No abstract

Ferricytochrome c binds imidazole and 1-methylimidazole in reactions with 1:1 stoichiometry. The pH and temperature dependence of imidazole binding indicate that the reaction is analogous to the binding of the enzyme by ligands such as cyanide and azide. At pH 7.4 and 18°the observed thermodynamic parameters for imidazole binding are: AF = -1.9 kcal/mole, AH = +1.1 kcal/mole, and AS = +10.2 eu. The

Proteinase 3 (PR3), the target autoantigen of antineutrophil cytoplasmic antibodies in the autoimmune vasculitis, Wegener's granulomatosis, is a serine proteinase stored in granules of human neutrophils. As previously shown, PR3 is expressed also on the plasma membrane of unactivated neutrophils, and this expression increases in primed or stimulated cells. The current study demonstrates that membrane-bound PR3 colocalizes with the adhesion molecule CD11b/CD18 (beta2 integrin). Immunoprecipitation experiments using plasma membranes of phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils revealed coimmunoprecipitation of PR3 with CD11b/CD18, indicating their location in the same complex. PR3 was also detected in TritonX-100-insoluble cytoskeleton of plasma membranes isolated from unactivated and activated neutrophils. Release of cytoskeletal PR3 by salt treatment implied electrostatic interaction with the enzyme. The serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) augmented membrane expression of PR3 in unactivated and PMA-stimulated neutrophils. PMSF significantly reduced adhesion of neutrophils to fibrinogen-coated plates and their NADPH oxidase activity. Moreover, the addition of exogenous PR3 (1-5 microg/ml) augmented the CD11b/CD18-dependent adhesion of neutrophils. Taken together, these results implicate the beta2 integrin of neutrophils in their membrane association with PR3 and suggest a role of PR3 in the modulation of cell adhesion.

Cytochrome c was nitrated with tetranitromethane at pH 8.0.A modified enzyme nitrated specifically at the tyrosyl residue in position 67 was purified. This nitrocytochrome c was found Q kjpecific chemical modifications of amino acid side chains are often used to establish whether particular amino acid residues in a protein are required for its biological activity. Experimentally, tyrosine is one of the most accessible residues of proteins (Vallee and . Horse heart cytochrome c contains four tyrosyl residues. Their ionizations were studied by spectropolarimetry (Ulmer, 1966, and references cited therein), and their reactivities by acetylation reactions. The present communication reports the preparation using tetranitromethane, and the enzymic activity of nitrotyro sy 1-67-cy to chrome c. The accompanying paper describes the physicochemical properties of this modified enzyme (Schejter et al., 1970). A preliminary report has been given (Schejter and Sokolovsky, 1969) and similar studies were reported also by Skov etal. (1969). MaterialsA crystalline preparation of cytochrome c from horse heart was a gift of Dr. E. Margoliash. Commercial horse heart cytochrome c, type II, was obtained from the Sigma Chemical Co., and purified on Amberlite CG-50 (Margoliash and Walasek, 1967). Tetranitromethane was obtained from Fluka AG; -chymotrypsin, three-times crystallized, from Worthington; and Dowex AG 50-X2 (200-400 mesh) was obtained from Bio-Rad. All other chemicals were of the best grade available. MethodsConcentrations of native cytochrome c were determined by the absorbance of the reduced form at 550 m/i using a molar

Cytochrome c-557 from Crithidia oncopelti and cytochrome c-558 from Euglena gracilis are mitochondrial cytochromes c that have an atypical haem-binding site. It was of interest to know whether the loss of one thioether bond affected the physicochemical properties of these cytochromes. The thermodynamic parameters of the redox potential were measured. The reaction with imidazole, the kinetics and thermodynamics of the alkaline isomerization and the effect of heating on the visible spectrum are described for the ferricytochromes. The kinetics of the loss of cyanide, the spectral changes occurring on reduction with dithionite at alkaline pH values and the reactivity with CO are described for the ferrocytochromes. In many respects the cytochromes of the two protozoans are very similar to the cytochromes of horse and yeast. The ferricytochromes do, however, undergo a reversible transition to high-spin species on heating, which may be due to the more flexible attachment of the prosthetic group. Similarly the alkaline isomers of cytochromes c-557 and c-558 give rise to high-spin proteins above pH 11. The alkaline isomerization of cytochrome c-558, involves a pKobs. of 10 and kinetics which do not obey the model of Davis et al. [(1974) J. Biol. Chem. 249, 2624-2632] for horse cytochrome c. It is proposed that a model involving two ionizations, followed by a conformation change, may fit the data. Both cytochromes c-557 and c-558 combine slowly with CO at neutral pH values.