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1.
2.
1. The renal cell lines, JTC-12 and MDCK, not only synthesize galactosylceramide 3-sulfate and lactosylceramide 3'-sulfate in vivo, but also contain enzymes that catalyze the transfer of sulfate to galactosylceramide and lactosylceramide in vitro. 2. Concentration of cations necessary for maximum sulfotransferase activity occurred at 40 mM Ca2+ with galactosylceramide and 15 mM Ca2+ with lactosylceramide as the substrate. Na+ was also found to stimulate the sulfation of galactosylceramide, but was slightly inhibitory for the sulfation of lactosylceramide. 3. The products of the in vitro assay mixture were characterized as galactosylceramide 3-sulfate and lactosylceramide 3'-sulfate by a variety of TLC separations. 4. The apparent Km of JTC-12 cells for galactosylceramide was 17 microM, while that for lactosylceramide was 82 microM. The Km values of MDCK cells were comparable to those of JTC-12 cells. Competition studies suggested that galactosylceramide and lactosylceramide were sulfated by a single enzyme in both cell lines. 相似文献
3.
Some properties of the enzyme activity that catalyzes the transfer of N-acetylgalactosamine from UDP-N-acetylgalactosamine to exogenous lactosylceramide-II3-sulfate (SM3) and N-acetylneuraminosyllactosylceramide (GM3) were studied using the enzyme preparation solubilized from the 100,000 X g pellet of 6-day-old rat brain. The products from SM3 and GM3 were identified as gangliotriaosylceramide-II3-sulfate (SM2) and N-acetylneuraminosylgangliotriaosylceramide (GM2), respectively, by TLC-autoradiography. Optimal conditions for both activities were similar: pH (Hepes-NaOH), 7.0-7.5; detergent (heptylthioglucoside), 0.64% and Mn2+, 5-10 mM. The concentrations of the detergent optimal for both enzyme activities were also examined at various concentrations of the acceptors. The lower the amounts of acceptors, the less the amounts of detergent that were required, and vice versa, for the maximum activities. The acceptor-saturation curve for SM2 synthesis was triphasic, exhibiting a sigmoidal region at lower concentrations, a hyperbolic region and finally a descending region. For GM2 synthesis, the curve was biphasic without the descending region. The donor-saturation curves were classical hyperbolic ones for both syntheses. The Km values calculated for SM3 and GM3 were 0.37 and 0.19 mM, respectively, when the data corresponding to the hyperbolic regions were used for the double-reciprocal plots. The Km values for UDP-N-acetylgalactosamine in the SM2- and GM2-synthesis were 82 and 26 microM, respectively. SM3 and GM3 were the best acceptors for this enzyme preparation. From the results of the acceptor competition study, it was suggested that the two synthetic reactions are catalyzed by a single enzyme. 相似文献
4.
The present study was carried out in order to elucidate the metabolic pathway from 1 alpha,25-(OH)2D3 to 1 alpha,25-(OH)2D3-26,23-lactone. For that purpose, we stereospecifically synthesized the vitamin D3 derivatives 1 alpha,23(S),25-(OH)3D3, 1 alpha,23(S),25(R),26-tetrahydroxyvitamin D3, and 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-lactol. The in vitro metabolism of these compounds was examined in kidney homogenates and intestinal mucosa homogenates from 1 alpha,25-(OH)2D3-supplemented chicks. The naturally occurring 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone was produced (in increasing amounts) from 1 alpha,25-(OH)2D3, 1 alpha,25(R),26-(OH)3D3, 1 alpha,23(S),25-(OH),D3, 1 alpha,23(S),25(R),26-(OH)4D3, and 23(S),25(R)-1 alpha,25-(OH)2D3-26,23-lactol. These results indicated that there are two possible metabolic pathways from 1 alpha,25-(OH)2D3 to 1 alpha,23(S),25(R),26-(OH)4D3: the major one is by way of 1 alpha,23(S),25-(OH)3D3 and the minor one is by way of 1 alpha,25(R),26-(OH)3D3. 1 alpha,23(S),25(R),26-Tetrahydroxyvitamin D3 is further metabolized to 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone via 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactol. In the course of our studies, a new biosynthetic vitamin D3 metabolite was isolated in pure form. This metabolite was identified as 23(S),25(R)-1 alpha,25-(OH)2D3-26,23-lactol by UV spectrophotometry and mass spectrometry. Furthermore, we establish in this report that the lactonization of 1 alpha,23,25,26-(OH)4D3 and 1 alpha,25-(OH)2D3-26,23-lactol occurs in a stereo-retained and stereo-selective fashion. 相似文献
5.
6.
Y Itoh A Oguro K Nakamura Y Ishizuka K Yamane 《Bioscience, biotechnology, and biochemistry》1992,56(6):867-871
An artificially inserted extra peptide (21 amino acid peptide) between the B. subtilis alpha-amylase signal peptide and the mature thermostable alpha-amylase was completely cleaved by B. subtilis alkaline protease in vitro. The cleavage to form a mature enzyme was observed between pH 7.5 and 10, but not between pH 6.0 and 6.5, although a similar protease activity toward Azocall was observed between pH 6.0 and 7.5. To analyze the effects of pH on the cleavage, CD spectra at pH 6, 8, and 11 of the NH2-terminally extended thermostable alpha-amylase were analyzed and the results were compared with those of the mature form of the alpha-amylase. It is suggested that the cleavage of the NH2-terminally extended peptide is controlled by the secondary and tertiary structure of the precursor enzyme. Similar cleavage of different NH2-terminally extended peptides by the alkaline protease was also found in other hybrid thermostable alpha-amylases obtained. 相似文献
7.
T Ishizuka J Yoshitake T Yamada S Horie M Koshiishi S Nagai T Satou 《Acta cytologica》1988,32(5):658-662
Cytologic examination of sputum samples from an elderly patient revealed the presence of two cell populations: squamous cell carcinoma cells and rhabdomyosarcoma cells. The abnormal squamous cells showed both keratinizing and nonkeratinizing forms while some of the rhabdomyosarcoma cells showed cross striations. Sputum cytology was thus able to suggest a diagnosis of pulmonary carcinosarcoma. Histologically, the tumor was composed mainly of sarcomatous tissue showing various kinds of cells: fusiform or fibrous cells, round anaplastic cells, spindled cells with typical cross striations and myoblastic cells. A partially myxomatous degeneration was present. In addition, squamous cell carcinoma proliferated along the bronchi and formed small invasive cell nests in the sarcomatous tissue. No transition between the two components was noted. Both cellular constituents had metastasized to an interlobar lymph node. 相似文献
8.
Sequence and over-expression of subunits of adenosine triphosphate synthase in thermophilic bacterium PS3 总被引:8,自引:0,他引:8
S Ohta M Yohda M Ishizuka H Hirata T Hamamoto Y Otawara-Hamamoto K Matsuda Y Kagawa 《Biochimica et biophysica acta》1988,933(1):141-155
The primary structures of all the subunits of thermophilic ATP synthase were determined, and its alpha, beta and gamma subunits could be over-expressed in Escherichia coli, because these subunits were stable and reconstitutable. DNA of 7500 base pairs in length was found to contain a cluster of nine genes for subunits of ATP synthase. The order of their reading frames (size in base pairs) was: I(381): a(630): c(216): b(489): delta(537): alpha(1507): gamma(858): beta(1419): epsilon(396), I being a gene for a small hydrophobic, basic protein expressed in vitro. All the termini of TF0F1 subunits were confirmed by peptide sequencing. Large quantities of the overexpressed thermophilic alpha, beta and gamma subunits were prepared from the extract of E. coli, by a few purification steps. 相似文献
9.
F1-ATPase is the major enzyme for ATP synthesis in mitochondria, chloroplasts, and bacterial plasma membranes. F1-ATPase obtained from thermophilic bacterium PS3 (TF1) is the only ATPase which can be reconstituted from its primary structure. Its beta subunit constitutes the catalytic site, and is capable of forming hybrid F1's with E. coli alpha and gamma subunits. Since the stability of TF1 resides in its primary structure, we cloned a gene coding for TF1, and the primary structure of the beta subunit was deduced from the nucleotide sequence of the gene to compare the sequence with those of beta's of three major categories of F1's; prokaryotic membranes, chloroplasts, and mitochondria. The following results were obtained. Homology: The primary structure of the TF1 beta subunit (473 residues, Mr = 51,995.6) showed 89.3% homology with 270 residues which are identical in the beta subunits from human mitochondria, spinach chloroplasts, and E. coli. It contained regions homologous to several nucleotide-binding proteins. Secondary structure: The deduced alpha-helical (30.1%) and beta-sheet (22.3%) contents were consistent with those determined from the circular dichroism spectra. Residues forming reverse turns (Gly and Pro) were highly conserved among the F1 beta subunits. Substituted residues and stability of TF1: We compared the amino acid sequence of the TF1 beta subunit with those of the other F1 beta subunits mentioned above. The observed substitutions in the thermophilic subunit increased its propensities to form secondary structures, and its external polarity to form tertiary structure. Codon usage: The codon usage of the TF1 beta gene was found to be unique. The changes in codons that achieved these amino acid substitutions were much larger than those caused by minimal mutations, and the third letters of the optimal codons were either guanine or cytosine, except in codons for Gln, Lys, and Glu. 相似文献
10.
T Hasegawa S Izumi S Kurihara Y Ishizuka Y Ito A Takechi M Tsuchiya 《Acta cytologica》1985,29(3):363-366
In a high-volume cytology laboratory in the metropolitan Tokyo area, the incidence of cytologically diagnosed herpes infection in cervical scraping smears of the female genital tract was studied according to the year-by-year changes, age distribution, seasonal variation and types of cytologic alteration. The overall incidence over the 12 years studied was 0.007% (87 cases among 1,230,773 examined). The incidence varied from 0.003% to 0.005% in the early 1970s (except for 1973) and increased to 0.011% in the last three years (1980 to 1982). A large increase was noted in younger age groups in comparison with middle and older age groups. There was a tendency for the infection rate to be higher in the spring (0.011%) and lower in the fall (0.005%). Multinucleation and a ground-glass appearance were observed in the infected cells in almost every case while eosinophilic inclusion bodies were found in 20.7% of the cases. 相似文献