and subtilisin. The cystin-containing peptides were isolated from the hydrolysates by chromatography on Sephadex, ion-exchange columns, preparative paper chromatography, thin-layer chromatography, electrophoresis or by a combination of these methods. They were characterized by amino acid analyses and by determination of the N-terminal amino acids using the Dansyl-Edman procedure. Thus all the disulfide bridges of an IgA1 immunoglobulin could be established. The monomer has all together 48 cysteins, seven in each L- and seventeen in each H-chain; all these are covalently bonded by SS-bridges. Free SH-groups were not detected. The J-chain could only be identified serologically in the polymeric form of the protein. It is shown that the subunits of the polymers are covalently attached through either Cysl3, Cysl7 or both these residues of the H-chain.     

Expression and analysis of COOH-terminal deletions of the human thrombospondin molecule

Thrombospondin (TSP) is a homotrimeric extracellular glycoprotein with a subunit molecular mass of 140 kD. The subunits have a modular or domain-like structure and are held together by interchain disulphide bonds. A number of domains have been identified including those for the binding of collagen, fibrinogen, and heparin. Due to the trimeric form of the TSP molecule, the various domains are trivalent in nature and this contributes to the ability of TSP to mediate cell-substrate interactions. Indeed, TSP has recently been shown not only to promote cell adhesion but also to be intimately involved in cell growth and migration. The adhesive function of TSP is attributable to the "solid-phase" or matrix-bound form of the molecule. There is some evidence that the heparin-binding domain mediates incorporation of soluble TSP into the insoluble matrix form. The heparin-binding domain of TSP is a compact globular amino-terminal moiety that contains two clusters of basic amino acids and a single intrachain disulphide bond. To delineate the role of the heparin-binding domain in matrix assembly and to define further the precise region of interchain disulphide bonding that results in trimer formation, we have expressed deleted forms of the cDNA encoding TSP in SV-40-transformed. African green monkey kidney cells. The proteins synthesized from the various deleted TSP cDNAs were examined for (a) secretion into the culture medium and incorporation into the extracellular matrix; (b) binding to heparin-Sepharose; (c) immunoprecipitability by a conformation-specific monoclonal antibody; and (d) ability to form trimers. This analysis allowed us to draw the following conclusions. (a) A 218 amino acid NH2-terminal protein that preserves the intrachain disulphide bridge of the heparin-binding domain is capable of binding to heparin-Sepharose and incorporating into the extracellular matrix. (b) A shorter 164 amino acid NH2-terminal peptide that does not contain the intrachain disulphide bridge of the heparin-binding domain is neither able to bind to heparin-Sepharose nor able to incorporate into the extracellular matrix. (c) The region of interchain disulphide bridging necessary for trimer assembly resides within a cluster of seven cysteine residues immediately adjacent to the heparin-binding domain.     

The complete amino acid sequence of a mouse κ light chain

The complete amino acid sequence of the kappa-chain of the mouse myeloma protein MOPC 21 was established. The protein was reduced and alkylated with iodo[2-(14)C]acetic acid, and 21 tryptic peptides were isolated, mainly by paper electrophoresis and paper chromatography. Three large tryptic peptides (of 35, 36 and 42 residues), which were difficult to isolate in this manner, were obtained pure and in excellent yields by a combination of Sephadex G-50 gel filtration in 1% (w/v) NH(4)HCO(3) and chromatography on a DEAE-cellulose column in ammonium acetate buffer, pH8.1. Peptides overlapping the tryptic peptides were isolated from a chymotryptic digest. The chain is 214 residues long. Microheterogeneity of two peptides was observed and is believed to be due to deamidation. It was not excluded that such deamidation could occur in serum from which the protein was isolated. The sequence is compared with the sequences of two other mouse kappa-chains, and with the human kappa-chain basic sequences.     

The structure of a small collagenous fragment isolated from chicken hyaline cartilage

In previous experiments, two collagenous fragments were isolated from pepsin digests of chicken hyaline cartilage and called the high molecular weight, (HMW) and low molecular weight (LMW) fractions [3]. In the present experiments, the chains of LMW were isolated after denaturation and subsequent reduction and alkylation of interchain disulfide bridges and were further fractionated by carboxymethyl-cellulose chromatography. Four peaks were resolved during chromatography and were designated LMW 1, 2A, 2B, and 3. Amino acid analyses and peptide mapping after cleavage with trypsin, V8 protease, and cyanogen bromide showed that three genetically distinct chains must be present in LMW. Fractions 2A and 2B were very similar, but not identical, in structure. LMW 1, 2A plus 2B, and 3 were consistently isolated in approximately equal proportions, suggesting that the probable chain organization of LMW is [1][2A + 2B][3]. This suggestion was supported further by experiments that attempted to fractionate LMW by carboxymethyl-cellulose chromatography after denaturation but without reduction and alkylation of interchain disulfide bridges. No fractionation of LMW was achieved, the single peak subsequently being shown to contain LMW 1, 2A plus 2B, and 3.     

An amino acid sequence in the active site of lipoamide dehydrogenase from pig heart

1. The two cysteine residues forming the disulphide bridge that comprises part of the active site of lipoamide dehydrogenase from pig heart were specifically labelled with iodo[2-(14)C]acetic acid. 2. A tryptic peptide containing these carboxymethylcysteine residues was isolated from digests of reduced and S-carboxymethylated lipoamide dehydrogenase and its amino acid sequence of 23 residues was determined. 3. The sequence is highly homologous with a similar sequence containing the active-site disulphide bridge of lipoamide dehydrogenase derived from the 2-oxoglutarate dehydrogenase complex of Escherichia coli (Crookes strain) and it is probable that, as in the bacterial enzyme, the disulphide bridge forms an intrachain loop containing six residues. The results indicate that the bacterial and mammalian proteins have a common genetic origin. 4. Amino acid sequences containing six other unique carboxymethylcysteine residues were also partly determined. 5. The analysis of the primary structure thus far is consistent with the view that the enzyme (mol.wt. approx. 110000) is composed of two identical polypeptide chains.     

Determination of disulfide array and subunit structure of taste-modifying protein, miraculin

The taste-modifying protein, miraculin (Theerasilp, S. et al. (1989) J. Biol. Chem. 264, 6655-6659) has seven cysteine residues in a molecule composed of 191 amino acid residues. The formation of three intrachain disulfide bridges at Cys-47-Cys-92, Cys-148-Cys-159 and Cys-152-Cys-155 and one interchain disulfide bridge at Cys-138 was determined by amino acid sequencing and composition analysis of cystine-containing peptides isolated by HPLC. The presence of an interchain disulfide bridge was also supported by the fact that the cystine peptide containing Cys-138 showed a negative color test for the free sulfhydryl group and a positive test after reduction with dithiothreitol. The molecular mass of non-reduced miraculin (43 kDa) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was nearly twice the calculated molecular mass based on the amino acid sequence and the carbohydrate content of reduced miraculin (25 kDa). The molecular mass of native miraculin determined by low-angle laser light scattering was 90 kDa. Application of a crude extract of miraculin to a Sephadex G-75 column indicated that the taste-modifying activity appears at 52 kDa. It was concluded that native miraculin in pure form is a tetramer of the 25 kDa-peptide and native miraculin in crude state or denatured, non-reduced miraculin in pure form is a dimer of the peptide. Both tetramer miraculin and native dimer miraculin in crude state had the taste-modifying activity.     

Comparison of the dimensions of the combining sites of the dinitrophenyl-binding immunoglobulin A myeloma proteins MOPC 315, MOPC 460 and XRPC 25 by spin-label mapping

The mouse immunoglobulin A myeloma proteins MOPC 315, MOPC 460 and XRPC 25 all possess dinitrophenyl (Dnp)-binding activity. Differences in specificities were shown by measuring the affinities of a variety of haptens. By using a series of Dnp-spin-labelled haptens, the dimensions of the binding sites of the three myeloma proteins were compared by the method described for protein MOPC 315 [Sutton, Gettins, Givol, Marsh, Wain-Hobson, Willan & Dwek (1977) Biochem. J.165, 177-197]. The dinitrophenyl ring is rigidly held in all three sites. The depths of the sites are all 1.1-1.2nm, but there are differences in the lateral dimensions at the entrance to the sites. For protein XRPC 25 these dimensions are 0.75nmx0.8nm, which may be compared with 0.85nmx1.1nm for protein MOPC 315 and >/=1.0nmx1.1nm for protein MOPC 460. The site in protein MOPC 460 is more symmetrical with respect to the plane of the dinitrophenyl ring than in either of the other two myeloma proteins and also allows greater penetration of solvent. In protein XRPC 25 a positively charged residue was located at the entrance to the site, similarly positioned to that reported for protein MOPC 315 [Sutton, Gettins, Givol, Marsh, Wain-Hobson, Willan & Dwek (1977) Biochem.J.165, 177-197]. All three proteins possess lanthanide-binding sites, but only in protein MOPC 315 is there antagonism between lanthanide and hapten binding. However, the effects of the diamagnetic La(III) on the electron-spin-resonance spectra of bound Dnp spin labels in both proteins MOPC 460 and XRPC 25 suggest an interaction between the two sites. Comparison of this effect with that caused by the addition of the paramagnetic Gd(III) enables the distance between the lanthanide- and hapten-binding sites to be calculated. In both proteins MOPC 460 and MOPC 315 the metal site is approx. 1.0nm from the nitroxide moiety of the spin-labelled hapten, but in protein XRPC 25 this distance is at least 2.0nm.     

Interchain and intrachain disulphide bonds in human platelet glycoprotein IIb. Localization of the epitopes for several monoclonal antibodies.

The single interchain disulphide bond in platelet glycoprotein IIb (GPIIb) is accessible to extracellular reductants, and selective cleavage does not liberate GPIIb alpha from platelet plasma membrane, confirming that non-covalent interactions contribute to maintaining attachment of this subunit to the membrane. Eosin-maleimide labelling of isolated GPIIb after selective cleavage of this interchain disulphide bond, followed by full reduction and alkylation, CNBr cleavage, and analysis of the cleavage products allowed us to establish that this interchain disulphide bridge is formed between GPIIb beta (GPIIb beta-subunit) Cys-9 and GPIIb alpha Cys-826, and this conclusion was confirmed by independent routes. The other two cysteines of GPIIb beta (Cys-14 and Cys-19) form the single intrachain disulphide bond in this subunit. Last, the intrachain disulphides in GPIIb alpha (GPIIb alpha-subunit) are distributed in four main peptide domains which are not disulphide-bonded among themselves. The linear epitope for monoclonal antibody M1 is localized between Pro-4 and Met-24 (or Met-31) of GPIIb beta. The linear epitope for M3 is situated between Cys-826 and the C-terminus of GPIIb alpha. The M4 epitope is also linear and localized somewhere between residues 115 and 285 of GPIIb alpha. Finally, the epitopes for M5 and M6 are somewhere between Cys-608 and Met-704, within a 35 kDa membrane-bound chymotryptic product of digestion of GPIIb in whole platelets. The N-terminal amino acid sequences determined for eight different cleavage products of GPIIb alpha and GPIIb beta agree with the corresponding amino acid sequences predicted by cDNA sequence for human-erythroleukaemic-cell GPIIb [Poncz, Eisman, Heindenreich, Silver, Vilaire, Surrey, Schwartz & Bennett (1987) J. Biol. Chem. 262, 8476-8482].     

Density comparisons of heavy chains of membrane and secreted immunoglobulins of mouse.

In order to explore structural differences between membrane and secreted immunoglobulins the buoyant densities of mouse immunoglobulin (Ig) heavy (H) chains were compared by isopycnic centrifugation in CsCl containing guanidine hydrochloride. The buoyant densities, under denaturing conditions, of mouse myeloma protein MOPC 21 IgG, MOPC 315 IgA and MOPC 104E IgM H chains were consistent with their carbohydrate contents. Mouse membrane IgM and MOPC 104E-secreted IgM H chains were of equal density. The buoyant densities of MOPC 104E-secreted IgM and spleen-cell-secreted IgM H chains were indistinguishable. The IgD-like membrane H chain was denser than membrane IgM H chain, and its carbohydrate content was calculated to be 15.5%. The resolution of the technique was sufficient to conclude that the apparent 1500 mol.wt. difference, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, between membrane and secreted IgM H chains was due to peptide rather than to carbohydrate. The results also imply that intact membrane IgM and IgD bind detergent and are thus integral membrane proteins.