Involvement of the Binuclear Copper Site in the Proteolytic Activity of Polyphenol Oxidase

Plant polyphenol oxidase (PPO) is apt to degrade during andeven after purification. We developed a method to stabilizePPO by 0.3 M NaCl, 0.1% (w/v) Tween 20, and 50% (w/v) ethyleneglycol at pH 6.5. The protein slowly degraded by itself whenthe stabilizing reagents were removed. Ascorbate and/or H₂O₂accelerated the degradation. The ascorbate-induced degradationwas inhibited by catalase, suggesting that H₂O₂ is generatedthrough reduction of PPO by ascorbate. It is likely that dissolvedoxygen is converted to peroxide through two-electron reductionby the reaction center of PPO, binuclear Cu site, and a Fenton-typereaction occurred on it. This understanding was supported bythe finding that the H₂O₂-induced degradation was inhibitedby metal-chelators as well as by polyphenolic substrate of PPO.Considering the postulated mechanism of the self-degradationof PPO, we re-examined the degradation of the 23-kDa proteinof PSII by PPO [Kuwabara et al. (1997) Plant Cell Physiol. 38:179]. The obtained results suggested that the 23-kDa proteintriggers the active oxygen production by the binuclear Cu site,probably as reductant, and receives the radical species preferentiallyto the polypeptide moiety of PPO. (Received April 15, 1999; Accepted July 21, 1999)     

The effects of deuterium oxide (2H2O) on the polymerization of tubulin in vitro

The initial rate and final extent of polymerization of both bovine brain tubulin and sea urchin egg tubulin were enhanced in the presence of ²H₂O. The yields were increased in association with the elevation of the ²H₂O concentration. ²H₂O also reduced the critical concentration for polymerization of brain tubulin. Thermodynamic analysis was attempted using the temperature dependence of the critical concentration for polymerization in the presence of ²H₂O. We obtained linear van 't Hoff plots and calculated thermodynamic parameters which were positive and were increased with the elevation of the ²H₂O concentration. The enhancement of the polymerization of tubulin by ²H₂O could, therefore, be the result of the strenghening of intra-and/or inter-molecular hydrophobic interactions of the tubulin molecules. We believe that the increase in lenghth and number of microtubules of the mitotic spindles in the dividing cells of the eukaryotes with ²H₂O may be caused by the direct involvement of ²H₂O in the polymerization of tubulin.     

Partial purification and characterization of taurodeoxycholate 7α-monooxygenase

Taurodeoxycholate 7α-monooxygenase was partially purified from rat liver microsomes. The enzyme was solubilized with cholate, fractionated with polyethylene glycol and chromatographed on a Sepharose 4B column with cholate as ligand. The enzyme activity was eluted from the column into the fraction eluted with 50 mM phosphate buffer containing cholate and KCl, whereas the benzphetamine demethylase activity was eluted in the non-bound fraction. Thus it was established that both enzymes are different entities. The taurodeoxycholate 7α-monooxygenase activity was reconstituted from the partially purified cytochrome P-450, highly purified NADPH-cytochrome P-450 reductase, dilauroylglyceryl-3-phosphorylcholine and NADPH.     

Commitment to Ploidy Conversion of 3y1 Cells During Metaphase Arrest By Colcemid

Abstract. Diploid rat 3Y1 fibroblasts proliferate to a saturation density, where they are arrested with a 2N DNA content. After treatment to induce ploidy conversion, the conversion rate can be estimated by determining the fraction of cells with a 4N DNA content in the confluent culture using flow cytometry. Using this method it was found that during mitotic inhibition with colcemid, 3Y1 cells were converted to tetraploids with a high efficiency (above 80%); the optimum colcemid concentration and exposure period were 40 ng/ml and 8 hr, respectively. When metaphase cells were reseeded with 40 ng/ml of colcemid, they delayed anchorage to a dish; 6 hr was required for complete adhesion (in the absence of colcemid only 1 hr was required). When reseeded metaphase cells were exposed to 40 ng/ml of colcemid for 5 hr followed by its removal, a greater fraction of the cells anchored to the substratum were converted to tetraploids, whereas most of the floating cells were not. A greater fraction of the anchored cells had formed nuclei, whereas most of the floating cells preserved condensed metaphase chromosomes. These results indicate that the cells which have formed nuclear structure without chromosome separation during mitotic inhibition are irreversibly committed to ploidy conversion, with restoration of anchorage.     

Effects of winter flooding on phosphorus dynamics in rice fields

Limnology - Controlling phosphorous (P) loads from rice fields is important for the conservation of aquatic ecosystems, in part because P is relatively concentrated at its sources. Recently, winter...     

Structural characterization and dynamics of globotetraosylceramide in vascular endothelial cells under TNF-α stimulation

In several vascular inflammatory reactions (i.e. immunity and thrombosis) inflammatory mediators lead to the activation of vascular endothelial cells (EC). To date, a number of functional molecules induced on the surface of activated-EC have been identified. We report here that Globotetraosylceramide (Gb4), a glycosphingolipid expressed in EC, is a novel inducible molecule on EC activated by TNF-α. The cell surface expression of Gb4 is increased in a time-dependent manner under TNF-α stimulation, which shows distinct expression kinetics of major proteins induced by TNF-α on EC. MALDI-TOF-MS analysis revealed that the enhanced Gb4 predominantly contains C24:0 fatty acid in the ceramide moiety. Isolated caveolae/lipid raft-enriched detergent insoluble membrane domains in activated-EC predominantly contain this molecular species of Gb4. Gb4 containing C16:0 fatty acid in the ceramide moiety, which is known to constitute the major species of Gb4 in plasma, is also found as a major molecular species in EC. These observations indicate that Gb4, especially with very long fatty acid, is enhanced in EC during its inflammatory reaction, and suggest the potential utility of Gb4 as a biomarker for monitoring inflammation status of EC involving its related diseases.     

Seasonal pattern of filial cannibalism by Apogon doederleini mouthbrooding males

In temperate waters of southern Japan, mouthbrooding males of the cardinal fish Apogon doederleini sometimes ate their entire brood within a day of spawning. In spite of annual variation in age structure and length of the breeding season, however, the annual cannibalism rate (broods cannibalized to total broods spawned) was nearly constant, 12–16%. Fish 1 year old frequently cannibalized early broods of the season, especially the first brood, whereas the cannibalism rate by 2 year-old fish did not vary within the season. In contrast, the cannibalism by older males increased late in the season. This tendency was attributed to their different allocation patterns to growth and reproduction. For 1 year-old fish, which can enhance future reproductive success by growth and cannibalizing on early broods instead of mouthbrooding (at relatively low temperatures), this may be a tactic for investing energy in growth. For fish 3 years, which have more breeding cycles and grow little, cannibalizing late broods may be energetic compensation for their poor somatic condition. No seasonal variation of cannibalism by 2 year-old fish can be explained by their intermediate nature of growth and reproduction. Multiple mate availability is one factor facilitating the cannibalism by 2 and 3 + year-old fish whose operational sex ratio is female-biased.     

Modification in Cell Shape Unrelated to Cellulose Microfibril Orientation in Growing Thallus Cells of Chaetomorpha moniligera

Cellulose microfibril orientation patterns in thallus cellsof Chaetomorpha moniligera were studied, and the relationshipbetween the microfibril and the peripheral microtubule arrangementsduring cell-shape modification by colchicine was examined. Inthe cuttings from growing thalli, linearly arranged cylindricalcells developed into cask-shaped cells during 4–6 daysof culture at 27?C. In the cylindrical cells, microfibrils formingthe innermost portion of the wall were arranged alternatelyin longitudinal and transverse directions, but peripheral microtubuleswere always arranged only in a longitudinal direction. Thesefeatures were also noted in the cask-shaped cells. Colchicineat 10^–3M and 3?10^–3M accelerated both cell expansionand wall thickening with matrix deposition, but the directionsin which both microfibrils and microtubules were arranged werethe same as those of the cylindrical cells. These results indicatethat (1) the microfibril and microtubule arrangements of Chaetomorphaare not necessarily correlated, (2) changes in cell shape ofChaetomorpha are not necessarily accompanied by changes in thearrangement of cell-wall microfibrils, and (3) colchicine playsa role in the loosening and thickening of cell walls by enhancingmatrix deposition. (Received June 2, 1986; Accepted February 13, 1987)     

Purification and characterization of cholesterol 7 alpha-hydroxylase from rat liver microsomes

Cholesterol 7 alpha-hydroxylase (cholesterol, NADPH: oxygen oxidoreductase, 7 alpha-hydroxylating, EC 1.14.13.17) was purified from liver microsomes of cholestryramine-fed male rats by using high-performance ion-exchange chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,000), and its dithionite-reduced CO complex exhibited an absorption maximum at 450 nm. The specific content of the enzyme was 9 nmol of cytochrome P-450/mg of protein. Upon reconstitution with NADPH-cytochrome P-450 reductase, the enzyme showed a high activity of cholesterol 7 alpha-hydroxylation with the turnover number of 50 min-1 at 37 degrees C. The reaction was inhibited neither by aminoglutethimide nor by metyrapone, but inhibited markedly by iodoacetamide and disulfiram. The reaction was also inhibited significantly by CO. The enzyme catalyzed hydroxylation of cholesterol with strict regio- and stereoselectivity and was inert toward other sterols which are intermediates in the conversion of cholesterol to bile acids, i.e. 7 alpha-hydroxy-4-cholesten-3-one (12 alpha-hydroxylation), 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol (25-hydroxylation), and taurodeoxycholate (7 alpha-hydroxylation). Unlike other cytochromes P-450 isolated from rat liver microsomes, the enzyme showed no activity toward testosterone and xenobiotics such as 7-ethoxycoumarin and benzo[a] pyrene. The NH2-terminal amino acid sequence of the enzyme was Met-Phe-Glu-Val(Ile)-Ser-Leu-, which was distinct from those of any other cytochromes P-450 of rat liver microsomes hitherto reported. These results indicate that the enzyme is a novel species of cytochrome P-450 so far not isolated from liver microsomes.