Kinetics of dimerization of the bence-jones protein Au

Maéda, Hiroshi; Steffen, Erna; Engel, Jürgen

doi:10.1016/0301-4622(78)87015-x

Cited by 17 publications

(11 citation statements)

References 13 publications

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“…These values are comparable to those that have been determined for K and X chains by large-zone gel filtration, circular dichroism, and sedimentation equilibrium (24)(25)(26)(27)(28)(29) The KD calculated from a computer-simulated analysis of protein AU was comparable to the KD, 6.6 X l04 M-1, obtained experimentally (24). Gel filtration through Sephadex G-75 of protein AU at concentrations of 20, 10, and 2 mg/ml indicated that the elution positions of both the monomer and VL fragment were concentration dependent; as expected, sample dilutions resulted in a shift of these species to an apparently lower Mr (Fig.…”

Section: Resultssupporting

confidence: 87%

“…The virtually identical primary structure of the CL portions of these proteins (and of the other K chains studied) implicates the VL portion of the polypeptide chain as the region responsible for the observed heterogeneity in self-association of light chains. The KDs of the native X chain TOD and the KI protein AU were slightly less than that of their respective VL-related fragments (24,28,32); the C domain therefore has little influence on the association of these light chains.…”

Section: Discussionmentioning

confidence: 87%

See 1 more Smart Citation

Self-association of human immunoglobulin kappa I light chains: role of the third hypervariable region.

Stevens¹,

Westholm²,

Solomon³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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Gel electrophoresis and molecular sieve chromatography were used to compare 17 different human KI type Bence Jones proteins including 5 for which the amino acid sequence is known. Although electrophoresis in the presence of NaDodSO4 showed uniformity of covalent dimer and monomer molecular weights, Sephadex chromatography under nondissociating conditions showed that monomers eluted with different apparent molecular weights. These differences were attributed to heterogeneity in light chain self-association; dimerization constants of the 17 proteins, calculated from a computer simulation of their behavior upon gel filtration, ranged from <103 to >106 M-1. The variable region, more specifically the third hypervariable region, appears to be responsible for the variation in the dimerization constant. Association properties of light chains of known sequence suggest that the presence of an aromatic or hydrophobic residue at position 96 enhances dimer formation whereas a charged residue at that position results in light chains remaining stable monomers. The location of hypervariable residue 96 within the amino-terminal portion of the joining segment of the variable region suggests that the joining region may account for the variability of selfassociation of light chains and, moreover, that it has a function in determining the selective association of immunoglobulin polypeptide chains.Studies on the physical and chemical properties of Bence Jones proteins-the homogeneous counterparts of immunoglobulin light chains-have provided considerable information concerning the structural basis for the generation of antibody diversity (1). Sequence analysis of K and X type Bence Jones proteins has revealed that light chains possess a primary structure characterized by variation in sequence among the first 107 amino-terminal residues, the variable half of the light chain (VL), and by a relatively invariant sequence of the ;107 carboxy-terminal residues, the constant half (CL). The VL consists of framework and hypervariable regions. Four subgroups of K chains and five subgroups of X chains have been defined structurally and immunochemically by the framework sequence homologies. The three hypervariable segments (HV1, HV2, HV3) contribute to the antigen-binding site (2).The VL and CL are under separate genetic control; recent studies of murine Bence Jones proteins indicate that the VL is encoded by two gene segments. One, the V gene, determines the sequence of the first t95 amino-terminal residues; the second, the joining (J) gene, determines the sequence of -10-12 carboxyl-terminal VL residues (3-6). The joining (J) segment of the V region contains a portion of segment HV3; thus, it contributes directly to the sequente diversity of the antigenbinding site. Its other functions, if any, have not as yet been determined. Whereas X Bence Jones proteins and light chains exist primarily as disulfide-linked covalent dimers, K Bence Jones proteins and light chains occur more commonly in the form of noncovalent dimers, stable monomers, ...

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Section: Resultssupporting

confidence: 87%

Section: Discussionmentioning

confidence: 87%

Self-association of human immunoglobulin kappa I light chains: role of the third hypervariable region.

Stevens¹,

Westholm²,

Solomon³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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“…of 35000. This compares favourably with a KD of 6.6 x 104M-1 determined spectrophotometrically for protein Au at 20°C by Maeda et al (1978). The simulated gel filtration of a mixture of two proteins is of interest in this laboratory for experiments in which the heterologous association of Bence-Jones proteins is examined.…”

Section: Fig 2 Simulated Elution Patterns Demonstrating Effect Ofcosupporting

confidence: 57%

“…The association constant (KD) governing protein self-association is typically determined by equilibrium sedimentation (Steiner, 1952;Klotz et al, 1975) or chromatographically by large-zone (plateau) gel filtration (Winzor & Scheraga, 1963, 1964; Ackers, 1975). Certain cases in which the environment of an aromatic amino acid residue is altered by the aggregation of the polypeptides permit the use of spectrophotometric methods to monitor changes in circular dichroism, fluorescence polarization or absorbance (Azuma et al, 1978;Maeda et al, 1976Maeda et al, , 1978 to characterize the association process. The value of computer simulation to assist the interpretation of sedimentation equilibrium was illustrated by Cox (1965a,b), and simulation techniques have been extensively applied to large-zone gel filtration in studies employing numerical solution to transport equations characterizing the chromatography process (for reviews, see Ackers, 1970Ackers, , 1975.…”

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confidence: 99%

Computer simulation of protein self-association during small-zone gel filtration. Estimation of equilibrium constants

Stevens¹,

Schiffer²

1981

Biochemical Journal

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A simulation is developed that qualitatively describes the small-zone-gel-filtration behaviour of a reversibly associating protein. The results reflect the dependence of the apparent molecular weight of a reversibly associating protein on the equilibrium constant (KD) and initial concentration of the protein as well as the column length. The behaviour of a protein on an individual column is characterized and thus a means is provided for estimation of KD. The procedure is extended to describe the behaviour of a mixture of two proteins capable of heterologous as well as homologous association. This computer simulation has been applied in association studies of immunoglobulin light chains [Stevens, Westholm, Solomon & Schiffer (1980) Proc. Natl. Acad. Sci. 77, 1144--1148]. The KD value determined for the Bence--Jones protein Au (10(5) M-1) is close to the value (6.6 X 10(4) M-1) determined by other methods [Maeda, Steffen & Engel (1978) Biophys. Chem. 9, 57-64].

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“…The calculated dimerization constant for rREC was unusually high, IO' M-', approximately two orders of magnitude higher than that of the nonpathologic LEN and rLEN proteins. The dimerization constants of rLEN, IO5 M-', and rSMA, IO6 M-I, were in the range of the selfassociation constants observed for other human Ig light chains, lo3 M" to IO6 M" (Maeda et al, 1978;Stevens et al, 1980;Kolmar et al, 1994).…”

Section: Light-chain Dimer Formationmentioning

confidence: 90%

Recombinant immunoglobulin variable domains generated from synthetic genes provide a system for in vitro characterization of light‐chain amyloid proteins

et al. 1995

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The primary structural features that render human monoclonal light chains amyloidogenic are presently unknown. To gain further insight into the physical and biochemical factors that result in the pathologic deposition of these proteins as amyloid fibrils, we have selected for detailed study three closely homologous protein products of the light-chain variable-region single-gene family VKIV. Two of these proteins, REC and SMA, formed amyloid fibrils in vivo. The third protein, LEN, was excreted by the patient at levels of 50 g/day with no indication of amyloid deposits. Sequences of amyloidogenic proteins REC and SMA differed from the sequence of the nonpathogenic protein LEN at 14 and 8 amino acid positions, respectively, and these amino acid differences have been analyzed in terms of the three-dimensional structure of the LEN dimer. To provide a replenishable source of these human proteins, we constructed synthetic genes coding for the REC, SMA, and LEN variable domains and expressed these genes in Escherichia coli. Immunochemical and biophysical comparisons demonstrated that the recombinant VKIV products have tertiary structural features comparable to those of the patient-derived proteins. This welldefined set of three clinically characterized human KIV light chains, together with the capability to produce these KIV proteins recombinantly, provide a system for biophysical and structural comparisons of two different amyloidogenic light-chain proteins and a nonamyloidogenic protein of the same subgroup. This work lays the foundation for future investigations of the structural basis of light-chain amyloidogenicity.Keywords: amyloid proteins; immunoglobulin light chains; kappa IV domains; recombinant human VL; synthetic genes; variable-domain dimerization Over the past 30 years, detailed analyses of the structure and biomutagenesis to investigate effects of the changes on biological physical properties of immunoglobulin molecules have probed activities; synthetic Ig genes have been generated for the promany aspects of Ig interactions and effector functions (Padlan, duction of unique antibody reagents for medical and diagnos-1994). More recently, Ig genes have been cloned and altered by tic purposes (Borrebaeck, 1992 its include the assembly of a primarily p-structure protein intoThe first two authors contributed equally to this project.nonbranching, insoluble fibrils of diameter 7-10 nm and a char-IPTG, isopropyl P-D-thiogalactopyranoside; TES, Tris-EDTA-sucrose lead to death' Defining features Of amy1oid depos-42 1

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Kinetics of dimerization of the bence-jones protein Au

Cited by 17 publications

References 13 publications

Self-association of human immunoglobulin kappa I light chains: role of the third hypervariable region.

Self-association of human immunoglobulin kappa I light chains: role of the third hypervariable region.

Computer simulation of protein self-association during small-zone gel filtration. Estimation of equilibrium constants

Recombinant immunoglobulin variable domains generated from synthetic genes provide a system for in vitro characterization of light‐chain amyloid proteins

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