Gas-tissue autoradiography using [15O] molecular oxygen for visualization of mitochondrial oxygen fixation in brain slices.

We have developed a novel in vitro autoradiographic method of visualizing oxygen fixation using [15O]O(2). Brain slices (400 microm) are preincubated in Krebs-Ringer phosphate buffer and exposed to [15O]O(2) with carrier O(2) for 10 min on slide glass or on a Millicell-CM membrane in a chamber. After the 15O(2) exposure, the brain slices are placed in contact with an imaging plate. The autoradiograms are analyzed with MacBas to obtain images of the distribution of radioactivity. The [15O]O(2) fixation was more extensive in the gray matter (striatum, thalamus and cerebral cortex) than in the white matter. The fixation of [15O]O(2) in brain slices decreased dose dependently with NaCN concentration. These results indicate that [15O]O(2) is converted into [15O]H(2)O in the brain tissue according to the activity of mitochondrial electron transfer and remains in the tissue. This method provides information regarding the basic oxygen consumption of brain slices of rat and mouse under conditions of sufficient O(2) delivery, which reflects mitochondrial electron transport.

Efficient HPLC separation of [11C]beta-CFT or [11C]beta-CIT from an N-desmethyl precursor on a semipreparative reversed phase ODS column.

HPLC separation of either [11C]beta-CFT or [11C]beta-CIT from the N-desmethyl precursor greatly depended on the ODS column used and the pH of the phosphate buffer in the mobile phase. The separation was accomplished on the semipreparative reversed phase ODS column without end-capping treatment over a pH ranging from 6.4 to 9.2, but failed on the end-capped ODS column. This suggests that the presence of residual silanol groups on the ODS is an important factor for the separation.

Specific cleavage at fibers of a bacteriophage-tail-like bacteriocin, pyocin R1, by successive treatment with organomercurial compounds and trypsin.

When pretreated with the organomercurial compound that reversibly blocks the adhesion of pyocin R1 to sensitive cells, this bacteriocin became susceptible to trypsin. Trypsinization resulted in the exclusive fragmentation of subunit protein no. 2 among 20 different subunit components and the disappearance of distal knobs from the fibers as well as in irreversible inactivation.

Bacteriolytic enzyme induced from pyocinogenic Pseudomonas aeruginosa. Purification and characterization of PR1-lysozyme.

A bacteriolytic enzyme, PR1-lysozyme, has been purified from the lysate of mitomycin C-induced pyocinogenic Pseudomonas aeruginosa, by acrinol treatment, Amberlite CG-50 chromatography, ammonium sulfate fractionation, Sephadex G-100 gel filtration and two cycles of SP-Sephadex C-50 chromatography. Homogeneity of the preparation was demonstrated by three electrophoretic techniques. PR1-lysozyme is a basic protein (pI, 9.4) and consists of a single polypeptide chain having a molecular weight of 24,000. The amino acid composition of the protein was analyzed, and no cystein residue was found among more than 210 amino acid residues. The optimum pH for enzymatic activity was 6.4 and the enzyme exhibited about 50 to 70 times greater specific activity than hen egg-white lysozyme when assayed with chloroform-killed P. aeruginosa as a substrate. By analyzing the products of enzymatic action on purified peptidoglycan of P. aeruginosa, the enzyme was identified as an N-acetylmuramidase, i.e., the same classification as hen-egg-white lysozyme. PR1-lysozyme did not show any activity towards intact cells of gram-positive and gram-negative bacteria tested. However, the enzyme was able to lyse chloroform-killed gram-negative and gram-positive bacteria.

Affinity chromatography of trypsin and related enzymes. IV. Quantitative comparison of affinity adsorbents containing various arginine peptides.

In order to study the mechanism of substrate binding of trypsin by affinity chromatography, we synthesized various L-arginine-terminated oligopeptides having different chain length and amino acid sequences, and immobilized them on agarose gel. The interaction of beta-trypsin with these adsorbents was studied by a quantitative affinity chromatographic procedure which gave the dissociation constant (Kd) of the trypsin-immobilized ligand complex. This procedure proved to be very useful and to give information equivalent to that obtained by kinetic procedures. The contribution of the amino acid residue at P2 of the ligands to the affinity was studied by using tripeptide (Gly-X-Arg) Sepharoses, and alanine was found to be more effective than glycine or valine. This conclusion was supported by a kinetic experiment in which Ki values of the corresponding soluble tripeptides (Ac-Gly-X-Arg) were determined. A significant decrease in Kd was observed when the ligand was elongated from dipeptide to tripeptide. However, Kd decreased only slightly when the ligand was elongated further. This suggests that a tripeptide is sufficiently long as a ligand. On the basis of these results, the mode of substrate binding of trypsin is discussed.

Comparison of the catalytic properties of thrombin and trypsin by kinetic analysis on the basis of active enzyme concentration.

The interaction of bovine thrombin [EC 3.4.21.5] with synthetic substrates and products was studied. The enzyme was purified from Parke-Davis topical thrombin. The purification process afforded some preparations with different clottin specific activities but with similar esterase specific activities. The preparation having highest clotting specific activity and that having lowest clotting activity were tentatively named thrombin-C and thrombin-E, respectively. Kinetic parameters for the hydrolysis of synthetic substrates and normality titrants were determined on the basis of active enzyme quantity, which was assayed by means of a fluorometric normality titrant. It was shown that thrombin-E was acylated by the substrates more slowly than thrombin-C, while deacylation proceeded at similar rates in the two preparations. The results were also compared with those obtained with bovine trypsin [EC 3.4.21.4]. The acylation rates of both thrombin preparations were markedly lower than that of trypsin, while the deacylation rates of the former were only slightly lower than that of the latter. The effects of various product-type inhibitors, such as benzyloxycarbonyl-, benzoyl-, and tosyl-L-arginine, were also examined. Thrombin was affected by these inhibitors not competitively, though trypsin was inhibited competitively.