Crystal structure of cytochrome c peroxidase compound I

We have compared the 2.5-A crystal structure of yeast cytochrome c peroxidase (CCP) with that of its semistable two-equivalent oxidized intermediate, compound I, by difference Fourier and least-squares refinement methods. Both structures were observed at -15 degrees C. The difference Fourier map reveals that formation of compound I causes only small positional adjustments of a few tenths of an angstrom. The map's most pronounced feature is a pair of positive and negative peaks bracketing the heme iron position. Least-squares refinement shows that the iron atom moves about 0.2 A toward the distal side of the heme. No significant difference density is evident near the side chains of Trp-51 or Met-172, each of which has been proposed to be the site of the electron paramagnetic resonance (EPR) active radical in compound I. However, the second most prominent feature of difference density is a negative peak near the side chain of Thr-180, which, according to the results of least-squares refinement, moves by 0.15 A in the direction of Met-230. These observations, together with the results of mutagenesis experiments [Fishel, L. A., Villafranca, J. E., Mauro, J. M., & Kraut, J. (1987) Biochemistry 26, 351-360; Goodin, D. B., Mauk, A. G., & Smith, M. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1295-1299] in which Trp-51 and Met-172 have been replaced without loss of the EPR radical signal in compound I, lead us to consider the possibility that the radical site lies within a cluster composed of the side chains of Met-230, Met-231, and Trp-191.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generation of H2O2 is regulated by cytoplasmic free calcium in cultured porcine thyroid cells

Effects of calcium ionophore A23187 and BAY-K-8644, a calcium channel agonist, on cytoplasmic free calcium ([Ca2+]i) and H2O2 generation were studied in cultured porcine thyroid cells. We monitored continuously the effects of A23187 and BAY-K-8644 on [Ca2+]i and H2O2 generation, using the intracellularly trapped fluorescent Ca2+ indicator, fura-2, and homovanillic acid, respectively. A23187 and BAY-K-8644 induce an immediate increase in [Ca2+]i and H2O2 generation. The A23187- and BAY-K-8644-induced [Ca2+]i responses and H2O2 generation occur immediately, reach a maximum within several seconds, and then slowly decline. The minimum doses of A23187 or BAY-K-8644 to increase [Ca2+]i stimulate H2O2 generation. H2O2 generation is regulated by [Ca2+]i.     

Depolarization induced by injection of cyclic nucleotides into frog taste cell

In order to identify the intracellular transmitter involved in the taste transduction process, cyclic nucleotides were iontophoretically injected into the frog taste cells while membrane potentials were recorded intracellularly. Injection of either cyclic GMP or cyclic AMP induced a depolarization response of about 5 mV in the taste cells, but injection of Cl- had no effect. The rate of a repolarization after the depolarization elicited by cyclic GMP was larger than that after cyclic AMP. The possible role of cyclic nucleotide in the taste transduction was discussed.     

Effects of some alcohols on the conformation of mitochondrial H+-ATPase complex and F1-ATPase from pig heart

The conformations of the H+-ATPase complex and F1-ATPase in low concentrations of methanol, ethanol, n-propanol, iso-propanol and t-butanol were studied by circular dichroism. For F1-ATPase, all but methanol first increased and then decreased the circular dichroism magnitude of helical bands as the alcohol concentration was increased. With ethanol, n-propanol, iso-propanol and t-butanol, the alpha-helix content reached a maximum at about 5% alcohol and began to decrease at 10%. The content of beta-sheet showed the opposite effect, reaching a minimum at 5% and increasing slightly at higher concentrations. None of the alcohols studied had a significant effect on the conformation of the H+-ATPase complex. This difference implies that the alcohols had a greater effect on free F1-ATPase than on the membrane-bound F1-ATPase. The hydrophobic protein F0 and the membrane lipids in the H+-ATPase complex may stabilize and protect F1 from the effects of the alcohols.     

Latrotoxin-induced fusion of liposomes with bilayer phospholipid membranes

Liposomes containing amphotericin B as ionophoric marker were used to investigate the fusion of bilayer phospholipid membranes with liposomes. It was found that latrotoxin isolated from black widow spider venom induced the fusion of liposomes with planar bilayer when liposomes and latrotoxin were administered at opposite sides of the membrane.     

Enhanced release and synthesis of lipoprotein lipase in rat heart cell cultures exposed to high concentrations of Hepes

While attempting to optimize conditions for synthesis of lipoprotein lipase by cultured heart cells, we encountered an unexpected rise in enzyme activity when media were supplemented inadvertently with 100 mM Hepes buffer (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid). This finding was further investigated and optimal results were obtained at pH 7.0-7.2. The increase in lipoprotein lipase activity was time dependent; after 3-6 h there was a rise in medium activity but cellular activity increased only after 24 h. The increased enzyme activity was defined as lipoprotein lipase by inhibition with antiserum to rat adipose tissue lipoprotein lipase. A 72-h exposure to Hepes resulted in a 30% increase in the incorporation of [35S]methionine into cellular proteins and a 2-fold increase into heparin-releasable proteins. Using heparin Sepharose chromatography and stepwise elution, a lipoprotein lipase enriched fraction was recovered with 2 M NaCl. The amount of [35S]methionine and [3H]galactose incorporated into protein of this fraction derived from Hepes-treated cells was 2-6-fold that of controls. A 4-fold increase in cellular lipoprotein lipase mass in Hepes-treated cells was shown by immunoblotting. Results obtained with Hepes-conditioned medium suggest the presence of cell-derived compounds that enhance release and subsequent synthesis of lipoprotein lipase. The effect of Hepes-conditioned medium on lipoprotein lipase resembled to some extent that of the addition of heparin. Therefore, it appears that when Hepes is first added to the culture medium, it might promote a release of heparan sulfate or related compounds, possibly by virtue of its negatively charged sulfonic acid residue. The accumulated heparan sulfate could then promote a sustained release of lipoprotein lipase into the culture medium which in turn leads to increased enzyme synthesis.     

Reduction in antiviral activity of human interferon-gamma in acidic media with reference to structural change

The fluorescence intensity of a unique tryptophan 36 in human interferon-gamma was drastically decreased below pH 4 with a concomitant decrease of antiviral activity. The region of residues 32-42 of human interferon-gamma was found by calculation to have a low hydrophobicity together with a high helical hydrophobic moment, and the net electric charge of this region having an amphiphilic helical structure changed significantly near pH 4. These results suggest that the region of residues 32-42 plays an important role in exhibiting antiviral activity.     

Conformation between the substrate-binding site and heme of cytochrome P-450 studied by excitation energy transfer

The conformation between the substrate-binding site and heme of cytochrome P-450 was studied by excitation energy transfer. Cytochrome P-450 was obtained from the hepatic microsomes of polychlorinated biphenyl-treated male rats, and ten polycyclic aromatic hydrocarbons were used as substrates. The energy transfer from the substrate to the heme of the enzyme was measured according to the F?rster equation. On the basis of the assumption that the substrates are bound at different positions in the plane of the same substrate-binding site, the position of the heme in relation to the substrate-binding site was determined in solution and in the presence of synthetic phospholipid. The results demonstrated that the distance between the substrate-binding site and the heme of cytochrome P-450 was greater when the enzyme was incorporated into micelles of phospholipid than when in solution, and that the conformational relationship of the substrate-binding site towards the heme was changed by an angle of 21 degrees on incorporation of the enzyme into phospholipid micelles.     

Existence of multiple forms of cytochrome P-450scc purified from bovine adrenocortical mitochondria

Three fractions of cytochrome P-450scc (denoted as fractions a, b, and c) were purified by a new procedure from bovine adrenocortical mitochondria. The amino-acid content analyses of these three fractions showed no difference. NH2-terminal amino-acid sequences of cytochrome P-450scc fractions, a and b agreed completely with the sequence deduced by nucleotide sequence of cDNA of cytochrome P-450scc mRNA (Morohashi, K., Fujii-Kuriyama, Y., Okada, Y., Sogawa, K., Hirose, T., Inayama, S. and Omura, T. (1984) Proc. Natl. Acad. Sci. USA 81, 4647-4651), whereas the sequence of fraction c showed a missing of isoleucine at the NH2-terminal. COOH-terminal ámino-acid sequences of fractions a, b and c were -Gln-Ala-COOH, identical with the deduced sequence from the cDNA. Measurements of the enzymatic activities of cholesterol side-chain cleavage reaction revealed no distinct difference among these three fractions. Although each of these fractions appeared as a single protein staining band upon SDS-polyacrylamide gel electrophoresis, these fractions showed heterogeneities upon two-dimensional electrophoresis and chromatofocusing. Fraction a contained the major form of cytochrome P-450scc, and its isoelectric point was estimated to be pH 7.8 by isoelectric focusing under both native and denatured conditions, and this value was confirmed by chromatofocusing. Neither of the carbohydrate-specific stainings (such as periodic acid-Schiff staining and lectin-peroxidase stainings using concanavalin A, wheat-germ agglutinin, and soybean agglutinin) of purified cytochrome P-450scc fractions after the electrophoretic resolution on SDS-polyacrylamide gel could show cytochrome P-450scc fractions as glycoproteins, suggesting that the heterogeneities were not due to the glycosylation state.     

Distribution of phospholipid molecular species containing arachidonic acid and cholesterol in V79-UF cells

V79-UF cells were isolated from Chinese hamster V79 cells as a cell line that requires exogenous unsaturated fatty acids for growth. V79-UF cells incorporated arachidonic acid into phospholipids. The molecular species of diacyl phosphatidylcholine and phosphatidylethanolamine containing arachidonic acid comprised 61.4 and 70.5% of the total phospholipid molecular species in total membranes and 58.1 and 64.7% in plasma membrane, respectively. Polyunsaturated molecular species were distributed in a higher amount in the intracellular membranes than in the plasma membrane. No significant difference was seen in the diffusion coefficient between the plasma membranes from cells supplemented with oleic and arachidonic acids in spite of a distinct difference in the degree of unsaturation between the molecular species of these plasma membranes. The amount of cholesterol in the plasma membrane was higher in the cells grown in the presence of arachidonic acid than in those grown in the presence of oleic acid.