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51.
The beta subunit of tryptophan synthase. Clarification of the roles of histidine 86, lysine 87, arginine 148, cysteine 170, and cysteine 230 总被引:3,自引:0,他引:3
E W Miles H Kawasaki S A Ahmed H Morita H Morita S Nagata 《The Journal of biological chemistry》1989,264(11):6280-6287
Our studies, which are aimed at understanding the catalytic mechanism of the beta subunit of tryptophan synthase from Salmonella typhimurium, use site-directed mutagenesis to clarify the functional roles of several putative active site residues. Although previous chemical modification studies have suggested that histidine 86, arginine 148, and cysteine 230 are essential residues in the beta subunit, our present findings that beta subunits with single amino acid replacements at these positions have partial activity show that these 3 residues are not essential for catalysis or substrate binding. These conclusions are consistent with the recently determined three-dimensional structure of the tryptophan synthase alpha 2 beta 2 complex. Amino acid substitution of lysine 87, which forms a Schiff base with pyridoxal phosphate in the wild type beta subunit, yields an inactive form of the beta subunit which binds alpha subunit, pyridoxal phosphate, and L-serine. We also report a rapid and efficient method for purifying wild type and mutant forms of the alpha 2 beta 2 complex from S. typhimurium from an improved enzyme source. The enzyme, which is produced by a multicopy plasmid encoding the trpA and trpB genes of S. typhimurium expressed in Escherichia coli, is crystallized from crude extracts by the addition of 6% poly(ethylene glycol) 8000 and 5 mM spermine. This new method is also used in the accompanying paper to purify nine alpha 2 beta 2 complexes containing mutant forms of the alpha subunit. 相似文献
52.
Further characterization and immunochemical studies on the carbohydrate specificity of jackfruit (Artocarpus integrifolia) lectin 总被引:12,自引:0,他引:12
The lectin from jackfruit (Artocarpus integrifolia) seeds has been purified by Rivanol (6,9-diamino-2-ethoxyacridine lactate) treatment. The specific activity, molecular weights of parent lectin and its subunit, its glycoprotein nature, and hemagglutination-inhibition assays suggest that this preparation is identical to that obtained by affinity chromatography on melibiose-agarose adsorbent (Ahmed, H., and Chatterjee, B. P. (1986) in Lectins, Biology, Biochemistry, Clinical Biochemistry (B?g-Hansen, T. C., and van Driessche, E., eds) Vol. 5, pp. 125-133, Walter de Gruyter, New York). The lectin strongly agglutinates human and several animal erythrocytes. The lectin contains five isolectins of pI values 7.1, 6.85, 5.5, 5.3, and 5.1. It is thermally stable and loses its activity above 75 degrees C. The hemagglutinating activity remains unchanged in the presence of bivalent cations viz., Ca2+, Mg2+, Mn2+, etc. It is a metalloprotein. The lectin retains its activity by dialysis with acetic acid followed by EDTA. It agglutinates Ehrlich ascites cells. Equilibrium dialysis of lectin with melibiose and quenching of fluorescence of 4-methylumbelliferyl-alpha-D-galactopyranoside by the lectin show that homotetrameric jackfruit lectin has two sugar-binding sites. The lectin precipitates well several galactomannans and glycoproteins having terminal D-Gal-alpha-(1----6)- or D-Gal-beta-(1----3)-D-GalNAc residues. It hardly or does not precipitate polysaccharides having terminal D-Gal-alpha-(1----3) residues. Quantitative precipitin-inhibition studies using various haptens suggest that the -OCH2- group at C-1 and -OH groups at C-4 and partially at C-6 in the alpha-glycoside of D-galactose configuration are important for lectin-sugar interaction. 相似文献
53.
The phosphate adsorption maximum as calculated by the Langmuir equation was used to predict the fertilizer P requirement of
wheat (Triticum aestivium, cv. Caldwell) on both virgin and cultivated Decatur clay loam (clayey, kaolinitic, thermic, Rhodic Paleudult) and Hartsells
sandy loam soils (fine-loamy, siliceous, thermic, Typic Hapludult). Soils with higher adsorption maximum were found to require
more fertilizer P than soils with lower adsorption maximum. For soils 25% saturation of the adsorption maximum gave the optimum
dry matter yield. This corresponded to equilibrium P concentration of 0.45 mg L−1 for Decatur cultivated and 0.31 mg L−1 for Decatur and Hartsells virgin soils for optimum dry matter yield. These values are within the range of those reported
previously by other investigators working with different soils. 相似文献
54.
Casein kinase II (CK-2) is a ubiquitous serine/threonine protein kinase, and is localized to both the cell nucleus and cytoplasm. Despite extensive biochemical similarities in their properties, there is evidence that the two forms of the enzyme exhibit certain distinctions (1). This prompted us to produce antibodies against CK-2, which could be utilized as a possible tool for investigations of the various forms of this enzyme. Specific polyclonal antibodies against the rat liver cytosolic CK-2 were raised in egg yolk of laying hens; the enzyme had repeatedly failed to elicit an immunogenic response in rabbits. The purified polyclonal antibody (egg yolk immunoglobulin, IgY) recognized all three subunits (42, 38, and 28 kDa) of the enzyme in immunoblots. The antibody when bound to a matrix was capable of removing CK-2 from solution, and the bound enzyme could be recovered from the immunoaffinity matrix with 0.1 M diethylamine. The antibody exhibited a high affinity towards CK-2 prepared from cytosol of liver, ventral prostate, and several other rat tissues, but no immunoreactivity was detected towards a number of other protein kinases tested. The subunits of the nuclear form of CK-2 (PK-N2) migrated differently when electrophoresed in parallel in the same gel. However, the antibody did cross-react with the various subunits of PK-N2 suggesting a significant homology in the immunogenic domains in the various subunits of the two forms of the enzyme. 相似文献
55.
56.
Ahmed El Tamer Igor Prokopenko Ernst Wülfert Israel Hanin 《Journal of neurochemistry》1996,67(2):636-644
Abstract: In this study, we have investigated the effect of mivazerol, [3-(1H-imidazol-4-yl)methyl-1]-2-hydroxy-benzamide hydrochloride, a new α2-agonist lacking hypotensive properties and a potential anti-ischemic drug, on the evoked release of norepinephrine, aspartate, and glutamate in tissue preparations from hippocampus, spinal cord T1–T5 section, rostrolateral ventricular medulla, and nucleus tractus solitarii of the brainstem of rat. A simple and efficient in vitro procedure to study pharmacologically the release of norepinephrine and glutamate is described. Tissues were chopped into (0.3 × 0.2 × 0.2 mm3) sections and the resulting minces were used for this study. Exposure to KCl (10–75 mM) for 5 min served as a stimulus for the release response. One, S (for aspartate and for glutamate release), or two such stimuli, S1 and S2 (for norepinephrine release) were conducted. The release of norepinephrine (+150% above baseline) was inhibited in a dose-dependent manner by mivazerol in hippocampus (IC50 = 1.5 × 10?8M), spinal cord (IC50 = 5 × 10?8M), rostrolateral ventricular medulla (IC50 = 10?7M), and nucleus tractus solitarii (IC50 = 7.5 × 10?8M), and by clonidine in hippocampus (IC50 = 5 × 10?8M), spinal cord (IC50 = 4.5 × 10?8M), rostrolateral ventricular medulla (IC50 = 2.5 × 10?7M), and nucleus tractus solitarii (IC50 = 10?7M). This effect was counteracted by the selective α2-antagonists yohimbine and rauwolscine. A significant glutamate and aspartate release response was also induced by KCl (35 mmol/L) in hippocampus (+250 and +135%, respectively) and spinal cord (+120 and +55%, respectively), in vitro. However, neither mivazerol nor clonidine, at doses up to 10 µM, had any significant effect on KCl-induced glutamate release in spinal cord, whereas mivazerol blocked completely the release of both amino acids in hippocampus and only the release of aspartate in spinal cord. On the other hand, clonidine (1 µM) was only effective in reducing by 40% the release of aspartate in hippocampus. These data indicate that (1) inhibition of KCl-induced norepinephrine release by mivazerol is mediated by its action on α2-adrenergic receptors; (2) at concentrations selective for α2-adrenergic receptors, only mivazerol was effective in blocking the KCl-induced glutamate release in hippocampal tissue; and (3) at the same concentrations, both mivazerol and clonidine were unable to inhibit glutamate release in the spinal cord. These data suggest that prevention of hyperadrenergic activity by mivazerol in perioperative patients may be mediated through its effect on the release of norepinephrine and/or the release of glutamate and aspartate in regions of the CNS that are involved in the control of cardiovascular homeostasis. 相似文献
57.
Kazushige Morimoto Eisuke Tsuda Ahmed Abdu Said Eriko Uchida Satoshi Hatakeyama Masatsugu Ueda Takao Hayakawa 《Glycoconjugate journal》1996,13(6):1013-1020
Ten erythropoietin (EPO) fractions differing in sialic acid content, ranging from 9.5 to 13.8 mol mol–1 of EPO, were obtained from baby hamster kidney cell-derived recombinant human EPO by Mono Q column chromatography. The mean pI values of the EPO fractions determined by IEF-gel electrophoresis systematically shifted from 4.11 to 3.31, coinciding with the sialic acid content, without a change in the constitution of asialo N-linked oligosaccharides of each fraction. Although a linear relationship between thein vivo bioactivity and the sialic acid content of the fractionated, samples was observed until 12.1 mol mol–1 of EPO, there was no further increase in their activity over 12.4 mol mol–1 of EPO. On the other hand, an inverse relationship between thein vitro bioactivity and sialic acid content of EPO was observed. Also, we showed that thein vivo bioactivity of some fractions with low sialic acid contents was increased after treatment with 2,6-sialyltransferase, but thein vivo bioactivity of the other fractions with high sialic acid contents was either decreased or not affected.Abbreviations EPO
erythropoietin
- rHuEPO
recombinant human erythropoietin
- hCG
human chorionic gonadotropin
- BHK
baby hamster kidney
- CHO
Chinese hamster ovary
- NeuAc
N-acetyl neuraminic acid
- Gal
galactose
- HRCs
hemolyser-resistant cells
- WST-1
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Na
- IEF
isoelectric focusing
- pI
isoelectric point 相似文献
58.
Ahmed S. Rahman Mieko Kimura Katsuhiko Yokoi Tanvir-E Naher Yoshinori Itokawa 《Biological trace element research》1996,53(1-3):57-64
Three groups of rats were fed two types of synthetic diets for 52 d. The—A group was allowed free access to a vitamin A-deficient
diet and showed classical signs of vitamin A deficiency. The brain was the only organ in our experiment where no significant
weight difference was present among the three groups. In the brain, calcium concentration was significantly higher in the—A
group when compared with the PF (Pair-fed; allowed restricted amount of control diet) and +A groups (allowed free access to
control diet). In the tibia, calcium and magnesium concentrations were significantly lower in the—A group when compared with
other two groups. Excessive accumulation of calcium in brain and apparently similar unbalance in bone, mineral concentration
were observed in central nervous system (CNS) degenerative diseases. Our results suggest that abnormal metabolism of calcium
and magnesium in some tissues and excessive accumulation of calcium in brain may be responsible for the development of neurological
disorders in vitamin A-deficient rats. 相似文献
59.
Isteaq Ahmed Shameem Hiroaki Kurisu Hideyasu Matsuyama Tomoyuki Shimabukuro Katsusuke Naito 《Cancer immunology, immunotherapy : CII》1994,38(6):353-357
Although the present experimental use of recombinant human granulocyte-colony-stimulating factor (rG-CSF) has been proven to alleviate the myelosuppression induced by antitumor chemotherapy, it is also believed to stimulate growth of some nonhematopoietic tumor cells. We investigated both the direct and indirect effects of rG-CSF on in vitro colony formation of human bladder cancer cell lines using a modified human tumor clonogenic assay. Peripheral blood mononuclear cells (PBMC) were used as feeder cells (a mixture of 5×104 monocytes/dish and 5×105 lymphocytes/dish obtained from healthy donors). Human bladder cancer cell lines KK-47, TCCSUP and T24, all derived from human transitional-cell carcinomas, were incubated continuously with various concentrations of rG-CSF ranging from 0.01 ng/ml to 10 ng/ml both with and without PBMC for 7–21 days. The concentrations of rG-CSF used were chosen as being in the range of achievable serum concentrations in patients treated with rG-CSF. At the end of incubation, colonies were counted under an inverted phase-contrast microscope, and an increase in the number of colonies in comparison with the control was used to evaluate the effects of rG-CSF. Results were expressed as a percentage of controls. rG-CSF in the upper layer at concentrations ranging from 0.1 ng/ml to 10 ng/ml stimulated the colony formation of all the cancer cell lines tested in the absence of PBMC in the feeder layer, whereas cells with PBMC in the feeder layer were significantly stimulated more than those without PBMC in the feeder layer (P<0.05) up to a certain concentration, which varied from cell line to cell line. At higher concentrations of rG-CSF, no further stimulation but, on the contrary, a decrease in colony formation was observed in cells with PBMC in the feeder layer in all the cell lines tested. Colony formation in KK-47 and T24 cell lines was significantly inhibited at 5 ng/ml and/or 10 ng/ml rG-CSF compared with cells without PBMC in the feeder layer. Our results suggest that rG-CSF may have both direct and indirect stimulatory effects on the growth of human bladder cancer cell lines in vitro. The results obtained also raise the possibility of adverse effects of rG-CSF in bladder cancer patients whose malignant cells may be directly and indirectly stimulated by this factor while it is being used clinically to alleviate the myelosuppression induced by antitumor chemotherapy. 相似文献
60.
Summary The uterine cervix, urinary bladder and rectum of guinea pigs were injected with Fast Blue dye for retrograde transport studies. Dye-laden neuronal perikarya were detected for each viscus in the paracervical ganglion. These same perikarya also exhibited immunoreactivities for tyrosine hydroxylase, aromatic amino acid decarboxylase, dopamine -hydroxylase, neuropeptide Y, or vasoactive intestinal peptide, though the perikarya projecting to the urinary bladder did not exhibit immunoreactivity for aromatic amino acid decarboxylase. The results of this study indicate that the guinea-pig paracervical ganglion projects to viscera in addition to the uterus, and that the ganglion contains a range of immunoreactivities related to adrenergic and non-adrenergic neurotransmitters. 相似文献