Isolation and characterization of copper-binding sites of human ceruloplasmin

The quaternary structure of f:!-galactosidase, which consists of four identical subunits, has been studied by the isolation and characterization of appropriate mutants of Escherichia coli. Of 146 mutants examined, 19 were found to have enzymes with reduced subunit association. These altered enzymes are especially sensitive to inactivation by urea which, at concentrations that do not affect the normal enzyme, causes dissociation into subunits. Mapping with overlapping deletions showed that the mutations affecting quaternary structure are not distributed continuously, but occur in five or six groups within the gene. The mapping indicates that polypeptide sequences involved in subunit association and in substrate binding are contiguous. A model for f:!-galactosidase structure is suggested in which substrate binding sites are provided by the clefts formed between subunits when they associate.

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Isolation And Characterization Of The Vitamin K Dependent Domain Of Human Prothrombin

10.1055/s-0038-1652317 ◽

1981 ◽

Author(s):

C Dodé ◽

A Thiesce ◽

D Labie ◽

J Elion

Keyword(s):

Conformational Change ◽

Vitamin K ◽

Disulfide Bond ◽

Binding Sites ◽

Terminal Portion ◽

Isolation And Characterization ◽

Cooperative Process ◽

Dependent Domain ◽

Stimulation Of

Chymotryptic cleavage of human prothrombin (hPt) produces two fragments of 68000 and 5000 MW respectively. These two species were separated by Ba citrate adsorption and Sephadex G100 chromatography. The 68000 MW species corresponds to hPt (des 1-44) and has lost all the vitamin K dependent properties of hPt : adsorption on Ba citrate, Ca+2 and phospholipid dependent stimulation of the activation by FXa, presence of strong Ca+2 binding sites (as shown by dialysis equilibrium) and Ca+2 induced fluorescence quenshing. The 5000 MW species corresponds to the N-terminal portion of hPt (residues 1-41). It contains the 10 Gla residues and the 17-22 disulfide bond. This peptide is quantitatively adsorbed on Ba citrate. Activation of hPt in a mixture containing FXa, Ca+2 and phospholipid is drastically inhibited by the addition of peptide 1-41 (≃ 50 % inhibition for a 1/1 peptide/Pt ratio, ≃ 7 % for a 40/1 ratio).Ca+2 produces a quenshing of the intrinsic fluorescence of peptide 1-41 (λ emission = 355 nm). This quenshing plateausat 40 % of the initial fluorescence at 3 mM Ca+2. The plot of the fluorescence quenshing versus Ca+2 concentration however is not cooperative. Mn+2 and Mg+2 also induced fluorescence quenshing but to a lesser extent. Hence peptide 1-44 represents a functionnal domain in itself interacting with Ca+2 and phospholipid. It contains only one Trp (residue 41), showing directly the involvment of this residue in the Ca+2 induced fluorescence quenshing observed for Pt fragment (FI) and Pt. This isolated vitamin K dependent domain therefore retains many of the vitamin K dependent properties of FI or Pt, but shows differences in the Ca+2 induced conformational change in that it is no longer a cooperative process.

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Specific oxidation of copper binding sites in copper(II)-oligopeptide complexes

Biochemistry ◽

10.1021/bi00777a010 ◽

1971 ◽

Vol 10 (1) ◽

pp. 64-66 ◽

Cited By ~ 2

Author(s):

Arieh Berger ◽

Alexander Levitzki

Keyword(s):

Binding Sites ◽

Copper Binding ◽

Specific Oxidation ◽

Copper Binding Sites

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