Two structurally different types of rabbit light chains

Disulphide bonds of rabbit γ-G-globulin and the antibody of the γ-G-globulin type against the 2,4-dinitrophenyl group were split both by the oxidative sulphitolysis at pH 8.6 and by the reduction with 2-mercaptoethanol followed by carboxymethylation. The fractionation was carried out in 0.05 m formic acid containing 6m urea, in 1m propionic acid or in 6m guanidine hydrochloride. Both heavy (H) and light) (L) chains are released from the I+J fraction preceding on an elution diagram H chains when rechromatographed in a stronger desaggregation medium. A small amount of the L chains is also released on rechromatography of the H chains (isolated from 1m propionic acid) in 6m guanidine hydrochloride. The separation of the degraded γ-G-globulin in 0.05m formic acid containing 6m urea or in 6m guanidine hydrochloride showed a separation of the L chains to two fractions differing by electrophoretic properties, peptide maps and N-terminal amino acids. However, these chains exhibit a similar molecular weight, immunoelectrophoretic behaviour and similar properties on reactivation of the antibody H chain.     

Living macrophages phosphorylate the 20,000 dalton light chains and heavy chains of myosin

Brief incubation of rabbit alveolar macrophages in medium containing ³²Pi results in the incorporation of radioactivity into the 20 KD light chains and into the 220 KD heavy chains of myosin. Phosphorylation of the heavy chain is mediated by a kinase that is probably not myosin light chain kinase. Limited proteolysis of the phosphorylated myosin shows that radioactivity is associated with the rod portion of the heavy chain.     

Co-translational modification of nascent immunoglobulin heavy and light chains

We have investigated the in vivo co-translational covalent modification of nascent immunoglobulin heavy and light chains. Nascent polypeptides were separated from completed polypeptides by ion-exchange chromatography of solubilized ribosomes on QAE-Sephadex. First, we have demonstrated that MPC 11 nascent heavy chains are quantitatively glycosylated very soon after the asparaginyl acceptor site passes through the membrane into the cisterna of the rough endoplasmic reticulum. Nonglycosylated completed heavy chains of various classes cannot be glycosylated after release from the ribosome, due either to rapid intramolecular folding and/or intermolecular assembly, which cause the acceptor site to become unavailable for the glycosylation enzyme. Second, we have shown that the formation of the correct intrachain disulfide loop within the first light chain domain occurs rapidly and quantitatively as soon as the appropriate cysteine residues of the nascent light chain pass through the membrane into the cisterna of the endoplasmic reticulum. The intrachain disulfide loop in the second or constant region domain of the light chain is not formed on nascent chains, because one of the cysteine residues involved in this disulfide bond does not pass through the endoplasmic reticulum membrane prior to chain completion and release from the ribosome. Third, we have demonstrated that some of the initial covalent assembly (formation of interchain disulfide bonds) occurs on nascent heavy chains prior to their release from the ribosome. The results are consistent with the pathway of covalent assembly of the cell line, in that completed light chains are assembled onto nascent heavy chains in MPC 11 cells (IgG₂b), where a heavy-light half molecule is the major initial covalent intermediate; and completed heavy chains are assembled onto nascent heavy chains in MOPC 21 cells (IgG₁), where a heavy chain dimer is the major initial disulfide linked intermediate.     

Phosphorylation of vertebrate nonmuscle and smooth muscle myosin heavy chains and light chains

In this article we review the various amino acids present in vertebrate nonmuscle and smooth muscle myosin that can undergo phosphorylation. The sites for phosphorylation in the 20 kD myosin light chain include serine-19 and threonine-18 which are substrates for myosin light chain kinase and serine-1 and/or-2 and threonine-9 which are substrates for protein kinase C. The sites in vertebrate smooth muscle and nonmuscle myosin heavy chains that can be phosphorylated by protein kinase C and casein kinase II are also summarized.Original data indicating that treatment of human T-lymphocytes (Jurkat cell line) with phorbol 12-myristate 13-acetate results in phosphorylation of both the 20 kD myosin light chain as well as the 200 kD myosin heavy chain is presented. We identified the amino acids phosphorylated in the human T-lymphocytes myosin light chains as serine-1 or serine-2 and in the myosin heavy chains as serine-1917 by 1-dimensional isoelectric focusing of tryptic phosphopeptides. Untreated T-lymphocytes contain phosphate in the serine-19 residue of teh myosin light chain and in a residue tentatively identified as serine-1944 in the myosin heavy chain.Abbreviations MLC myosin light chain - MHC myosin heavy chain - Tris tris(hydroxymethyl)aminomethane - EGTA [ethylenebis(oxyethylenenitrilo)]tetraacetic acid - EDTA ethylenediaminetetraacetate - TPCK N-tosyl-L-phenylalanine chloromethyl ketone - PMA phorbol 12-myristate 13-acetate     

Partial amino acid sequence of the heavy and light chains of botulinum neurotoxin type A

The dichain (nicked) type A botulinum neurotoxin is a protein (mol. wt. 145,000) composed of a heavy and a light chain (mol. wt. 97,000 and 53,000, respectively) that are held together by disulfide bond(s). We report here the sequence of the first 17 amino acid residues of the light chain, and the first 10 residues of the heavy chain. The heavy chain was isolated from the neurotoxin by two different methods, while the light chain was isolated by the only available method. The identical amino acid sequence was found in both preparations of heavy chain. Two samples of the light chain isolated from two separately prepared batches of the neurotoxin also had identical sequences.     

Mouse pre-B cells synthesize and secrete mu heavy chains but not light chains

The immunoglobulins produced by the earliest recognizable B cell precursors (pre-B cells) were characterized in the mouse and human. Immunofluorescent analysis revealed no evidence of surface IgM components, and only mu heavy chains could be detected intracytoplasmically in pre-B cells. Surface IgM components could not be isolated from intact fetal liver cells that lacked sIgM+ B lymphocytes but possessed pre-B cells. Pre-B cells were shown to synthesize and secrete mu heavy chains but not light chains by immunochemical analysis. These mu chains constituted less than 0.01% of TCA precipitable protein synthesized and secreted by fetal liver cells during an 8 hr labelling period. Migration of both intracellular and secreted mu chains on SDS-PAGE suggested that they were smaller than mu chains secreted by mouse and human plasmacytomas. These data indicate that mu chain synthesis precedes light chain expression during B cell ontogeny and suggest a new role for pre-B cells in the generation and expression of a diverse immunoglobulin repertoire.     

Synthesis of myosin heavy and light chains in muscle cultures

The weight ratio of myosin/actin, the myosin heavy chain content as the percentage of total protein (wt/wt), and the kinds of myosin light chains were determined in (a) standard muscle cultures, (b) pure myotube cultures, and (c) fibroblast cultures. Cells for these cultures were obtained from the breast of 11-day chick embryos. Standard cultures contain, in addition to myotubes, large numbers of replicating mononucleated cells. By killing these replicating cells with cytosine arabinoside, pure myotube cultures were obtained. The myosin/actin ratio (wt/wt) for pure myotube, standard muscle, and fibroblast cultures average 3.1, 1.9, and 1.1 respectively. By day 7, myosin in myotube cultures represents a minimum of 7% of the total protein, but about 3% in standard cultures and less than 1.5% in fibroblasts cultures. Myosin from standard cultures contains light chain LC1, LC2, and LC3, with a relative stoichiometry of the molarity of 1.0:1.9:0.5 and mol wt of 25,000, 18,000 and 16,000 daltons, identical to those in adult fast muscle. Myosin from pure myotubes exhibits light chains LC1 and LC2, with a molar ratio of 1.5:1.6. Myosin from fibroblast cultures possesses two light chains with a stoichiometry of 1.8:1.8 and mol wt of 20,000 and 16,000 daltons. Clearly, the faster migrating light chain, LC3, found in standard cultures is synthesized not by the myotubes but ty the mononucleated cells. In myotubes, both the assembly of the sarcomeres and the interaction between thick and thin filaments required for spontaneous contraction occur in the absence of light chain LC3. One set of structural genes for the myosin light and heavy chains appears to be active in mononucleated cells, whereas another set appears to be active in multinucleated myotubes.     

A proteoglycan related to the urinary trypsin inhibitor (UTI) links the two heavy chains of inter-alpha-trypsin inhibitor

cDNA studies have suggested that inter-alpha-trypsin inhibitor (ITI) is a complex of several different peptide chains; the sequence of the inhibitory part of ITI is in excellent agreement with that of the urinary trypsin inhibitor (UTI). The present report demonstrates that a compound immunologically related to UTI is released by digestion with porcine pancreatic elastase or human leucocyte elastase. Since UTI has been shown to be a proteoglycan, ITI has been treated by chondroitinase. In these conditions, ITI is dissociated and gives rise to two heavy chains (78 and 85 kDa) and one light chain (26 kDa) immunologically related to UTI and which in PAGE moves close to UTIc (produced by chondroitinase treatment of UTI). We suggest that ITI is a non-covalent complex comprising two heavy chains and one light chain immunologically related to UTI and which is also a proteoglycan.     

Studies on the alkali light chains of vertebrate skeletal muscle myosin. Effect of tyrosyl modification on the ability to reassociate to heavy chains

The structures of the alkali light chain subunits A1 and A2 have been studied by examining the effect of the conformationally sensitive reagent tetranitromethane, which reacts specifically with tyrosyl residues. Whereas reaction in the presence of 6 M guanidine hydrochloride results in modification of the three tyrosyl residues of both these light chains, only two tyrosyl residues are exposed to the reagent in the native conformations of these proteins. By gel chromatography of the CNBr-cleaved chains it was demonstrated that the two reactive tyrosyls are those located in the CB-1 and CB-3 segments and that these tyrosyl residues are modified simultaneously and not sequentially. The unreactive tyrosyl residue is in the CB-6 segment and is separated by two residues from the single cysteinyl residue of these chains. It is found that the modified light chains cannot be made to reassociate with the heavy chains by the NH4Cl hybridization procedure of Wagner and Weeds [J. Mol. Biol. 109, 455-470 (1977)] or by the thermal hybridization procedure [Burke and Sivaramakrishnan (1981) Biochemistry 20, 5908-5913]. Furthermore, reduction of the nitrotyrosyl groups to aminotyrosyl residues by sodium dithionite does not restore this effect. The data suggest that regions of the light chains at CB-1 and CB-3 are involved in the association to the heavy chains.     

Allotypically related sequences in the Fd fragment of rabbit immunoglobulin heavy chains

The sequence has been completed of the N-terminal 94 residues of the variable section of the Fd fragment of heavy chains from rabbit immunoglobulin G (IgG) of allotype As1. Most of the sequence of the same section from IgG of allotype Aa3 is also reported. These results, in conjunction with a substantial sequence of the variable region of allotype Aa2 reported elsewhere (Fleischman, 1971), show the presence of 16 positions (including six consecutive positions) in which the residue present correlates with the allotype. No allotype-related sequence variation has been found in the constant section of the Fd fragment. This evidence supports the view that two genes code for the heavy chain and it can be used as evidence in favour of somatic mutation as the origin of the variability in the sequence of the N-terminal section. The evolutionary origin of the ;a' locus allotypes of rabbit immunoglobulins remains obscure.     

Protein kinase C phosphorylates both the light chains and the head portion of the heavy chains of brain myosin

Protein kinase C phosphorylated both the 19/21-kDa regulatory light chains and heavy chains of bovine brain myosin. The major phosphorylation sites of the light chains were on their threonyl residues, while those for myosin light chain kinase were on their seryl residues. Whereas several non-muscle regular myosins have been reported to be phosphorylated by different types of protein kinases at the non-helical small segments at the tail ends of the heavy chains, the phosphorylation sites for protein kinase C were localized on the head portion of the heavy chains of brain myosin. The possible role of phosphorylation of brain myosin by protein kinase C in the regulation of motility of neural cells is discussed.     

Role of the heavy and light chains of botulinum neurotoxin in neuromuscular paralysis

Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum in any of seven antigenically distinct forms called types A-G. NT, when fully active, is a dichain protein, composed of two polypeptides, a heavy (H) and a light (L) chain (approximately 100,000 and approximately 50,000 Da, respectively) that are held together by noncovalent bonds and at least one disulfide bond. Two types of dichain NT, A and B, and their respective H and L chains were applied to nerve-muscle (NM) preparations (phrenic nerve-hemidiaphragm of the mouse), in order to develop a broader, comparative understanding of the neuroparalytic actions of NT types. It was found that the paralysis induced by dichain NT was delayed or antagonized if NM preparations were incubated with isolated and purified H chain prior to, or during, incubation with the parent, dichain NT. NM preparations preincubated with H chain and then washed free of unbound H chain became paralyzed after subsequent incubation with L chain. Paralysis did not occur if NM preparations were incubated first with L chain, washed, and then incubated with H chain. These observations suggest that the H chain binds with specific sites on the nerve terminal. This binding appears to permit the L chain, or some combination of the L and H chain, to bring about neuroparalysis through a mechanism very similar to that of the parent, dichain NT.     

Noncovalent association of heavy and light chains in Rana catesbeiana immunoglobulins

The unreduced immunoglobulins (Ig) in the bullfrog, Rana catesbeiana, dissociate into two components when subjected to electrophoresis or molecular sieving in dissociating solvents. One of these components is monomeric light chain and the other is a disulfide-bonded complex of heavy chains. This unusual behavior has been observed with all classes of bullfrog Ig that have been isolated and characterized previously: a high m.w. Ig that resembles mammalian IgM and two antigenically distinct varieties of low m.w. Ig. Light chains, isolated from the high m.w. Ig by gel filtration in 8 M urea, 1 M acidic acid, were found to contain, on average, 5.7 residues of half-cystine. None of these residues were in the free sulfhydryl form nor were they blocked by half-cystine. Moreover, none was alkylated after mild reduction of the high m.w. Ig. These findings indicate that none of the light chain half-cystine residues participate in an interchain disulfide bridge, and that most of the light chains contain three intrachain bridges. This unusual pattern of disulfide bonding appears to be responsible for the noncovalent association of heavy and light chains in this species.     

Amino terminal sequence of heavy and light chains from ratfish immunoglobulin

The ratfish,Callorhinchus callorhinchus, a representative of the Holocephali, has a natural serum hemagglutinin (M _r 960 000), composed of heavy (M _r 71000), light (M _r 22 500), and J (M _r 16 000) chains. To approach the mechanisms that generate diversity at this level of evolution, the amino terminal sequence of the heavy and light chains was determined by automated microsequencing. The chains are unblocked and have modest internal sequence heterogeneity. The heavy chains show sequence similarity with the terminal region of the heavy chain from the horned shark,Heterodontus francisci, and other species. In contrast to the heavy chain, the ratfish light chains display low sequence similarity with their shark kappa counterparts. However, their similarity with the variable region of the chicken lambda light chains is about 75%.