Effects of Science Classes Promoting Active Learning on Junior High School Students’ Motivation for Experimentation and their Experimental Strategies : Focusing on Gender Differences

This study practically examined, with a focus on gender differences, the effects of science classes promoting active learning on junior high school students’ motivation for experimentation and experimental strategies. First, we designed a science lesson that incorporates active learning by using the “Force and Effect” unit of junior high school science as a case study. We then conducted the lesson with 57 first-year students at a public junior high school. The results suggested that among boys, the use of the understanding-oriented strategy and the repeat strategy was encouraged. Furthermore, the understanding-oriented strategy was encouraged more among boys when there was increased utility value.本研究は，平成27～31年度科学研究費補助金（基盤研究C：研究代表者：草場実）（課題番号15K04448，研究課題「メタ認知能力を基盤とした科学的思考力育成のための理科学習指導法の開発」）により行った

Intermediate and mechanism of hydroxylation of o-iodophenol by salicylate hydroxylase

金沢大学自然科学研究科　　金沢大学理工研究域自然システム学系Salicylate hydroxylase [EC 1.14.13.1] from Pseudomonas putida catalyzes the hydroxylation of salicylate, and also o-aminophenol, o-nitrophenol, and o-halogenophenols, to catechol. The reactions with these o-substituted phenols comprise oxygenative deamination, denitration, and dehalogenation, respectively. The reaction try, as to NADH oxidized, oxygen consumed, and catechol formed, is 2 : 1 : 1, respectively. The mechanisms for the deiodination and oxygenation of o-iodophenol were investigated in detail by the use of I+-trapping reagents such as DL-methionine, 2-chlorodimedone, and L-tyrosine. The addition of the traps did not change the molar ratio of catechol formed to NADH oxidized, nor iodinated traps produced were in the incubation mixture. The results suggest that I+ was not produced on the deiodination in the hydroxylation of o-iodophenol. On the other hand, L-ascorbate, L-epinephrine, and phenylhydrazine increased the molar ratio. o-Phenylenediamine decreased it, being converted to phenazine. This suggests that o-benzoquinone is formed in the oxidation of o-iodophenol as a nascent product. The quinone was detected spectrophotometrically by means of the stopped-flow method. Kinetic analysis of the reactions revealed that o-benzoquinone is reduced nonenzymatically to catechol by a second molecule of NADH. A mechanism of elimination for the ortho-substituted groups of substrate phenols by the enzyme is proposed and discussed

Identification of a lysine residue in the NADH-Binding site of salicylate hydroxylase from Pseudomonas putida S-1

金沢大学自然科学研究科　　金沢大学理工研究域自然システム学系Salicylate hydroxylase from Pseudomonas putida S-1 was irreversibly inactivated by trinitrobenzenesulfonic acid (TNBS). The reaction was linearly dependent on TNBS concentration and the second-order rate constant was 120 M-1.min-1 for the holoprotein at pH 8.5. Modification of one mole of lysine residue per mole of enzyme caused a large loss of the activity, and the enzyme was no longer able to show NADH-dehydrogenase activity after uncoupling. The presence of NADH, NAD+, ATP, or AMP afforded protection against the inactivation. The enzyme modified at a single lysine residue was isolated by hydrophobic chromatagraphy as an apoprotein form and characterized. It could bind FAD with the same K(d) value for that of native apoprotein. The apparent Michaelis constant of the enzyme was increased 13-fold for NADH, but not for salicylate. V(max) for NADH oxidation was decreased to one-fifth of that of the native enzyme. A peptide containing one trinitrophenyl-lysine residue was isolated from the chymotryptic digest of the modified enzyme and its amino acid sequence was determined to be TADVAIAADGIKSSM, which is homologous to the sequence from R-154 to I-168 of salicylate hydroxylase from P, putida PpG7. The lysine in the peptide may represent a basic residue interacting with an anionic group of NADH in the binding site of the enzyme

Hydroxylation of o-halogenophenol and o-nitrophenol by salicylate hydroxylase

金沢大学自然科学研究科　　金沢大学理工研究域自然システム学系Salicylate hydroxylase [EC 1.14.13.1] from Pseudomonas putida catalyzed the formation of catechol from substrate analogues such as o-nitro-, o-amino-, o-iodo-, o-bromo-, and o-chloro-phenol by removing the ortho-substituted groups.They are converted into nitrite, ammonia, and halide ions, respectively. Kinetic parameters of these reactions were determined by spectrophotometric and polarographic methods. Hydroxylation of o-nitro- or o-iodophenol proceeds with the unusual stoichiometry of 2 : 1 : 1 for consumed NADH, O2-uptake, and catechol formed. Other ortho-substituted phenols examined also gave the same results. Like salicylate, these substrates perturb the absorption spectrum of salicylate hydroxylase in the visible region, indicating the formation of enzyme·substrate complexes. Titration experiments with ortho-substituted phenols gave the dissociation constants of the complexes. The complexes were quantitatively reduced with NADH or dithionite without detectable formation of the intermediates. The fact that one atom of 18O2 was incorporated into the produced catechol in hydroxylation of o-nitrophenol indicates that the reaction is of monooxygenase nature. It is concluded that salicylate hydroxylase cleaves the C-N and C-X bonds of ortho-substituted phenols

Crystallization and preliminary X-ray analysis of salicylate hydroxylase from Pseudomonas putida S-1

金沢大学自然科学研究科　　金沢大学理工研究域自然システム学系Apo-salicylate hydroxylase from Pseudomonas putida S-1 has been crystallized by the dialysis method, using ammonium sulfate as the precipitant. The crystals belong to hexagonal space group P62 or P64 with unit cell dimensions of a = b = 142.8 Å and c = 63.8 Å, and diffract X-rays at higher than 3.5 Å resolution. A heavy-atom derivative has been prepared by soaking a crystal in an ammonium sulfate solution containing p-chloromercuriphenylsulfonate