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R E Lippoldt

Publications and source records attributed to R E Lippoldt.

32 records · Page 2Linked to original sources
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↗
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↗
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↗
PubMed1977

Characterization of human thyroxine-binding globulin. Evidence for a single polypeptide chain.

Thyroxine-binding globulin (TBG) was purified from fresh human plasma by affinity, anion exchange, and gel filtration chromatography. The protein gave a single band in overloaded analytical disc gel electrophoresis. The molecular weight was 54,000 and E1%/1 cm at 280 nm, corrected for thyroxine (T4) absorbance, was 6.17. Six preparations of TBG contained from 0.09 to 0.64 mol of T4/mol; the TBG used in this study contained 0.19 mol of T4 and was able to bind an additional 0.85 mol. The carbohydrate composition was determined and accounted for 23% of the molecular weight. Four lines of chemical and physical evidence failed to demonstrate subunits. These included quantitative COOH-terminal amino acid analysis, peptide mapping and amino acid composition, treatment with sodium dodecyl sulfate, and denaturation of the reduced, alkylated protein with guanidine. From these data, we conclude that TBG is a single polypeptide chain.

Amino Acids↗
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↗
PubMed1975

A zinc protein isolated from human parotid saliva.

A zinc protein has been isolated and purified to apparent homogeneity from subjects with normal taste acuity by gel filtration and ion-exchange chromatography. The protein has a molecular weight of 37,000 and does not appear to have subunits. It is composed of 8% histidine residues and has 2 moles of zinc per mole of protein.

Chromatography, Gel↗