Search PubMed⌕ Search

Biomedical subjects

I M Chaiken

Publications and source records attributed to I M Chaiken.

At least 73 records · Page 4Linked to original sources
PubMed1981

Semisynthetic peptides and proteins.

Semisynthesis provides a flexible approach for using chemical synthesis to produce large, biologically active polypeptides and analogues. Currently developing improvements in the basic methods used, including polypeptide fragmentation, peptide synthesis, and reconstitution of synthetic and native components, make this overall approach applicable to a variety of species. Sequence modification through semisynthesis thus provides a flexible route to explore the code of rules whereby primary structure directs higher order properties of folded conformation and biological function of large peptides and proteins. The fruits of this endeavor, an understanding of how these macromolecules work, and therein, a basis for design of new structures that ultimately may be produced directly or by recombinant DNA methods, have begun to emerge.

Amino Acid Sequence↗
PubMed1980

Enzyme-catalyzed formation of semisynthetic staphylococcal nuclease using a new synthetic fragment, [48-glycine]synthetic-(6-49).

While trypsin can catalyze resynthesis of the peptide bond between fragments in the noncovalent complex of nuclease-T-(6-48) and nuclease-T-(49-149), this reaction leads to excision of Lys 49 and formation of inactive [des Lys 49]-nuclease-(6-149). To provide a method for making active active covalent semisynthetic nuclease, we chemically synthesized the fragment of residues 6 to 49 in which lysine 48 was replaced by glycine. This peptide was made using the recently described solid phase support, 4-(oxymethyl)phenylacetamidomethyl-polystyrene. The resultant crude polypeptide exhibited 30-50% of native nuclease-T enzymatic activity when added to native nuclease-T-(50-149). When the non-covalent complex formed by native nuclease-T-(50-149) and a 10-fold molar excess of [Gly 48]synthetic-(6-49) was equilibrated with trypsin in 90% glycerol, an increase in enzymatic activity from 8 to 32% (versus nuclease) was observed. Simultaneously, approximately 20% conversion of nuclease-T-(50-149) to nuclease-molecular weight material was observed by gel electrophoretic analysis. These data indicate that a covalent semisynthetic species is formed with activity about equal to that of native nuclease. The results confirm the importance of loop integrity on catalytic site organization. The Gly-48-containing fragment system defined above can allow preparation of semisynthetic nuclease sequence analogs.

Micrococcal Nuclease↗
PubMed1979

Expression of multivalency in the affinity chromatography of antibodies. Appendix: Derivation and evaluation of equations for independent bivalent interacting systems in quantitative affinity chromatography.

The expression of multivalency in the interaction of antibody with immobilized antigen was evaluated by quantitative affinity chromatography. Zones of radioisotopically labeled bivalent immunoglobulin A monomer derived from the myeloma protein TEPC 15 were eluted from columns of phosphorylcholine-Sepharose both in the absence and presence of competing soluble phosphorylcholine. At sufficient immobilized phosphorylcholine concentration, the variation of elution volume of bivalent monomer with soluble ligand was found to deviate from that observed for the univalent binding of the corresponding Fab fragment. In addition, the apparent binding affinity of the bivalent monomer increased with immobilized antigen density. Use of equations relating the variation of elution volume with free ligand concentration for a bivalent binding protein allowed calculation of microscopic single-site binding parameters for the bivalent monomeric antibody to both immobilized and soluble phosphorylcholine. The chromatographic data not only demonstrate the effect of multivalency on apparent binding affinity but also offer a relatively simple means to measure microscopic dissociation constants for proteins participating in bivalent interactions with their ligands.

Antibodies↗
PubMed1979

Immunological and chemical identification of a neurophysin-containing protein coded by messenger RNA from bovine hypothalamus.

The biosynthetic origin of the 10,000 molecular weight neurophysins, carriers of the peptide hormones oxytocin and vasopressin, has been studied by cell-free synthesis, Poly(A)-RNA was isolated from bovine hypothalamus and translated in a wheat germ system containing (35)S- or (3)H-labeled amino acids. A number of unique [(35)S]cysteine- but few [(35)S]-methionine-labeled proteins were coded by hypothalamic mRNA. A single, major, isotopically labeled protein (molecular weight 23,000-25,000) was immunoprecipitated from these translation mixtures by addition of purified antibodies against bovine neurophysin II and subsequent addition of Cowan I strain of Staphylococcus aureus. Specificity of the immunoprecipitation was demonstrated by competition with unlabeled authentic neurophysins and the absence of competition with structurally unrelated ovalbumin. Furthermore, neither nonimmune serum nor purified antibodies against ribonuclease immunoprecipitated the protein. The [(35)S]cysteine-labeled protein that was specifically immunoprecipitated was oxidized with performic acid and digested with trypsin in the presence of unlabeled, authentic bovine neurophysin II. Peptide mapping revealed that most of the major [(35)S]cysteine-labeled peptides (of the translation product) were identical to major cysteine-containing peptides of authentic neurophysin. The data show that hypothalamic mRNA directs the translation of several unique cysteine-rich proteins in an in vitro cell-free system. Furthermore, one of these proteins, which has a higher molecular weight than authentic neurophysin, is recognized by purified antibodies to bovine neurophysin II and has cysteine-containing tryptic peptides in common with those of authentic neurophysin. The data suggest that this protein is the primary translation product, pre-pro-neurophysin.

Animals↗
PubMed1978

Design of a photoaffinity label for the hormone binding site of neurophysin.

The photolabile peptide, L-methionyl-L-tyrosyl-p-azido-L-phenylalaninamide, was synthesized by solution methods. This peptide, as well as the analogous species containing tritiated methionine, were found to bind reversibly and specifically, in the dark, to bovine neurophysin II. The dissociation constant, stoichiometry, and pH-dependence of this noncovalent interaction are typical of those properties for hormone (oxytocin) and hormone-like ligand binding to neurophysin II. Under photolytic conditions, methionyl-tyrosyl-p-azidophenylalaninamide causes irreversible inhibition of the noncovalent ligand binding activity of neurophysin II. This inactivation was achieved to the extent of about 90%. Both the dark and light (photolytic) interactions of the photolabile peptide with neurophysin II indicate its reaction at the hormone binding site of the protein and thus its potential use to identify amino acid residues at this site by covalent photoaffinity labelling.

Animals↗
PubMed1976

The effects of ligands on enzymic carboxyl-methylation of neurophysins.

The methyl-acceptor activities of bovine neurophysins I and II for the enzyme protein carboxymethylase (EC 2.1.1.24) were found to be similar and as high as for other previously identified, biologically active protein substrates. Effects on the rate of methylation of these neurophysins were investigated with the posterior pituitary hormone ligands, oxytocin and vasopressin, and the hormone-related tripeptide ligand, methionyl-tyrosyl-phenylalaninamide. An increase in the rate of neurophysin II methylation was observed with both oxytocin and tripeptide. This ligand-induced response did not occur with either native neurophysin I or disulfide-scrambled neurophysin II.

Animals↗
PubMed1976

Analysis of ribonuclease-nucleotide interactions by quantitative affinity chromatography.

Quantitative affinity chromatography on uridine-5'-(Sepharose-4-aminophenylphosphoryl)-2'(3')-phosphate was developed for the study of binding of ribonuclease species to nucleotide ligands. Elution of the native species ribonuclease-A and -S on the afffinity matrix in 0.4 M ammonium acetate, pH 5.2, containing various amounts of the soluble competing ligand 2'-cytidine monophosphate, reveals an inverse response of elution volume to concentration of soluble ligand. This response conforms to behavior expected for the competing binding equilibria enzyme-soluble ligand and enzyme-insoluble ligand. A-NALYSIS OF ELUTION DATA ALLOWS CALCULATION OF KI and KIM, the dissociation constants, respectively, for the soluble and insoluble protein-ligand complexes. The values of these chromatographically derived constants are similar to values of dissocation constants determined in solution by kinetics of inhibition by 2'-cytidine monophosphate and uridine-5'-(j-aminophenylphosphoryl)-2'(3')-phosphate. Successful competitive elution experiments with [p-F-Phe8]semisynthetic ribonuclease-S' and individual elution trials for [4-F-His12]semisynthetic ribonuclease-S' indicate the utility of the quantitative affinity chromatographic technique for determination of ligand binding properties of ribonuclease derivatives, including inactive species. Nonbiospecific aspects of the interaction of ribonuclease with the affinity matrix in ammonium acetate buffers of concentrations 0.1 M and below were noted, delinating limits of conditions allowing the biospecificity needed for ligand-binding analyses by competitive elution. The dependence of ribonuclease competitive elution behavior on the amount of protein eluted also was examined and related to theoretical considerations in the quantitative application of affinity chromatography.

Binding Sites↗
PubMed1976

Crystalline semisynthetic ribonuclease-S'.

Crystals of solid phase-derived semisynthetic ribonuclease-S' were prepared and compared with those for native ribonuclease-S' and -S. The semisynthetic species used was the noncovalent complex of synthetic fragment-(1-20), corresponding to residues 1 through 20 of bovine pancreatic ribonuclease-A (ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22), and native ribonuclease-S-(21-124); the fragment containing residues 21 through 124 of ribonuclease-A. This semisynthetic complex was completely active enzymatically, was homogeneous as judged by polyacrylamide gel electrophoresis, and had no greater than trace amounts of excess ribonuclease-s(21-124) as judged by affinity chromatography. Crystallization of both semisynthetic and native ribonuclease-s' at pH 5.3 resulted in well-formed crystallseater than trace amounts of excess ribonuclease-S-T21-124) as judged by affinity chromatography. Crystallization of both semisynthetic and native ribonuclease-S' at pH 5.3 resulted in well-formed crystals with the symmetry of space group P3121 and unit cell dimensions a=b=44.82, c=97.3 A. This crystal form corresponds to the Y form of native ribonuclease-S previously reported [Wyckoff et al. (1967) J. Biol. Chem. 242, 3749-3753]. X-ray diffraction patterns of the crystals were indistinguishable, indicative of the structural identity of semisynthetic and native ribonuclease-S'.

Crystallography↗