著者

Sakamoto Taiichi Kim Mi Hee Kurihara Yasuyuki SASAKI Nobuyuki NOGUCHI Tomoaki KATAHIRA Masato UESUGI Seiichi

出版者

社団法人 日本生化学会

雑誌

The journal of biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.121, no.2, pp.288-294, 1997-02-01

参考文献数

48

被引用文献数

1

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The properties of a mutant hammerhead ribozyme system, which consists of two RNA oligomer strands and in which stem II is deleted (replaced with a UUUU loop), are described. The effects of temperature, pH, and metal ions on the cleavage reaction were similar to those for the parent ribozyme with stem II. The mutant ribozyme showed a much lower cleavage rate (k_cat=0.04min^-1) in the presence of 10mM MgCl₂, where the parent ribozyme showed full cleavage activity. However, increasing the concentration of MgCl₂ from 10 to 100mM restored the cleavage activity of the mutant ribozyme to the original level (k_cat=0.2min^-1). CD titration experiments with MgCl₂ using a noncleavable substrate were carried out. Deletion of stem II resulted in an about 20-fold reduction of the apparent Mg²⁺ binding affinity when the Mg²⁺ concentrations of half-saturation are compared. The results were analyzed by curve-fitting analysis and compared with those for the parent ribozyme. The analysis showed that the Mg²⁺ concentration dependence data in CD and cleavage experiments for the mutant enzyme can be explained by a two Mg²⁺ ion binding mechanism. These results suggest that stem II is important for maintaining the conformation of the catalytic core suitable for Mg²⁺ binding.

著者

Ono Yasuko Kinouchi Tadatoshi Sorimachi Hiroyuki ISHIURA Shoichi SUZUKI Koichi

出版者

社団法人 日本生化学会

雑誌

The journal of biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.121, no.3, pp.585-590, 1997-03-01

参考文献数

39

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Alzheimer's disease amyloid precursor protein (APP) generates a &beta;-amyloid protein (A&beta;) that is a main component of the senile plaques found in the brains of Alzheimer's disease patients. APP is thought to undergo proteolysis via two different pathways, the amyloido-genic pathway which produces A&beta;, and the non-amyloidogenic pathway which releases a large N-terminal fragment into the medium. The proteases that mediate these processes remain unidentified. The physiological function of APP is not clear yet. Therefore, the cytoplasmic region of APP has attracted much interest, because this region is highly conserved among species, and members of the amyloid precursor-like protein (APLP) family. Several potentially functional sequences exist in the region, including signal sequences for protein sorting and a Go-protein binding sequence. We constructed two mutants, 695<i>&Delta;</i>NPTY and 695<i>&Delta;</i>GYEN. They lack potential endosome/lysosome targeting signals, NPTY and GY, in the cytoplasmic domain of APP695, respectively. The mutant APPs had longer half-lives and were secreted more easily into the medium than the wild type, suggesting that these sequences are important for the secretion and metabolism of APP.

著者

KOBAYASHI Toshihide OHTA Akinori SHIBUYA Isao

出版者

社団法人 日本生化学会

雑誌

The Journal of Biochemistry

巻号頁・発行日

vol.99, no.5, pp.1393-1400, 1986

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<i>Escherichia coli</i> mutants harboring the <i>pss-1</i> allele (coding for a temperature-sensitive phosphatidylserine synthase) are temperature sensitive for growth and synthesize less phosphatidylethanolamine at higher temperatures, giving rise to abnormal membrane phospholipid compositions. To obtain information concerning the determinant for the phospholipid polar headgroup composition and the lethal factor in the defective membranes, we have examined the effect of increased supply of <i>sn</i>-glycerol 3-phosphate on the phospholipid synthesis and the growth ability of a <i>pss-1</i> mutant. For this purpose, a pair of <i>E. coli</i> K-12 derivatives isogenic except for the <i>pss-1</i> allele was constructed from strain BB26-36 to harbor the mutations related to glycerol metabolism (<i>glpD3</i>, <i>glpR2</i>, <i>glpK</i>¹, and <i>phoA8</i>). Pulse- and uniform-labeling of phospholipids with ³²P at 42&deg;C in a synthetic medium with (0.2%) or without glycerol showed that glycerol further lowered the temperaturesensitive formation of phosphatidylethanolamine, removed the phosphatidate and CDP-diacylglycerol accumulated in the absence of glycerol, and resulted in an increase in cardiolipin content in the <i>pss-1</i> mutant. The phospholipid synthesis and contents in the pssr⁺ strain were not significantly affected by glycerol. Glycerol in the medium markedly enhanced the growth defect of the <i>pss-1</i> mutant, which was remediable by sucrose. The results indicate that the intracellular pool of <i>sn</i>-glycerol 3-phosphate is the limiting factor for acidic phospholipid synthesis in the <i>pss-1</i> mutant, and cardiolipin unusually accumulated is injurious to the functional E. coif membranes. Possible determinants for the phospholipid composition of the wild-type <i>E. coli</i> cells are also discussed on the basis of the present observations.

著者

Hayakawa Hiroshi Kumura Keiko Sekiguchi Mutsuo

出版者

社団法人 日本生化学会

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.84, no.5, pp.1155-1164, 1978

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Uracil-DNA glycosylase, which acts specifically on uracil-containing DNA, was purified 250-fold from an extract of <i>Escherichia coli</i> 1100. The enzyme releases free uracil from DNA, producing alkali-labile apyrimidinic sites in the DNA. The enzyme is active on both native and heat-denatured DNA of phage PBS1, which contains uracil in place of thymine. &phi;X174 DNA which had been treated with bisulfite and then at alkaline pH was susceptible to the action of uracil-DNA glycosylase. Since DNA treated with bisulfite alone was less susceptible to the enzyme, it is likely that the enzyme recognizes deaminated cytosine, namely uracil, but not bisulfite adducts of uracil and cytosine in the treated DNA. DNA treated with nitrite or hydroxylamine was not attacked by the enzyme. Enzyme activity acting on bisulfite-treated DNA was absent from an extract of <i>E. coli</i> mutant BD10 (<i>ung</i>). The mutant exhibited higher sensitivity to bisulfite than did the wild-type strain and was unable to reactivate phage T1 pre-exposed to bisulfite and weak alkali.

著者

KARAZAWA SUGIZO

出版者

社団法人 日本生化学会

雑誌

The Journal of Biochemistry

巻号頁・発行日

vol.10, no.3, pp.389-394, 1929

著者

Hara Atsushi Taketomi Tamotsu

出版者

社団法人 日本生化学会

雑誌

The Journal of Biochemistry

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 (207&mu;g NeuAc/g tissue) was markedly increased, being about 12-fold the normal level (17&mu;g 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 &mu;g 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.