Journal ArticleDOI
Third component of human complement: purification from plasma and physicochemical characterization.
B D Tack,James W. Prahl +1 more
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Alanine is the ultimate carboxy-terminal amino acid of at least one of the chains, as indicated by the action of carboxypeptidases on C3 in the presence of sodium dodecyl sulfate.Abstract:
The third component of complement has been purified from fresh human plasma employing an initial fractionation with poly(ethylene glycol) followed by sequential depletion of plasminogen by affinity adsorbents, chromatography on diethylaminoethylcellulose, gel filtration on agarose, and batch adsorption/desorption on hydroxylapatite. Final recoveries of C3 were 33% of the initial protein, as quantitated by radial immunodiffusion, and 31% of the initial hemolytic activity. Apparent homogeneity is indicated by immunological criteria and by polyacrylamide gel electrophoresis. A partial specific volume of 0.736 +/- 0.003 mlgm-1 was determined for C3 by the mechanical oscillator technique. "Low speed" sedimentation equilibrium yielded an apparent weight average molecular weight for the protein of 187 650 +/- 5650. Based upon this molecular weight, a molar extinction coefficient of 1.82 X 10(5) 1. mole-1 cm-1 at 280 nm was calculated from boundary-spreading experiments in the ultracentrifuge and as assumed refractive index increment. Amino acid analyses revealed no unusual or distinctive characteristics. Automated Edman degradation revealed a double N-terminal sequence, Ser-Val,Pro-Glx,Met-Lee,Tyr-Thr,Ser-Glx,Ile-Lys,Gly-Arg,Thr-Met,Pro-Asx, in agreement with the two chain structure observed on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and revealing both chains are available to degradation. Serine is postulated as the initiating sequence in both chains based upon high recoveries of dinitrophenylserine upon hydrolysis of dinitrophenylated C3, and our inability to identify any other dinitrophenyl or phenylthiohydantoin derivatives in this position. Alanine is the ultimate carboxy-terminal amino acid of at least one of the chains, as indicated by the action of carboxypeptidases on C3 in the presence of sodium dodecyl sulfate.read more
Citations
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Journal ArticleDOI
Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte.
TL;DR: Direct evidence for the identity of gp205 as the C3b receptor of the four cell types was obtained when detergent-solubilized membrane proteins of the surface-radioiodinated cells were reacted with anti- gp205 and the immunoprecipitate was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.
Journal ArticleDOI
Protein volumes and hydration effects. The calculations of partial specific volumes, neutron scattering matchpoints and 280-nm absorption coefficients for proteins and glycoproteins from amino acid sequences.
TL;DR: In this paper, an intermediate consensus volume set of amino acid-residue volumes is proposed in order to predict experimental v values using sequence information, and the method is extended to carbohydrates and glycoproteins.
Book ChapterDOI
Anaphylatoxins: C3a and C5a.
TL;DR: This chapter discusses the structural and functional characterization of anaphylatoxins, spasmogenic substances released during complement activation that elicit a variety of cellular responses, which implies that they play a significant role in inflammation and acute allergic reactions.
Journal ArticleDOI
Formation of the initial C3 convertase of the alternative complement pathway. Acquisition of C3b-like activities by spontaneous hydrolysis of the putative thioester in native C3.
TL;DR: The hypothesis that spontaneous hydrolysis of the thioester generates a derivative of C3 that is responsible for the formation of the initial C3 convertase of the alternative pathway of complement is examined.
Journal ArticleDOI
Large scale isolation of functionally active components of the human complement system.
TL;DR: The utility and effectiveness of this preparative scheme have allowed isolation of C3, C5, and C7 as pure components with full hemolytic activity as judged by functional, immunochemical, and physicochemical criteria.
References
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Journal ArticleDOI
The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis
Klaus Weber,Mary Osborn +1 more
TL;DR: The results show that the polyacrylamide gel electrophoresis method can be used with great confidence to determine the molecular weights of polypeptide chains for a wide variety of proteins.
Journal ArticleDOI
Immunochemical quantitation of antigens by single radial immunodiffusion
TL;DR: By standardizing the technical conditions of the experiment it is possible to use this principle for the immunochemical determination of antigens, and the lower limit of the method was found to correspond to 0·0025 μg of antigen, and to an antigen concentrations of 1·25 μg per ml.
Journal ArticleDOI
Determination of protein: A modification of the lowry method that gives a linear photometric response
TL;DR: Under the new conditions there is direct proportionality between absorbance at 650 nm and weight of protein within the range 15–110 μg.
Journal ArticleDOI
Plasminogen: Purification from Human Plasma by Affinity Chromatography
Dale G. Deutsch,Edwin T. Mertz +1 more
TL;DR: Plasminogen was prepared from human plasma by affinity chromatography on L-lysine-substituted Sepharose with a specific activity of 100 caseinolytic units per milligram of nitrogen.