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Biomedical subjects

I M Chaiken

Publications and source records attributed to I M Chaiken.

At least 55 records · Page 3Linked to original sources
PubMed1984

High-performance liquid chromatography and studies of neurophysin-neurohypophysial hormone pathways.

High-performance liquid chromatography (HPLC) is being used extensively to characterize active polypeptides, precursor processing mechanisms, and cooperative peptide-protein noncovalent complexes in neuroendocrine pathways for neurohypophysial peptide hormones, oxytocin and vasopressin, and the hormone-associated proteins, neurophysins. Reversed-phase and ion-exchange HPLC polypeptide mapping have been used to detect the hormones, associated proteins, and other molecular forms containing these. This mapping has provided a means not only to isolate these molecules when present in micro amounts but also ultimately to identify anatomical sites which contain the neurophysin/hormone molecular pathways and to define the relatedness of polypeptide forms contained in different pathways. Reversed-phase HPLC also has provided a means to study proteolytic precursor processing, both to isolate synthetic and semisynthetic polypeptides prepared for use as substrates in processing reactions and eventually to study the polypeptides and intermediates produced by these reactions. Finally, bioaffinity HPLC is being evaluated as a separatory and analytical tool. The latter includes its use to characterize the noncovalent peptide-protein and protein-protein interactions which occur among the molecular forms of the neurophysin/hormone pathways. These experiments typify the impact of HPLC for both analytical and preparative separations in studies of biologically active peptides and proteins.

Animals↗
PubMed1984

Characterization of the size and 5' cap of messenger RNA encoding neurophysin precursors.

The coding activity of bovine hypothalamic poly A+ mRNA for neurophysin I and II immunoreactive proteins was characterized with respect to size and 5' cap. The mRNA was fractionated by methylmercuric hydroxide agarose gel electrophoresis and subsequently translated in vitro in rabbit reticulocyte lysates. Alternatively, mRNA was fractionated by gel exclusion HPLC and translated in wheat germ extracts. Immunoprecipitated translation products were analyzed by gel exclusion HPLC. Neurophysin-immunospecific protein of approximately 17,000 daltons, the size expected for the neuropeptide hormone-neurophysin precursors, was encoded by mRNA species of two size classes. The smaller class of mRNA's was of the size expected from the size of the precursor proteins. The larger class was 5-10 times larger. The low K+ concentration optimum for translation of unfractionated mRNA encoding neurophysin I immunoreactive proteins and the inability of a cap analogue to inhibit this translation suggest that mRNA species encoding neurophysin I-immunoreactive translation products are incompletely capped. By contrast, the mRNA encoding neurophysin II-immunoreactive products appear to contain a normal cap structure.

Animals↗
PubMed1984

Purification and characterization of ornithine transcarbamoylase from Saccharomyces cerevisiae.

Ornithine transcarbamoylase (OTCase) has been purified in 100-mg quantities from a plasmid-containing, enzyme-overproducing strain of Saccharomyces cerevisiae. The specific activity of the homogeneous enzyme is 2.5-fold above that previously reported. The molecular weight and partial specific volume of OTCase were determined by sedimentation equilibrium in solutions containing H2O and D2O. Data from two rotor speeds were simultaneously fit using nonlinear least squares analysis with multiple independent variables giving a molecular weight of 110,000 +/- 2,200 and a partial specific volume of 0.732 +/- 0.006 ml g-1. The ultraviolet absorption spectrum of OTCase gives a specific absorbance at 280 nm of 0.36. This low value is consistent with a small number of aromatic residues. Amino acid analysis, fluorescence, and multicomponent analysis yield 1 tryptophan, 4 tyrosine, and 24 phenylalanine/polypeptide chain. From an analysis of the circular dichroic spectrum, it was determined that OTCase contained 22% alpha-helix, 43% beta-sheet, 8% beta-turn, and 27% random structure. The fluorescence of the single tryptophan/polypeptide chain has an emission maximum at 320 nm, indicating a hydrophobic environment.

Amino Acids↗
PubMed1984

High-performance liquid chromatographic mapping and structural characterization of neurophysin isoforms.

Both ion-exchange and reverse-phase HPLC protocols for micromapping of neurophysins have been examined and the structural relationships among the major isoforms identified in the maps have been characterized. Reverse-phase HPLC was found to be especially useful for obtaining fingerprints of the isoforms within each of the two major families of neurophysins, I (oxytocin-related) and II (vasopressin-related), for both bovine and human neurophysins from posterior pituitary sources. From fractionation of the bovine proteins on octylsilyl columns, at least four neurophysins I were identified, one of which corresponds to the intact sequence of 93 residues and three of which vary from the parent by various degrees of carboxyl-terminal truncation. For bovine neurophysin II, two isoforms were identified in the reverse-phase HPLC maps, both of 95 residues, which vary from one another by the residue, either Ile or Val, at position 89. Isoforms were also detected for human neurophysins, including a carboxyl-terminal truncated form of human neurophysin II. All of the major neurophysin isoforms and several of the minor forms were shown to be functionally active as expressed by their binding to peptide ligand affinity matrices. Reverse-phase HPLC mapping on the octylsilyl matrix allowed neurophysin fingerprinting of crude tissue extracts by providing a narrow "window" within which the neurophysins elute but many other polypeptides expected to be present are excluded. The reverse phase HPLC method provides a useful way to obtain isolated neurophysin isoforms for physicochemical characterizations now usually carried out with mixtures of isoforms obtained by ion-exchange chromatography. The method also has characteristics amenable both for high-sensitivity fingerprinting of neurophysin isoforms, from different species and anatomical sources, and as a prelude to microstructural and -functional characterization of the isoforms so isolated.

Amino Acids↗
PubMed1984

Cooperative interactions in neurophysin-neuropeptide hormone complexes. Analytical affinity chromatography of native and covalently-modified neurophysins.

The structural interdependence between neurophysin (NP) self-association and ligand binding surfaces has been studied by analytical affinity chromatography of several NP sequence variants and derivatives on Met-Tyr-Phe-aminobutyl-agarose and bovine NP-II Sepharose. Elutions of radiolabeled NP's from both matrices show that hybrid dimers can form between major bovine NP's (I and II, or VLDV- and MSEL-NP's, respectively), as well as between human and bovine NP's, with affinities close to that for homologous dimer formation. Such evidence supports the view that the region of NP involved in NP-NP contact is composed primarily of conserved structural elements of the protein. NP antibodies which recognize surfaces close to or in the NP-NP contact region have been detected by their effects on bovine NP-II elution on NP-II Sepharose. Elutions of [3-nitro-Tyr 49] BNP-II from Met-Tyr-Phe-aminobutyl-agarose showed that nitration has little effect on the chromatographic properties of NP-II. This evidence substantiates previous arguments (Angal, S. & Chaiken, I.M. (1982) Biochemistry 21, 1574-1580) that the chromatographic behavior of native NP's on the affinity matrices is an expression of the interdependence of NP self-association and ligand binding surfaces and not due to bivalent peptide binding by NP monomer. The affinity chromatographic properties of NP derivatives, including bovine NP-II photolabeled in the ligand binding site and tryptic fragments of bovine NP-I (NP-I-(9-93) and [des 19-20] NP-I-(9-93)), support the view that the surfaces for ligand binding and NP-NP contact are conformationally linked. The data argue that conformational changes that ensue upon noncovalent ligand binding and lead to enhanced NP self-association cannot occur favorably with the protein modified by either covalent ligand attachment or limited amino-terminal proteolysis.

Animals↗
PubMed1983

Onset of neurophysin self-association upon neurophysin/neuropeptide hormone precursor biosynthesis.

The potential of the common biosynthetic precursor of neurophysin and neuropeptide hormones to self-associate has been assessed by quantitative affinity chromatographic analysis. The precursor form, with the hormone sequence in the amino terminal region and assumed able to interact intramolecularly with the hormone binding site of the neurophysin domain of the folded precursor, exhibits an affinity for neurophysin-agarose which is intermediate between those of unliganded neurophysin and non-covalently hormone-liganded neurophysin. The results lead to a prediction that neurophysin self-association is established upon precursor synthesis and prior to limited proteolysis of the precursor to release mature neurophysin and hormone components. Such self-association could play a role in packaging of the precursor into secretory granules and in regulating subsequent precursor processing events within the granules.

Animals↗
PubMed1983

An activity from Escherichia coli membranes responsible for the modification of nitrate reductase to its precursor form.

An enzymatic activity which modifies nitrate reductase has been identified in the cytoplasmic membrane of Escherichia coli. This activity changes subunit B to a form with a slightly greater electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels (B'). The B' polypeptide produced by this modifying enzyme was compared to an apparently identical polypeptide identified in the precursor form of nitrate reductase which can be found in the cytoplasm of all strains and in the membrane of mutants defective in the insertion of nitrate reductase. These B' polypeptides were all identical with respect to mobility on gradient sodium dodecyl sulfate gels and peptides produced by limited digests using trypsin, papain, and Staphylococcus aureus V8 protease. When compared to subunit B, the proteolytic gel maps of B' polypeptides showed minor differences. From the identity of the modified B' with precursor B', the ability to convert B into B' in vitro and the in vivo nature of B' as a precursor of B, it was concluded that the modification of B to B' is a reversible process and is due to the removal of one or more small nonprotein molecules.

Amino Acids↗
PubMed1983

Minimum information content and formation of interacting ribonuclease fragment complexes.

The degree to which amino acid sequence can be simplified with retention of conformational and functional properties has been investigated by semisynthesis using non-covalent fragment complexes of bovine pancreatic ribonuclease as test cases. Based on the ribonuclease S system, a set of synthetic model sequences was defined for the S-peptide (1-20) region which interacted productively with native S-protein (21-124). The most simple sequence, an eicosapeptide containing helix-favoring Ala residues at all positions except Glu 1 and 14, Phe 8, His 12, and Met 13, effected at least 15% of ribonuclease catalytic activity (versus native ribonuclease S) when added to S-protein in saturating amounts. The data for model S-peptides define an alpha-helical framework and specific side chains at positions 8, 12, and 13 as the core of sequence information necessary for S-peptide to effect a productive non-covalent complex with S-protein. Previous ribonuclease fragment studies also were used as a basis for making the productive, non-overlapping complex, (1-15):(21-111):(116-124). Addition of synthetic (1-15) and (116-124) to (21-111) led to a 3 degrees increase in Tm and 4% (versus ribonuclease A) catalytic activity. The three-fragment complex, with the beta-bend residues 112-115 deleted, exhibited significantly lower stability to thermal denaturation than did related two-fragment complexes. The potential use of three-fragment complexes related to the above is discussed for semi-synthetic sequence modeling concomitantly in the N- and C-terminal regions of ribonuclease.

Amino Acid Sequence↗
PubMed1982

Effects of limited tryptic proteolysis of bovine neurophysins on molecular properties of hormone binding, self-association, and antigenicity.

Limited tryptic fragmentation of disulfide-intact bovine neurophysins I and II (NP-I and -II, respectively) has been found to cause selective disruption of both hormone binding and neurophysin self-association. Loss of binding interactions, measured as a loss of ability to stimulate retardation of 125I-labeled neurophysin on Met-Tyr-Phe-amino-butylaminoagarose, is complete within 3 h at 37 degrees C. Reverse-phase high-performance liquid chromatography (HPLC) analysis of tryptic digests of neurophysin I allows detection of two major protein products and the peptide fragment 1-8. Release of the latter N-terminal piece occurs at about the same rate as loss of binding interactions. Reverse-phase HPLC elution behavior before and after performic acid oxidation and amino acid composition of the protein products led to their identification as NP-I-(9-93) (the 9-93 sequence) and [des-19,20]NP-I-(9-93) (the 9-93 sequence with the dipeptide 19-20 missing) for the more rapidly and more slowly formed species, respectively. NP-I-(9-93), unlike intact neurophysin I, is not retarded strongly by either Met-Tyr-Phe-amino-butylaminoagarose or neurophysin II-Sepharose. In contrast, both NP-I-(9-93) and [des-19,20]NP-I-(9-93) are equally as effective as intact NP-I in binding neurophysin I antibodies. The role of amino-terminal residues in promoting hormone binding, self-association, and antigenic recognition interactions is considered.

Amino Acid Sequence↗
PubMed1982

Enzymatic condensation of nonassociated peptide fragments using a molecular trap.

We have tested the feasibility of achieving protease-catalyzed condensation between nonassociating peptide fragments through mediation of a molecular trap. In this study, two subfragments of bovine pancreatic ribonuclease S-peptide, containing residues 1-10 and 11-15, were rejoined by clostripain catalysis to form the 1-15 peptide. The extent of this stereospecific condensation was enhanced by adding ribonuclease S-protein (residues 21-124), which acts as a trap in binding 1-15 but not 1-10 or 11-15 and which thus shifts the equilibrium to favor 1-15 formation. The resultant (1-15) X (21-124) noncovalent complex, defined as [des-16-20]ribonuclease S, was detected by the enzymatic activity characteristic of the naturally derived ribonuclease S complex. Reaction of 1 mM S-protein and 20 mM fragments leads to 80% of the ribonuclease activity expected from the amount of 21-124 present. This indicates that 4% of the fragments 1-10 and 11-15 were condensed, compared to a maximal condensation of 5% based on the amount of trap. The less than theoretical yield is due largely to slow proteolytic degradation of 21-124 to a form which is no longer able to bind the condensation product 1-15. Yields were increased to 15% by addition of further trap. The successful synthesis of 1-15 emphasizes the usefulness of molecular traps to promote stereospecific fragment condensation between nonassociating peptide fragments for the synthesis and semisynthesis of polypeptides.

Amino Acids↗
PubMed1982

Domain characteristics of the carboxyl-terminal fragment 206-316 of thermolysin: immunochemical studies.

The extent of nativeness of the stable conformation of the thermolysin fragment containing the carboxyl-terminal third of the protein (from residues 206 to 316, denoted fragment FII) was examined by its immunogenic and antigenic characteristics. Antisera elicited in rabbits by either intact thermolysin or fragment FII were fractionated serially on two affinity columns, containing either the isolated fragment or intact protein. Both sera gave rise to substantial antibody populations which recognized the fragment FII region in native thermolysin. The relative affinities of these specific antibodies for isolated fragment FII and intact thermolysin were evaluated by radioimmunoassay, by assessing the relative extents of competition by these for binding of either 14C-labeled thermolysin or 14C-labeled fragment FII to each antibody population. Competition by fragment FII was substantial, though generally weaker than that for intact thermolysin, for antibody binding of both labeled antigens. The data demonstrate that the stable structure of fragment FII as observed spectroscopically likely is one which possesses conformational features similar to those of this region in intact thermolysin, but with perhaps less conformational rigidity. The results support the view that the region of thermolysin composed primarily of residues 206-316 is a conformational domain of the intact protein and that isolated fragment FII retains domain-like characteristics of stable and native-like conformation.

Animals↗
PubMed1982

Interdependence of neurophysin self-association and neuropeptide hormone binding as expressed by quantitative affinity chromatography.

The reciprocal modulation of neurophysin self-association and noncovalent peptide--protein interaction between neurophysin and the hormones oxytocin and vasopressin has been assessed by quantitative affinity chromatography. Competitive elutions of radiolabeled bovine neurophysin II (NPII) from the affinity matrices Met-Tyr-Phe-omega-(amino-hexyl)- [and (aminobutyl)-] agarose were performed with increasing concentrations of either of the soluble ligands oxytocin or lysine-vasopressin. Also, the dependence of NPII retardation by the same adsorbents on the concentration of applied protein was investigated in the absence of soluble ligand. The affinity constant of NPII for the immobilized peptide increased markedly with increasing amounts of applied protein and with the addition of small amounts of soluble ligand, the latter being more pronounced at higher protein concentrations. The affinity constant of the protein for the soluble ligand showed a smaller increase. The variation of l/(V - V0) (where V = the NPII elution volume and V0 = the elution volume of noninteracting control protein) with soluble ligand concentration was linear except near [ligand] = 0. The quantitative affinity chromatographic results on the tripeptidyl affinity columns are consistent with the view that NPII exists in a monomer in equilibrium dimer equilibrium, with the dimer exhibiting a stronger interaction with both neuropeptide and tripeptide analogues. The data also indicate that the self-associated protein dimer itself exhibits cooperativity, that is, stronger binding of the immobilized ligand at one site when a second site is occupied with a molecule of the soluble ligand than when no soluble ligand is bound. The deduction from the above of ligand-induced dimerization is evident also in the increased retardation of NPII on neurophysin--Sepharose when the eluting buffer contains soluble peptide hormone.

Chromatography, Affinity↗
PubMed1982

Sequence modeling using semisynthetic ribonuclease S.

We have designed and synthesized a model pentadecapeptide predicted to have the essential sequence information needed to form a stable and enzymatically active noncovalent complex with bovine pancreatic ribonuclease S-protein. The model peptide sequence, based on the conformational approach of simplifying the native sequence in a manner consistent with retention of essential noncovalent contacts and of secondary structure features, contained alanine at all positions except for Glu 2, Lys 7, Phe 8, Arg 10, His 12, and Met 13. The peptide was synthesized by the Merrifield solid phase method. The circular dichroism spectra of the purified model peptide in water and trifluoroethanol indicated a tendency to form an alpha-helical structure similar to that found for native S-peptide. The model peptide formed a stable complex with ribonuclease S-protein. With 12-fold excess of the peptide, the complex exhibited 36% of the specific activity of fully native ribonuclease S against the substrate cyclic cytidine 2':3'-monophosphate at pH 7.15. The dissociation constant of the model peptide for S-protein was found to be 1.1 x 10(-6) M, compared with 0.1 x 10(-6) M for native S-peptide. Crystals grown of the model peptide-S-protein complex were found to be isomorphous with those of native complex. The activity, stability, and structural integrity of the model complex verify the deductions made about essential sequence information in the NH2-terminal region of ribonuclease. Further, the results emphasize the general usefulness of the conformational approach in designing simplified sequences for other peptides and proteins.

Amino Acid Sequence↗
PubMed1982

Photoaffinity labeling of the hormone binding site of neurophysin.

The chemical structure of the hormone binding region of the neurophysins has been investigated by photoaffinity labeling with the photolabile tripeptide, L-[methyl-3H]Met-L-Tyr-p-azido-L-Phe amide. Photolysis of the photoaffinity tripeptide in the presence of bovine neurophysin I and II and a human neurophysin II led to approximately equal extents of covalent incorporation of radioactivity into protein. Photolabeled bovine neurophysin II was fractionated into binding site derivatized protein and nonbinding site derivatized protein by affinity chromatography, with results of amino acid and radiolabel analysis of the hormone binding site blocked protein indicating that 1 mol of tripeptide was covalently incorporated/mol of protein. Tyrosine 49 was the only protein amino acid modified in the binding site photolabeling reaction as assessed by peptide mapping of the performic acid oxidized and trypsin-digested photolabeled protein using reverse phase high performance liquid chromatography. Modification of the single neurophysin tyrosine also was found by amino acid analysis of performic acid oxidized photolabeled bovine neurophysin II. The covalent bond formed in neurophysin upon photolysis was cleaved by either exhaustive acid hydrolysis or reduction-carboxymethylation without loss of the protein amino acid residues and by performic acid oxidation with loss of both protein and tripeptide tyrosine residues. These overall data indicate that tyrosine 49 is the probable site for specific covalent attachment of the photoaffinity tripeptide. Assuming that the tripeptide binding site is the high affinity hormone binding site reported for the neurophysins, this conclusion argues that tyrosine 49 is close to or within this site.

Affinity Labels↗
PubMed1981

Interactions of free and immobilized myelin basic protein with anionic detergents.

The interaction of free and immobilized myelin basic protein (MBP) with sodium deoxycholate (DOC) and sodium dodecyl sulfate (NaDodSO4) was studied under a variety of conditions. Free MBP formed insoluble complexes with both detergents. Analysis of the insoluble complexes revealed that the molar ratio of detergent/MBP in the precipitate increased in a systematic fashion with increasing detergent concentration until the complex became soluble. At pH 4.8, equilibrium dialysis studies indicated that approximately 15 mol of NaDodSO4 could bind to the protein without precipitation occurring. Regardless of the surfactant, however, minimum protein solubility occurred when the net charge on the protein-detergent complex was between +18 and -9. Complete equilibrium binding isotherms of both detergents to the protein were obtained by using MBP immobilized on agarose. The bulk of the binding of both detergents was highly cooperative and occurred at or above the critical micelle concentration. At I = 0.1, saturation levels of 2.09 +/- 0.15 g of NaDodSO4/g of protein and 1.03 /+- 0.40 g of DOC/g of protein were obtained. Below pH 7.0 the NaDodSO4 binding isotherms revealed an additional cooperative transition corresponding to the binding of 15-20 mol of NaDodSO4/mol of protein. Affinity chromatography studies indicated that, in the presence of NaDodSO4 (but not in its absence), [125I]MBP interacted with agarose-immobilized histone, lysozyme, and MBP but did not interact with ovalbumin-agarose. These data support a model in which the detergent cross-links and causes precipitation of MBP-anionic detergent complexes. Cross-linking may occur through hydrophobic interaction between detergents electrostatically bound to different MBP molecules.

Animals↗