共查询到20条相似文献,搜索用时 31 毫秒
1.
J R Firca K R Ely P Kremser F A Westholm K J Dorrington A B Edmundson 《Biochemistry》1978,17(1):148-158
Previous crystallographic studies in this laboratory demonstrated that immunoglobulin light chains with the same amino acid sequence can have at least two and probably three or more conformations, depending on whether the second member of an interacting pair is a light or heavy chain. If a heavy chain is not available in the assembly medium, a second light chain plays the structural role of the heavy chain in the formation of a dimer. In the present work, the lambda-type light chains were dissociated from the heavy chains of a serum IgG1 immunoglobulin from the patient Mcg and reassembled noncovalently into a dimer. The reassembly process was completed by allowing the penultimate half-cystine residues to form an interchain disulfide bond. The covalently linked dimer was compared with the Mcg urinary Bence-Jones dimer, for which an atomic model has been fitted to a 2.3-A electron density map. The assembled dimer and the native Bence-Jones protein were indistinguishable in their chromatographic and electrophoretic properties, as well as in their activity in the binding of bis(dinitrophenyl)lysine. These results indicate that the light chains can be converted into the two types of Bence-Jones conformational isomers. The procedure was also reversed: the two Bence-Jones isomers were dissociated and reassembled as the single type of isomer associating with each of two heavy chains in the IgG1 protein. The change in activity occurring when a light chain associates with a heavy chain instead of a second light chain is illustrated by the fact that the Mcg IgG1 immunoglobulin does not bind dis(dinitrophenyl)lysine in measurable amounts. 相似文献
2.
Exchange of the fluorescence-labeled 20,000-dalton light chain of smooth muscle myosin. 总被引:3,自引:0,他引:3
The 20,000-dalton light chain of smooth muscle myosin was exchanged with exogenous light chain in a solution containing 0.5 M NaCl and 10 mM EDTA at 40 degrees C. The light chain was almost completely exchanged within 30 min under the above conditions. The exchange was markedly inhibited either below 37 degrees C or in the presence of Mg2+ concentrations higher than 10 microM. The 20,000-dalton light chain was selectively labeled of a single thiol (Cys-108) with 5-[[2-[(iodoacetyl)amino]ethyl]amino-naphthalene-1-sulfonic acid (1,5-IAEDANS). The labeled light chain was exchanged stoichiometrically into myosin and was used as a probe to investigate the conformation of smooth muscle myosin. The resulting myosin hybrids showed enzymatic properties virtually identical with those of the control, untreated myosin; i.e., actin-activated ATPase activity was dependent on the 20,000-dalton light-chain phosphorylation catalyzed by myosin light chain kinase, and the 10S-6S conformational transition of myosin correlating with the changes in ATPase was also affected either by the light-chain phosphorylation or by the change in the ionic strength. Steady-state fluorescence antisotropy measurements were performed by varying the temperature. The Perrin-Weber plots were constructed in order to obtain information about the average rotational mobility of the probe and to estimate the rotational correlation time for the AEDANS-myosin head. The fluorescence probe on the 20,000-dalton light chain was found to be quite immobile as indicated by its limiting anisotropy (A0 = 0.33).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
3.
Binding of the 5-(iodoacetamido)fluorescein (IAF)-labeled high molecular weight (HMW) kininogen light chain to prekallikrein and D-Phe-Phe-Arg-CH2Cl-inactivated kallikrein was monitored by a 0.040 +/- 0.002 increase in fluorescence anisotropy. Indistinguishable average dissociation constants and stoichiometries of 14 +/- 3 nM and 1.1 +/- 0.1 mol of prekallikrein/mol of IAF-light chain and 17 +/- 3 nM and 0.9 +/- 0.1 mol of kallikrein/mol of IAF-light chain were determined for these interactions at pH 7.4, mu 0.14 and 22 degrees C. Prekallikrein which had been reduced and alkylated in 6 M guanidine HCl lost the ability to increase the fluorescence anisotropy of the IAF-kininogen light chain, suggesting that the native tertiary structure was required for tight binding. The kallikrein heavy and light chains were separated on the basis of the affinity of the heavy chain for HMW-kininogen-Sepharose, after mild reduction and alkylation of kallikrein under nondenaturing conditions. Under these conditions, alkylation with iodo [14C]acetamide demonstrated that only limited chemical modification had occurred. Binding of the IAF-kininogen light chain to the isolated alkylated kallikrein heavy chain, when compared to prekallikrein and kallikrein, was characterized by an indistinguishable increase in fluorescence anisotropy, average dissociation constant of 14 +/- 3 nM, and stoichiometry of 1.2 +/- 0.1 mol of kallikrein heavy chain/mol of IAF-light chain. In contrast, no binding of the D-Phe-Phe-Arg-CH2Cl-inactivated kallikrein light chain was detected at concentrations up to 500 nM. Furthermore, 300 nM kallikrein light chain did not affect IAF-kininogen light chain binding to prekallikrein, kallikrein, or the kallikrein heavy chain. The binding of monomeric single chain HMW-kininogen to prekallikrein, kallikrein, and the kallikrein heavy and light chains was studied using the IAF-kininogen light chain as a probe. Analysis of the competitive binding of HMW-kininogen gave average dissociation constants and stoichiometries of 12 +/- 2 nM and 1.2 +/- 0.1 mol of prekallikrein/mol of HMW-kininogen, 15 +/- 2 nM and 1.3 +/- 0.1 mol of kallikrein/mol of HMW-kininogen, 14 +/- 3 nM and 1.4 +/- 0.2 mol of kallikrein heavy chain/mol of HMW-kininogen, and no detectable effect of 300 nM kallikrein light chain on these interactions. We conclude that a specific, nonenzymatic interaction between sites located exclusively on the light chain of HMW-kininogen and the heavy chain of kallikrein or prekallikrein is responsible for the formation of 1:1 noncovalent complexes between these proteins. 相似文献
4.
B N Manjula E B Mushinski C P Glaudemans 《Journal of immunology (Baltimore, Md. : 1950)》1977,119(3):867-871
Murine myeloma immunoglobulin (IgA, K) J539, which shows enhanced tryptophanyl fluorescence on ligand binding, and S10, which shows reverse-sign changes in tryptophanyl fluorescence on ligand binding (RLIF, see below), have been reduced, alkylated, and dissociated into their light (L) and heavy (H) chains. Two hybrid recombinants, H10L539 and H539L10, have been prepared and the 7S material has been isolated by chromatography. The binding behavior of these recombinants was studied with a number of ligands. Both recombinants showed activity with beta(1 leads to 6) linked galactose ligands comparable to the native immunoglobulins. The ligand-induced fluorescence changes of the recombinants paralleled those of the heavy chain donor. For the recombinant H10L539, two different galactose-ligands caused fluorescence changes in opposite directions. It was quantitatively shown that binding of these ligands, nevertheless, took place in the same combining region. The idiotype of each recombinant resembled that of the heavy chain donor. 相似文献
5.
Myosin light chain kinase is activated by Ca2+/calmodulin. Insights into the kinetic mechanism of this activation by Ca2+/calmodulin have now been obtained using extrinsically labeled fluorescent calmodulin, a fluorescent peptide substrate, and a stopped-flow spectrophotofluorimeter. We employed spinach calmodulin labeled with the sulfhydryl-selective probe, 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid, to measure changes in the fluorescence intensity of the 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin upon binding to rabbit skeletal muscle myosin light chain kinase. The fluorescent peptide substrate KKRAARAC(sulfobenzo-furazan)SNVFS-amide was used to measure kinase activity. Our results showed that the binding interaction could be modeled as a two-step process: a bimolecular reaction with an association rate of 4.6 x 10(7) M-1 s-1 followed by an isomerization with a rate of 2.2 s-1. Phosphorylation of the peptide during stopped-flow experiments could be modeled by a two-step process with a catalytic association rate of 6.5 x 10(6) M-1 s-1 and a turnover rate of 10-20 s-1. Our results also indicated that kinase activity occurred too rapidly for the slower isomerization rate of 2.2 s-1 to be linked specifically to the activation process. 相似文献
6.
Rapid isolation of cloned isotype switch variants using fluorescence activated cell sorting 总被引:7,自引:0,他引:7
We have used highly specific, directly fluorescein-conjugated heterologous (conventional) and monoclonal antibodies directed against mouse immunoglobulin isotypes in conjunction with the fluorescence activated cell sorter (FACS) to enrich and clone hybridoma cells producing new immunoglobulin heavy chain constant regions. Each variant retains the parental heavy chain variable region and the parental immunoglobulin light chain; thereby each variant binds the same dansyl (DNS) hapten. These isotype switch variants occur at frequencies of approximately 10-5 to 10-6. We were able to isolate the variants by first sorting for an approximate 1000-fold enrichment of the desired immunoglobulin-producing cells, growing these cells for five to nine days, followed by a second 1000-fold enrichment and direct cell cloning into 96 well culture trays. Clones were screened only 3-5 weeks after the original selection for secretion of dansyl-binding immunoglobulin of the selected isotype. Judicious combination of existing methods permits improved analytical techniques using the cell sorter. These include: first, "red" fluorescence staining of dead cells with ethidium bromide or propidium iodide and using the red fluorescence measurement to exclude dead cells from the green fluorescence selection; and second, the use logarithmic amplification of fluorescence signals, allowing for more succinct selection of fluorescence parameters for sorting. 相似文献
7.
Light chains associated with normal serum immunoglobulin can be resolved into a finite number of discrete focusing bands by isoelectric focusing. Four distinct light chain patterns can be distinguished among the inbred mouse strains. In the present studies inheritance of the characteristic light chain patterns has been studied in the AKXL recombinant inbred lines (derived from C57L/J and AKR/J parental lines) and in the inbred Ly-2a,3a congenic line B6.PL-Ly-2aLy-3a/Cy as well as in individual backcross animals of an incipient Ly-2a,3a congenic strain. Virtually complete concordance was observed for the expression of light chains characteristic of phenotype B (AKR-J-like) and the expression of the Ly-2a,3a allele. This observation indicates that a locus controlling light chain structure and/or expression is closely linked (less than 2.6 map units) to the Ly-2,3 locus on mouse Chromosome 6. The locus controlling normal light chain IF-patterns has been designated Ef1. 相似文献
8.
Morstadt L Bohm A Yüksel D Kumar K Stollar BD Baleja JD 《Protein science : a publication of the Protein Society》2008,17(3):458-465
The surrogate light chain (SLC) is a key regulator of B cell development in the bone marrow, resulting in mature B cells that produce antibodies that are capable of interacting with antigens. The SLC comprises two noncovalently interacting proteins: VpreB and 14.1. We engineered a construct to represent the complete immunoglobulin-like domain of the SLC variable domain in a single protein chain that could be bacterially expressed. In this construct, the incomplete immunoglobulin domain of VpreB (residues 1-102) was linked to the J-segment of 14.1 (residues 40-53), which provided one beta-strand to complete the V-like domain (VpreBJ). Because VpreBJ has the interface to VH chains, but lacks the unique region of 14.1, which is important for SLC signaling, we predict that a properly folded VpreBJ would have the potential to act as a dominant negative mutant of the surrogate light chain. X-ray crystallography of VpreBJ at 2.0 A resolution showed that the engineering was successful. With its two beta-pleated sheets, packed face-to-face, the single chain VpreBJ resembles a mature light chain immunoglobulin V-domain (VL). The surface that would normally interact with the VH chain interacts with a crystallographically related VpreBJ molecule. The presence of dimeric species in solution was verified by analytical ultracentrifugation. VpreBJ is easily overexpressed in bacteria, while retaining the native conformation of an immunoglobulin domain, and thus may serve as an important reagent for future studies in B-cell development. 相似文献
9.
Human myeloma cell line RPMI 8226 synthesizes and secretes a lambda immunoglobulin light chain. Total cellular RNA, obtained from cells grown in culture, was used for the isolation of poly(A)-containing mRNA by oligo(dT)-cellulose chromatography. The poly(A)-containing mRNA was translated in an Ehrlich ascites extract. Immunoprecipitation of the cell-free products showed that a polypeptide chain antigenically related to human lambda chain was synthesized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the cell-free product was larger than the secreted light chain. On tryptic digestion the cell-free product and the secreted light chain exhibited essentially identical peptide patterns except for an additional peptide derived from the cell-free product. We conclude that the lambda mRNA from this human myeloma cell line codes for a precursor to the secreted lambda chain as described for immunoglobulin mRNAs from murine plasmacytomas. 相似文献
10.
Unfolding and refolding of a type kappa immunoglobulin light chain and its variable and constant fragments 总被引:5,自引:0,他引:5
By limited proteolysis of a type kappa immunoglobulin light chain (Oku) with clostripain, both the VL and CL fragments were obtained with a high yield. The unfolding and refolding by guanidine hydrochloride of light chain Oku and its VL and CL fragments were studied at pH 7.5 and 25 degrees C with circular dichroism and tryptophyl fluorescence. The concentration of guanidine hydrochloride at the midpoint of the unfolding curve was 1.2 M for the VL fragment and 0.9 M for the CL fragment. As in the case of the CL fragment of light chain Nag (type lambda) [Goto, Y., & Hamaguchi, K. (1982) J. Mol. Biol. 156, 891-910], the unfolding and refolding kinetics of the CL fragment could be explained in principle on the basis of the three-species mechanism U1 in equilibrium U2 in equilibrium N, where N is native protein and U1 and U2 are the slow-folding and fast-folding species, respectively, of unfolded protein. The unfolding and refolding kinetics of the VL(Oku) fragment were described by a single exponential term. Double-jump experiments, however, showed that two forms of the unfolding molecule exist. The equilibrium and kinetics of unfolding of light chain Oku were explained by the sum of those of the VL and CL fragments. On the other hand, the refolding kinetics of light chain Oku were greatly different from the sum of those of the VL and CL fragments.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
11.
Immunoglobulins are tetrameric molecules consisting of two heavy and two light chains linked by disulfide bonds. Single light chains are normally secreted in the plasma under soluble form. These immunoglobulin free light chains circulating in the blood may hold unexpected roles in diseases.Minimal change disease is defined as a renal disease with massive proteinuria and no obvious damage on light microscopy. We hypothesize that minimal change disease is not a primary renal disease but an immune disease due to a defect in B cells mediated by a special immunoglobulin chain. The efficiency of drugs targeting the immune system and the association to Hodgkin disease as well as: (1) the efficiency of B cell depletion to prevent relapse; (2) the association with B leukemia; and (3) the activation of CD23 during relapse point up a primary involvement of B cells. We hypothesize that an immunoglobulin chain with special polymorphism might be the link between the immune system and the dysfunction of the glomerular wall while immunoglobulin depletion leads to a transient remission of proteinuria in graft recurrence of the disease and nephropathy mediated by a monoclonal immunoglobulin chain may feature minimal change disease.Other diseases where free light chains may be involved include atopy, thromboembolism, glomerular inflammatory diseases and autoimmune diseases. We conclude that free circulating light chains, through infinite possibilities of polymorphisms determined by the variable domain are potential disturbing agents of many biological cascades or structures and could likely play the first role in multiple diseases. 相似文献
12.
F J Walker 《The Journal of biological chemistry》1992,267(28):19896-19900
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. 相似文献
13.
There has been controversy in the past about whether or not antibody-forming cells have antigen-specific receptors. Evidence is presented here which clearly demonstrates that some antibody-forming cells possess receptors while others show no evidence of receptors. Moreover, in this report the receptors are characterized in terms of their light and heavy chain immunoglobulin determinants. Receptors were found on ~50% of the direct PFC 6 days after one injection of sheep red blood cells (SRBC). On Day 4 of the secondary response only 20–33% of the direct PFC were found to possess receptors. Receptors could not be detected on indirect PFC in either the primary or secondary response. Results similar to the above were observed for the primary and secondary anti-hapten response to 3-nitro-4-hydroxy-5-iodophenyl acetic acid coupled to diphtheria toxoid (NIP-TOX). The receptors on the antibody-forming cells were shown to possess a class-specific heavy chain determinant (TgM) as well as two allotypic determinants, one a product of the a locus on the heavy chain and the other a product of the b locus on the light chain. The antigen-binding receptors were modulated to cap formation by antibody directed toward either light or heavy chain immunoglobulin determinants. The antigen-binding activity of the receptors was completely destroyed by trypsin. 相似文献
14.
Limited proteolysis of high molecular weight kininogen by kallikrein resulted in the generation of an inactive heavy chain of Mr = 64,000 and active light chains of Mr = 64,000 and 51,000 when analyzed by sodium dodecyl sulfate (SDS)-gel electrophoresis under reducing conditions. Starting with kininogen from outdated plasma, a light chain with an apparent molecular weight of 51,000 on 7.5% SDS gels was purified and characterized. Molecular weights of 28,900 +/- 1,100 and 30,500 +/- 1,600 were obtained by gel filtration of the reduced and alkylated protein in 6 M guanidine HCl and equilibrium sedimentation under nondenaturing conditions in the air-driven ultracentrifuge, respectively. The light chain stained positively with periodic acid-Schiff reagent on SDS gels indicating that covalently attached carbohydrate may be responsible for the anomalously high molecular weight estimated by SDS-gel electrophoresis. A single light chain thiol group reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) in the presence and absence of 6 M guanidine HCl. Specific fluorescent labeling of the thiol group with 5-(iodoacetamido)fluorescein (IAF) occurred without loss of clotting activity. Addition of purified human plasma prekallikrein to the IAF-light chain resulted in a maximum increase in fluorescence anisotropy of 0.041 +/- 0.001 and no change in the fluorescence intensity. Fluorescence anisotropy measurements of the equilibrium binding of prekallikrein to the IAF-light chain yielded an average Kd of 17.3 +/- 2.5 nM and stoichiometry of 1.07 +/- 0.07 mol of prekallikrein/mol of IAF-light chain. Measurements of the interaction of prekallikrein with iodoacetamide-alkylated light chain using the IAF-light chain as a probe gave an average Kd of 16 +/- 4 nM and stoichiometry of 1.0 +/- 0.2 indicating indistinguishable affinities for prekallikrein. 相似文献
15.
John F. Kennedy David Catty Patricia A. Keep 《International journal of biological macromolecules》1980,2(3):137-142
The purification of rabbit immunoglobulin molecules expressing kappa (κ) light chains, utilizing the allotypic specificity b4, has been achieved in stages involving isolation of specific antibody, preparation of a solid phase immunoadsorbent of coupled antibody, and subsequent isolation of b4 (κ) IgG. Cellulose trans-2.3-carbonate is shown to be an effective matrix enabling chemical coupling of antibodies and antigens to the support at neutral pH thus preservng immunological activity. The trans-2,3-carbonate derived from microcrystalline cellulose is more effective as a matrix than the trans-2,3-carbonate derived from macroporous cellulose for the chemical coupling of rabbit a1a3/b4 IgG antigen and binding of specific anti-b4 antibody. The microcrystalline celulose carbonate is also more efficient for the coupling of rabbit anti-b4 antibody and the subsequent binding and elution of rabbit b4 (κ) IgG, thus separating immunoglobulin, expressing kappa light chain, from that expressing lambda light chain. The purification technique has potential application in other allotypic systems and antibody- antigen populations. 相似文献
16.
The first chicken immunoglobulin light (L) chain allotypic specificity (L-1.1) to be described that was present on IgM, 7S Ig, Fab, and L chains was detected by radioimmunoassay. The gene controlling the expression of L-1.1 is inherited in a simple Mendelian fashion at an autosomal locus and is unlinked to a constant region heavy chain locus, four blood group loci and three loci determining lymphocyte cell surface alloantigens. 相似文献
17.
Immunoglobulin light chain amyloidosis (AL) is a cancer of plasma cells that secrete unstable full-length immunoglobulin light chains. These light chains misfold and aggregate, often with aberrant endoproteolysis, leading to organ toxicity. AL is currently treated by pharmacological elimination of the clonal plasma cells. Since it remains difficult to completely kill these cells in the majority of patients, we seek a complementary drug that inhibits light chain aggregation, which should diminish organ toxicity. We discovered a small-molecule binding site on full-length immunoglobulin light chains by structurally characterizing hit stabilizers emerging from a high-throughput screen seeking small molecules that protect full-length light chains from conformational excursion-linked endoproteolysis. The x-ray crystallographic characterization of 7 structurally distinct hit native-state stabilizers provided a structure-based blueprint, reviewed herein, to design more potent stabilizers. This approach enabled us to transform hits with micromolar affinity into stabilizers with nanomolar dissociation constants that potently prevent light chain aggregation. 相似文献
18.
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. 相似文献
19.
Sequence analysis of the 3'' non-coding region of mouse immunoglobulin light chain messenger RNA. 总被引:3,自引:3,他引:0
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Using an oligonucleotide d(pT10-C-A) as primer, cDNA has been transcribed from the 3' non-coding region of mouse immunoglobulin light chain mRNA and sequenced by a modification1 of the 'plus-minus' gel method2. The sequence obtained has partially corrected and extended a previously obtained sequence3. The new data contains an unusual sequence in which a trinucleotide is repeated seven times. 相似文献
20.
Melittin is a 26-residue peptide which undergoes high-affinity calcium-dependent binding by calmodulin [Barnette, M.S., Daly, R., & Weiss, B. (1983) Biochem. Pharmacol. 32, 2929; Comte, M., Maulet, Y., & Cox, J.A. (1983) Biochem. J. 209, 269; Anderson, S.R., & Malencik, D.A. (1986) Calcium Cell Funct. 6, 1]. The results in this paper show that three different types of myosin light chain--the smooth muscle regulatory light chain, the smooth muscle essential light chain, and the skeletal muscle regulatory 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) light chain--also associate with melittin. The resulting complexes have dissociation constants ranging from 1.1 to 2.5 microM in the presence of 0.10 M NaCl and from approximately 50 to approximately 130 nM in solutions of 20 mM 3-(N-morpholino)propanesulfonic acid alone. The regulatory smooth muscle myosin light chain exhibits two equivalent melittin binding sites while each of the others displays only one. The myosin light chains evidently contain elements of structure related to the macromolecular interaction sites present in calmodulin and troponin C but not in parvalbumin. The association of melittin and other peptides with the light chains requires consideration whenever assays of the calmodulin-dependent activity of myosin light chain kinase are used to determine peptide binding by calmodulin. The binding measurements performed on the DTNB light chain and melittin necessitated derivation of the equation relating complex formation to the observed fluorescence anisotropy of a solution containing three fluorescent components. This analysis is generally applicable to equilibria involving the association of two fluorescent molecules emitting in the same wavelength range. 相似文献