著者

MAEDA Iori SHIMOHIGASHI Yasuyuki IKESUE Koichi NOSE Takeru IDE Yuzuru KAWANO Keiichi OHNO Motonori

出版者

The Japanese Biochemical Society

雑誌

The journal of biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.119, no.5, pp.870-877, 1996-05-01

参考文献数

25

被引用文献数

5

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The dipeptide benzyl amide H-D-Thr-Phe-NH-CH₂-C₆H₅ was found to inhibit chymotrypsin strongly (K_i=4.5×10^-6M) in a competitive manner. When a series of phenyl amides H-D-Thr-Phe-NH-(CH₂)<i>_n</i>-C₆H₅ (<i>n</i>=0-4) were tested, inhibitory potency peaked at n=1 (benzyl amide). Incorporation of a methyl group into the benzyl methylene resulted in formation of stereoisomers, H-D-Thr-Phe-NH-(<i>R</i> or <i>S</i>)-CH(CH₃)-C₆H₅, with considerably different inhibitory potencies. The <i>R</i>-isomer was as active as the benzyl amide, while the <i>S</i>-isomer was about 30-fold less active than the benzyl amide. Furthermore, when a fluorine atom was introduced into the para-position of the amide-benzyl group, the resulting H-D-Thr-Phe-NH-CH₂-C₆H₄(<i>p</i>-F) showed considerably enhanced inhibitory activity (about 5-fold, <i>K</i>_i=9.1×10^-7M). In conformational analysis by 400MHz ¹H-NMR, all dipeptides having D-Thr-Phe backbone structure showed large upfield shifts of D-Thr-βO<i>H</i> (shifts in ppm, 0.09-0.17), D-Thr-βC<i>H</i> (0.23-0.32), and D-Thr-γC<i>H</i>₃ (0.38-0.53), indicating the presence of shielding effects from the benzene ring. In addition, NOE enhancements between the D-Thr-γCH₃ and Phe-phenyl groups were evidenced by measurements of two-dimen-sional NOESY spectra and NOE difference spectra. These observations demonstrated the spatial proximity of these side chains, which is due to side chain-side chain CH/π interaction. All these results support the idea that the amide-benzyl group binds at the chymotrypsin S₁, site, while the hydrophobic core with CH/π interaction binds at the S₂ or S₁' site.

著者

Satoru Oshiro Masahiro Kawahara Shirao Mika Kazuyo Muramoto Kazuo Kobayashi Ryuta Ishige Koji Nozawa Makoto Hori Cai Yung Shigetaka Kitajima Yoichiro Kuroda

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.123, no.1, pp.42-46, 1998 (Released:2008-11-18)

参考文献数

27

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We previously demonstrated that cultured human fibroblasts internalize iron via transferrin-independent iron uptake (Tf-IU), redox, and receptor-mediated endocytosis uptake systems [Oshiro, S., Nakajima, H., Markello, T., Krasnewich, D., Bernardini, I., and Gahl, W. A. (1993) J. Biol. Chem. 268, 21586-21591]. Of these iron transport systems, the Tf-IU system is involved in the accumulation of transition metals in various mammalian cells. It is also known that in experimental animals fed aluminum (Al), Al at micromolar level selectively accumulates in the brain. In the present study, we examined the effects of Al accumulated in the brain cells on iron transport by the Tf-IU system and iron metabolism, using primary cultures from fetal rat cerebral cortex. Pretreatment of cells with 200 μM Al-nitrilotriacetate upregulated the Tf-IU system for iron. Moreover, of various metals tested, Al markedly upregulated the Tf-IU activity. To examine the influence of Al on iron metabolism, the interaction between Al accumulated in the cells and iron-responsive element binding protein (IRE-BP), a cellular iron regulator, was examined by Northern blot analysis, and activity assay: Al decreased the Tf receptor mRNA level and increased the aconitase activity of IRE-BP. The increase of aconitase activity by Al was also observed in vitro. These results suggest that Al accumulated in cortical cells affects iron metabolism.

著者

Hara Atsushi Taketomi Tamotsu

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.109, no.6, pp.904-908, 1991

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Characterization and elucidation of the changes of glycosphingolipids in the aorta along with the progression of atherosclerosis were performed in the Watanabe hereditable hyperlipidemic (WHHL) rabbit, an animal model for human familial hypercholesterolemia, as compared with in the normal rabbit. Neutral glycosphingolipids in aortae of both normal and WHHL rabbits were composed of glucosylceramide, galactosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, and galactosylneolactotetraosylceramide. The total amount of neutral glycosphingolipids in the aorta of the WITHL rabbit (557nmol/g tissue) was increased about 5-fold compared to the normal level (107nmol/g tissue). Prominent increases were observed in glucosylceramide (13-fold the normal level) and lactosylceramide (12-fold the normal level). The amount of total gangliosides in the aorta of the WHHL rabbit (207μg NeuAc/g tissue) was markedly increased, being about 12-fold the normal level (17μg NeuAc/g tissue). GM3 ganglioside was increased about 11-fold compared to normal. GD3 ganglioside, which was almost undetectable in normal aorta, also showed a marked increase in that of the WHHL rabbit (51.7 μg NeuAc/g tissue). Sulfatide, which was absent in the aorta of the normal rabbit, was markedly accumulated in that of the WHHL rabbit (280nmol/g tissue). The fatty acid composition of neutral glycosphingolipids of WHHL rabbit was found to include a higher amount of C23:0, which is the major fatty acid of glycolipids in serum lipoproteins. Gangliosides in the aorta of the WHIZ, rabbit contained more C16:0 than in the normal rabbit. Sphingosine of sulfatide in the aorta of the WHHL rabbit was composed of sphingenine (86%), sphinganine (9%), 4-D-hydroxysphinganine (4%), and 4-D-hy-droxyeicosasphinganine (less than 1%). The results of fatty acid analysis of glycosphin-golipids in the aorta of WHHL rabbit suggested that the various glycosphingolipids mostly derived from serum lipoproteins were accumulated in the aorta of the WHEL rabbit along with the progression of atherosclerosis, and that most of these glycolipids were hydrolyzed into less polar glycolipids such as glucosylceramide or lactosylceramide. On the other hand, the moderate increases in globotriaosylceramide, globotetraosylceramide, and galactosylneolactotetraosylceramide, which are ordinary constituents of the normal aorta, indicated the marked intimal thickening of the aorta of the WHITE, rabbit. It is also suggested that GM3 and GD3 gangliosides were derived not only from sera but also from new-type cell populations, such as foam cells or macrophages in the atherosclerotic lesions, because the fatty acids of these gangliosides included more palmitic acid than those of either serum lipoproteins or the normal aorta. The most interesting finding was that the occurrence of sulfatide and GD3 ganglioside in the aorta of the WHHL rabbit could be a useful indicator of the degree of progression of atherosclerosis, since these glycosphingolipids were hardly detected in the normal aorta.

著者

Kazuo UMETSU Suezo KOSAKA Tsuneo SUZUKI

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.95, no.1, pp.239-245, 1984 (Released:2008-11-18)

参考文献数

20

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A lectin was purified from the hemolymph of Allomyrina dichotoma larvae by affinity chromatography on acid-treated Sepharose 4 B. The purified lectin showed two protein bands on polyacrylamide gel electrophoresis. These two lectin bands (allo A-I and -II) were separated by DEAE-Cellulofine column chromatography. By gel filtration on Sephadex G-100, the molecular weights of alto A-I and -II were estimated to be 65, 000 and 66, 500, respectively. On the other hand, by SDS-polyacrylamide gel electrophoresis after cross-linking of subunits with glutaraldehyde, they are estimated to be 38, 000 and 39, 000, respectively. On SDS-polyacrylamide gel electrophoresis, it was proved that both allo A-I and -II lectin consisted of two subunits, respectively. The molecular weights were 17, 500 and 20, 000 for allo A-I, and 19, 000 and 20, 000 for allo A-II. The isoelectric points of alto A-I and -II were estimated to be 6.4 and 5.9, respectively. On double immunodiffusion, allo A-I and -II gave single precipitin lines, which fused completely with each other, against the antibody to crude allo A. The hemag-glutinating activity of allo A-I and -II was inhibited only by β-linked D-galactose such as lactose and lactulose.

著者

Ohnishi Yasuo Beppu Teruhiko Horinouchi Sueharu

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.121, no.5, pp.902-913, 1997

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A serine protease (SSP) of <i>Serratia marcescens</i> is one of the extracellular enzymes secreted from this Gram-negative bacterium. SSP is produced as a large precursor and converted to a mature protein by cleavages removing an NH₂-terminal signal sequence and a COOH-terminal pro-region. This COOH-terminal pro-region is integrated into the outer membrane and has a functional role for the export of the mature protein across the outer membrane. Southern hybridization analysis with a DNA fragment encoding the COOH-terminal pro-region as the probe showed a wide distribution of nucleotide sequences encoding SSP exporter-like proteins among <i>Serratia</i> species. Moreover, S. marcescens IFO 3046, from which the ssp gene had been cloned, was found to contain two ssp homologues (<i>ssp-h1</i> and <i>ssp-h2</i>). They were cloned and their nucleotide sequences were determined. The two ssp homologues were found to exist in tandem on the genome and their amino acid sequences showed 81% identity to each other. Both of them showed 55% identity in amino acid sequence to preproSSP. In addition, both showed end-to-end similarity to the 100 kDa serotype-specific antigen (Ssa1) of Pasteurella haemolytica. Escherichia coli JM105 containing <i>ssp-h1</i> gene produced a 53 kDa protein corresponding to the NH₂terminal portion and a 49 kDa protein corresponding to the COOH-terminal portion, both of which were rigidly integrated in the outer membrane. Consistent with the significant similarity of the COOH-terminal portions of the homologues to that of SSP, they showed the ability to translocate the mature SSP part across the outer membrane into the medium. Furthermore, the NH₂-terminal portion of the homologue was not translocated into the outer membrane without its COOH-terminal part. All of these data show that the SSP homologues are outer membrane proteins that are translocated into the outer membrane with the aid of the translocator function of their COOH-terminal part.

著者

Masato Otsuka Tomoharu Mine Kentarou Ohuchi Shinji Ohmori

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.119, no.2, pp.246-251, 1996 (Released:2008-11-18)

参考文献数

19

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The metabolism of diacetyl (2, 3-butanedione), acetoin (3-hydroxy-2-butanone), and 2, 3-butanediol, which are metabolites of acetaldehyde was quantitatively investigated using rat liver homogenate, liver perfusion, and in vivo experiments. Diacetyl and acetoin were reduced to 2, 3-butanediol in these experiments, but acetoin and 2, 3-butanediol were scarcely oxidized to diacetyl, indicating that the reduction reaction to 2, 3-butanediol from diacetyl occurs actively in rat liver. The formation of acetoin from diacetyl required either NADH or NADPH as a reductant, while the reduction of acetoin to 2, 3-butanediol required NADH. Acetoin and 2, 3-butanediol were more readily accumulated than diacetyl in brain tissue.