Modulation of the alkaline transition in cytochrome <i>c</i> and cytochrome <i>c</i>-T by full or specific partial acetimidylation

Wallace, C J A

doi:10.1042/bj2170601

Cited by 21 publications

(9 citation statements)

References 18 publications

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“…The protected fragments may be combined to form a stable complex (1-38)-(39-104) (the cytochrome c-T described by Harris & Offord, 1977) that exhibits physical properties similar to those of the intact protein but somewhat depressed redox potential and biological activity (Wallace, 1984a,b). Similar results have been obtained in studies on the formation and properties of the complex between the unmodified peptide fragments and (Parr et al, 1978;Westerhuis et al, 1979;Wallace, 1984a,b) The complexes formed between fragments of cytochrome c have been used in studies on the residues involved in the biological activity of the protein (Wallace, 1984a), in studies on the spectroscopic properties of the protein (Wallace, 1984b) and in investigations of the residues involved in cytochrome c conformation and folding (Parr et al, 1978;Juillerat & Taniuchi, 1982). The association of modified tryptic fragments has been used to prepare semisynthetic analogues of cytochrome c to investigate the role of residues at the site of cleavage on the formation and properties of peptide complexes (Harris & Offord, 1977;Proudfoot et al, 1986).…”

Section: Introductionsupporting

confidence: 63%

A new non-covalent complex of semisynthetically modified tryptic fragments of cytochrome c

Proudfoot¹,

Wallace²,

Harris³

et al. 1986

Biochemical Journal

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We have prepared a semisynthetic analogue of fully acetimidylated horse cytochrome c, a complex in which the peptide bond between residues glycine-37 and arginine-38 is lacking. In contrast with the complex that we have previously described [Harris & Offord (1977) Biochem. J. 161, 12-25], in which the break in continuity is between residues arginine-38 and lysine-39, the new analogue has a nearly normal redox potential, and can more fully restore succinate oxidation to mitochondria depleted of cytochrome c. Studies of this and other analogues lead us to propose an explanation for the low biological activity of complex (1-38)-(39-104) and a role for the invariance of arginine-38.

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Section: Introductionsupporting

confidence: 63%

A new non-covalent complex of semisynthetically modified tryptic fragments of cytochrome c

Proudfoot¹,

Wallace²,

Harris³

et al. 1986

Biochemical Journal

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“…The only small peptide generated in the first few minutes was Table 1. pK of the alkaline transition in cytochrome c and its analogues Values were determined as described by Wallace (1984b) Charge density (net charge divided by Mr) was plotted against the ionic strength necessary to elute it from a column (1 cm x 10 cm) of SP-Trisacryl (in phosphate/urea buffer, pH 7.0). The fragments studied are listed below.…”

Section: Resultsmentioning

confidence: 99%

On the relationship between oxidation-reduction potential and biological activity in cytochrome c analogues. Results from four novel two-fragment complexes

Wallace

Proudfoot

1987

Biochemical Journal

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We have confirmed the propensity of fragments of cytochrome c to form complexes that reproduce the structure and, in part, the functionality, of the native protein by preparing four novel complexes. We have used trypsin under three different sets of conditions in sequence to prepare a contiguous two-fragment complex (1-55).(56-104). One of the intermediates is a stable overlapping complex (1-65).(56-104). Conditions for limited acid hydrolysis of peptide bonds in cytochrome c have been developed that optimize the yield of fragments (1-50) and (51-104). These two fragments also form a stable association, as do (1-50) and (56-104). These complexes are potentially useful for the semisynthesis of analogues modified in the region of the cleavage sites, which include a number of highly conserved amino acid residues, and are being used for studies of protein folding, interactions with oxidase, cytochrome c immunogenicity and of artificially induced spontaneous resyntheses between complexing fragments. Like other known two-fragment complexes of cytochrome c, they exhibit normal visible spectra, including the presence of the 695 nm band, indicative of a functional haem crevice. Studies of their biological activities and redox potentials lead to a number of conclusions on structure-function relationships in cytochrome c. Most significantly there is a linear relationship between the logarithm of electron-transfer rates from cytochrome c reductase and redox potential in this series of analogues, indicating that such transfer is thermodynamically controlled. This discovery contributes to our understanding of the interaction of cytochrome and reductase. Since the relationship is obeyed by other types of analogues, except for those that involve modification of the active site of cytochrome c, we have a useful diagnostic for those residues that participate directly in electron transfer.

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“…Another conformer, the alkaline form, is known to differ from the native protein in heme ligation (Davis et al, 1974;Gupta & Koenig, 1971; Hettinger & Harbury, 1964;Shechter & Saludjian, 1967; Smith & Millett, 1980;Wallace, 1984; Wilgus & Stellwagen, 1974) but retains much of the secondary structure of the native state (Dohne et al, 1989). Comparisons of binding rates yield the result that the rate constants are the same for binding of one MAb1 to the oxidized or reduced native forms while a second MAb has a slightly smaller rate constant for binding reduced rather than oxidized cytochrome c. Similarly, the rate constant for association differs in the alkaline conformation for one epitope, but not the other.…”

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confidence: 99%

Diffusion-limited rates for monoclonal antibody binding to cytochrome c

Raman¹,

Jemmerson²,

Nall³

et al. 1992

Biochemistry

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The kinetic and spectroscopic changes accompanying the binding of two monoclonal antibodies to the oxidized form of horse heart cytochrome c have been investigated. The two epitopes recognized by the antibodies are distinct and noninteracting: antibody 2B5 binds to native cytochrome c near a type II turn (residue 44) while antibody 5F8 binds on the opposite face of the protein near the amino terminus of an alpha-helical segment (residue 60). Antibody-cytochrome c binding obeys a simple bimolecular reaction mechanism with second-order rate constants approaching those expected for diffusion-limited protein-protein interactions. The association rate constants have small activation enthalpies and are inversely dependent on solvent viscosity, as expected for diffusion-controlled reactions. There is a moderate ionic strength dependence of the rate of association between the 2B5 antibody and cytochrome c, with the rate constant increasing about 4-fold as the ionic strength is varied between 0.14 and 0 M. Comparison of the rates for antibody-cytochrome c complex formation for binding to the reduced-native, oxidized-native, and alkaline conformations shows that for MAb 2B5 the forward rate constant depends slightly on cytochrome c conformation. Investigation of the pH-induced transition between the native and alkaline conformational states for free cytochrome c and for antibody-cytochrome c complexes shows that antibody binding stabilizes the native form of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

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Modulation of the alkaline transition in cytochrome c and cytochrome c-T by full or specific partial acetimidylation

Cited by 21 publications

References 18 publications

A new non-covalent complex of semisynthetically modified tryptic fragments of cytochrome c

A new non-covalent complex of semisynthetically modified tryptic fragments of cytochrome c

On the relationship between oxidation-reduction potential and biological activity in cytochrome c analogues. Results from four novel two-fragment complexes

Diffusion-limited rates for monoclonal antibody binding to cytochrome c

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