Search PubMed⌕ Search

Biomedical subjects

H Edelhoch

Publications and source records attributed to H Edelhoch.

At least 37 records · Page 2Linked to original sources
PubMed1982

Effects of thyroxine binding on the stability, conformation, and fluorescence properties of thyroxine-binding globulin.

The effects of thyroxine (T4) on several molecular properties of human thyroxine-binding globulin (TBG) have been evaluated. Changes in the sedimentation constant and relaxation time indicate that TBG becomes more symmetric and compact when T4 is bound. This modification in structure is associated with an increase in the stability of TBG to denaturation by either acid or guanidinium chloride. T4 binding also produces changes in the emission and excitation spectra of TBG, reflecting different environments of the four tryptophanyl residues. T4 preferentially quenches residues in a less polar environment. In addition, it alters the effect of the collisional quencher, acrylamide, so as to indicate a shift in the environment of some of the exposed tryptophanyl residues.

Acrylamides↗
PubMed1982

Instability of coated vesicles in concentrated sucrose solutions.

A method of preparing homogeneous coated vesicles that eliminates the high sucrose concentrations heretofore used is presented. It is shown that sucrose at high concentrations dissociates the coat from coated vesicles. This reaction can explain the presence of empty coats observed with preparations obtained with high concentrations of sucrose. The protein and membrane lipid components have been analyzed by the intrinsic tryptophan and extrinsic diphenylhexatriene fluorescence, respectively. Analysis of mixtures of coated vesicles and baskets resolved the contributions of the two species to the fluorescence curves.

Animals↗
PubMed1981

Effect of basic compounds on the polymerization of clathrin.

The effects of several divalent cations, various polybasic amines, and lysozyme on the rate of polymerization of 8S clathrin to the 300S coat structure have been evaluated by turbidimetric procedures. Ca2+ and Mn2+ strongly enhance the rate of polymerization. Only spermine among the naturally occurring polybasic amines had an important effect. Of the several basic proteins evaluated, only lysozyme stimulated the rate of polymerization. Some of these substances were able to increase the rate sufficiently so that polymerization occurred at physiological pH values. Without these compounds, clathrin will only polymerize at pH values of 6.8 or less.

Animals↗
PubMed1981

Polymerization of clathrin protomers into basket structures.

The effects of pH, ionic strength, temperature, and protein concentration on the rate of clathrin (8 S) polymerization to form coat (or basket) structures (approximately 300 S) have been measured by turbidity. The extent of polymerization has also been evaluated under the same experimental conditions by analytical centrifugation. The characteristic polygonal structure of the re-formed coat was confirmed by electron microscopy. The rate of polymerization is sensitive to all the variables investigated. The reaction is very slow at pH approximately 7 and becomes very rapid by pH approximately 6. The polymerization is readily reversed by increasing the pH slightly. The time dependence of the polymerization does not conform to either a first- or a second-order reaction but to a higher order. Increasing temperature increases the rate but decreases the extent of reaction. Increasing the salt concentration decreases the rate. The effects of several salts on the rate follow the Hofmeister ranking, with the exception of sulfate.

Animals↗
PubMed1981

Molecular characterization of human clathrin.

Clathrin extracted from coated vesicles at pH 8.0 sediments as a single boundary with 8.1S sedimentation constant (s020,w) of 8.1 +/- 0.1 S. Sedimentation equilibrium gave a molecular weight (Mr) of 610 000 +/- 30 000. The clathrin frictional ratio (pH 7.5) computed from s020,w and Mr is very large, i.e., 3.06 +/- 0.18. Analysis of the circular dichroic spectrum in the far-ultraviolet showed that about half of the peptide residues are in a alpha-helical conformation. The molecular weight of a preparation of clathrin purified to homogeneity on a Sepharose CL-4B column in 6 M guanidine hydrochloride was 170 000 +/- 26 000 by sedimentation equilibrium, which is in agreement with the values we and others obtained by sodium dodecyl sulfate gel electrophoresis. The 8.1S clathrin species may be regarded as the "native" promoter since (1) it is extracted from coated vesicles by an extremely mild procedure, (2) it is stable over considerable ranges of pH, temperature, and ionic strength, and (3) it readily polymerizes into characteristic closed lattice structures resembling those observed in coated vesicles in the electron microscope. The 8.1S clathrin molecule self-associates at pH 6.3 to form two very high molecular weight species with average sedimentation coefficients of 150 and 300 S. The sedimenting boundaries of both of these species have been analyzed to reveal their molecular heterogeneity. The two species observed by sedimentation velocity may correspond to the two sizes of coated vesicles previously reported to be present in some cells when observed by electron microscopy. Analysis of the sedimentation pattern in the ultracentrifuge also gives the amount of unreacted 8.1S clathrin from which the yield of polymerizable clathrin is obtainable. This methodology can therefore be employed to estimate the quality of the 8.1S preparation of clathrin and thereby affords an assay of its activity.

Circular Dichroism↗
PubMed1981

Enzymatic conversion of the major polypeptide chains of thyroglobulin.

The subunit composition of fully reduced bovine thyroglobulin consists of two polypeptide chains with molecular weights near 300,000. These two chains have been separated on sodium dodecyl sulfate gels by electrophoresis and were referred to as S and F. The effect of incubating native bovine thyroglobulin with a commercial preparation of horseradish peroxidase resulted in the conversion of S to F (and faster migrating polypeptides). Since this reaction was independent of the presence of H2O2 and iodide, it was necessary to demonstrate that the activity was present in the peroxidase enzyme which could be identified by its heme group. When the enzyme preparation was fractionated on Sephadex G-100, the activity responsible for the conversion of S to F was not associated with the heme peak but with another protein peak which eluted later on the column. Proteolytic inhibitors, such as phenylmethyl sulfonyl fluoride and pancreatic trypsin inhibitor, caused partial inhibition of the activity responsible for the conversion of S to F, whereas inhibitors of peroxidase activity had no effect. Guinea pig thyroglobulin was less susceptible to the proteolytic activity present in the peroxidase preparation than bovine thyroglobulin. The subunit composition of guinea pig thyroglobulin is different from that of bovine, since three bands of different sizes are present. The 300,000 mol wt subunit of guinea pig thyroglobulin (band A) ws largely degraded, whereas the 210,000 mol wt subunit (band B) was partially degraded and the 100,000 mol wt subunit (band C) was unaffected.

Animals↗
PubMed1980

Molecular properties of the reassembled coat protein of coated vesicles.

Clathrin has been prepared from human and bovine brains by a rapid technique which does not require sucrose gradient centrifugation. The promoter molecule which is obtained has the ability to polymerize and form protein coats, i.e., so-called cages or baskets, which resemble the structures observed in coated vesicles. The polymerization of clathrin to form cage structures in 0.2 M ammonium acetate, pH 6.8, results in two distributions of sedimenting particles in the ultracentrifuge, one centered near 300S and the other near 150S. Equilibrium sedimentation gives molecular weights of the 150S and 300S particles near 25 million and 100 million, respectively. The turbidities of the two species have been measured during centrifugation in the ultracentrifuge. When the turbidity values are combined with the molecular weight values, the radii of the 150S and 300S species can be obtained, assuming a hollow sphere as a model for the clathrin polyhedral molecules.

Animals↗
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↗