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At least 523 records · Page 29Linked to original sources
PubMed1975

Self-association of the major protein component of bovine serum high density lipoprotein.

The major protein component of bovine high density lipoprotein was investigated in solution by fluorescence polarization and ultracentrifugal techniques. A fluorescent derivative of this protein with 1-dimethylaminonaphthalene-5-sulfonyl chloride was employed in the fluorescence experiments. Over the concentration range from 5-10(-7) M to 5-10(-4) M of the protein monomer at pH values from 2 to 11 and ionic strengths from 0.03 to 2.0, at 23 degrees C, the major protein of bovine high density lipoproteinapoprotein (Apo-HOL-I) was found to exist in a stable aggregated form. The aggregate was not affected by dioxane additions of up to 20% nor by Triton X-100 to 0.2%, but dissociated readily in the presence of 0.07% sodium dodecylsulfate or 6 M urea. At concentrations below 5-10(-7) M, dissociation of the protein aggregate started spontaneously and continued down to 10(-8) M, the lowest measurable concentration. Several physiocochemical properties of the major protein of bovine high density lipoprotein were determined in the stable aggregate form. Molecular weight was 104 000 from ultracentrifugal analysis and 80 000 from gel-filtration. Rotational relaxation time was 115 ns at 25 degrees C, and s-0 20,w was 4.78 s. The results suggest very strong protein-protein interactions (Kd less than 10(-7) M) that are not electrostatic in nature. Hydrophobic interactions of a magnitude that could be affected by 20% dioxane or 0.2% Triton X-100 detergent are also excluded. There is saturation of the interaction sites by the aggregation of a few protein monomer units possibly to form a tetramer which is moderately asymmetric (1:4 axial ratio, assuming an ellipsoid of revolution) and relatively rigid. The strong protein-protein interactions in this pure apolipoprotein suggest the possibility of competition of inter-protein associations with protein-lipid interactions in in vitro lipid binding or lipoprotein reconstitution experiments.

Animals↗
PubMed1975

Structure of N-terminal cyanogen bromide fragment of human plasma albumin.

The complete amino acid sequence of 87 residues of cyanogen bromide fragment CB1 (Asp), the N-terminal fragment of human plasma albumine molecule, has been established. The sequence was determined from the characterization of all tryptic peptides and of chymotryptic arginine-containing peptides in the fragment digested. Overlaps were obtained by tryptic and chymotryptic cleavage of the maleylated S-sulfo derivative of fragment CB1(Asp). Residue 34 is the only cysteine residue in the albumin molecule and it was determined in the form of S-carboxymethyl-cysteine. Edman and dansyl-Edman degradation were used for the sequential analysis.

Amino Acid Sequence↗
PubMed1975

The N-terminal heterogeneity of edta-extractable erythrocyte membrane proteins.

N-terminal analysis of the proteins extracted from ox (Bos taurus) erythrocyte membranes by dilute EDTA is used as a means of estimating the heterogeneity of the protein fractions. Dinitrophenylation and dansylation reveal up to 8 different N-terminal amino acids in fractions with electrophoresis after dodecylsulphate treatment shows as having far fewer polypeptide chains. These fractions are prepared by gel electrophoresis in the presence and absense of detergent. Molecular weight estimation by the Ferguson procedure of the components revealed in the EDTA extract by electrophoresis in the absence of detergent confirms the large size concluded from electrophoresis with dodecylsulphate. The N-terminal analyses indicate that the high molecular weight of the complexes, both in the presence and absence of detergent, is more probably due to their being complexes of several polypeptides than exceptionally long single polypeptide chains.

Amino Acid Sequence↗
PubMed1976

Fluorescence depolarization in a model system: carbonic anhydrase-dansyl sulphonamide.

The model system, carbonic anhydrase to which dansyl sulphonamide is specifically adsorbed, was chosen to test the fundamentals of the fluorescence depolarisation method. Fluorescence decay was shown to be satisfactorily represented by a single exponential decay term and the derived lifetime did not seem to vary significantly with mean excitation or emission wavelength. Depolarisation measurements were quite reproducible and those made with varying temperature were consistent with those performed with varying solution viscosity, thus excluding localised dye rotation. The calculated relaxation time, which was independent of wavelength of excitation or emission, was in conformity with a rigid spherical molecule of molecular weight 29 000 with 30% hydration.

Carbonic Anhydrases↗
PubMed1976

Specific fluorescent derivatives of macromolecules. Reaction of dansyl fluoride with serine proteinases.

5-Dimethylaminonaphthalene-1-sulfonyl fluoride was evaluated as a reagent for the selective labeling of proteins. In a comparative study with Dns-chloride a greatly increased selectivity of the fluoride was found with a number of proteins. The reaction of Dns-fluoride with alpha-chymotrypsin, subtilisin Carlsberg and trypsin was found to be highly specific, resulting in a stoichiometric incorporation of the Dns label with concomitant loss of enzymatic activity. The reaction of Dns-chloride with the same proteinases is unspecific. Evidence was obtained to indicate that reaction of the serine esterases with Dns-fluoride occurs exclusively at the active serine residue. The stability of Dns-fluoride labeled chymotrypsin was investigated. The conjugate was found to be fairly stable in the pH range from 3 to 9 at 25 degrees C and is therefore suitable for fluorescence investigations of the chymotrypsin active-site. Molar extinction coefficients for Dns-labeled serine proteinases were determined using radiocative label.

Binding Sites↗
PubMed1980

Purification and characterization of the proteolipid of rabbit sarcoplasmic reticulum.

The proteolipid of rabbit sarcoplasmic reticulum was isolated and characterized. Tyrosine was identified as the C-terminal amino acid by hydrazinolysis and carboxypeptidase A digestion. The N-terminal sequence of proteolipid is: Met-Glx-Arg-Ser-Thr-Arg-Glx-Leu-Cys-Leu-Asp-Phe. The hydrophilic character of the N-terminal portion suggests that it is exposed on the membrane surface.

Amino Acid Sequence↗
PubMed1981

Fluorescent labeling of the carbohydrate moieties of human chorionic gonadotropin and alpha 1-acid glycoprotein.

A method for covalent attachment of a fluorescent molecule to the carbohydrate moieties of glycoproteins is described. The glycoproteins were oxidized with periodate under mild conditions selective for sialic acid (Van Lenten, L. and Ashwell, G. (1971) J. Biol. Chem. 246, 1889--1894). The resulting aldehydes were condensed with either dansylhydrazine, dansylethylenediamine, or fluoresceinamine followed by reduction with NaCNBH3 and NaBH4. Conjugates prepared with dansylhydrazine were found to be insufficiently stable for spectroscopic analysis, whereas the primary amines produced stable conjugates whose fluorescence polarization (P) was constant for several hours at 37 degrees C. The degree of labeling correlated roughly with the sialic acid contents of the vaious glycoproteins. Very little covalent incorporation was observed with albumin (which is devoid of carbohydrate) or with asialo alpha 1-acid glycoprotein. Exclusion chromatography in the presence of a dissociating agent was sometimes required to remove significant amounts of noncovalently adsorbed dye. Fluorescent-labeled alpha subunits of human chorionic gonadotropin were shown to recombine normally with native beta subunits. However, the labeling procedure appeared to compromise the ability of the beta subunits to recombine. Electrophoretic analysis produced evidence of covalent cross-linking between subunits following periodate oxidation of the intact gonadotropin. The possibility that primary amine groups of the protein compete with added fluorescent amines for reaction with periodate-generated aldehydes is discussed.

Chemical Phenomena↗