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991.
Localization of xanthine oxidoreductase activity using the tissue protectant polyvinyl alcohol and final electron acceptor Tetranitro BT 总被引:3,自引:0,他引:3
A Kooij W M Frederiks R Gossrau C J Van Noorden 《The journal of histochemistry and cytochemistry》1991,39(1):87-93
We have detected xanthine oxidoreductase activity in unfixed cryostat sections of rat and chicken liver, rat duodenum, and bovine mammary gland using the tissue protectant polyvinyl alcohol, the electron carrier 1-methoxyphenazine methosulfate, the final electron acceptor Tetranitro BT, and hypoxanthine as a substrate. Enzyme activity was localized in rat duodenum at lateral membranes and brush borders of enterocytes and in goblet cells and mucus. Hepatocytes in pericentral areas and especially sinusoidal cells showed high activity in rat liver. Xanthine oxidoreductase was also detected in epithelial cells and milk lipid globules of lactating bovine mammary gland, which is known to contain large quantities of the oxidase form of the enzyme. Chicken liver, which contains an inconvertible dehydrogenase form, also showed high activity in sinusoidal cells. Therefore, we conclude that the tetrazolium reaction demonstrates both the dehydrogenase and the oxidase form of xanthine oxidoreductase. Control activity, in the absence of hypoxanthine or in the presence of the competitive inhibitor allopurinol, was low in all tissues studied. Addition of O2 or NAD to the incubation medium did not change the specific reaction in bovine mammary gland or chicken liver, implying that the dehydrogenase and the oxidase form are not dependent on their natural electron acceptors in this tetrazolium salt reaction. We conclude that the present light microscopic method gives specific and precise localization of xanthine oxidoreductase activity in situ. 相似文献
992.
F Chirat A Martinage G Briand M Kouach A Van Dorsselaer M Loir P Sautière 《European journal of biochemistry》1991,198(1):13-20
The ram transition protein 1 (TP1) is present in spermatid cell nuclei in the nonphosphorylated, monophosphorylated and diphosphorylated forms. Its primary structure was determined by automated Edman degradation of S-carboxamidomethylated protein and of peptides generated by cleavage with thermolysin and endoproteinase Lys-C. The ram TP1 is a small basic protein of 54 residues and structurally very close to other mammalian TP1. The mass spectrometric data obtained from the protein and its fragments reveal that ram TP1 is indeed a mixture (approximately 5:1) of two structural variants (Mr 6346 and 6300). These variants differ only by the nature of the residue at position 27 (Cys in the major variant and Gly in the minor variant). The study of phosphorylation sites has shown that four different serine residues could be phosphorylated in the monophosphorylated TP1, at positions 8, 35, 36 or 39. From previous physical studies, it has been postulated that the Tyr32 surrounded by two highly conserved basic clusters was responsible for the destabilization of chromatin by intercalation of its phenol ring between the bases of double-stranded DNA. The presence of three phosphorylatable serine residues in the very conserved sequence 29-42 is another argument for the involvement of this region in the interaction with DNA. 相似文献
993.
F J Van de Ven H W Van den Hooven R N Konings C W Hilbers 《European journal of biochemistry》1991,202(3):1181-1188
Nisin is a posttranslationally modified protein of 34 amino acids, and is a member of the class of bacteriocidal polypeptides known as lantibiotics, that contain the unusual amino acid lanthionine. Its structure in aqueous solution has been determined on the basis of NMR data, i.e. interproton distance constraints derived from nuclear Overhauser enhancement spectroscopy and torsion angle constraints derived from double-quantum-filtered correlated spectroscopy. Translation of the NMR constraints into a three-dimensional structure was carried out with the distance-geometry program DISMAN, followed by restrained energy minimization using CHARMm. The internal mobility of the peptide chain prohibited the determination of a precise overall folding of the molecule, but parts of the structure could be obtained, albeit sometimes with low resolution. The structure of nisin can best be defined as follows. The outermost N-terminal and C-terminal regions of nisin appear quite flexible, the remainder of the molecule consists of an amphiphilic N-terminal fragment (residues 3-19), joined by a flexible 'hinge' region to a rigid double-ring fragment formed by residues 23-28. The latter fragment has the appearance of a somewhat overwound alpha-helix. It is suggested, by assuming the presence of a (transient) alpha-helical structure in this part of prenisin, that the coupling between residues 23 and 26, as well as between 25 and 28, by thioether bridges, and the inversion of the C alpha chiralities at positions 23 and 25, can be rationalized. 相似文献
994.
A. A. W. M. Van Loon P. J. Den Boer G. P. Van Der Schans P. Mackenbach J. A. Grootegoed R. A. Baan P. H. M. Lohman 《Experimental cell research》1991,193(2)
An immunochemical method has been used to detect quantitatively DNA damage caused by ionizing radiation in germ cells. With this method, DNA strand breaks as well as lesions converted into breaks in alkaline medium are measured as a function of controlled partial unwinding of the DNA, a time-dependent process starting at each breakage site, followed by the determination of the relative amount of single-stranded regions by use of a single-strand specific monoclonal antibody. With this method the induction and repair of DNA damage in different cellular stages of spermatogenesis (spermatocytes, round and elongated spermatids) of the hamster were investigated. Germ cells were irradiated in vitro with 60Co-γ-rays, at doses between 0 and 5 Gy. A linear dose-response relationship was observed. Spermatocytes and round spermatids had normal, fast repair of the lesions when compared with the repair of these sites in cultured V79 or CHO cells and human lymphocytes. The elongated spermatids, however, showed hardly any repair. Similar results were obtained after the in vivo γ-irradiation of hamsters with doses of 0, 4, and 8 Gy and subsequent isolation of germ cells. The damage was still detectable in the elongated spermatids at 24 h after exposure. The results of the experiments show substantial differences in repair capacity between different stages of germ cell development. Because DNA is the major target for mutation induction, this assay may be useful for assessment of the genetic risk of exposure of male germ cells to ionizing radiation, in relation to the stage of development. 相似文献
995.
E Prinsen N Chauvaux J Schmidt M John U Wieneke J De Greef J Schell H Van Onckelen 《FEBS letters》1991,282(1):53-55
Flavonoids activate nod gene expression in Rhizobium resulting in the synthesis of Nod signals which trigger organogenesis in the host plant. This paper shows that nod-inducers also stimulate the production of the phytohormone IAA (indole-3-acetic acid). 相似文献
996.
A B Van Kuilenburg H L Dekker C Van den Bogert P Nieboer B F Van Gelder A O Muijsers 《European journal of biochemistry》1991,199(3):615-622
The subunit pattern and the steady-state kinetics of cytochrome-c oxidase from human heart, muscle, kidney and liver were investigated. Polyacrylamide gel electrophoresis of immunopurified cytochrome-c oxidase preparations suggest that isoforms of subunit VIa exist, which show differences in staining intensity and electrophoretic mobility. No differences in subunit pattern were observed between the other nucleus-encoded subunits of the various cytochrome-c oxidase preparations. Tissue homogenates, in which cytochrome-c oxidase was solubilised with laurylmaltoside, were directly used in the assays to study the cytochrome-c oxidase steady-state kinetics. Cytochrome-c oxidase concentrations were determined by immunopurification followed by separation and densitometric analysis of subunit IV. When studied in a medium of low ionic strength, the biphasic kinetics of the steady-state reaction between human ferrocytochrome c and the four human cytochrome-c oxidase preparations revealed large differences for the low-affinity TNmax (maximal turnover number) value, ranging from 77 s-1 for kidney to 273 s-1 for liver cytochrome-c oxidase at pH 7.4, I = 18 mM. It is proposed that the low-affinity kinetic phase reflects an internal electron-transfer step. For the steady-state reaction of human heart cytochrome-c oxidase with human cytochrome c, Km and TNmax values of 9 microM and 114 s-1 were found, respectively, at high ionic strength (I = 200 mM, pH 7.4). Only minor differences were observed in the steady-state activity of the various human cytochrome-c oxidases. The interaction between human cytochrome-c oxidase and human cytochrome-c proved to be highly specific. At high ionic strength, a large decrease in steady-state activity was observed when reduced horse, rat or bovine cytochrome c was used as substrate. Both the steady-state TNmax and Km parameters were strongly affected by the type of cytochrome c used. Our findings emphasize the importance of using human cytochrome c in kinetic assays performed with tissues from patients with a suspected cytochrome-c oxidase deficiency. 相似文献
997.
The Bacillus subtilis cytochrome-c oxidase. Variations on a conserved protein theme. 总被引:16,自引:0,他引:16
M Saraste T Metso T Nakari T Jalli M Lauraeus J Van der Oost 《European journal of biochemistry》1991,195(2):517-525
The structural genes of cytochrome-c oxidase in Bacillus subtilis have been isolated and sequenced. Five genes, ctaB-F, are closely spaced. ctaC, ctaD, ctaE and ctaF are the genes for subunits II, I, III and IVB, respectively, ctaB, which may encode an assembly factor, is separated and upstream from the others. In comparison to its mitochondrial counterparts, subunit I has an extended C-terminus with two additional transmembrane segments, whereas subunit III has lost two such segments from its N-terminus. The C-terminal extension in subunit II is a covalent cytochrome-c domain, previously characterized only in the thermophilic oxidases. Subunit IVB, a small hydrophobic protein, is a novel subunit. These predictions suggest that the B. subtilis cytochrome-c oxidase is structurally more related to the four-subunit Escherichia coli cytochrome-bo complex than, for instance, to the Paracoccus denitrificans enzyme. Cytochrome aa3, which was previously isolated from B. subtilis [de Vrij, W., Azzi, A. & Konings, W. N. (1983) Eur. J. Biochem. 131, 97-103] is not encoded by the ctaC-F genes; thus, there seems to be two different cytochrome-aa3-type oxidases in this Gram-positive bacterium. 相似文献
998.
The segregation of the 75K gamma secalin locus (Sec-2) in combination with five interchanges (reciprocal translocations) and two marker genes was analyzed. The translocations involved chromosome arms 1RL, 1RS, 2RL, 2RS, 4RL, 5RL, 5RS, 6RL and 6RS. The gene loci were both on 2R, but the arm was not known. Although the Sec-2 locus was expected to be on chromosome 2RS, no linkage between Sec-2 and any of the markers was found. This is concluded to be the result of exceptionally frequent recombination between Sec-2 and the break point of one of the translocations, which is the only marker in 2RS. 相似文献
999.
Isolation and characterization of the moxJ, moxG, moxI, and moxR genes of Paracoccus denitrificans: inactivation of moxJ, moxG, and moxR and the resultant effect on methylotrophic growth.
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R J Van Spanning C W Wansell T De Boer M J Hazelaar H Anazawa N Harms L F Oltmann A H Stouthamer 《Journal of bacteriology》1991,173(21):6948-6961
By using the moxF gene encoding the large fragment of methanol dehydrogenase as a probe, a downstream linked chromosomal fragment was isolated from a genomic bank of Paracoccus denitrificans. The nucleotide sequence of the fragment was determined and revealed the 3' part of moxF, four additional open reading frames, and the 5' part of a sixth one. The organization and deduced amino acid sequences of the first three frames downstream from moxF were found to be largely homologous to the moxJ, moxG, and moxI gene products of Methylobacterium extorquens AM1. Directly downstream from these three genes, a new mox gene was identified. The gene is designated moxR. By using the suicide vector pGRPd1, the moxJ, moxG, and moxR genes were inactivated by the insertion of a kanamycin resistance gene. Subsequently, suicide vector pRVS1 was used to replace the marker genes in moxJ and moxG for unmarked deletions made in vitro. As a result, the three insertion strains as well as the two unmarked mutant strains were unable to grow on methanol, even in the presence of pyrroloquinoline quinone. Growth on succinate and on methylamine was not affected. In all five mutant strains, synthesis of the large subunit of methanol dehydrogenase and of inducible cytochrome c553i was observed. The moxJ and moxG insertion mutant strains were unable to synthesize both the cytochrome c551i and the small subunit of methanol dehydrogenase, and this lack of synthesis was attended by the loss of methanol dehydrogenase activity. The moxJ deletion mutant strain partly synthesized the latter two proteins, cytochrome c551i. Partial synthesis of the small subunit of methanol dehydrogenase observed with the latter strain was attended by a corresponding extent of methanol dehydrogenase activity. The moxR insertion mutant strain was shown to synthesize cytochrome c551i as well as the large and small subunits of methanol dehydrogenase, but no methanol dehydrogenase activity was observed. The results show that periplasmic cytochrome c551i is the moxG gene product and the natural electron acceptor of methanol dehydrogenase in P. denitrificans. In contrast to earlier suggestions, this cytochrome was found to be different from membrane-bound cytochrome c552. In addition, it is demonstrated that moxI encodes the small subunit of methanol dehydrogenase. It is suggested that MoxJ is involved in the assemblage of active methanol dehydrogenase in the periplasm and, in addition, that MoxR is involved in the regulation of formation of active methanol dehydrogenase. 相似文献
1000.
Summary The transepithelial water permeability in frog urinary bladder is believed to be essentially dependent on the ADH-regulated apical water permeability. To get a better understanding of the transmural water movement, the diffusional water permeability (P
d) of the basolateral membrane of urinary bladder was studied. Access to this post-luminal barrier was made possible by perforating the apical membrane with amphotericin B. The addition of this antibiotic increasedP
d from 1.12±0.10×10–4 cm/sec (n=7) to 4.08±0.33×10–4 cm/sec (n=7). The effect of mercuric sulfhydryl reagents, which are commonly used to characterize water channels, was tested on amphotericin B-treated bladders. HgCl2 (10–3
m) decreasedP
d by 52% andpara-chloromercuribenzoic acid (pCMB) (1.4×10–4
m) by 34%. The activation energy for the diffusional water transport was found to increase from 4.52±0.23 kcal/mol (n=3), in the control situation, to 9.99±0.91 kcal/mol (n=4) in the presence of 1.4×10–4
m
pCMB. Our second approach was to measure the kinetics of water efflux, by stop-flow light scattering, on isolated epithelial cells from urinary bladders.pCMB (0.5 or 1.4×10–4
m) was found to inhibit water exit by 91±2%. These data strongly support the existence of proteins responsible for water transport across the basolateral membrane, which are permanently present. 相似文献