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1.
The interactions of diethylpyrocarbonate (DEP) with the various forms of cytochrome b5 were studied to gain a better understanding of the factors that influence the extent of modification of the axial histidines of cytochrome b5. Very low concentrations of DEP were able to decrease the heme binding capacity of apocytochrome b5. Moreover, it was shown that two additional histidines, presumed to be the axial ligands (His 39 and 63), were modified in the apo but not the holo form of a given preparation of cytochrome b5. Trypsin-solubilized bovine cytochrome b5 was resistant to the effects of DEP. A 200-fold molar excess of DEP displaced only 15% of the heme in the trypsin-solubilized protein in contrast to an 84% displacement of the heme in the detergent-solubilized protein. However, detergent-solubilized cytochrome b5 which had been incorporated into phospholipid vesicles exhibited the same reactivity with DEP as did the trypsin-solubilized protein. This is attributed to the fact that the two resistant preparations of cytochrome b5 are monomeric in their respective environments while detergent-solubilized cytochrome b5 is known to exist as an octamer in aqueous solutions. Our studies suggest that dissociation of the octamer to the monomer results in a conformational change that decreases the reactivity of the axial ligands of the hydrophilic heme-containing domain of cytochrome b5. Examination of the cytochrome b5 molecule by computer graphics indicates that a tunnel leads from the surface of the molecule to axial histidine 63 and that axial histidine 39 is buried.  相似文献   

2.
E Amat  J Palou  F Garcia Blanco 《Enzyme》1985,34(2):83-93
The different effects induced by AMP and its analogues on the tertiary structure and the coenzyme environment of phosphorylase b were studied by titration of the slowly reacting thiol groups and by quenching of the coenzyme fluorescence, respectively, to determine the possible differences that activate phosphorylase b. The following results were obtained: The coenzyme environment depends upon the nucleotide studied. AMP, when bound to its first site, opens the coenzyme pocket. The slow cysteines were shielded by the nucleotides against their DTNB titration depending on the nucleotide studied. The enzyme difference spectra in presence of the nucleotide showed that the negative band of 260 nm is similar for all nucleotides possessing the same base, but the positive band obtained in the presence of AMP was diminished when other nucleotides were present.  相似文献   

3.
We present a fully automatic structural classification of supersecondary structure units, consisting of two hydrogen-bonded β strands, preceded or followed by an α helix. The classification is performed on the spatial arrangement of the secondary structure elements, irrespective of the length and conformation of the intervening loops. The similarity of the arrangements is estimated by a structure alignment procedure that uses as similarity measure the root mean square deviation of superimposed backbone atoms. Applied to a set of 141 well-resolved nonhomologous protein structures, the classification yields 11 families of recurrent arrangements. In addition, fragments that are structurally intermediate between the families are found; they reveal the continuity of the classification. The analysis of the families shows that the α helix and β hairpin axes can adopt virtually all relative orientations, with, however, some preferable orientations; moreover, according to the orientation, preferences in the left/right handedness of the α–β connection are observed. These preferences can be explained by favorable side by side packing of the α helix and the β hairpin, local interactions in the region of the α–β connection or stabilizing environments in the parent protein. Furthermore, fold recognition procedures and structure prediction algorithms coupled to database-derived potentials suggest that the preferable nature of these arrangements does not imply their intrinsic stability. They usually accommodate a large number of sequences, of which only a subset is predicted to stabilize the motif. The motifs predicted as stable could correspond to nuclei formed at the very beginning of the folding process. Proteins 30:193–212, 1998. © 1998 Wiley-Liss, Inc.  相似文献   

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The interaction of two Burkitt lymphoma lines, Raji and Rael, with human C and NK cells was analyzed. Raji cells activate the alternative C pathway (ACP) and then bind C3 fragments. Consequently, the cells become more sensitive to lysis by CR3-bearing NK cells but not to C lysis. In contrast, Rael cells are poor ACP activators, do not bind C3 fragments, and are therefore resistant to C-dependent NK lysis. As suggested earlier, the difference between Raji and Rael could be attributed to the presence or absence of CR2, respectively, on their surface. To potentiate C- and NK-dependent lysis of target cells, we generated heteroconjugates composed of a murine antitransferrin receptor mAb and of human C C3b or iC3b. Antibody-C3b conjugates induced C3 deposition on Rael cells and elevated C3 deposition on Raji cells in human serum. Both Raji and Rael cells coated with antibody-C3b conjugates were efficiently lyzed by the cytolytic ACP in human serum. This conjugate had a small enhancing effect on target cell lysis by NK cells which could be markedly increased by combined treatment of the target cell with antibody-C3b conjugate and C5-depleted human serum. On the other hand, antibody-iC3b conjugates efficiently potentiated lysis of target cells by NK cells in the absence of serum. The iC3b-directed cytotoxicity was mediated by CR3-bearing NK effector cells. Anti-C3 but not anti-mouse Ig antibodies abrogated the activity of the antibody-iC3b conjugate. These results further demonstrate that NK cytotoxicity may be potentiated by opsonizing the target cells with C3 fragments and suggest that antibody-C3b/iC3b conjugates could be potent tools for targeting and potentiation of the lytic action of both C and NK cells against tumor cells.  相似文献   

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Cytochrome b of isolated chloroplasts   总被引:3,自引:0,他引:3  
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17.
Rabbit skeletal muscle phosphorylase b was separated into two fractions by column chromatography on AMP-Sepharose. The first fraction protein was eluted by glucose-6-phosphate while the second fraction protein was eluted in an AMP concentration gradient. The bulk of the protein eluate was represented by the first fraction protein. Chromatography of phosphorylase b from bovine skeletal muscle under identical conditions also resulted in two fractions, however, with a reverse correlation: the bulk protein of this fraction was eluted by AMP. It was shown that the two phosphorylase b forms eluted by glucose-6-phosphate and AMP differ by their kinetic and physico-chemical properties as well as by the SH-group reactivity. The phosphorylase b forms eluted by the nucleotide were practically uninhibited by glucose-6-phosphate. It can thus be assumed that the equilibrium between the "active" (R) and "inactive" (T) conformations of the protein changes depending on metabolic peculiarities of a given tissue used as a source for enzyme isolation.  相似文献   

18.
The crystal state conformations of three peptides containing the α,α-dialkylated residues. α,α-di-n-propylglycine (Dpg) and α,α-di-n-butylglycine (Dbg), have been established by x-ray diffraction. Boc-Ala-Dpg-Alu-OMe (I) and Boc-Ala-Dbg-Ala-OMe (III) adopt distorted type II β-turn conformations with Ala (1) and Dpg/Dbg (2) as the corner residues. In both peptides the conformational angles at the Dxg residue (I: ? = 66.2°, ψ = 19.3°; III: ? = 66.5°. ψ = 21.1°) deviate appreciably from ideal values for the i + 2 residue in a type II β-turn. In both peptides the observed (N…O) distances between the Boc CO and Ala (3) NH groups are far too long (1: 3.44 Å: III: 3.63 Å) for an intramolecular 4 → 1 hydrogen bond. Boc-Ala-Dpg-Ata-NHMe (II) crystallizes with two independent molecules in the asymmetric unit. Both molecules HA and HB adopt consecutive β-turn (type III-III in HA and type III-I in IIB) or incipient 310-helical structures, stabilized by two intramolecular 4 → 1 hydrogen bonds. In all four molecules the bond angle N-Cα-C′ (τ) at the Dxg residues are ≥ 110°. The observation of conformational angles in the helical region of ?,ψ space at these residues is consistent with theoretical predictions. © 1995 John Wiley & Sons, Inc.  相似文献   

19.
Reduction kinetics of cytochromes b   总被引:2,自引:0,他引:2  
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