ノシメマダラメイガのProteaseに及ぼすイネ種子糊粉層Trypsin Inhibitorの影響

イネ種子(Orayza sativa L.cv. Nihonbarre)中のtrypsin inbibitor(TI)の貯穀害虫であるノシメマダラメイガの腸内proteaseへの影響の検討を行った。試料には、5齢の幼虫全体を用い、リン酸ナトリウム緩衝液pH7.0でホモジナイズした上清を酵素液とした。イネ種子由来TIがtrypsinを約35%阻害するのと同じタンパク質量を酵素反応系に添加したところ、害虫酵素活性はTIによって約10%阻害され、メイガの消化液はserine protease単独ではないと考えられたため、幼虫酵素液のpHによる影響を検討した。活性のピークはpH3、6および9.5の3ケ所に認められ、どの群のproteaseかを特定することができなかった。次に各クラスを代表するprotease inhibitor存在下での幼虫酵素活性への影響を検討したところ、aspartic protease inhibitorで90%、cysteine protease inhibitorで10-30%活性が低下したが、metallo protease inhibitorでは活性は維持されたままであった。また、CaseinおよびBSAを基質に用い、pH3、6、8および9.5のbuffer中で一晩幼虫酵素液とともに反応させた。得られたペプチド断片を逆相HPLCにより分画し、それぞれの画分についてアミノ酸配列分析を行った結果と、既知のBSAアミノ酸配列から切断部位を推定した。切断部位のうち、pH3、6ではaspartic proteaseの反応部位と、pH8、9.5ではaspartic protease、cysteine protease、serine proteaseの反応部位と一致した。以上のことから、メイガ幼虫酵素液中には、aspartic proteaseが最も多く存在し、反対にmetallo proteaseは存在しないと考えられた。Caseinolytic activities in homogenates from the indian-mealmoth, P.interpunctella, were inhibited about 10% by trypsin inhibitor from rice aleurone, described earlier, which has a same dose of the ability to inhibit about 35% the activity of trypsin. Secondary, proteolytic activity in the extracts of the P.interpunctella decreased in the presence of commercially available protease inhibitors of each classes! Decreased 90% by aspartic protease inhibitor and 10 to 30% in cysteine protease inhibitor, however, was not inhibited by metallo protease inhibitor. Furthermore, the optimum pH\u27s of the protease of P.interpunctella were detected at pH 3,6 and 9.5. These results suggested that the digestive enzymes of the noxious insect may be consisted of a few classes of proteases. Casein or bovine serum albumin was digested by protease in four given pH, pH 3, 6, 8 and 9.5, respectively. Amino acid sequencis of the peptide fractions obtained from the digests by high-performance liquid chromatography, were analyzed. We considered that N-terminal amino acid of peptides fractionated were reactive sites of the midgut protease. From these results, it was confirmed that the cleaved sites were coincided with those of aspartic protease at pH 3 and 6 and with those of aspartic, cysteine and serine protease at pH 8 and 9.5. Consequently, the proportion of aspartic protease in the protease of P.interpunctella were high level, but metallo protease was not detected

ミネラル含有量が異なる塩の官能評価

When we made salt a water solution,a difference of taste of a component ratio of a main element depended on the sodium concentration,but the possibility that a mineral except sodium participated in "taste" was shown in a rice ball when we seasoned it

Induced-fit motion of a lid loop involved in catalysis in alginate lyase A1-III.

The structures of two mutants (H192A and Y246F) of a mannuronate-specific alginate lyase, A1-III, from Sphingomonas species A1 complexed with a tetrasaccharide substrate [4-deoxy-L-erythro-hex-4-ene-pyranosyluronate-(mannuronate)(2)-mannuronic acid] were determined by X-ray crystallography at around 2.2 Å resolution together with the apo form of the H192A mutant. The final models of the complex forms, which comprised two monomers (of 353 amino-acid residues each), 268-287 water molecules and two tetrasaccharide substrates, had R factors of around 0.17. A large conformational change occurred in the position of the lid loop (residues 64-85) in holo H192A and Y246F compared with that in apo H192A. The lid loop migrated about 14 Å from an open form to a closed form to interact with the bound tetrasaccharide and a catalytic residue. The tetrasaccharide was bound in the active cleft at subsites -3 to +1 as a substrate form in which the glycosidic linkage to be cleaved existed between subsites -1 and +1. In particular, the O(η) atom of Tyr68 in the closed lid loop forms a hydrogen bond to the side chain of a presumed catalytic residue, O(η) of Tyr246, which acts both as an acid and a base catalyst in a syn mechanism

Crystallization and preliminary crystallographic analysis of endo-1,3-β-glucanase from Arthrobacter sp.

Endo-1,3-β-glucanase from Arthrobacter sp. has been crystallized and X-ray diffraction data have been collected to 1.66 Å resolution