Precursor sequence of MOPC-315 mouse immunoglobulin heavy and light chains.

Partially purified mRNA coding for the MOPC-315 heavy (alpha) or light (lambda 2) immunoglobulin chain was translated in a nuclease-treated reticulocyte lysate containing 20 labeled amino acids. Radiolabeled precursor heavy and light chains, purified by immunoprecipitation and preparative gel electrophoresis, were subjected to Edman degradation. The labeled phenylthiohydantoin derivatives obtained in each degradative cycle were identified and quantitated by high pressure liquid chromatography. Both heavy and light chain precursor segments were hydrophobic in nature; however, they were not homolgous in sequence. To establish whether COOH-terminal proteolytic processing of the heavy chain might also be occurring during secretion, the cyanogen bromide peptides of the heavy chain precursor were compared to those of the mature secreted heavy chain. The results indicated that the COOH termini of the two chains were identical.     

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     

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.     

Temporal relationship of translation and glycosylation of immunoglobulin heavy and light chains.

The initial glycosylation of MPC 11 gamma 2b heavy chains occurs quantitatively in vivo when the nascent heavy chains reach a size of approximately 38 000 daltons. Nonglycosylated, completed MPC 11 heavy chains cannot be glycosylated in these cells. Other classes of mouse heavy chains (i.e., mu, alpha, and gamma 1) also appear to be glycosylated as nascent chains; nonglycosylated, completed heavy chains cannot be glycosylated by the cell in any of these cases. In contrast, variant MPC 11 cells synthesizing a heavy chain with a carboxy-terminal deletion appear to glycosylate some heavy chains prior to chain completion and some heavy chains after chain completion and release from the polysomes. Similar to the variant MPC 11 cells, MOPC 46B cells (which synthesize a kappa light chain containing an oligosaccharide attached to an asparagine located 28 residues from the amino terminus) glycosylate the majority of light chains after prior to chain completion but also some light chains after chain completion and release from the polysomes. In addition, it appears that, although completed MOPC 46B light chains can be glycosylated if they are present in a monomeric form, they cannot be glycosylated if they are present in a covalent dimeric form.     

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.     

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.     

Kinetics of interaction of heavy and light chains in immunoglobulins. Use of a sulfhydryl-specific light chain fluorescent probe.

The fluorescent probe N-(iodoacetylaminoethyl)-8-naphthylamine-1-sulfonic acid (1,8-I-AEDANS) reacts stoichiometrically with the COOH-terminal cysteine residue of a human immunoglobulin light (L) chain. The absorption and fluorescence spectral properties of the L-AEDANS product and the environmental sensitivity of the fluorescence suggest the utility of L-AEDANS in the study of the subtle conformational changes accompanying interactions between light chain and other proteins or ligands. Its applicability to the problem of immunoglobulin assembly is illustrated by the association of L-AEDANS with heavy chain dimers, which results in an enhancement of fluorescence. The data indicate that L-AEDANS binds to kinetically equivalent, noninteracting sites with an apparent second order rate constant of 6 X 10(6) liters mol(-1) s(-1) at 20 degrees, pH 7.5.     

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%.     

A component of the fraction of porcine light chains structurally related to heavy chains

In the previous paper (Rejneket al., 1967) we described the fractionation of light chains (L) by Zn ions resulting in an accumulation of antigenic determinants of the heavy chain (H) in the Zn precipitate. Peptide maps of the obtained fractions of the L chains differ considerably from each other. Peptides of the L chains, the position of which corresponds within the experimental error to peptides of the H chain may be detected by comparing them with the peptide map of the H chains. The number of such peptides increases with qualitatively assayed accumulation of the component precipitated with anti-H serum during fractionation. The concentration of N-terminal glutamic acid, characteristic for the H chains increases at the same time.     

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.     

Characterization of the interaction between the heavy and light chains of bovine factor Va.

Bovine factor Va has been previously been shown to consist of heavy (M(r) = 94,000) and light chains (M(r) = 81,000), that interact in a manner dependent upon the presence of either calcium or manganese ions. In an attempt to understand the mechanism of subunit interaction we have studied the effects of temperature and ions on factor Va stability. The rates of formation of factor Va from isolated chains and dissociation were temperature-dependent with an energy of activation of 6.2 and 1.3 kcal mol-1, respectively. The yield of factor Va from isolated chains was inversely related to the amount of time the chains were incubated at 4 degrees C. Incubation of individual chains revealed that the heavy chain is cold-labile, an effect that is reversible. Manganese ion was observed to prevent the conversion to the inactive form. High salt tends to stabilize the two-chain structure of factor Va, but is inhibitory to its formation from isolated chains. High concentrations of either manganese or calcium ions also inhibited reconstitution of activity. The light chain, in particular, was sensitive to the presence of manganese or calcium ion. Heavy chain that had been cleaved by activated protein C had a weakened interaction with the light chain, and the resulting complex had no procoagulant activity. Cooling of the heavy chain to 4 degrees C enhanced its intrinsic fluorescence. Manganese ion prevented some of this enhancement. The heavy chain fluorescence returned to the room temperature value with a half-life of approximately 10 min. In the presence of manganese ion relaxation was accelerated. The intrinsic fluorescence of activated protein C-cleaved heavy chain was not increased when the temperature was decreased. These data suggest that the heavy chain can exist in two forms. Elevated temperature converts it to a form that can bind ions and have a productive interaction with the light chain. However, conditions that prevent the heavy chain from combining with the light chain also stabilize the two subunit structure, suggesting that the high affinity of the complex is due to conformational changes that occur after chain interaction.     

Prediction of the secondary structure of myosin light chains from comparison of homologous sequences. Implications for the interaction between myosin heavy and light chains

The primary sequences of seventeen essential and seventeen regulatory myosin light chains were analyzed and compared, using algorithms based on the different structural properties of their amino acid residues. This process allowed estimation of the structural homology between the proteins studied, and improved the prediction of their mean secondary structure and functionally important segments or residues. On the basis of the crystal structure of troponin C, a model of the myosin essential light chain with a fairly compact form is proposed. The possible sites of interaction between myosin light and heavy chains from rabbit skeletal muscle were also investigated by a complementarity method adapted to helix-rich proteins. Segments 139-149 and 65-75 in the essential light chain and segments 27-37, 67-77 and 97-107 in the regulatory light chain are suggested to constitute some of these sites, as most of them were found to have the features of surface-seeking helices.     

The multiplicity of combinations of myosin light chains and heavy chains in histochemically typed single fibres. Rabbit tibialis anterior muscle.

1. Combined histochemical and biochemical single-fibre analyses [Staron & Pette (1987) Biochem. J. 243, 687-693], were used to investigate the rabbit tibialis-anterior fibre population. 2. This muscle is composed of four histochemically defined fibre types (I, IIC, IIA and IIB). 3. Type I fibres contain slow myosin light chains LC1s and LC2 and the slow myosin heavy chain HCI, and types IIA and IIB contain the fast myosin light chains LC1f, LC2f and LC3f and the fast heavy chains HCIIa and HCIIb respectively. 4. A small fraction of fibres (IIAB), histochemically intermediate between types IIA and IIB, contain the fast light myosin chains but display a coexistence of HCIIa and HCIIb. 5. Similarly to the soleus muscle, C fibres in the tibialis anterior muscle contain both fast and slow myosin light chains and heavy chains. The IIC fibres show a predominance of the fast forms and the IC fibres (histochemically intermediate between types I and IIC) a predominance of the slow forms. 6. A total of 60 theoretical isomyosins can be derived from these findings on the distribution of fast and slow myosin light and heavy chains in the fibres of rabbit tibialis anterior muscle.     

Phosphorylation of smooth muscle myosin heavy and light chains. Effects of phorbol dibutyrate and agonists

A number of different protein kinases phosphorylate purified heavy chains or the 20-kDa light chain of smooth muscle myosin. The physiological significance of these phosphorylation reactions has been examined in intact smooth muscle. Myosin heavy chain was slightly phosphorylated (0.08 mol of phosphate/mol) under control conditions in bovine tracheal tissue. Treatment with carbachol, isoproterenol, or phorbol 12,13-dibutyrate resulted in no significant change. In contrast, heavy chain was phosphorylated to 0.30 mol of phosphate/mol of heavy chain in tracheal smooth muscle cells in culture. This value increased significantly with ionomycin treatment. In control tissues, 9% of the light chain was monophosphorylated with 32P in the serine site phosphorylated by myosin light chain kinase. Carbachol (0.1 microM) alone resulted in contraction and 42% monophosphorylated light chain with 32P only in the serine site phosphorylated by myosin light chain kinase. Similarly, stimulation with histamine, 5-hydroxytryptamine, or KCl resulted in 32P incorporation into only the myosin light chain kinase serine site. Phorbol 12,13-dibutyrate (1 microM) alone resulted in 22% monophosphorylated light chain. However, only 25% of the 32P was in the myosin light chain kinase serine site, whereas 75% was in a serine site phosphorylated by protein kinase C. Phorbol 12,13-dibutyrate plus carbachol resulted in 27% monophosphorylated light chain; 75% of the 32P was in the myosin light chain kinase serine site, with the remainder in the protein kinase C serine site. These results indicate that phorbol esters act to increase phosphorylation of myosin light chain by protein kinase C. However, receptor-mediated stimulation or depolarization leading to tracheal smooth muscle contraction results in phosphorylation of myosin light chain by myosin light chain kinase alone.