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Biomedical subjects

A N Schechter

Publications and source records attributed to A N Schechter.

At least 163 records · Page 9Linked to original sources
PubMed1977

Genetic control of the immune response to staphylococcal nuclease. IV. H-2-linked control of the relative proportions of antibodies produced to different determinants of native nuclease.

The relative proportions of antibodies of different specificities within antisera raised to native staphylococcal nuclease have been studied in several strains of mice in which the antibody response has been shown to be under H-2-linked Ir-gene control. A method was developed in which binding to different radiolabeled fragments of nuclease was titrated against increasing fragment concentration until the binding capacity of the antiserum for that fragment was saturated. In comparing the low responder (H-2b) strain C57BL/10 with its congenic high responder counterpart B10.A (H-2a), it was found that the two strains made markedly and reproducibly different proportions of antibodies to different determinants on native nuclease. Since these two strains differ only at H-2, and therefore have identical immunoglobulin structural gene repertoires, we conclude that H-2-linked Ir genes can control the response to different determinants on the same antigen molecule independently of one another. This result suggests a possible role of H-2-linked genes in the selection of specific B cells.

Animals↗
PubMed1977

Covalent attachment of fluorescent probes to the X-base of Escherichia coli phenylalanine transfer ribonucleic acid.

tRNA PheE, coli was labeled with the N-hydroxysuccinimide esters of 1-dimethylaminonaphthalene-5-sulfonyl glycine and N-methylanthranilic acid through reaction with the amino acid moiety of its X-base, whereby yields of 66% and 24%, respectively, were obtained. The purified dimethylaminonaphthalene-sulfonate derivative could not be aminoacylated and was found to be a strong competitive inhibitor of phenylalanine-tRNA synthetase [Ki=8X10(-7) M]. The N-methylanthraniloyl derivative could be charged to an extent of 5% as compared to native tRNA Phe. The fluorescence emission spectra of the derivatives are indicative of a slightly hydrophobic environment for both fluorophores. The results suggest that the integrity of the polar amino acid group of the X-base is required for the maintenance of the biologically active conformation.

Dansyl Compounds↗
PubMed1977

Properties of tRNA-specific antibodies from NZB/NZW mice.

Antibodies that bind tRNA are produced spontaneously in New Zealand Black/New Zealand White (NZB/NZW) F1 hybrid female mice. An assay for the detection of these antibodies has been developed by using gel filtration and radioactive tRNA. This assay was found superior to the widely used ammonium sulfate precipitation assay because of the nature of the interaction between the protein and the tRNA. The ant-bodies bound native tRNA preferentially to tRNA denatured by cross-linking with formaldehyde. This conformational specificity was confirmed in competition experiments. The antibodies to native tRNA had an average association constant of 5 x 10(7) leter/mole at 4 degrees C and could bind to more than one site per tRNA molecule. Experiments with immunoglobulin class-specific anti-mouse antisera, in solution and by radioimmunoelectrophoresis, showed that the antibodies were heterogeneous, but were predominantly of the IgG class. These antibodies may be useful for detection, localization, and conformational analysis of tRNA in solution as well as for understanding the pathogenesis of the lupus-like syndrome in these mice.

Animals↗
PubMed1976

The NH2-terminal region of the beta chain of sickle hemoglobin. I. Synthesis and purification of oligopeptides.

Five peptides from the NH2-terminal region of the beta chain of hemoglobin S, betaS (1-13), betaS (3-13), betaS (1-8), betaS (4-10), And betaS (4-8), have been synthesized by a rapid solid phase method based on the Merrifield procedure. In addition, one peptide, betaS (3-13), has also been synthesized by the original Merrifield method. We have shown that the products of the two methods are comparable, that gel filtration is a useful method for removing truncated fragments of the desired oligopeptide, and that measurement of the efficiency of coupling at each step is an important adjunct to amino acid analysis in determining purity. Peptides of the purity achieved by these methods may be used to fractionate antibodies to the native hemoglobin S, in the characterization of antigen-binding properties of specific antibodies, and in other studies of peptide-protein interactions.

Amino Acid Sequence↗
PubMed1976

The NH2-terminal region of the sickle hemoglobin beta chain. II. Characterization of monospecific antibodies.

We have previously shown that antibodies specific for hemoglobin S could be fractionated by absorption of an antiserum to hemoglobin S to Sepharose containing a synthetic oligopeptide. betaS (1-13), corresponding to the first 13 amino acid residues of the beta chain of hemoglobin S. We report here that this antibody population, anti-betaS (1-13), shows considerable restriction of heterogeneity in isoelectric focusing studies and monospecificity on velocity ultracentrifugation in the presence of hemoglobin S. The binding of various hemoglobin species to anti-betaS (1-13) was studied using a double antibody radioimmunoassay with [14C]carbamoylated hemoglobin S. Carbonmonoxy-, oxy-, met-, and cyanmethemoglobin S reacted equally with the antibody, but deoxyhemoglobin (with or without organic phosphates) reacted differently. Hemoglobin A and several hemoglobin mutants with alterations in the NH2-terminal region of the beta chain did not displace labeled hemoglobin S from anti-betaS (1-13). BETAS chains reacted with the antibody, but less well than hemoglobin S, while betaA and alpha chains, and globins did not react with the antibody. The synthetic peptide, betaS (1-13), used for fractionation, reacted with the antibody about 300-fold less efficiently than hemoglobin S. BetaS (3-13) was even less reactive, while smaller peptides which included the valine residue at position 6 displaced little of the tracer [14C]carbamoylated hemoglobin S at concentrations as high as 10(-2) M. We interpret these results to indicate that this method of immunoabsorption has produced a monospecific subfraction of antibodies which is specific for the NH2-terminal region of the beta chain of hemoglobin S in its native conformation.

Amino Acid Sequence↗
PubMed1976

Probability analysis of the interaction of antibodies with multideterminant antigens in radioimmunoassay: application to the amino terminus of the beta chain of hemoglobin S.

A simple theory, based on probability, is developed for the analysis of the interaction of multideterminant antigens with multispecific antisera in radioimmunoassays. The theory is completely general except for the assumptions that the determinants be unique and bind antibodies independently of one another. The analysis shows that the shape of the curve of bound/free as a function of the antigen concentration is very sensitive to the multiplicity of determinants. The predictive ability of the theory is illustrated for the case of antibodies to subregions of the N-terminal third of the beta chain of sickle hemoglobin, studied using antisera fractionated on affinity chromatographic columns of synthetic peptides. The implications for obtaining quantitative binding data by radioimmunoassay for natural antigens, which almost universally have more than one antigenic determinant on the same molecule, are discussed.

Amino Acid Sequence↗
PubMed1976

Antibodies to an NH2-terminal fragment of betaS globin. I. Preparation and radioimmunoassay.

Polypeptide fragments corresponding to the NH2-terminal 55 amino acids of betaS and betaA globins were prepared by cyanogen bromide treatment of globin and isolated by gel filtration on Sephadex G-50. Sheep were immunized with the isolated NH2-terminal fragments, and one of these sheep produced precipitating antibodies to the NH2-terminal fragment of betaS globin. These antibodies also reacted with betaA and betaS globin and hemoglobins A and S, as shown by immunodiffusion and quantitative precipitation studies. A radioimmunoassay was developed using the radioiodinated NH2-terminal fragment as tracer, and dextran-coated charcoal for separating bound and free peptide. The radioimmunoassay was used to characterize the interaction of the antibodies and the NH2-terminal fragment of betaS globin.

Amino Acids↗
PubMed1976

Antibodies to an NH2-terminal fragment of betaS globin. II. Specificity and isolation of antibodies for the sickle mutation.

The immunochemical specificity of an antiserum produced to an NH2-terminal 55-residue polypeptide fragment of the betaS globin, betaS(1-55), was analyzed with a radioimmunoassay using the radioiodinated fragment as a tracer. These studies show that most of the antibodies have comparable reactivity with betaS(1-55), betaA(1-55), betaS globin, betaA globin, HbS, and HbA. However, the antiserum contains some antibodies which react only with the species derived from HbS. These "S" -specific antibodies were isolated by absorption of the serum on a column of betaA(1-55) coupled covalently to Sepharose. The S-specific antibodies have markedly diminished reaction with betaA(1-55) and HbA. The S specificity was localized to the valine substitution at position 6 of the beta globin, as shown by inhibition of the binding of the radiolabeled fragment to S-specific antibodies by the synthetic peptide betaS(1-13). These antibodies, which appear monospecific, can be used to study the conformation of the NH2-terminal region of the beta chain of HbS.

Amino Acid Sequence↗
PubMed1976

Histidyl transfer ribonucleic acid synthetase from Salmonella typhimurium. Interaction with substrates and ATP analogues.

Structural requirements for substrate binding to histidyl-tRNA synthetase from Salmonella typhimurium have been investigated using ATP analogues. Ki values and the relative binding affinity of the enzyme for these analogues have been determined in the tRNA aminoacylation reaction. The enzyme is highly specific for ATP: no binding was found for GTP, CTP, TTP and UTP. dATP is a very poor substrate for acylation of tRNA, with a Km 40-fold higher than that of ATP. Binding of adenosine 5'-triphosphate requires interactions of the amino group of adenosine and the sugar moiety; the 2' and the 5' positions of the ribose appear to be essential for recognition; the phosphate groups enhance the binding. AMP is a noncompetitive inhibitor with ATP. The interaction of histidyl-tRNA synthetase, a dimeric enzyme, with histidine and ATP was examined by fluorescence measurements at equilibrium and by equilibrium dialysis. Binding with L-histidine is significantly tighter at pH 6 than at pH 7, while the ATP binding is independent of pH. The stoichiometry was measured at pH 6 than at pH 7, while the ATP binding is independent of pH. The stoichiometry was measured at pH 7.5 by equilibrium dialysis and is 1 mol ATP/mol enzyme and, variably, close to 2 or 1 mol histidine/mol enzyme.

Adenosine Triphosphate↗
PubMed1976

Does Freund's adjuvant denature protein antigens? EPR studies of emulsified hemoglobin.

Use of complete Freund's adjuvant for production of antibodies to study protein conformation is valid only if emulsification in adjuvant does not denature protein antigens. Using electron paramagnetic resonance to observe directly the protein in situ in the opaque emulsions, we demonstrate that hemoglobin is not denatured by emulsification or storage in adjuvant for 24 hr at 4 degrees C, conditions comparable to the usual handling of antigens before immunization.

Antigens↗
PubMed1975

Application of nuclear magnetic resonance spectroscopy to proteins.

The active sites of enzymes can be studied in great detail using nuclear magnetic resonance spectroscopy. The determination of pKa values of active site histidine residues in bovine pancreatic ribonuclease and the characterization of the binding of peptide hormones to carrier proteins are two such examples. The study of the active site of staphylococcal nuclease is another example and is presented in detail in this paper. The structure of 3'5'-thymidine diphosphate bound in the active site of staphylococcal nuclease has been studied by measuring the relaxation rate enhancement of substrate analog nuclei by a paramagnetic metal ion. The lanthanide ion, Gd(III), was substituted for Ca(II) in the formation of the ternary complex of nuclease: Gd(III) : 3'5'-thymidine diphosphate. Measurements were made of the transverse relaxation rates of protons and the longitudinal and transverse relaxation rates of the phosphorus nuclei of bound nucleotide. Internuclear distances between the metal ion and atoms of the 3'5'-thymidine diphosphate nucleotide were determined from these data by using the Solomon-Bloembergen equation. In general, these distances corresponded closely to those determined by previous X-ray crystallography of the thymidine diphosphate complex. These internuclear distances were also used with a computer program and graphics display to solve for metal : nucleotide geometries which were consistent with the experimental data. A geometry similar to the structure of the metal : nucleotide complex bound to nuclease determined by X-ray analysis was one of the solutions to this computer modeling process. For staphylococcal nuclease the NMR and X-ray methods yield compatible high resolution information about the structure of the active site.

Animals↗
PubMed1975

Isolation of antibodies specific to sickle hemoglobin by affinity chromatography using a synthetic peptide.

Antibodies to hemoglobin have been studied with a radioimmunoassay which employs [14C]carbamylated (= carbamolyated) hemoglobin S. An antiserum raised against hemoglobin S, which initially discriminated poorly between hemoglobins S and A, was fractionated by absorption to a column of Sepharose to which a synthetic peptide corresponding to the first 13 amino-acid residues of the beta chain of sickle hemoglobin had been covalently bound. A subpopulation of the antiserum was eluted from this column with 4 M guanidine - HC1. These antibodies showed binding to hemoglobin S but not to hemoglobin A and this interaction could be inhibited by the synthetic peptide. These antibodies, of demonstrated fine structural specificity, may be useful in the detection of sickle hemoglobin and in the study of its structure in solution.

Amino Acids↗