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S Beychok

Publications and source records attributed to S Beychok.

At least 19 recordsLinked to original sources
PubMed1986

Proteolytic dissection of a hapten binding site.

IgG Gar, a human myeloma protein that binds riboflavin with a high affinity, was used to derive variable region fragments from the heavy chain and the light chain. Riboflavin binding ability of the active site generated by V(H) and light chain and the active site generated by V(H) and V(L) was compared to riboflavin binding by the F(ab) fragment. The riboflavin binding ability of the F(ab) fragment is the same as the intact molecule, while the binding ability of the active site formed by V(H) and light chain is lowered by two to three orders of magnitude, indicating that the removal of C(H1) domain decreases the interaction between riboflavin and the amino acids that is important in tight binding of riboflavin. Removal of the third hypervariable region and the constant region domain from the light chain further lowers the binding constant by one order of magnitude. The results indicate that the V(H) and V(L) segments of IgG Gar can reconstitute a riboflavin binding site. The decrease in affinity probably reflects a decrease in the rigidity with which the hypervariable loops are held together to place the contact amino acid residues in optimal contact with the hapten.

Binding Sites, Antibody↗
PubMed1985

cDNA of the immunoglobulin kappa chain of an Epstein-Barr virus-transformed human lymphoid cell line: partial sequence determination and bacterial expression.

We report the isolation, nucleotide sequence determination, and bacterial expression of a partial cDNA for the immunoglobulin kappa chain from the Epstein-Barr virus-transformed human lymphoid cell line GM131. The cDNA, cloned in pBR322 by use of oligo(dG) X oligo(dC) tails, yields two Pst I fragments of 250 and 600 base pairs (bp). Various restriction enzyme fragments of the cDNA were subcloned in the vectors M13 mp10 and M13 mp11 for sequence analysis. As a result of instability of the 250-bp M13 subclones, the base sequence of the 250-bp Pst I fragment could not be determined. The 600-bp Pst I fragment contains coding sequences for part of the variable (V) region (residues 78-95) and all of the joining (J) (residues 96-108) and constant (C) regions (residues 109-212) and extends 148 bp into the 3' flanking region. Although the C- and 3'-flanking-region sequences are identical to germ-line sequences, the J-region sequence does not correspond to any of the five human germ-line J regions. The sequence is most similar to that of J4, with three base changes resulting in one silent mutation and two amino acid substitutions, at residues 103 (Lys----Tyr) and 106 (Ile---Met). The silent mutation appears to be the result of RNA splicing between the J and the C regions. The V-region sequence differs from published V-region germ-line sequences at several codons and from the more common amino acid sequences at two positions, residues 91 and 93. At these positions, histidine residues are found in place of the more common tyrosine and serine, respectively. None of the four amino acid substitutions observed for the GM131 kappa-chain are unique, suggesting that the changes, which most likely contribute to antigenic specificity, are compatible with antibody structure and function. The 600-bp Pst I fragment was subcloned in two prokaryotic expression vectors, pATH11 and pUC8. In both instances, a kappa-chain fusion protein detectable by immunoblotting was produced.

Amino Acid Sequence↗
PubMed1984

A heme binding site on myelin basic protein: characterization, location, and significance.

Myelin basic protein (MBP), an extrinsic membrane protein from the myelin sheath, binds dicyanohemin. The binding generates absorption bands in the Soret region and quenches the fluorescence emitted by the sole tryptophan residue. The absorption titration curves in the Soret demonstrate that the binding is stoichiometric, one heme per protein, with a large value of the extinction coefficient (8 X 10(4) M-1 cm-1 at 420 nm). Fluorescence quenching titration curves indicate an identical stoichiometry and a low quenching efficiency of 20%. From the heme titration curve the association constant between dicyanohemin and MBP is estimated to be greater than or equal to 10 nM-1 in 50 mM 4-morpholinepropanesulfonic acid buffer, pH 7.0, at 20 degrees C. Digestion of MBP by Staphylococcus aureus V8 protease yields a peptide (38-118) whose heme binding properties are identical to those of MBP. In contrast, peptides obtained by digestion of MBP with cathepsin D do not exhibit any specific binding of dicyanohemin. The cleavage of the Phe-Phe (42-43) bond appears to be critical in this respect. A comparison of the sequence immediately preceding, including these residues with a probable heme binding site of a mitochondrial cytochrome b, reveals a high degree of homology. The possible significance of heme binding is discussed.

Amino Acids↗
PubMed1984

A human myeloma immunoglobulin G binding four moles of calcium associated with asymptomatic hypercalcemia.

A calcium-binding immunoglobulin G (IgG1 lambda RUP) was identified in the serum of a patient with multiple myeloma, asymptomatic hypercalcemia, and a normal ionized serum calcium. Calcium binding by IgGRUP was confirmed by two-dimensional electrophoresis with calcium-45 and equilibrium dialysis. Amino acid analyses indicated an unusually high number of glutamic (or glutamine) residues in the L chain and Fab fragment but no detectable gamma-carboxyglutamic acid. As determined by equilibrium dialysis with 45Ca, the intact IgGRUP and its Fab fragments bound calcium at an optimum pH of 7.4. There was minimal binding of calcium to H chains and no binding by L chains or the Fc fragment. Recombination of H and L chains partially restored the binding activity. By Scatchard analysis, the binding affinity (Kd) of IgGRUP was 1.7 X 10(-3) M and the binding capacity was 4 mol of calcium/mol of IgG. The binding of 4 mol of calcium/mol of IgG is twice that reported previously for two other calcium-binding myeloma proteins and suggests unique properties of IgGRUP.

Aged↗
PubMed1981

Further studies of the riboflavin-binding immunoglobulin IgGGar. Resolution into fractions of different riboflavin content and aspects of reassembly.

A previously described human immunoglobulin with unusual flavin-binding activity, IgGGar [Farhangi, M., & Osserman, E. F. (1976) N. Engl. J. Med. 294, 177], is further characterized. The protein can be fractionated into two subpopulations, one of which is nearly completely saturated with riboflavin and one in which the binding sites are largely vacant. Possible differences between these fractions and/or their binding sites are explored. While electrophoretically distinct, the IgGGar-riboflavin complexes possess a basic similarity in the binding sites of both fractions as evidenced by spectroscopic examination. However, an important difference exists in that added riboflavin equilibrates reversibly with the vacant sites of native IgGGar, while the riboflavin in the occupied sites is essentially irreversibly bound. The tight association may be due to an in vivo combination of riboflavin with protein of different conformation than occurs in vitro, such as an incompletely assembled or folded tetramer. Accordingly, in vitro renaturation was examined. Studies of renaturation revealed that the reduced interchain disulfides within a tetramer reoxidize smoothly, although inter-heavy-chain bonds form less readily than inter-heavy-light-chain disulfides. Renaturation of IgGGar, unlike previously studied IgG molecules, does not proceed under conditions in which the protein structure had previously been significantly disrupted. The assembly defect is localized in the inability of the denatured heavy chain to refold into a stable species capable of combining with the light chain.

Binding Sites↗
PubMed1981

Further studies on the riboflavin-binding immunoglobulin IgGGar. Equilibrium and kinetic aspects of the interaction.

The subpopulation of flavin-binding protein IgGGar which differ with respect to the degree of saturation with riboflavin [Chang, M. Y., Friedman, F. K., Beychok, S., Shyong, J. S., & Osserman, E. F. (1981) Biochemistry (preceding paper in this issue) are characterized in terms of hapten binding activity. Riboflavin fluorescence is used as a sensitive probe of the rate and extent of complex formation. The vacant sites on native IgGGar reversibly bind up to a total of 2 equiv of riboflavin/mol of protein. Kinetic and equilibrium measurements yield an affinity constant of (1.7 +/- 0.1) x 10(9) M-1. This value is the highest found to date for a hapten-monoclonal antibody system and is a consequence of an unusually low dissociation rate for the complex. The system exhibits maximal binding efficiency between pH 6 and 8, van't Hoff analysis of binding yields delta H = -15 kcal mol-1 and delta S = -9.5 cal deg-1 mol-1. The occupied sites on native IgGGar irreversibly bind riboflavin under normal reaction conditions. Irreversible denaturation of protein with urea affects the release of riboflavin. The dissociation mechanism is complex and is partially characterized.

Binding Sites↗
PubMed1981

O2 binding properties of the product of the central exon of beta-globin gene.

The hypothesis that the exons of eukaryotic structural genes code for functional domains and that the partitioned arrangement of coding information may thus serve to mediate the rapid evolution of new and unique proteins from pre-existing exons is also supported by our recent studies which demonstrate that the product of the central exon of the human beta-globin gene is a complete functional domain capable of binding haem tightly and specifically. Moreover, an analysis of the structure/function changes induced by mutations in different parts of the haemoglobin molecule suggests that each of the three exon-encoded segments is primarily associated with different functions of haemoglobin (for example, haem-binding, haem-haem interaction). We have now extended our studies to determine whether the central fragment is sufficient for maintenance of a stable complex of ferrous haem with molecular oxygen and, if not, what are the minimal requirements for the expression of this activity. The results of our reconstitution experiments indicate that the isolated central exon peptide is unable to maintain a ferrous haem-dioxygen complex unless the side exon products and the complementary haem-containing subunit are present. A conformational change which accompanies the noncovalent association of fragments may account for the restoration of reversible oxygen binding.

Ferrous Compounds↗
PubMed1981

Kinetics and mechanism of heme-induced refolding of human alpha-globin.

Hemoglobin alpha and beta chains are tightly packed, highly (75%) helical stable molecules. Removal of the heme results in unfolded (30% helical) unstable globin chains that can be refolded to the native conformation by recombination with heme. We have studied the kinetics of heme binding and the ensuing conformational changes by using three stopped-flow techniques: (i) fluorescence quenching, which monitors the spatial orientation and distance between the bound heme and the A12(14)alpha tryptophan; (ii) absorption at the Soret band maxima, whose position and intensity depend on the local environment of the heme and the nature of the axial ligands; and (iii) far-UV circular dichroism, which directly gauges the recovery of secondary structure. The fluorescence quenching was biphasic: An initial second-order decay, representing 80-85% of the total amplitude, marked the binding of hemin dicyanide to a relatively well-defined site at a rate constant of 3.3 x 10(7) M(-1) sec(-1), corresponding to a half-time of 10 msec at 2.4 muM reactants. The Soret absorption and circular dichroism were also multiphasic, all three probes detecting a first-order process of half-time 25-40 sec, during which the final secondary and tertiary structures of the heme pocket were established, and the spatial relationship between the heme and the A12 tryptophan was fixed. A slower circular dichroism change, representing two-thirds of the total backbone refolding, with a half-time of 116 sec, marked the full acquisition of the native subunit conformation. The results show that the residues of the heme pocket achieve or closely approach their final three-dimensional structure well before the entire chain is folded. These measurements represent a direct observation of the rate of prosthetic group-induced secondary structure formation and illustrate the advantages of multiple probe analysis in outlining a protein folding pathway.

Carbon Monoxide↗
PubMed1980

Physicochemical properties and interactions of Escherichia coli ribonucleic acid polymerase holoenzyme, core enzyme, subunits, and subassembly alpha 2 beta.

We have investigated several physicochemical properties of Escherichia coli DNA-dependent RNa polymerase, its constituent subunits alpha, beta, beta', and sigma, and the subassembly alpha 2 beta. These included ultraviolet (UV) absorption, isoelectric points, sulfhydryl content, extinction coefficients, and circular dichroism (CD). Among the most notable results is the observation, based on CD measurements, that the sigma subunit, free and combined in holoenzyme, is a highly structured protein with approximately 75% of its residues folded in alpha-helical conformation and little or no detectable beta sheet. No secondary structure changes in either sigma or core accompany formation of holoenzyme. In contrast to the conformational independence of the subunits in assembly of holoenzyme, the protein and its components exhibit conformational flexibility as glycerol concentration is varied and in their interaction with DNA. The effect of glycerol on the conformation of sigma, core, and holoenzyme was monitored by circular dichroism measurements. In the far-ultraviolet, the residue ellipticity at 220 nm ([theta]220) increased approximately 15% from 0 to 10% glycerol for both core and holoenzyme. In the near-ultraviolet, the residue ellipticity at a peak near 280 nm also varied with glycerol concentration, decreasing in intensity by about 50% with holoenzyme, when glycerol was raised from 5 to 10%, then increasing at still higher glycerol contents. Electrophoretic and molecular sieve anaysis showed core and sigma to have greater affinity for each other in 50% glycerol than in 10% glycerol. The presence of 10% glycerol in the assay buffer increased the activity of the enzyme. The effect of various DNA templates on the conformations of core, holoenzyme, alpha 2 beta subassembly, and beta' subunit was also monitored by far-ultraviolet circular dichroism. All the protein samples showed between 10 and 20% decrease in secondary structure upon the addition of the DNA.

Bacterial Proteins↗
PubMed1980

Characterization of globin domains: heme binding to the central exon product.

We have prepared and isolated the peptide fragments coded for by the three exons of the human beta-globin gene, using the arginine-specific protease clostripain (EC 3.4.22.8). The region encoded by the central exon (amino acid residues 31-104) contains an arginine at position 40. This site was less susceptible to cleavage than the two sites that correspond to the exon-intron boundaries, and the isolated central fragment was an approximately equimolar mixture of the entire central fragment, beta(o) (31-104), and the somewhat smaller fragment contained within it, beta(o) (41-104). This central fragment mixture bound heme stoichiometrically and tightly at micromolar concentrations, generating a strong Soret absorption band as well as a characteristic absorption band in the visible spectrum. The Soret band occurred at the same wavelength and had the same shape as in hemoglobin, exhibiting an intensity greater than (2/3) that achieved when native intact beta globin is reconstituted with heme. Nearly the full intensity was regained when an equivalent of heme was added to the unfractionated digest, suggesting that the noncovalently associated side fragments add precision to the fit of the heme pocket. Three controls were used in establishing the specificity of heme binding to the central fragment mixture. Similar, but preliminary, experiments have also been undertaken with alpha globin. A clostripain digest containing alpha(o) (1-31) and alpha(o) (32-141) bound heme, yielding a Soret band identical to that observed in alpha subunits reconstituted from the native globin chains and heme. Measurements of circular dichroism spectra as indices of secondary structure suggested a role for the side exon products in the acquisition of the native three-dimensional structure of hemoglobin. These experiments confirm a prediction of W. Gilbert that the product of the central exon of the globin gene is a complete functional domain that binds heme tightly and specifically.

Amino Acid Sequence↗
PubMed1978

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.

Dansyl Compounds↗
PubMed1978

Covalent immunoglobulin assembly in vitro: reactivity of light chain covalent dimers (L2) and blocked light chain monomers.

Covalent light chain dimers (L2) and cysteine-blocked L chain monomers readily react with partially reduced heavy (H) chains. A rapid disappearance of these blocked L chain species is followed by the appearance of covalent intermediates-HL, H2, and H2L-leading to fully assembled H2L2. The mechanism of initial disulfide bond formation between heavy and light chains is disulfide interchange.

Bence Jones Protein↗
PubMed1978

Far-ultraviolet stopped-flow circular dichroism.

A stopped-flow circular dichroism instrument, with a total accessible wavelength range of 200 to 750 nanometers, has been constructed and provides a spectroscopic method for kinetic investigations of a wide array of fast reactions in which optical activity changes in absorbing regions are involved. An important biochemical application depends on the far-ultraviolet capability, which allows observation of the rapid alterations in backbone conformation associated with folding and unfolding reactions of proteins. Results obtained by following two such reactions at 222 nanometers represent direct monitoring by circular dichroism of rapid secondary structure changes in proteins.

Circular Dichroism↗