Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0026764 (multiple myeloma)
 36,148 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This paper reports the initial progress in a research programme to identify and obtain the relative orientations, in solution, of the amino acid residues that constitute the combining site of the myeloma protein MOPC 315. This protein has a molecular mass of 150,000, but enzymic digestion yields the Fv fragment of molecular mass 25,000 which still has the combining site intact, as judged by the affinity for dinitrophenyl haptens. Analysis of the e.s.r. spectra of a series of dinitrophenyl spin labelled haptens has allowed the dimensions, rigidity and polarity profile of the combining site to be determined. The combining site is a cleft of overall dimensions 1.1 nm x 0.9 nm x 0.6 nm which has considerable structural rigidity. One of these spin labels has also been used to perturb the n.m.r. spectrum of the Fv and using difference spectroscopy the 270 MHz proton n.m.r. spectrum of the amino acid residues in and around the combining site has been obtained. This spectrum contains only the equivalent of about 30 aromatic and 21 aliphatic protons. Comparison of this difference spectrum with that obtained using a diamagnetic analogue suggests that any conformational changes on hapten binding are mainly localized to the combining site. By the use of (n.m.r.) difference spectroscopy the protons of the three histidine residues in the Fv are observed to titrate with pH and have pKa values of about 8.1, 6.9 and 6.1. The histidine resonances with pKa values 6.9 and 6.1 alter slightly in the presence of haptens and also appear in the spin label difference spectrum, and must therefore be in or near to the combining site. These are assigned to His 102H and His 97L. The existence of lanthanide binding sites on the Fv, necessary for the mapping studies, has been demonstrated by measurements of Gd III water relaxation rates in Fv solutions and also by the changes in the Fv tryptophan fluorescence on addition of Gd III. At pH 5.5 there is one tight binding site for the lanthanides (KD approximately 80 muM) but in the presence of hapten this is weakened 10-20 fold with a reciprocal effect on the hapten binding. Measurements of the Gd III quenching of the e.s.r. spectrum of a spin labelled hapten bound to Fv indicate that the lanthanide site is ca. 1.5 nm from the nitroxide moiety.
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PMID:Antibody--hapten interactions in solution. 0 18

In order to test the concepts that aminoacyl-tRNAs in plasmacytomas may on the one hand modulate the protein synthesized or on the other hand reflect the structure of the synthesized protein, the RPC-5 chromatographic profiles of aminoacyl-tRNAs for all 20 amino acids were studied in tRNA prepared from normal mouse liver and 11 plasmacytomas. The patterns of isoaccepting tRNA were compared with the structure of the myeloma protein being synthesized. The elution profiles of aminoacyl-tRNAs for nine of the amino acids were constant, i.e. they were the same for liver and all plasmacytomas. Significant variability was observed in the profiles of the other 11 families of aminoacyl-tRNAs: asparagine, serine and tryptophan, had peaks of isoaccepting tRNAs found in tumors and not in liver; glutamic acid, histidine and lysine, had different patterns of aminoacyl-tRNAs in plasmacytomas which could be distinguished from the elution profile of liver; and isoleucine, proline, threonine and tyrosine, showed pattern variability in only a few of the tumors. Valyl-tRNA uniquely had one isoacceptor present in liver but absent in the tumors. This variability is thought to be associated with different posttranscriptional modification of the tRNAs rather than regulation of individual tRNA genes in response to particular amino acid sequences in secreted myeloma proteins. Similarily, the lack of correlation of isoacceptors with sequence differences makes the modulation of protein fine structure by tRNA availability unlikely.
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PMID:Transfer ribonucleic acids from eleven immunoglobulin-secreting mouse plasmacytomas. Constant and variable chromatographic profiles compared with the myeloma protein sequences. 25 44

The binding of Tnp (2,4,6-trinitrophenyl) derivatives to the Fv fragment (variable region of heavy and light chains) of the mouse myeloma IgA protein MOPC 315 was investigated by 270MHz proton nuclear magnetic resonance. Two of the haptens, Tnp-glycine and Tnp-l-aspartate, are in fast exchange with the Fv fragment, and the changes in chemical shifts for both protein and hapten resonances were determined by titrations. For the tightly binding hapten epsilon-N-Tnp-alpha-N-acetyl-l-lysine, which is in slow exchange with the Fv fragment, the changes in chemical shifts for the hapten H(3)+H(5) resonances were determined by cross-saturation. By using these data and the known structure of the combining site of protein MOPC 315 [Dwek, Wain-Hobson, Dower, Gettins, Sutton, Perkins & Givol (1977), Nature (London) 266, 31-37] the mode of binding of Tnp derivatives is deduced by ring-current calculations. The trinitrophenyl ring stacks with tryptophan-93(L) (light chain) in the ;aromatic box' formed by tryptophan-93(L), tyrosine-34(L) and phenyl-alanine-34(H) (heavy chain). Further evidence for the stacking interaction with a tryptophan residue is provided by the similarity of the optical-difference spectra observed with Tnp-aminomethylphosphonate in the presence of either the Fab fragment (light chain and N-terminal half of heavy chain) of protein MOPC 315 or tryptophan. These data show that the modes of binding of all the Tnp derivatives are very similar, despite a 100-fold range in their affinities. It is also concluded that the modes of binding of Dnp (2,4-dinitrophenyl) and Tnp derivatives to protein MOPC 315 are very similar, and that the structural basis for this is that the aromatic box is large enought to allow the trinitrophenyl ring to stack with tryptophan-93(L) while still forming hydrogen bonds to asparagine-36(L) and tyrosine-34(L).
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PMID:The binding of 2,4,6-trinitrophenyl derivatives to the mouse myeloma immunoglobulin A protein MOPC 315. 62 44

To provide information on the tertiary structure of the antibody molecule we have investigated the luminescent properties of the light polypeptide chain of human immunoglobulins. The fluorescence and phosphorescence yields, spectra, lifetimes, and anisotropies of a large number of homogeneous light chains, i.e., Bence-Jones proteins and light chains derived from myeloma proteins, were measured. No two proteins gave identical tyrosyl or tryptophyl fluorescence spectra in comparative studies on over 75 proteins belonging to the four basic subgroups of kappa chains and of lambda chains. Spectral differences were apparent even among proteins exhibiting more than 85% amino acid sequence identity. The fluorescence yields of tyrosine and tryptophan vaired 10- and 100-fold, respectively; the Stokes' shift of tryptophan ranged from 328 to 365 nm, but that for tyrosine was apparently invariant (305-307nm). Emission as well as excitation spectra showed tyrosyl and tryptophyl redidues interact minimally or not at all. Fluorescence lifetimes of the tyrosyl and tryptophyl contributions were measured spearately, and the apparent natural lifetimes were calculated. Proteins could be grouped in accordance with similarities in fluorescence lifetimes and fluorescence yields; there was no evident relationship between these groupings and the light chain type (kappa or lambda), amino acid sequence, or tryptophan content. Also apparent were individual differences among kappa light chains and among lambda light chains in respect to their tyrosyl and trptophyl phosphorescence spectra and phosphorescence lifetimes. Certain proteins showed an atypical, short-lived tryptophan phosphorescence decay time. Such variance in the luminescent behavior of the tryptophyl residue(s) indicates a conformational interaction between the V and C domains of light chains. Selective proteolytic cleavage of the light chain into VL and CL fragments permitted the comparison to be made of the luminescent properties of the V and C domains with those of the whole protein. The V domain and intact protein have luminescent features in common, whereas the C domain possesses features distinctive from that of the native protein. Data derived from fluorescence anisotropy spectral studies of intact light chains and their VL-related fragments indicate that energy transfer between tryptophyl residues occurs in the C domain. The results of emission spectroscopic measurements performed at 220 and at 77 K indicate that the observed phophorescence of light chains is mainly from a tryptophyl residue contiguous to a disulfide link. The potential for interdomain interaction in light chains is evidenced by the finding that the orientation of the tryptophyl residue(s) in the V domain can influence the tryptophyl-disulfide ling interactions in the C domain; this interaction may account further for the extensive structural diversity of antibody molecules.
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PMID:Luminescence studies on Bence-Jones proteins and light chains of immunoglobulins and their subunits. 82 15

Magnetic-resonance techniques are used to refine the model of the combining site of the Fv fragment of the dinitrophenyl-binding mouse myeloma protein MOPC 315 constructed by Padlan, Davies, Pecht, Givol & Wright (1976) (Cold Spring Harbor Symp. Quant. Biol.41, in the press). Light-absorption studies indicate a dinitrophenyl-tryptophan interaction in the Fv fragment of the type occurring in free solution. The Dnp-aspartate-tryptophan complex is therefore used as a starting point for the n.m.r. (nuclear-magnetic-resonance) analysis of the dinitrophenyl-Fv fragment interaction. Ring-current calculations are used to determine the geometry of the complex. The specificity of complex-formation between dinitrophenyl and tryptophan is confirmed by the lack of ring-current shifts of the dinitrophenyl resonances when tryptophan is replaced by any other aromatic amino acid. Proton n.m.r. difference spectra (at 270MHz), resulting from the addition of a variety of haptens to the Fv fragment, show that the combining site is highly aromatic in nature. Calculations on the basis of ring-current shifts define the geometry of the combining site, which involves a dinitrophenyl ring in van der Waals contact with four aromatic amino acid residues on the protein. The observation of a nuclear Overhauser effect on the H((3)) resonance of the dinitrophenyl ring provides additional constraints on the relative geometry of the H((3)) proton and an aromatic amino acid residue on the Fv fragment. The specificity of the Fv fragment for dinitrophenyl ligands arises from a stacking interaction of the dinitrophenyl ring with tryptophan-93(L), in an ;aromatic box' of essentially tryptophan-93(L), phenylalanine-34(H) and tyrosine-34(L); asparagine-36(L) and tyrosine-34(L) also contribute by forming hydrogen bonds with the nitro groups on the dinitrophenyl ring. The n.m.r. results also confirm that the antibody-hapten reaction may be visualized as a single encounter step. An Appendix shows the method of calculation of ring currents for the four aromatic amino acids and their use in calculating structures.
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PMID:The combining site of the dinitrophenyl-binding immunoglobulin A myeloma protein MOPC 315. 92 44

A recombinant myeloma NS1-derived clone was grown in chemostat cultures in Dulbecco's MEM/Ham's F12 (1:1) medium containing various concentrations of glucose, at a dilution rate of 0.028 h-1. Serum-supplemented cultures were virtually glucose-limited at a large range of glucose feed concentrations (0.7-5 mM). True glucose-limited cultures, however, were only established at low glucose supply levels to 1.3 mM at a maximum. In cultures obtained at higher glucose concentrations methionine was shown to be the growth-limiting compound. The pattern derived for serum-free chemostat cultures was similar, except that growth yields on glucose were much lower. Glucose was shown to be the growth-limiting substrate in cultures fed with media containing less than 4.5 mM glucose. Upon supplying glucose at higher concentrations such cultures presumably run into methionine and/or tryptophan limitation.
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PMID:Physiology of myeloma cells grown in glucose-limited chemostat cultures. 136 65

Hybridomas secreting monoclonal antibodies to Nippostrongylus brasiliensis antigens were generated by hybridization of IR983F myeloma cells with spleen cells from Lou/M/Wol rats infected with living third-stage larvae. Antibodies specific either for larval or worm antigens were identified by enzyme-linked immunosorbent assays with Nippostrongylus brasiliensis fragments, homogenates and secretions as antigens. The results demonstrate that all antibodies which recognized larval antigens (38 antibodies) also reacted with worm surfaces. Ten antibodies were specific only for worm antigens. Ten antibodies reacted with worm homogenate, three antibodies recognized components of worm secretion and 17 antibodies combined with acetylcholinesterase. The epitope specificity was investigated by the capacity of various glycosides, aminoacids, N-acetylneuraminic acid and phosphorylcholine to inhibit the binding to worm fragments. The analysis revealed that alpha-methylglucoside, alpha-methylmannoside, N-acetylglucosamine, N-acetylgalactosamine, fucose and the amino acids leucine, phenylalanine, tyrosine, serine, tryptophan did not combine with the antigen-binding sites of the antibodies. Proline, arginine and histidine, however, displayed inhibitory effects. With N-acetylneuraminic acid as inhibitor three groups of antibodies could be discriminated. At a concentration of 10-20 mM, phosphorylcholine was a potent inhibitor for all antibodies.
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PMID:Characterization of antigens of the nematode Nippostrongylus brasiliensis by monoclonal antibodies. 241 42

Phosphorescence spectroscopy on mouse myeloma IgA J539 in rigid solution at 77K revealed the type of anomalous short-lived component in the tryptophan decay originally observed with lysozyme (Churchich, J.E., 1966. Biochim. Biophys. Acta. 120:406-412) and seen in a large number of Bence Jones proteins (Longworth, J.W., C.L. McLaughlin, and A. Solomon. 1976. Biochemistry. 15:2953-2958). The decay time of the anomalous component that results from the interaction between tryptophan side chains and disulfide linkages in proteins was observed to significantly lengthen in J539 in response to binding of a galactan antigen. With hen egg-white lysozyme in which the type of fluorescence enhancement on ligand binding seen with J539 has also been observed, phosphorescence measurements revealed a similar lengthening of the decay time of the disulfide-induced anomalous component in the tryptophan decay. These perturbations are interpreted as ligand-induced changes to the tryptophan-disulfide proximities that have been shown to exist in these structures. Given the short-range nature of the disulfide perturbation (see following article) the observations suggest, in particular when combined with x-ray crystallographic data, that phosphorescence decay-time measurements of disulfide perturbations can serve as a sensitive spectroscopic indicator of subtle conformational changes in immunoglobulins and other tryptophan-disulfide containing proteins.
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PMID:Evidence for ligand-induced conformational changes in proteins from phosphorescence spectroscopy. 277 30

To explore the possibility that the affinity of some myeloma proteins for 2,4-dinitrophenyl (DNP) ligands is the consequence of a "strange" (i.e., unexpected) cross-reaction for more natural ligands, a variety of substances (primarily derivatives of purines, pyrimidines, naphthaquinone) were tested for ability to block the binding of [(3)H]-epsilon-DNP-L-lysine by protein 315, an IgA mouse myeloma protein with high affinity for DNP ligands. The most impressive inhibiting activity was observed with 2-methyl-1,4-napthaquinone (menadione, vitamin K(3)). The affinity (intrinsic association constant) of protein 315 for menadione was 5 x 10(5) L/M (at 4 degrees C). Because the same affinity was measured in direct-binding assays (e.g., equilibrium dialysis) and in an indirect one based on the assumption of competitive binding with DNP-lysine, it is likely that menadione and DNP bind at overlapping sites in the protein's combining region. This conclusion is supported by molecular models which reveal some common structural features in these ligands. Hence it is not surprising that antinitrophenyl antibody preparations, raised by conventional immunization procedures (anti-2,4-DNP; anti-2,6-DNP; anti-2,4,6-TNP) also bind menadione with considerable affinity. As with DNP ligands, when menadione binds to protein 315 or to conventional antinitrophenyl antibodies, some of the protein's tryptophan fluorescence is quenched, there is a change in the ligand's absorption spectrum (hypochromia and/or red shift), and the binding is temperature-dependent (exothermal).
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PMID:The strange cross-reaction of menadione (vitamin K3) and 2,4-dinitrophenyl ligands with a myeloma protein and some conventional antibodies. 413 7

A mouse helix-destabilizing protein (HD protein-1) has been purified and characterized, and controlled tryptic digestion has been used to generate two large fragments of this protein and to study structural changes accompanying DNA binding. HD protein-1, a DNA-binding protein that has higher affinity for single-stranded DNA (ssDNA)-cellulose than for double-stranded DNA (dsDNA)-cellulose and is resistant to a dextran sulfate elution from ssDNA-cellulose, was purified from mouse myeloma by the method described by Herrick and Alberts (Herrick, G., and Alberts, B. M. (1976) J. Biol. Chem. 251, 2124-2132). HD protein-1 was heterogeneous with regard to apparent molecular weight (range of Mr = 24,000 to 33,000), but individual Mr species shared extensive primary structure homology as revealed by tryptic peptide mapping. The predominant species of this protein, Mr = 27,000, was resolved from other species and obtained in nearly homogeneous form by preparative isoelectric focusing. Mouse HD protein-1 was capable of lowering the Tm of poly[d(A-T)] by 25 degrees C, indicating that it is a helix-destabilizing protein. Sedimentation boundary analysis revealed that binding to ssDNA was noncooperative and that the binding site covered about 6 nucleotide residues. There was a 35% increase in the intrinsic tryptophan fluorescence of the protein in the presence of ssDNA, suggesting that structural change accompanies binding. Subcellular localization studies indicated that 75% of mouse HD protein-1 is nuclear, but not chromatin-associated, and primary structure analysis indicated that HD protein-1 is distinct from high mobility group proteins 1 and 2, histones, and P8 protein. Tryptic hydrolysis of HD protein-1 produced discrete, large fragments whose apparent molecular weights ranged from 19,000 to 24,000, and whose relative abundance was changed by the presence of ssDNA during the digestion. Thus, a Mr = 22,000 fragment (22 HDP*) predominated in the absence of ssDNA, and a Mr = 19,000, fragment (19 HDP*) predominated in the presence of ssDNA. Poly(dT) and denatured calf thymus DNA were more effective than were other polynucleotides tested in promoting accumulation of 19 HDP*; (dT)8 was as effective as were longer molecules of (dT)n, but (dT)4 and (dT)6 were much less effective, indicating that the binding site involved in 19 HDP* accumulation covered between 6 and 8 residues of (dT)n. Both 19 HDP* and 22 HDP* have the same COOH-terminal end and the same affinity for ssDNA-cellulose as does the native HD protein-1, indicating that a Mr = 8,000 sequence at the NH2-terminal end of HD protein-1 is not required for binding to ssDNA. Even though 22 HDP* retained the ability to bind to ssDNA, it could not be converted to 19 HDP* by further trypsin digestion.
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PMID:Studies on the structure of mouse helix-destabilizing protein-1. DNA binding and controlled proteolysis with trypsin. 625 73


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