Portion Of Heavy Chain Research Articles

A new function of kininogens as thiol-proteinase inhibitors: inhibition of papain and cathepsins B, H and L by bovine, rat and human plasma kininogens

The amidolytic activities of papain and rat liver cathepsins B, H and L were strongly inhibited by high (HMM) and low (LMM) molecular mass kininogens from bovine, human and rat plasmas, and their K i values were estimated to be in the order of 10 −10–10 −11 M for papain and 10 −8–10 −9M for cathepsins. The derivatives of bovine kininogens, HMM kinin-free protein, HMM kinin- and fragment 1 ·2-free protein, and LMM kinin-free protein also showed strong inhibitory activity toward these thiol-proteinases. These results suggest that a reactive site which interacts with thiol-proteinases is contained in the heavy chain portion in kininogens. Kininogen Thiol-proteinase inhibitor Papain Cathepsin B Cathepsin H Cathepsin L

Cleavage of the four human IgG subclasses with cathepsin G.

Cathepsin G, the chymotrypsin-like serine proteinase from human polymorphonuclear leucocytes, cleaves human IgG. The relative susceptibilities of the four IgG subclasses to the action of this enzyme were studied kinetically and showed the following decreasing order of susceptibility: IgG3 much greater than IgG4 greater than IgG1 greater than IgG2. IgG1 and IgG2 produced primarily F(ab')2 and traces of Fc-related fragments. IgG4 gave rise to both Fab and F(ab')2 as major products, and small amounts of an Fc-related fragment were detected. The cleavage of IgG3 produced various fragments, depending on the experimental conditions: The primary fragments were Fab and Fch (Fc covalently joined to the extended hinge-region polypeptide of IgG3) and an intermediate Fab-Fch species. Both Fab and Fch were further degraded by cathepsin G. Fch was gradually split, giving rise to three subfragments that were finally degraded to dialysable peptides. The enzyme further cleaved the Fab fragment in the heavy-chain portion and released a polypeptide probably representing the VH domain.

The effect of cleavage at site 1 of gizzard HMM in the interaction with skeletal muscle actin.

Two different classes of gizzard heavy meromyosins (HMMs) were prepared from phosphorylated myosin by chymotryptic digestion in the presence and absence of ATP and were compared with respect to their actin-activated Mg2+-ATPase reactions. One class of HMM, named HMM(+), had a cleavage at site 1 in the N terminal portion of the heavy chain and the other class of HMM, named HMM(-), had no cleavage at this site. Maximum turnover rate (Vmax) of the skeletal acto-gizzard HMM Mg2+-ATPase reaction was obviously different between HMM(+) and HMM(-). The Vmax value of HMM(+) was 2.5-fold larger than that of HMM(-). On the other hand, the apparent association constants (Ka) of skeletal muscle actin for both HMMs which were deduced from double reciprocal plots (v-1 versus [actin]-1) seemed to be identical. The difference in Vmax value was attributed to the cleavage at site 1 since a following chymotryptic cleavage of HMM(-) at site 1 caused a 2.5-fold increase in the Vmax value. That site 1 in the N terminal portion of the gizzard myosin heavy chain was the key locus for the actin-myosin interaction was shown in addition to our previous finding of the effects of cleavage at site 1 on the ATPase activity and nucleotide binding ability of gizzard HMM (Okamoto, Y. & Sekine, T. (1981) J. Biochem. 90, 833-843; 843-949).

Characterization of the protein kinase activity of avian sarcoma virus src gene product.

The avian sarcoma virus src gene product, p60src, has been purified 650-fold from cytoplasmic extracts of the rat tumor cell line RR1022 by using ammonium sulfate fractionation, hydrophobic chromatography on omega-aminohexyl agarose, and ion exchange chromatography on phosphocellulose. Partially purified p60src is a monomer, with a native molecular weight of about 60,000 and an apparent pI of 6.0. In immunoprecipitates, p60src catalyzed phosphorylation of anti-p60src IgG heavy chains within the variable (VH) domain, which contains the heavy chain portion of the antigen combining site. Crude preparations of p60src contained phosphatase activity able to cleave phosphate from IgG heavy chains; this activity was removed by the purification procedure, and partially purified p60src could phosphorylate the heavy chain of specific antibody in solution. Furthermore, purified p60src catalyzed phosphorylation in solution of the general protein kinase substrate, alpha-casein, strengthening the hypothesis that it may in fact function as a protein kinase in vivo.

Structure of tetanus toxin. II. Toxin binding to ganglioside.

The interaction between tetanus toxin and ganglioside containing 2 N-acetylneuraminic acid residues linked in sequence to one another has been investigated using a new method involving radioactively labeled ganglioside and tetanus toxin adsorbed to Sephadex matrix. Binding between the two components was demonstrated, and it was calculated that in the nanomolar concentration range, tetanus toxin becomes half-saturated at about 5 X 10(-8) M concentration of ganglioside. Removal of the ceramide portion from the ganglioside resulted in the complete loss of binding activity, whereas removal of the terminal N-acetylneuraminic acid residue from the intact ganglioside had no effect. Among the fragments derived from tetanus toxin (Helting, T. B., and Zwisler, O. (1977) J. Biol. Chem. 252, 187-193), only the heavy chain polypeptide exhibited a binding activity of the same order of magnitude as that observed for the native toxin. The light chain polypeptide showed no interaction with ganglioside and among the fragments derived from the toxin by digestion with papain, only Fragment C, at a high protein concentration, displayed marginal binding activity. Using monovalent antibodies directed against specific regions of the tetanus toxin molecule, it was demonstrated that antibodies directed against Fragment C uniquely interfere with the binding process. Anti-light chain serum was ineffective, as well as antitetanus toxoid serum previously absorbed with Fragment C. It is concluded that the binding site for ganglioside is located on the heavy chain portion of tetanus toxin, possibly in or near the region comprised by Fragment C.

Isoelectric focusing of proteolytic fragments from rabbit anti-streptococcal antibodies of restricted heterogeneity

FEBS LettersVolume 39, Issue 2 p. 160-163 Full-length articleFree Access Isoelectric focusing of proteolytic fragments from rabbit anti-streptococcal antibodies of restricted heterogeneity Anne Vienne, Anne Vienne Laboratoire de Physiologie Animale, Université Libre de Bruxelles, 1640 Rhode-St-Genèse, BelgiumSearch for more papers by this authorMaurice Wikler, Maurice Wikler Laboratoire de Physiologie Animale, Université Libre de Bruxelles, 1640 Rhode-St-Genèse, BelgiumSearch for more papers by this author Anne Vienne, Anne Vienne Laboratoire de Physiologie Animale, Université Libre de Bruxelles, 1640 Rhode-St-Genèse, BelgiumSearch for more papers by this authorMaurice Wikler, Maurice Wikler Laboratoire de Physiologie Animale, Université Libre de Bruxelles, 1640 Rhode-St-Genèse, BelgiumSearch for more papers by this author First published: February 15, 1974 https://doi.org/10.1016/0014-5793(74)80041-4Citations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 B.A. Askonas, A.R. Williamson, B.E.G. Wright, Proc. Natl. Acad. Sci. U.S., 67, (1970), 1398– 1403. 2 Z.L. Awdeh, A.R. Williamson, B.A. Askonas, Biochem. J., 116, (1970), 241– 248. 3 D.R. Hoffman, A.L. Grossberg, D. Pressman, J. Immunol., 107, (1971), 325– 330. 4 M.D. Waterfield, J.W. Prahl, L.E. Hood, T.J. Kindt, R.M. Krause, Nature New Biol., 240, (1972), 215– 217. 5 C.K. Osterland, E.J. Miller, W.W. Karakawa, R.M. Krause, J. Exptl. Med., 123, (1966), 599– 614. 6 K. Eichmann, J. Greenblatt, J. Exptl. Med., 133, (1971), 424– 441. 7 R.R. Porter, Biochem. J., 73, (1959), 119– 126. 8 W.J. Mandy, M.M. Rivers, A. Nisonoff, J. Biol. Chem., 236, (1961), 3221– 3226. 9 Z.L. Awdeh, A.R. Williamson, B.A. Askonas, Natura, 219, (1968), 66– 67. 10 S. Utsumi, Karush, Biochemistry, 4, (1965), 1766– 1779. 11 D.S. Smyth, S. Utsumi, Nature, 216, (1967), 332– 335. 12 R. Heimer, S.H. Schnoll, J. Immunol., 100, (1968), 231– 237. 13 D. Frommel, R. Hong, Biochim. Biophys. Acta, 200, (1970), 113– 124. 14 R. Kornfeld, J. Keller, J. Baenziger, S. Kornfeld, J. Biol. Chem., 246, (1971), 3259– 3267. 15 J.W. Prahl, W.J. Mandy, C.W. Todd, Biochemistry, 8, (1969), 4935– 4940. 16 G. Florent, Vries G.M. De, R. Hamers, Immunochemistry, 10, (1973), 425– 430. Citing Literature Volume39, Issue2February 15, 1974Pages 160-163 ReferencesRelatedInformation

Immunochemistry and Genetics of a “New” Allotypic Specificity Ae14 of Rabbit γG Immunoglobulins: Recombination in Somatic Cells

Abstract A “new” allotypic specificity of rabbit immunoglobulins (A14) is described. The determinant is localized on the Fc portion of heavy chains of rabbit γG. The specificity is inherited in a simple Mendelian manner and the gene controlling its synthesis is localized at the locus Ae, which is distinct from Aa and Ab loci; the product of the gene allelic to the Ae14 gene is not yet known. There is a strong indication that the loci Ae and Aa are closely linked in the germline, but that they may undergo recombination in the somatic cells, thus causing the synthesis of recombinant molecules.

Studies on the amino acid sequence of heavy chains from rabbit immunoglobulin G.

It is now generally agreed that the four-chain subunit structure of Immunoglobulins which was first proposed by Porter (1962), accurately represents the gross structure of immunoglobulin G (IgG) and specific antibodies (Fleischman, Porter & Press 1963; Edelman & Gally 1964; Marler, Nelson & Tanford 1964; Nelson et al . 1965). However, an understanding of the structural basis of antibody specificity requires greater insight into the amino acid sequence of the polypeptide chain components of specific antibodies. Isolated light chains from specific antibodies and inert IgG, show a considerable degree of electrophoretic heterogeneity (Edelman & Gally 1964; Cohen & Porter 1964; Poulik 1964). Tryptic peptide maps of light chains (Nelson et al . 1965) have suggested that this heterogeneity may be accounted for by differences in amino acid sequence. This view has received considerable support from the observation that Bence-Jones proteins, which may be regarded as light chains, vary significantly in amino acid sequence (Hilschman & Craig 1965; Milstein 1966; Titani, Whitley & Putman 1966). A similar but less well-defined sequence heterogeneity has been suggested to exist in the heavy chains of specific antibodies (Feinstein 1964). However, the Fc fragment of the heavy chains has been thought to possess a regular amino acid sequence which may be similar, if not identical, among all specific antibodies (Porter 1959; Nelson et al . 1965). This paper summarizes the results of studies on the amino acid sequence of heavy chains and that portion of heavy chain, Fc fragment, which is obtained on treatment of rabbit IgG with papain (Porter 1959). These studies were designed to determine how much of the amino acid sequence of heavy chain could be accounted for by a unique, regular amino acid sequence which was common to most, if not all, IgG antibodies. In addition, attempts were made to locate regions of heavy chains which varied in amino acid sequence. Although structural variants appear to occur among the heavy chains found in non-specific IgG, it would be desirable to know what portion of the heavy chain sequence is invariant among all antibodies. If antibody specificity results from sequence heterogeneity in light and heavy chains, then knowledge of the variant and invariant portions of these chains may provide insight into the nature of specific binding sites in anti-­bodies.

A gamma-globulin genetic factor related to Gm(a) but localized to a different portion of the same heavy chains.

PREVIOUS investigations have indicated that Gm(a)* and Gm(f)* are genetic characters localized in various areas of the major heavy chain class1–3 of human γG globulins. These determinants, detected as antigens, segregate in most populations, as if controlled by allelic genes, and occur independently in myeloma proteins from Caucasians. A previous report indicated that specific reagents for the human genetic factors can be produced by immunizing rabbits with myeloma proteins4. In order to analyse in a more satisfactory way the genetic control of different portions of the heavy chains, it seemed desirable deliberately to immunize with critical protein fragments in order to disclose additional genetic sites believed to be present but not detected by available antisera.

CORROBORATION OF RECENT MODELS OF THE GAMMA-G IMMUNOGLOBULIN MOLECULE.

The relationships between the polypeptide chains of gammaG immunoglobulin and fragments of the molecule produced by papain and pepsin have been investigated. Specific procedures were employed including peptide mapping of tryptic hydrolysates and analysis of molecules reconstituted from chains labeled with different iodine isotopes. By these means, the Fab fragment was shown unequivocally to consist of the light chain and a portion of the heavy chain, the Fd fragment. The Fc fragment was found to be comprised of the residual portions of the heavy chain. These findings support the gross arrangement of chains embodied in recent models of the gammaG immunoglobulin molecule. The present studies have also provided additional information on the susceptibility of gammaG immunoglobulin to proteolytic cleavage. It was found that the portion of heavy chains corresponding to the Fd fragment was extensively cleaved by papain.