scholarly journals Fragments produced by digestion of human immunoglobulin G subclasses with pepsin in urea

1977 ◽  
Vol 165 (2) ◽  
pp. 303-308 ◽  
Author(s):  
D M Parr
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Light Chains ◽  
Constant Region ◽  
Heavy Chains ◽  
Myeloma Proteins ◽  
Chain Cleavage ◽  
Peptic Digestion ◽  
Human Igg1 ◽  
Kappa Light Chains

It was previously shown that digestion of human IgG1/kappa myeloma proteins with pepsin in the presence of 8 M-urea produces fragments which differ from other proteolytic fragments of IgG, including those produced by peptic digestion in aqueous buffers. The two large urea/pepsin fragments each consist of three peptides, and together account for all of the constant region of the light chains and most of the constant region of the heavy chains. Myeloma proteins of subclasses IgG2, IgG3 and IgG4 with kappa light chains were digested with pepsin in 8 M-urea, and the resulting fragments compared with those produced from IgG1/kappa proteins. Gel filtration, starch- and polyacrylamide-gel electrophoresis and sequence analysis have shown that the peptides from each subclass are analogous with those from IgG1. A brief investigation of the products of urea/pepsin digestion of myeloma proteins with lambda light chains has shown that in these proteins light-chain cleavage occurs at residue leucine-182, instead of or as well as at residue 117, where cleavage takes place in kappa chains. Comparison of sequences around sites of urea/pepsin cleavage has shown that pepsin has quite restricted specificity under these conditions.

scholarly journals Characterization of upFc, a fragment of human immunoglobulin G1 produced by pepsin in urea

1976 ◽  
Vol 157 (3) ◽  
pp. 535-540 ◽  
Author(s):  
D M Parr ◽  
T Hofmann ◽  
G E Connell
Keyword(s):  
Amino Acid ◽  
Light Chains ◽  
Constant Region ◽  
Amino Acid Sequencing ◽  
Chain Fragment ◽  
Heavy Chains ◽  
Myeloma Proteins ◽  
Peptic Digestion ◽  
Human Igg1

The digestion of human IgG1/K myeloma proteins with pepsin in the presence of 8 M-urea produces fragments that differ from those produced by aqueous peptic digestion, and from other characteristic immunoglobulin fragments. Fb′2, the larger urea/pepsin fragment, was previously shown to consist of the constant regions of the light chains, and the CH1 domains and hinge regions of the heavy chains. The smaller fragment, upFc, has now been characterized. After reduction, three peptides were released from fragment upFc. Amino acid sequencing, N- and C-terminal determinations and amino acid compositions have enabled these peptides to be identified as residues Ile-253 to Leu-306, residues Thr-307 to Asp-376 and residues Thr-411 to Gly-446 of the heavy chain. Fragment upFc therefore contains the entire Fc region, beginning at residue Ile-253, except for a 34-residue section from within the CH3-domain disulphide loop. Peptic digestion of IgG1/K proteins in 8M-urea therefore provides a method for isolating from gamma1 heavy chains five homogeneous peptides in good yield, which account for almost the entire constant region. Characterization of fragments Fb′2 and upFc has shown that the action of pepsin in urea is entirely different from that of aqueous pepsin. Two gamma1 heavy chains have been shown to differ in sequence at three positions from the sequence reported for protein Eu.

scholarly journals Fb′2, a new peptic fragment of human immunoglobulin G

1976 ◽  
Vol 155 (1) ◽  
pp. 31-36 ◽  
Author(s):  
D M Parr ◽  
G E Connell ◽  
D I C Kells ◽  
T Hofmann
Keyword(s):  
Amino Acid ◽  
Sedimentation Equilibrium ◽  
Light Chains ◽  
Amino Acid Sequencing ◽  
Equilibrium Studies ◽  
Myeloma Protein ◽  
Heavy Chains ◽  
C Terminus ◽  
Peptic Digestion ◽  
Human Igg1

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.

scholarly journals STUDIES ON HUMAN ANTIBODIES

1968 ◽  
Vol 127 (3) ◽  
pp. 633-646 ◽  
Author(s):  
William J. Yount ◽  
Marianne M. Dorner ◽  
Henry G. Kunkel ◽  
Elvin A. Kabat
Keyword(s):  
Teichoic Acid ◽  
Exclusion Principle ◽  
Light Chains ◽  
Allelic Exclusion ◽  
Human Antibodies ◽  
Heavy Chains ◽  
Myeloma Proteins ◽  
Quantitative Analyses ◽  
Kappa Light Chains ◽  
The One

The composition of various isolated antibodies was determined by quantitative analyses for heavy chain subgroups and light chain types. Certain antibodies such as anti-tetanus toxoid and anti-A isoagglutinins were predominantly of the major γG1-type. However, a high preponderance of molecules of the minor γG2-subgroup was found for antibodies to dextran, levan, and teichoic acid. These findings explain some unusual features previously noted for anti-dextrans such as weak PCA reactions and lack of Gm antigens. Studies of several isolated antibodies from single heterozygous individuals showed a selective absence of genetic markers in certain antibodies and their presence in others. The "allelic exclusion" principle was clearly evident in the isolated antibodies of two different individuals. Large differences in the ratio of kappa to lambda light chains were observed for the same type of antibody from different individuals. Subfractionation of dextran antibodies by affinity for specific glycosidic linkage or combining site size produced marked changes in the ratios. The isomaltohexaose eluates of the dextran antibodies from two subjects were primarily kappa and the isomaltotriose eluates were predominantly lambda. The one anti-levan antibody studied was uniquely homogeneous, consisting exclusively of γG2-heavy chains and kappa light chains. By these criteria as well as others, it closely resembled myeloma proteins.

scholarly journals Immunochemical Characterization of a Monoclonal γG4, λ Human Antibody to Factor IX

Blood ◽  
1972 ◽  
Vol 40 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Isadore M. Pike ◽  
William J. Yount ◽  
Elliot M. Puritz ◽  
Harold R. Roberts
Keyword(s):  
Gel Filtration ◽  
Factor Ix ◽  
Light Chains ◽  
Human Antibody ◽  
Inhibitor Activity ◽  
Heavy Chains ◽  
Human Factor Ix ◽  
Zone Electrophoresis ◽  
Kappa Light Chains

Abstract A circulating inhibitor specific for factor IX in a patient with hemophilia B was characterized with antisera to human immunoglobulins. Inhibitor-rich plasma was mixed with an excess of specific antiserum and then assayed for residual inhibitor activity. Inhibitor activity was completely removed by antisera specific for γG4 heavy chains and lambda light chains. Antisera specific for γA, γM, γD, γE, γG1, γG2, and γG3 heavy chains and kappa light chains had no effect on the inhibitor. On preparative zone electrophoresis, inhibitor activity was localized in a sharp band in the anodal portion of the γG peak, an electrophoretic distribution paralleling that of γG4. Precise localization of the inhibitor activity on calibrated gel filtration columns revealed a relatively narrow zone of fractions containing the inhibitor, wherein the proteins have an estimated molecular weight of 145,000. The relative homogeneity of the antibody may reflect specificity for a uniform, discrete portion of the human factor IX molecule.

scholarly journals Expression of a public idiotype by human monoclonal IgM directed to myelin-associated glycoprotein and characterization of the variability subgroup of their heavy and light chains.

1989 ◽  
Vol 170 (5) ◽  
pp. 1551-1558 ◽  
Author(s):  
J C Brouet ◽  
K Dellagi ◽  
M C Gendron ◽  
A Chevalier ◽  
C Schmitt ◽  
...  
Keyword(s):  
Nonhuman Primate ◽  
Light Chains ◽  
Antibody Activity ◽  
Rheumatoid Factors ◽  
Variable Regions ◽  
Cold Agglutinins ◽  
Heavy Chains ◽  
Myelin Associated Glycoprotein ◽  
Kappa Light Chains

Most studies using rabbit or mouse antisera failed to detect CRI between human IgM directed to MAG. We show here that 9 of 10 such IgM express a public CRI as defined by a nonhuman primate antiserum. Shared idiotype is likely involved in (or close to) the combining site of those IgM since antiidiotypic serum inhibited the binding of IgM to MAG and reacted with IgM having different variable regions of light and heavy chains. Partial aminoterminal sequence of heavy and light chains showed that anti-MAG IgM use either lambda chains (one IgM) or kappa light chains (six IgM) of different variability subgroups (V kappa IV in three instances, V kappa I in two, and V kappa II in one), whereas heavy chains belong to the VHIII (six IgM) or to the VHII (1 IgM) subgroup. These features distinguish these IgM from other human monoclonal IgM with a defined antibody activity, such as rheumatoid factors or cold agglutinins.

Studies on the Dissociation of Porcine Haptoglobin

1972 ◽  
Vol 50 (7) ◽  
pp. 775-781 ◽  
Author(s):  
W. L. Lockhart ◽  
W. P. Chung ◽  
David B. Smith
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Light Chains ◽  
Dilute Solution ◽  
Disulfide Bridges ◽  
Elution Volume ◽  
Reversible Dissociation ◽  
Electrophoretic Mobilities ◽  
Chain Composition

Porcine haptoglobin was tested for reversible dissociation in dilute solution by gel filtration. Elution volume in Bio-Gel P-150 was independent of concentration and shapes of elution profiles failed to show dissociation. Molecular weight in dilute solution was estimated as 96 500. Interchain disulfide bridges were assayed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Partially reduced samples showed a series of intermediates but fully reduced samples showed only heavy and light chains. Intermediates were tentatively identified as to chain composition from their electrophoretic mobilities.

One siRNA pool targeting the λ constant region stops λ light-chain production and causes terminal endoplasmic reticulum stress

Blood ◽  
2014 ◽  
Vol 123 (22) ◽  
pp. 3440-3451 ◽  
Author(s):  
Ping Zhou ◽  
Xun Ma ◽  
Lakshmanan Iyer ◽  
Chakra Chaulagain ◽  
Raymond L. Comenzo
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Light Chain ◽  
Antibody Production ◽  
Plasma Cells ◽  
Light Chains ◽  
Constant Region ◽  
Immunoglobulin Light Chain ◽  
Heavy Chains ◽  
Key Points

Key PointsImmunoglobulin light-chain and antibody production by plasma cells is significantly reduced by siRNA for the light-chain constant region. In plasma cells making intact antibodies, knockdown of light chains can cause terminal ER stress because of unpaired heavy chains.

scholarly journals Loss of an individual idiotype on chemical modification. A strategy for assigning idiotypic determinants.

1981 ◽  
Vol 153 (5) ◽  
pp. 1275-1285 ◽  
Author(s):  
J Dickerman ◽  
B Clevinger ◽  
B Friedenson
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Chemical Modification ◽  
Heavy Chain ◽  
Light Chains ◽  
Heavy Chains ◽  
Myeloma Proteins ◽  
Low Levels ◽  
The Individual ◽  
Complementary Strategy

Two dextran-binding myeloma proteins, J558 and Hdex 24, which possess the same individual idiotype (IdI) were diazotized to low levels (1-3.3 groups per subunit) with 1-[14C]-p-aminobenzoate. Both proteins lost the IdI idiotype under these conditions with most of the label incorporated on the heavy chains of each protein. When the diazotization ws carried out in the presence of the hapten 1-O-methyl-alpha-D-glucopyranoside the loss of idiotypic reactivity could be prevented for J558 but not for Hdex 24. Under these conditions most of the label was incorporated on the light chains of J558, but on the heavy chains of Hdex 24. For J558, these results show that a major determinant of the individual idiotype is within the hypervariable positions of the heavy chain. For Hdex 24 the determinant being modified is on the heavy chain but not involved in hapten binding. These results are consistent with previous work showing that J558 and Hdex 24 differ in amino acid sequence in the D and the J segments of the heavy chain and offer an alternative and complementary strategy for assigning idiotypic determinants.

Allotypic marker of kappa light chains of rat immunoglobulins localized in the constant region of the chain

1974 ◽  
Vol 11 (8) ◽  
pp. 517-518 ◽  
Author(s):  
R.S Nezlin ◽  
T.I Vengerova ◽  
O.V Rokhlin ◽  
H.K.G Machulla
Keyword(s):  
Light Chains ◽  
Constant Region ◽  
Kappa Light Chains ◽  
Allotypic Marker

scholarly journals Cytoplasmic myosin from Drosophila melanogaster.

1986 ◽  
Vol 103 (4) ◽  
pp. 1517-1525 ◽  
Author(s):  
D P Kiehart ◽  
R Feghali
Keyword(s):  
Molecular Weight ◽  
Drosophila Melanogaster ◽  
Cell Lines ◽  
Heavy Chain ◽  
Gel Filtration ◽  
Polyclonal Antiserum ◽  
Light Chains ◽  
Drosophila Cell ◽  
Heavy Chains ◽  
Muscle Myosin

Myosin is identified and purified from three different established Drosophila melanogaster cell lines (Schneider's lines 2 and 3 and Kc). Purification entails lysis in a low salt, sucrose buffer that contains ATP, chromatography on DEAE-cellulose, precipitation with actin in the absence of ATP, gel filtration in a discontinuous KI-KCl buffer system, and hydroxylapatite chromatography. Yield of pure cytoplasmic myosin is 5-10%. This protein is identified as myosin by its cross-reactivity with two monoclonal antibodies against human platelet myosin, the molecular weight of its heavy chain, its two light chains, its behavior on gel filtration, its ATP-dependent affinity for actin, its characteristic ATPase activity, its molecular morphology as demonstrated by platinum shadowing, and its ability to form bipolar filaments. The molecular weight of the cytoplasmic myosin's light chains and peptide mapping and immunochemical analysis of its heavy chains demonstrate that this myosin, purified from Drosophila cell lines, is distinct from Drosophila muscle myosin. Two-dimensional thin layer maps of complete proteolytic digests of iodinated muscle and cytoplasmic myosin heavy chains demonstrate that, while the two myosins have some tryptic and alpha-chymotryptic peptides in common, most peptides migrate with unique mobility. One-dimensional peptide maps of SDS PAGE purified myosin heavy chain confirm these structural data. Polyclonal antiserum raised and reacted against Drosophila myosin isolated from cell lines cross-reacts only weakly with Drosophila muscle myosin isolated from the thoraces of adult Drosophila. Polyclonal antiserum raised against Drosophila muscle myosin behaves in a reciprocal fashion. Taken together our data suggest that the myosin purified from Drosophila cell lines is a bona fide cytoplasmic myosin and is very likely the product of a different myosin gene than the muscle myosin heavy chain gene that has been previously identified and characterized.

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