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Biomedical subjects

H Edelhoch

Publications and source records attributed to H Edelhoch.

At least 19 recordsLinked to original sources
PubMed1979

The effects of thioureylene compounds (goitrogens) on lactoperoxidase activity.

The rates of oxidation of several goitrogens by lactoperoxidase and the rates of inactivation of lactoperoxidase by the same goitrogens have been measured. The influence of iodide on both reactions has also been evaluated. It has been shown by us that iodide acts catalytically in regulating lactoperoxidase activity at pH 8.8. The rate data have been analyzed by a computer program which solves the differential equations for the above mentioned reactions. From this computer analysis we have been able to obtain binding constants of the goitrogens and inactivation rate constants of lactoperoxidase. Iodide was shown to inhibit goitrogenic activity either by increasing the rate of drug oxidation or by reducing the rate of enzyme inactivation, or both, depending on the particular drug. Iodide had little or no effect on the goitrogen-binding constants. We have also shown that the relative rates of enzyme inactivation can be correlated with the potency of the goitrogen as an antithyroid drug.

Antithyroid Agents↗
PubMed1979

Iodide binding and regulation of lactoperoxidase activity toward thyroid goitrogens.

The effects of the antithyroid goitrogens, methylthiouracil and methylmercaptoimidazole, on the oxidation of N-acetyltyrosylamide at pH 8.8 by lactoperoxidase have been evaluated in the presence and the absence of iodide for the purpose of elucidating the effects of iodide. At pH 8.8, iodine is not oxidized. In the absence of iodide, the two antithyroid drugs inactivate lactoperoxidase by a second order process. When iodide is added before methylthiouracil or methylmercaptoimidazole, enzyme inactivation does not occur as rapidly and both goitrogens are readily oxidized. The kinetics of the oxidation reactions have been analyzed in order to obtain the equilibrium constant of the iodide . lactoperoxidase complex. Essentially the same iodide dissociation constant, i.e. 2 x 10(-5) M, was found by studying its effects on the kinetics of oxidation of the two antithyroid drugs. A large difference absorption spectrum is observed in the Soret region between native lactoperoxidase and lactoperoxidase inactivated by methylthiouracil.

Iodides↗
PubMed1979

Critical micelle concentrations of gangliosides.

The micellar properties of mixed, bovine gangliosides and purified galactosyl-N-acetylacetylgalactosaminyl (N-acetylneuraminyl) galactosylglucosylceramide were studied by gel filtration, equilibrium dialysis, and band and boundary centrifugation in sucrose gradients. The dissociation of micelles is very slow (days) in water and required us to approach equilibrium by association of monomers rather than by the dissociation of micelles. The gangliosides were therefore first converted into very low molecular weight aggregates (1-3 molecules) by dissolving them in Me2SO. Galactosyl-N-acetylgalactosaminyl(N-acetylneuraminyl)galactosylglucosylceramide was then diluted into aqueous sucrose gradients and sedimented by the boundary centrifugation technique. This gave a sedimenting micelle and a nonsedimenting monomer concentration of (1-2) x 10-10 M (or less) which corresponds to the critical micelle concentration value. The mixed gangliosides revealed two micellar sizes (i.e., 10 and 4.5 S), the slower sedimenting species being formed from the larger one with time (days). The critical micelle concentration of the mixed gangliosides was found to be approximately 10-8 M by a gel filtration, equilibrium dialysis, and band centrifugation.

Animals↗
PubMed1979

The structure and stability of human plasma cold-insoluble globulin.

The molecular properties of cold-insoluble globulin have been investigated by velocity centrifugation, circular dichroism, and fluorescence at neutral and alkaline pH. The stability of the protein to thermal and guanidine hydrochloride has been evaluated under both conditions. The close parallelism between the properties of cold-insoluble globulin and those of the cell surface protein (fibronectin) serve to establish the essential identity of the structures of the two proteins derived from different sources. It is suggested that the cold-insoluble globulin is composed of several domains connected by flexible polypeptide segments. The large increase in the frictional ratio observed between pH 7.0 and 11.0 can be explained by an expansion of the flexible segments without significant change in the domains. These domains are stable to about 55 degrees C at pH 7.0 but only to about 40 degrees C at pH 11.0.

Circular Dichroism↗
PubMed1978

Thyroxine-induced conformational changes in prealbumin.

The effects of thyroxine binding on the conformation of human prealbumin and bovine serum albumin have been examined. A blue shift in protein absorption was observed with prealbumin, whereas a red shift was observed with bovine serum albumin. In the case of prealbumin, where the two binding sites are identical, the total absorption change was confined to the binding of the first ligand and has been interpreted as resulting from a conformational change. A blue shift observed in the absorption spectrum of thyroxine, however, was the same for the first and second bound molecules. These data have been interpreted in terms of two identical and interacting sites on prealbumin and explain the origin of the difference in binding affinities between the first and second sites. Fluorescence quenching by thyroxine and thyroxine effects on tryptic hydrolysis of prealbumin are in accord with the above interpretation.

Humans↗
PubMed1978

The stability of cell surface protein to surfactants and denaturants.

The effects of several denaturants and detergents on the structure and stability of cell surface protein have been evaluated by circular dichroism and fluorescence measurements. Cell surface protein undergoes a single broad transition in both urea and guanidinium chloride. Although guanidinium chloride is twice as effective as urea on a molar basis, both appear to eliminate all of the organized structure present in the native molecule. Nonionic surfactants and lysolecithin have little effect on cell surface protein. However, sodium dodecyl sulfate increases the alpha helical content and cetyltrimethylammonium bromide increases the beta structure of cell surface protein. The reorganization of the polypeptide backbone requires the loss of certain restraints imposed by tertiary interactions as evidenced by a decrease in ellipticity in the far ultraviolet and in the polarization of tryptophanyl fluorescence. These results along with the data of a previous paper (Alexander, S. S., Jr., Colonna, G., Yamada, K. M., Pastan, I., and Edelhoch, H. (1978) J. Biol. Chem. 253, 5820--5824) suggest the presence of structural domains distributed along the flexible polypeptide chain of cell surface protein.

Animals↗
PubMed1978

Fractionation of human parotid saliva proteins.

A chromatographic procedure for purification of the proteins in human parotid saliva has been developed. The eluates of a Sephadex G-150 and two ion exchange columns have been analyzed simultaneously by several physical and chemical tests; these include three optical properties of proteins, assays for neutral sugars, sialic acid and zinc, and disc gel electrophoresis. The ratios of the different variables have been used to determine the homogeneity and complexity of the protein distribution in the various peaks of the chromatographic eluates. By chromatographic methods, it has been possible to purify a glycoprotein with unusual staining characteristics and amino acid composition. Glycoproteins with similar properties comprise a major portion of the proteins in parotid saliva and appear to constitute a family of related proteins which differ in molecular size, carbohydrate and sialic acid content, and electrophoretic mobility. The fractionation of several enzymes in parotid saliva is also reported.

Amino Acids↗
PubMed1978

Molecular properties of a major cell surface protein from chick embryo fibroblasts.

The molecular structure of chick embryo fibroblast cell surface protein has been investigated by ultracentrifugation, circular dichroism, and fluorescence. Most measurements were restricted to alkaline solutions because of the limited solubility of this protein at more neutral pH values. A very high frictional ratio for the protein suggests an asymmetric structure. However, there are elements of organized structure since typical thermal transition curves were found by several methods. Consequently, a model in which ordered domains are connected by flexible polypeptide chains seems to account for all the hydrodynamic and optical data.

Animals↗
PubMed1978

Effects of Hofmeister salts on the self-association of glucagon.

The trimerization constants of glucagon at pH 10.6 in 0.76 M K2HPO4 have been calculated from circular dichroism data between 5 and 50 degrees C. The free energy, enthalpy, and entropy of transfer have been evaluated from the current results and published data in 0.20 M phosphate. The free energies of transfer are derived completely from an increase in the entropy of transfer, since the enthalpy of transfer is less favorable at all temperatures. These parameters are compared with those of various model groups and compounds: CH2, peptide, methane, ethane, and the 1--13 N-terminal fragments of ribonuclease. The effects of fluoride and chloride on the self-association of glucagon have been compared with that of phosphate at 25 degrees C. These effects are consistent with the binding of approximately one molecule of salt to the trimer and a systematic decrease in the number of water molecules bound to the trimer compared to the monomer for the series K2HPO4, KF, and KCl.

Anions↗
PubMed1978

Evidence that thyroglobulin contains nonidentical half molecule subunits.

Bovine thyroglobulin was extracted from unfrozen glands, purified by sucrose gradient centrifugation, and fractionated into a narrow range in iodine content by RbCl isopycnic centrifugation. The subunit composition of these preparations was studied by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The extent of dissociation of 19 S into 12 S half-molecules followed the known relationship with iodine, i.e. decreased dissociability of 19 S with increased iodine content. The undissociated 19 S band always consisted of three closely spaced, equidistant bands. Reduction of the disulfide bonds of thyroglobulin by mercaptoethanol in SDS solution resulted in the formation of two major and one minor components (S, F, and A). The concentration of A was always less than 10% of the total. The ratio of S to F was, however, about equal in thyroglobulin preparations which ranged in iodine content from 0.2 to 1%. The final ratios were obtained before all the disulfides were reduced. The relative mobilitis of S, F, and A, decreased with increasing extent of reduction. Fully reduced S and F, but not A, migrated slower than unreduced 12 S. The three reduced alkylated polypeptides were purified by preparative SDS-polyacrylamide gel electrophoresis and their molecular weights were determined by sedimentation equilibrium in 8 M urea. Their Mw and Mz values agreed closely with that of the unreduced 12 S half-molecule subunit, thus indicating that reduction of the disulfide bonds changes the shape but not the molecular weights of the subunits.

Animals↗
PubMed1978

Self-association of glucagon as measured by the optical properties of rhodamine 6G.

The fluorescence of rhodamine 6G is completely quenched in glucagon solutions in 0.6 M K2HOP4 at pH 10.6. The absorption of rhodamine 6G is red-shifted by the same reaction. A single rhodamine 6G molecule appears to be bound to a hydrophobic patch in the center of the trimer of glucagon. Since the glucagon monomer has almost no organized structure this site exists only in the associated trimer form of glucagon. The self-association of glucagon to the trimer has been determined from the variation in rhodamine 6G fluorescence and absorption measured over a 60-fold range of dye concentration. The self-association constant agrees with values determined by other methods in the absence of dye. The binding isotherms of rhodamine 6G to glucagon shift with glucagon concentration and exhibit negative cooperativity.

Anilino Naphthalenesulfonates↗
PubMed1978

Protein-lipid interactions and the role of water.

The rigidity of the three-dimensional structure of a native protein is dependent on the network of hydrogen-bonded groups which provide the scaffolding for the other interactions. The structure is stabilized by the hydrophobic interactions of the nonpolar side chains. The latter are formed by the very unfavorable entropy change that occurs in water but not in less-polar solvents. It is unlikely that any solvent other than water can produce the same folding of a polypeptide chain to form the active native structure. Water plays a unique role, since it alone is responsible for the heat capacity changes observed when nonpolar groups are transferred from an aqueous to a nonaqueous environment, as exists in the interior of a protein. The need to juxtapose like groups and to avoid making contact among unlike groups imposes severe restrictions on the binding of small or large molecules to proteins. Consequently there must be proper pairing of polarities as well as close fitting of ligands for strong binding to occur. This is clearly evident from the x-ray studies of proteins containing subunits or prosthetic groups. The thermodynamic parameters observed in the most complex protein reactions--i.e., self-assembly systems--resemble rather well those observed in micelle association reactions or even in the solution of nonpolar gases in water. This interaction--hydrophobic--can be looked upon as the controlling reaction which stabilized the organized structures of most cellular entities aside from nucleic acids--i.e., membranes and organelles.

Butanes↗
PubMed1977

Structure and stability of human thyroxine-binding globulin.

The secondary and tertiary structure of human plasma thyroxine-binding globulin (TBG) was investigated by circular dichroism and fluorescence properties. The relaxation time of TBG indicated that it is a compact, symmetric molecule. It was calculated from the far ultraviolet CD spectrum that about one-half of the peptide groups are equally distributed in alpha helical and beta structures. In the near ultraviolet, the CD spectrum of TBG was modified when thyroxine was bound. TBG was stable at temperatures below 50 degrees at pH 9 and below 35 degrees at pH 10.5. Below pH 5 tryptophanyl fluorescence revealed a molecular transition which followed first order kinetics. The transition resulted in an irreversible loss of binding of the hormone. Acidification to pH 3.4 produced only a minor change in the CD spectrum, in which some of the alpha helical peptides were converted to beta structure.

Circular Dichroism↗
PubMed1977

Interaction of apoA-II from human high density lipoprotein with lysolecithin.

The effects of lysolecithin and hexadecyltrimethylammonium bromide on the structure and stability of apoA-II from human high density lipoprotein have been evalued by circular dichroism and fluorescence measurements. There is a profound enhancement in the stability of apoA-II to guanidinium hydrochloride denaturation when it forms a phospholipid complex with lysolecithin micelles. This complex is not only resistant to guanidinium hydrochloride denaturation, but it can be formed in a 6 M solution of this denaturant. The behavior of apoA-II in the native human high density protein is much closer to that of the lysolecithin apoA-II complex than to that of the free apoA-II.

Apoproteins↗
PubMed1977

Stability and subunit structure of human alpha2-macroglobulin.

The molecular weights of alpha2-macroglobulin and its non-covalent subunits have been determined by equilibrium centrifugation. The secondary structure of the native and the thermally denatured molecules has been analyzed by circular dichroic measurements. In contrast to most proteins the thermally denatured form contains a slightly more highly organized polypeptide chain than the native form. The relaxation time of the native protein, as determined by fluorescence polarization measurements, indicates that alpha2-macroglobulin is composed of domains smaller than that of the two subunits. The transitions in acid, alkali, and at high temperatures have been explored in order to establish the pH and thermal range of stability of alpha-macroglobin.

Drug Stability↗
PubMed1977

Thermodynamics of the self-association of glucagon.

In water, glucagon exists in an equilibrium between a trimer in which more than half of the peptide groups are in an alpha-helical configuration and a monomer which has a random coil configuration with few alpha-helical residues. The thermodynamics of this self-association have been evaluated by studying the temperature- and concentration-dependence of the mean residue ellipticity at 220 nm. The enthalpy and entropy changes of association were negative at all temperatures between 5 degrees and 50 degrees and had large negative temperature dependencies. Usually an association that involves nonpolar groups is considered to be driven by a positive entropy term. Such an explanation is not tenable in the case of glucagon. However, if the effects of nonpolar groups on the coil-to-helix transition of a polypeptide are included into the thermodynamic considerations of hydrophobic interactions, then the negative parameters observed for glucagon association can be readily understood. The hydrophobic interaction is therefore not necessarily controlled by the entropy change because, if there are significant conformational changes, the reaction may be controlled by the enthalpy change. Consequently, the more important parameter characteristic of all hydrophobic reactions is the heat capacity change.

Glucagon↗