Nitrobacter agilis Cytochrome c-550: Isolation, Physicochemical and Enzymatic Properties, and Primary Structure

Nitrobacter agilis cytochrome c-550 was purified to an electrophoreticallyhomogeneous state, and some of its properties were determined.The cytochrome showed an absorption peak at 410 nm in the oxidizedform, and peaks at 416, 521 and 550 nm in the reduced form.Its isoelectric point was 8.1 at 5?C. Analysis of the aminoacid composition showed that the cytochrome molecule was composedof 108 amino acid residues, 16 of which were lysine residues. The cytochrome reacted rapidly with N. agilis cytochrome c oxidaseand yeast cytochrome c peroxidase and more slowly with Pseudomonasaeruginosa nitrite reductase and bovine cytochrome c oxidase.The reactivities with these redox enzymes suggested that thecytochrome might be an evolutionary stage between bacterialand eukaryotic cytochromes c. The primary structure of the cytochrome from the N-terminusto the 85th residue was determined. The N-terminal sequencewas homologous to the corresponding portion of the primary structureof horse cytochrome c. ¹ Present adress: Department of Chemistry, Faculty of Science,Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo,152, Japan. (Received December 3, 1981; Accepted January 28, 1982)     

Effect of disulfiram on turnover of 5-hydroxytryptamine in rat brain.

Effect of disulfiram on 5-hydroxytryptamine (5-HT) turnover was studied. Treatment with disulfiram caused increases in 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in rat brain. Under the same condition, activity of brain mitochondrial aldehyde dehydrogenase was reduced, however, supernatant aldehyde dehydrogenase and monoamine oxidase activities remained unchanged. Disulfiram had no effect on synthesis rate of 5-HT, but decreased metabolism of 5-HT. Moreover, disulfiram impaired transport of 5-HIAA from brain tissue.     

Nitrogen fixation by immobilized Azotobacter chroococcum

A nitrogen-fixing bacterium, Azotobacter chroococcum, was immobilized in 2% agar gel. The optimum partial oxygen pressure, pO₂, of immobilized cells was 0.2 atm, wherea s that of native cells was 0.05 atm. When continual nitrogen fixation was performed under aerobic conditions, the nitrogenase activity of immobilized cells increased with increasing time. On the other hand, the activity of native cells decreased rapidly. Increase of nitrogenase activity was attributed to growth of the bacteria in the gel matrix. The production rate of total nitrogen compounds by the immobilized bacteria was also increased during the first 4 days. Nitrogen compounds produced by the immobilized cells were mainly amino acids such as γ-aminobutyrate, glutamate and arginine.     

Circadian alterations in tubular structures on the outer mitochondrial membrane of rat hepatocytes

Summary The ontogeny and distribution of immunoreactive motilin and secretin were studied in the gastro-entero-pancreatic (GEP) system of human fetuses, aged 5–24 weeks, using an indirect immunocytochemical method. Several controls to check for the specificity of the immunoperoxidase staining were performed. The first motilin- and secretin-containing cells were observed in the duodenal and jejunal mucosa in fetuses at a gestational age of 16 weeks. These immunoreactive cells were located in the glands of Lieberkühn and in the villi. No immunoreactive cells were present in the oxyntic and pyloric mucosa, ileum, colon and endocrine pancreas. These observations indicate that the motilin- and secretin-containing cells detected by our antisera appear (i) in the same organs of the fetus where they are also detectable in the adult, and (ii) after the completion of histogenesis of the gastro-entero-pancreatic (GEP) system.     

Effects of purine nucleotides on photosynthetic electron transport in isolated chloroplasts

The effects of guanylates and inosinates (and adenylates) on phosphorylation, ferricyanide reduction, and light-induced H⁺ uptake in spinach chloroplasts were studied. GDP, GTP, IDP, and ITP (but not GMP and IMP) stimulated the light-induced H⁺ uptake and partially inhibited ferricyanide reduction. Phosphate, arsenate, and phlorizin increased the extent of inhibition by these nucleotides and decreased the values of their apparent dissociation constants for the inhibition process. In the presence of phosphate (or arsenate), restoration of ferricyanide reduction from the level inhibited by guanylates and inosinates was observed as phosphorylation (or arsenylation) proceeded. These results suggest that phosphorylation of GDP and IDP as well as ADP takes place after two steps of nucleotide binding to the chloroplast coupling factor 1. The apparent dissociation constants of GDP and IDP for these two binding steps were estimated to be about 34 and 38 µM for the first and 110 and 160 µM for the second step, respectively (at pH 8.3, 15°C). Above pH 9, the ratio (P/e) of the extent of phosphorylation to the increment of electron transport from the basal level measured in the presence of [ATP + Pi] or [ADP + Pi + phlorizin], became increasingly large. When the electron transport level inhibited by dicyclohexylcarbodiimide was taken to be the basal activity, the P/e ratio remained almost constant ( 1) from pH 7.0 up to 10.     

Construction and characterization of the chimeric enzymes between the Bacillus subtilis cellulase and an alkalophilic Bacillus cellulase

The amino acid sequences of cellulase from Bacillus subtilis (BSC) and that from an alkalophilic Bacillus sp. N-4 (NK1) show significant homology in most parts except for the C-terminal portions. Despite the high homology, the pH activity profiles of the two enzymes are quite different; BSC has its optimum pH at 6-6.5, whereas NK1 is active over a broad pH range from 6 to 10.5. In order to identify the structural features which determine such pH activity profiles, chimeric cellulases between BSC and NK1 were constructed using four restriction sites commonly present within the homologous coding sequences, and were produced in Escherichia coli. The chimeric cellulases showed various chromatographic behaviors, reflecting the origins of their C-terminal regions. The pH activity profiles of the chimeric enzymes in the alkaline range could be classified into either the BSC or NK1 type mainly depending on the origins of the fifth C-terminal regions. In the acidic range, the profile was determined only by the origin of the fourth enzyme region from the N terminus. Comparison of the kinetic parameters between pH 5 and 6 using p-nitrophenyl cellobioside as a substrate indicated that the fourth region is responsible for the pH-dependent change of the kcat value. Only a limited number of amino acids in the fourth region may affect on deprotonation of catalytic residues of the cellulases and modulate the catalytic activity in the acidic pH values.     

A novel terminal oxidase, cytochrome baa3 purified from aerobically grown Pseudomonas aeruginosa: it shows a clear difference between resting state and pulsed state.

A novel type of cytochrome c oxidase was purified to homogeneity from Pseudomonas aeruginosa which was grown aerobically. The purified oxidase contained two molecules of heme a, two atoms of copper, and one molecule of protoheme per molecule. One of the two heme a molecules in the oxidase reacted with carbon monoxide, so that the enzyme was of baa3-type. The oxidase molecule was composed of three subunits with molecular weights of 38,000, 57,000, and 82,000. Although the oxidase oxidized ferrocytochrome c-550 obtained from the bacterial cells grown aerobically, the oxidizing activity was not high. The "resting form" and the "pulsed form" of the oxidase were observed clearly with this enzyme, and the transition from the resting form to the pulsed form was accompanied by a distinct change of the enzymatic activity. The difference in the kinetics of the catalytic reactions between the two forms is discussed.     

Thiobacillus novellus cytochrome c oxidase contains one heme alpha molecule and one copper atom per catalytic unit.

The minimal structural unit of cytochrome c oxidase purified from Thiobacillus novellus was composed of one molecule each of two subunits with molecular masses of 32 and 23 kDa, respectively, and the unit had one molecule of heme a and one atom of copper. In the presence of n-octyl-beta-D-thioglucoside, the oxidase existed as the monomeric form of the unit, while it occurred as the dimeric form of the unit in the presence of Tween 20. The monomeric form showed an active cytochrome c oxidizing activity and reduced molecular oxygen to water with ferrocytochrome c. Namely, it has been shown that the bacterial cytochrome c oxidase with one heme a molecule and one copper atom per molecule can catalyze oxidation of ferrocytochrome c with concomitant reduction of molecular oxygen to water.     

Parturition in a free-ranging Japanese monkey (Macaca fuscata)

Parturition behavior of a multiparous female and her interactions with group members throughout the birth process were recorded for a free-ranging Japanese monkey (Macaca fuscata). The female showed evidence of 18 contractions during the 35 min prior to delivery, with a mean duration and a mean intercontraction interval of 30 sec and 96 sec, respectively. These values were similar to those in individually caged Japanese monkeys. Some adult females remained in proximity to the female who was giving birth during the prepartum phase, and her 2-year-old daughter watched the delivery of the infant. Even during the prepartum phase the female moved in order to keep up with the group which traveled from the feeding site to a sleeping site in the forest.