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121.
Purification and characterization of p93fes- and p60src-related tyrosine protein kinase activities in differentiated HL-60 leukemia cells 总被引:1,自引:0,他引:1
Two tyrosine protein kinase activities have been identified previously to be present in HL-60 leukemia cells during induction of granulocytic and monocytic differentiation with a variety of differentiating agents. We have copurified a membrane-associated tyrosine kinase (p93) and an activity associated with both the cytosol and membrane fractions (p60). Triton X-100 extracts from HL-60 cells treated with dimethyl sulfoxide were subjected to tyrosine-agarose chromatography, polypropyl aspartamide high performance liquid chromatography (HPLC), and HPLC using an antiphosphotyrosine IgG-derivatized column. Overall purification was 2700-fold for p93 and 1800-fold for p60. p60 and p93 are phosphorylated exclusively on tyrosine residues and can use poly(Glu,Tyr)4:1, histone H1 and vasoactive intestinal peptide as substrates. Poly(Glu,Tyr)1:1 and poly(Glu,Ala,Tyr)6:3:1 were less effective substrates for p60 and p93. The activity of p93 was dependent on Mg2+ or Mn2+, whereas p60 was dependent on Mg2+; however, the activity of p60 was stimulated in a synergistic manner by the presence of both Mg2+ and Mn2+, whereas the activity of p93 was not enhanced further by the combination of divalent ions. Both p60 and p93 were immunoprecipitated by an anti-v-src monoclonal antibody but only p93 was immunoprecipitated by an anti-v-fps/fes antibody. V8 protease digestion of p60 revealed one major proteolytic fragment containing phosphotyrosine, whereas V8 protease digestion of p93 produced two major peptides that were phosphorylated on tyrosine residues. These results suggest that, although p93 and p60 may possess some epitopic similarities, they have distinguishing phosphorylation sites. Moreover, p93, in contrast to p60, appears to be strictly associated with granulocytic/monocytic differentiation and related to the cellular fps/fes protooncogene. 相似文献
122.
Characterization of tumor-associated fucogangliosides from PC 12 pheochromocytoma cells 总被引:3,自引:0,他引:3
T Ariga K Kobayashi Y Kuroda R K Yu M Suzuki H Kitagawa F Inagaki T Miyatake 《The Journal of biological chemistry》1987,262(29):14146-14153
PC 12h pheochromocytoma cells were subcutaneously transplanted into rat. We found the transplanted tumors accumulated some fucogangliosides associated with PC 12 cells. These gangliosides were isolated and purified by DEAE-Sephadex A-25 and Iatrobeads column chromatographies. Their structures were determined by fast atom bombardment mass spectrometry, proton nuclear magnetic resonance spectrometry, permethylation study, and sequential degradation using various exoglycosidases and mild acid hydrolysis. Two tumor-associated fucogangliosides were found to possess the blood group B determinant as follows: G6: IV2Fuc alpha, IV3Gal alpha, II3NeuAc, GgOse4Cer; G11: IV2Fuc alpha, IV3Gal alpha, II3 (NeuAc)2, GgOse4Cer. A ganglioside with the similar structure as ganglioside G6 was isolated from rat hepatoma cells (Holmes, E.H., and Hakomori, S-I. (1982) J. Biol. Chem. 257, 7698-7703). However, ganglioside G11 has not previously been reported in the literature. These fucogangliosides reacted with the monoclonal antibody prepared by immunizing mice with PC 12h cells. Other fucogangliosides were also found to accumulate in the transplanted tumor tissues. They were identified as fucosyl-GM1 and fucosyl-GDlb. These fucogangliosides did not react with the monoclonal antibody against PC 12h cells. 相似文献
123.
Properties of bovine heart mitochondrial cytochrome b560 总被引:2,自引:0,他引:2
A large-scale preparation of the two-subunit protein complex (QPs) that converts succinate dehydrogenase into succinate-ubiquinone reductase from cytochrome b-c1 particles is achieved by a procedure involving Triton X-100 solubilization and calcium phosphate column chromatography at different pH values. The isolated two-subunit QPs contains 25 nmol of cytochrome b560/mg of protein and is able to reconstitute with soluble succinate dehydrogenase to form a TTFA-sensitive succinate-ubiquinone reductase. The maximum reconstitutive activity is 100 mumol of succinate oxidized per min per mg of QPs protein at 23 degrees C. Although cytochrome b560 in isolated QPs is not succinate reducible and its dithionite reduced form is reactive to carbon monoxide, cytochrome b560 is shown to be physically associated with succinate dehydrogenase by the following observations. The dithionite reduced form of cytochrome b560 in isolated QPs has a symmetrical alpha-absorption peak, which upon reconstitution with succinate dehydrogenase becomes slightly broadened and shows a shoulder at around 553 nm, identical to that of cytochrome b560 in succinate-ubiquinone reductase. Upon addition of succinate dehydrogenase to QPs, about 50% of the reduced form of cytochrome b560 in the QPs becomes insensitive to carbon monoxide treatment. The redox potential of cytochrome b560 in QPs is -144 mV which is higher than that of cytochrome b560 in succinate-ubiquinone reductase (-185 mV). Upon addition of succinate dehydrogenase, the redox potential of about 46% of the cytochrome b560 in QPs preparation becomes identical to that of cytochrome b560 in succinate-ubiquinone reductase. Cytochrome b560 in the QPs preparation shows two epr signals, g = 3.07 and g = 2.92, whereas cytochrome b560 in succinate-ubiquinone reductase exhibits only one epr signal at g = 3.46. When QPs is reconstituted with succinate dehydrogenase to form succinate-ubiquinone reductase, the g = 3.46 epr signal reappears at the expense of the g = 3.07 signal. Based on epr measurement at liquid helium temperature, about 18% of the total cytochrome b in the isolated active succinate-cytochrome c reductase is cytochrome b560, indicating that cytochrome b560 is indeed a unique cytochrome b and not a denatured product of cytochrome b562 or b565. 相似文献
124.
125.
In order to develop liposomes for use in an immunoassay system, the preparation of immune liposomes and their characterization have been investigated. Liposomes have potential use in extremely sensitive analytical immunoassays, in addition to serving as an attractive drug delivery system. This liposome immunoassay system is based on membrane immunochemistry and an enzymatic reaction. An intense yellow color, easily detectable with the naked eye, was produced quite rapidly by the lysis of bovine serum albumin (BSA)-labeled, alkaline phosphatase-entrapped liposomes in the presence of anti-BSA rabbit serum and active complement under alkaline conditions. Sensitive detection is possible because of the antigen-antibody complex reaction, which leads to liposome lysis and an enzymatic reaction. The liposome immunoassay method offers a rapid, simple, and sensitive testing procedure which can quantitatively and qualitatively determine the presence or absence of antigenic materials and antibodies. 相似文献
126.
Molecular cloning of an endoglucanase gene from an alkalophilic Bacillus sp. and its expression in Escherichia coli. 总被引:1,自引:0,他引:1 下载免费PDF全文
One of the cellulase genes from alkalophilic Bacillus sp. strain N-4 was cloned in pBR322. A recombinant plasmid, pYBC107, expressing carboxymethyl cellulase (CMCase) was isolated, and the size of the cloned HindIII fragment was found to be 5.5 kilobases. The restriction map of pYBC107 showed a different pattern from those of pNKI and pNKII (N. Sashihara, T. Kudo, and K. Horikoshi, J. Bacteriol. 158:503-506, 1984). When the HindIII fragment from pYBC107 was subcloned into pYEJ001, there was a 3.8-fold increase in CMCase activity over that observed with pYBC107. Plasmid pYBC108 constructed by treatment of pYBC107 with HindIII and EcoRI expressed the CMCase activity, although to a limited extent. To verify the originality of cloned pYBC107 from Bacillus sp., we analyzed the restriction digest by Southern blotting. 相似文献
127.
Antimycin-insensitive succinate-cytochrome c reductase activity has been detected in pure, reconstitutively active succinate dehydrogenase. The enzyme catalyzes electron transfer from succinate to cytochrome c at a rate of 0.7 mumole succinate oxidized per min per mg protein, in the presence of 100 microM cytochrome c. This activity, which is about 2% of that of reconstitutive (the ability of succinate dehydrogenase to reconstitute with coenzyme ubiquinone-binding proteins (QPs) to form succinate-ubiquinone reductase) or succinate-phenazine methosulfate activity in the preparation, differs from antimycin-insensitive succinate-cytochrome c reductase activity detected in submitochondrial particles or isolated succinate-cytochrome c reductase. The Km for cytochrome c for the former is too high to be measured. The Km for the latter is about 4.4 microM, similar to that of antimycin-sensitive succinate-cytochrome c activity in isolated succinate-cytochrome c reductase, suggesting that antimycin-insensitive succinate-cytochrome c activity of succinate-cytochrome c reductase probably results from incomplete inhibition by antimycin. Like reconstitutive activity of succinate dehydrogenase, the antimycin-insensitive succinate-cytochrome c activity of succinate dehydrogenase is sensitive to oxygen; the half-life is about 20 min at 0 degrees C at a protein concentration of 23 mg/ml. In the presence of QPs, the antimycin-insensitive succinate-cytochrome c activity of succinate dehydrogenase disappears and at the same time a thenoyltrifluoroacetone-sensitive succinate-ubiquinone reductase activity appears. This suggests that antimycin-insensitive succinate-cytochrome c reductase activity of succinate dehydrogenase appears when succinate dehydrogenase is detached from the membrane or from QPs. Reconstitutively active succinate dehydrogenase oxidizes succinate using succinylated cytochrome c as electron acceptor, suggesting that a low potential intermediate (radical) may be involved. This suggestion is confirmed by the detection of an unknown radical by spin trapping techniques. When a spin trap, alpha-phenyl-N-tert-butylnitrone (PBN), is added to a succinate oxidizing system containing reconstitutively active succinate dehydrogenase, a PBN spin adduct is generated. Although this PBN spin adduct is identical to that generated by xanthine oxidase, indicating that a perhydroxy radical might be involved, the insensitivity of this antimycin-insensitive succinate-cytochrome c reductase activity to superoxide dismutase and oxygen questions the nature of this observed radical. 相似文献
128.
Resonance Raman evidence for the mechanism of the allosteric control of O2-binding in a cobalt-substituted monomeric insect hemoglobin. 下载免费PDF全文
The substitution of iron for cobalt in the monomeric insect hemoglobin CTT (Chironomus thummi thummi) III does not alter the Bohr effect for O2-binding. The cobalt substitution in this hemoglobin allows us to identify not only the O-O and Co-O2 stretching mode but also the Co-O-O bending mode by resonance Raman spectroscopy. The assignments were made via 16O2/18O2 isotope exchange. The modes associated with the Co-O-O moiety are pH-dependent. These pH-induced changes of the resonance Raman spectra are correlated with the t = r conformation transition. At high pH (high-affinity state) two unperturbed O-O stretching modes are observed at 1,068 cm-1 (major component) and 1,093 cm-1 (minor component) for the 18O2 complex. These frequencies correspond to split modes at 1,107 cm-1 and 1,136 cm-1 and an unperturbed mode at approximately 1,153 cm-1 for the 16O2 complex. At low pH (low-affinity state) the minor component becomes the major component and vice versa. The Co-O2 stretching frequency varies for approximately 520 cm-1 (pH 5.5) to 537 cm-1 (pH 9.5) indicating a stronger (hence shorter) Co-O2 bond in the high-affinity state. On the other hand, the O-O bond is weakened upon the conversion of the low- to the high-affinity state. The Co-O-O bending mode changes from 390 cm-1 (pH 9.5) to 374 cm-1 (pH 5.5). In the deoxy form the resonance Raman spectra are essentially pH-insensitive except for a vinyl mode at 414 cm-1 (pH 5.5), which is shifted to 416 cm-1 (pH 5.5). 相似文献
129.
Antimutagenic effect of garlic (Allium sativum L.) on 4NQO-induced mutagenesis in Escherichia coli WP2 总被引:1,自引:0,他引:1
Aqueous extract prepared from garlic bulbs markedly suppressed the mutagenesis in both E. coli WP2 trp- and E. coli WP2 trp- uvrA- induced by 4-nitroquinoline 1-oxide (4NQO), but not that induced by UV. Cellular toxicity, inhibition of the expression of the Trp+ phenotype and delay of the first cell division after 4NQO treatment were not observed in the presence of the extract. Since the extract showed identical antimutagenic effects against 4NQO in both test strains but no effect on the mutagenesis of UV, it seems that the extract might act by inactivating the electrophilic group(s) of 4NQO or inhibiting its metabolic activation. 相似文献
130.
Analysis of phylogenetic relations of durum,carthlicum and common wheats by means of comparison of alleles of gliadin-coding loci 总被引:1,自引:0,他引:1
E. V. Metakovsky A. M. Kudryavtsev Z. A. Iakobashvili A. Yu. Novoselskaya 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1989,77(6):881-887
Summary Polymorphism and inheritance of wheat storage protein, gliadin, of durum (macaroni) and carthlicum wheats have been studied. Analysis of gliadin in 78 cultivars and in F2 seeds of intercultivar crosses of durum wheat revealed three different chromosome 1A-encoded blocks of components similar to those found in common wheat (GLD1A2, GLD1A18, GLD1A19). Most of the durum cultivars studied had these three blocks; GLD1A2 was also frequent in common wheat. In contrast, all chromosome 1B-encoded blocks of durum clearly differed in component composition from those found in common wheat. Therefore, durum could not be an ancestor or a derivate of recent bread wheat. Analysis of gliadin in the collection of carthlicum wheat (14 accessions) revealed several suspected chromosome 1A, 1B, and 6A-controlled blocks, some of which were similar to those in common wheat, while others were different. Therefore, carthlicum is likely to be an ancestor or a derivate of some forms of bread wheat. There were also chromosome 1A and 6A-, but not 1B-encoded blocks which were identical in durum and carthlicum wheats. The results confirm that all three wheats share the same genome A, but emphasize the heterogeneity of genotypes among donors of this genome. Discovery of identical blocks in tetraploids and hexaploids indicates polyphyletic [from different genotypes of donor (s)] origin of these wheats. 相似文献