Studies on the precursor from of the first component of complement—I

Chapuis, Rose Marie; Isliker, H; Assimeh, S. N.

doi:10.1016/0019-2791(77)90229-4

Cited by 24 publications

(8 citation statements)

References 10 publications

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“…CIS was isolated by a rapid two-stage method involving affinity chromatography of serum or recalcified plasma on IgG-Sepharose according to the methods described by Chapuis et al [6] and Medicus et al [14], followed by the separation of the resulting mixture of Cls, Clr and amyloid (P-component) on DEAE-Sephacel.…”

Section: Discussionmentioning

confidence: 99%

“…1) was dialyzed against 20 mM acetate buffer, containing 50 mM NaCl and 1 mM EDTA, pH 5.0. Clr was then purified by ion-exchange chromatography, using the method of Chapuis et al [6] in a modified form. The conditions are described in the legend to Supplement Fig.…”

Section: Isolation Of C L Smentioning

confidence: 99%

“…For ]yophihation, 1 ml of 0.1 M Tris/HCl pH 8.6 and 20 mg sucrose were added and the pN adjusted to 8. 6 with NaOH, then the proteins were immediately lyophilized.…”

Section: Isolation Of C L Smentioning

confidence: 99%

“…40 mg) was dialyzed against 20 mM .Tris/HCl, 10 mM NaCl, 1 mM EDTA, 6 M urea, pH 7.5 and transferred to a column of DEAE-Sephacel equilibrated against the same buffer. The light chain appeared in the flow-through peak (pool 1, Supplement Fig.…”

Section: Isolation and Characterization Of Reduced And Carboxamidometmentioning

confidence: 99%

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Human complement component C1-s. Partial sequence determination of the heavy chain and identification of the peptide bond cleaved during activation

1986

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Human Cls proenzyme (Mr 83000) was isolated by a rapid two-stage method involving affinity chromatography of C1 on IgG-Sepharose and isolation of subcomponent Cls by ion-exchange chromatography on DEAE-Sephacel. Single-chain Cls proenzyme was activated to two-chain Cis with self-activated Cir. After reduction and S-carboxamidomethylation the heavy chain of Cis (Mr 57000) was isolated by ion exchange chromatography on DEAE-Sephacel. Cleavage of Cis heavy chain with CNBr yielded five fragments whose N-terminal sequences were determined. The alignment of the fragments within the heavy chain was established by tryptic peptides containing methionine. Cis heavy chain comprises about 470 amino acid residues and 42% of its sequence was determined. An intrachain sequence homology and a homology to the a2 chain of human haptoglobin were identified. The C-terminal CNBr fragment comprising 44 amino acid residues was completely sequenced. From BNPS-skatole cleavage of reduced and alkylated Cls proenzyme a fragment was isolated which overlaps the Cis heavy and light chain parts and which contains the peptide bond cleaved during activation. The results show that this is an Arg-Ile bond and that under standard conditions of activation no peptide material is liberated from this portion of the molecule. The sequence data and homology to two-chain serine proteases indicate a single interchain disulfide bond in Cis.

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Section: Discussionmentioning

confidence: 99%

Section: Isolation Of C L Smentioning

confidence: 99%

“…For ]yophihation, 1 ml of 0.1 M Tris/HCl pH 8.6 and 20 mg sucrose were added and the pN adjusted to 8. 6 with NaOH, then the proteins were immediately lyophilized.…”

Section: Isolation Of C L Smentioning

confidence: 99%

Section: Isolation and Characterization Of Reduced And Carboxamidometmentioning

confidence: 99%

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Human complement component C1-s. Partial sequence determination of the heavy chain and identification of the peptide bond cleaved during activation

1986

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“…These enzymes were prepared from human serum by a modification of the method of Chapuis et al [7] as follows. To about 1 1 of serum 200 ml of IgG-Sepharose 4B (10 mg human IgG/ml gel) was added.…”

Section: I S and C L Rmentioning

confidence: 99%

Kinetics of the Reaction between Human C1‐Esterase Inhibitor and C1r or C1s

Nilsson¹,

Wiman

1983

European Journal of Biochemistry

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The kinetics of the reactions of CT-esterase inhibitor with C i s or C i r has been studied in purified systems. Both reactions were found to proceed in two steps: a fast reversible second-order reaction followed by a slower irreversible first-order transition according to the following scheme: E + I A E I k ' . EI'. The rate constant kl for the reaction between C i s and Ci-esterase inhibitor was found to be 1 . 4~ lo5 M-' s-l at 25"C and 4 . 7~ 105 M -I S -~ at 37 "C, while the corresponding values for the reaction between C i r and Ci-esterase inhibitor were 2.5 x lo3 M-' s-' and 4.4 x lo4 M-' s-', respectively. The dissociation constant K of the initial complex was estimated as 1 -2 n M for the complex with C i s and 20-50 nM for the complex with Cir. Heparin was found to accelerate the rate of the Cfs/Cf-esterase inhibitor reaction by a 16-fold increase in kl at heparin concentrations above 100 mg/l, whereas no effect was obtained on the thermodynamic equilibrium. The inhibition of purified CIS added to dilutions of plasma or serum was found to proceed as expected from their known concentrations of Ci-esterase inhibitor and the rate constants determined in the purified system. Thus, the halflife of C i s in plasma under pseudo-first-order kinetics would be about 1 s A tThe classical pathway of the complement system is initially activated by limited proteolysis of the complement components by C l r and Cfs. These enzymes occur in plasma as single-chain zymogens in the so-called C1 complex [l]. Upon activation, C l r and C I S are cleaved to yield the active enzymes consisting of two disulphide-bonded polypeptide chains. The main inhibitor of these enzymes in human plasma is the CTesterase inhibitor [2]. During the reaction of CT-esterase inhibitor with CTr or Cis very stable stoichiometric 1 : 1 inactive complexes are formed involving the light chain of the enzymes [3].Recently an attempt to investigate the kinetics of these reactions in a purified system was performed [4]. It was suggested that the reactions were composed of two steps, the first of which was reversible and the second of which was irreversible. However, only the overall reaction was studied and n o experimental data was presented to supported the proposed mechanism. In the present investigation we have performed a mechanistic evaluation of the interaction between Ci-esterase inhibitor and C i s or C l r and determined the kinetic constants in purified systems as well as in plasma and serum. MATERIALS A N D METHODS C~-E s t t v m e InhibitorCi-esterase inhibitor was purified from freshly frozen human citrated plasma by poly(ethy1ene glycol) precipitation, DEAE-cellulose chromatography, and hydrophobic interaction chromatography on hexyl-Sepharose, as previously described [5]. The final product is a homogeneous protein which, when incubated with a molar excess of C i s (25"C, loinin), quantitatively forms a complex with the enzyme, as demon- strated by dodecylsulfate/polyacrylamide gel electrophoresis. The concentration was determined from the...

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Inorganic supports coated with N‐substituted polyacrylamides: Application to biospecific chromatography of proteins

Ivanov

Козлов

Shojbonov

et al. 1991

Biomedical Chromatography

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Wide porous glass (WPG) chemically coated with a poly-N-(2-hydroxyethyl)acrylamide layer is proposed as a carrier of biospecific ligands in affinity chromatography. The method of WPG chemical modification includes synthesis of the gamma-aminopropyl derivative followed by chemical adsorption of poly(p-nitrophenyl acrylate). Ester groups of the polyacrylate-coated WPG can be used for coupling the ligands bearing primary amino groups. Condensation of esters with ethanolamine yields a poly-N-(2-hydroxyethyl)acrylamide-coated support with non-specific adsorption properties resembling those of Sepharose 4B. Human IgG immobilized on the polyacrylate support was used for isolation of the first complement component from human serum and for its separation into subcomponents C1r, C1s and C1q by a one-step method. An unbound part of serum may be used as the R1 reagent for determining haemolytic C1 activity. The stepwise elution of C1r, C1s and C1q from the column reflects the course of C1 breakdown after its activation on immune complex formation.

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Studies on the precursor from of the first component of complement—I

Cited by 24 publications

References 10 publications

Human complement component C1-s. Partial sequence determination of the heavy chain and identification of the peptide bond cleaved during activation

Human complement component C1-s. Partial sequence determination of the heavy chain and identification of the peptide bond cleaved during activation

Kinetics of the Reaction between Human C1‐Esterase Inhibitor and C1r or C1s

Inorganic supports coated with N‐substituted polyacrylamides: Application to biospecific chromatography of proteins

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