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991.
Analysis of the chloroplast protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE) 总被引:5,自引:0,他引:5
Kügler Marion Jänsch Lothar Kruft Volker Schmitz Udo K. Braun Hans-Peter 《Photosynthesis research》1997,53(1):35-44
Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful procedure for the separation and characterization of the protein complexes from mitochondria. Membrane proteins are solubilized in the presence of aminocaproic acid and n-dodecylmaltoside and Coomassie-dyes are utilized before electrophoresis to introduce a charge shift on proteins. Here, we report a modification of the procedure for the analysis of chloroplast protein complexes. The two photosystems, the light-harvesting complexes, the ATP synthase, the cytochrome b
6
f complex and the ribulose-bisphosphate carboxylase/oxygenase are well resolved. Analysis of the protein complexes on a second gel dimension under denaturing conditions allows separation of more than 50 different proteins which are part of chloroplast multi-subunit enzymes. The resolution capacity of the blue-native gels is very high if compared to 'native green gel systems' published previously. N-terminal amino acid sequences of single subunits can be directly determined by cyclic Edman degradation as demonstrated for eight proteins. Analysis of chloroplast protein complexes by blue-native gel electrophoresis will allow the generation of 'protein maps' from different species, tissues and developmental stages or from mutant organelles. Further applications of blue-native gel electrophoresis are discussed. 相似文献
992.
993.
994.
CLAUDIO PEREIRA CRISTINA PAVETO JOAQUIN ESPINOSA GUILLERMO ALONSO MIRTHA M. FLAWÁ HECTOR N. TORRES 《The Journal of eukaryotic microbiology》1997,44(2):155-156
ABSTRACT. Trypanosoma cruzi epimastigote motility can be enhanced by addition of L-arginine, to the culture. This effect is blocked by N-methyl-L-arginine, a competitive inhibitor of the nitric oxide synthase. N-methyl-D-aspartate and L-glutamate, two agonists of the NMDALglutamate receptor, also enhanced motility. This stimulation is blocked by MK-801 a noncompetitive antagonist of the NMDA receptor. In addition, sodium nitroprusside, a guanylyl cyclase stimulator and 8-Br-cyclic GMP, an analog of cyclic GMP, also stimulated epimastigote motility. It is suggested that an increase of intracellular cyclic GMP levels mediated by nitric oxide may be responsible for the increase in epimastigote motility. 相似文献
995.
Ernst H. Oliw Lena Hrnsten Howard Sprecher 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1997,690(1-2):332-337
Prostaglandin H synthase-1 of ram vesicular glands metabolises 5,8,11-eicosatrienoic (Mead) acid to 13R-hydroxy-5,8,11-eicosatrienoic and to 11R-hydroxy-5,8,12-eicosatrienoic in a 5:1 ration. We wanted to determine the metabolism of this fatty acid by prostaglandin H synthase-2. Western blot showed that microsomes of sheep and rabbit placental cotyledons contained prostaglandin H synthase-2, while prostaglandin H synthase-1 could not be detected. Microsomes of sheep cotyledons metabolised [1-14C]5,8,11-eicosatrienoic acid to many polar metabolites and diclofenac (0.05 mM) inhibited the biosynthesis. The two major metabolites were identified as 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids. They were formed in a ratio of 3:2, which was not changed by aspirin (2 mM). 5,8,11-Eicosatrienoic acid is likely oxygenated by removal of the pro-S hydrogen at C-13 and insertion of molecular oxygen at either C-13 or C-11, which is followed by reduction of the peroxy derivatives to 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids, respectively. Prostaglandin H synthase-1 and -2 oxygenate 5,8,11-eicosatrienoic acid only slowly compared with arachidonic acid. 相似文献
996.
Hyperthyroidism enhances the prooxidant activity of the liver by elevating superoxide radical and/or hydrogen peroxide generation in microsomal, mitochondrial, and peroxisomal fractions, with an increased respiratory burst of Kupffer cells. In this study, the influence of daily doses of 0.1 mg 3,3′,5-triiodothyronine (T3)/kg for three consecutive days on liver nitric oxide (NO) synthase (NOS) was assessed, as a possible contributory mechanism to T3-induced liver prooxidant activity. Thyroid calorigenesis was paralleled by a progressive increment in the rate of NO generation, with significant increases after 2 (47%) and 3 days (70%) of T3treatment, and a net 45% (P< 0.05) enhancement in theNG-methyl-l-arginine-sensitive NO production, compared to control values. These enhancement effects were reversed to control levels after 3 days of hormone withdrawal, concomitantly with the normalization of hepatic respiration. Enhancement of liver NOS activity in hyperthyroid animals was diminished by 27% (P< 0.05) by the selectivein vivoinactivation of Kupffer cells by gadolinium chloride (GdCl3), without direct actions of GdCl3on the enzyme. These data demonstrate that hyperthyroidism leads to a significant and reversible enhancement in rat liver NOS activity, an effect that is exerted at hepatocyte and Kupffer cell levels, thus representing an additional source of prooxidants to those of reactive oxygen species. 相似文献
997.
998.
L. Ge J. Z. Liu W. S. Wong W. L. W. Hsiao K. Chong Z. K. Xu S. F. Yang S. D. Kung N. Li 《Plant, cell & environment》2000,23(11):1169-1182
Many abiotic environmental factors elicit the production of stress‐ethylene in higher plants. To elucidate the molecular mechanisms underlying the regulation of stress‐ethylene production and the physiological roles played by stress‐ethylene in stress responses of plants, we studied the gene expression of ACC synthase in tobacco plants that had been subjected to environmental stresses. Four new tobacco ACC synthase cDNA fragments, NT‐ACS2, NT‐ACS3, NT‐ACS4 and NT‐ACS5, were identified and sequenced. It was found that NT‐ACS2 could be induced by wounding, cold temperature and, especially, sunlight. NT‐ACS4 was induced at a faster kinetics by wounding. The multiple environmental stress‐responsive (MESR) NT‐ACS2 gene was found to contain three introns and four exons and encode a polypeptide of 484 amino acids, 54·6 kDa and pI 6·87. Computer analysis of the 3·4 kb 5 ′ flanking region upstream of the ACS coding region revealed the existence of a group of putative cis‐acting regulatory elements potentially conferring wounding, chilling, and UV light inducibility. Phylogenetic analysis of ACC synthase genes of different plant origins indicated that the chill‐inducible NT‐ACS2 gene is closely related to a chilling‐inducible citrus ACS gene. 相似文献
999.
Graciela L. Salerno 《Plant science》1985,42(1):5-8
The presence of sucrose and the enzymes related to sucrose metabolism, i.e. sucrose synthase (SS) (UDP-glucose: D-fructose-2-glucosyl transferase, EC 2.4.1.13), sucrose phosphate synthase (SPS) (UDP-glucose: D-fructose-6-phosphate-2-glucosyl transferase, EC 2.4.1.14) and invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) was demonstrated in Prototheca zopfii, a colorless alga. The levels of enzyme activities were lower than those obtained in Chlorella vulgaris, which is generally considered the photosynthetic counterpart of P. zopfii. Whem enzyme activities were measured in bleached cells of C. vulgaris, the levels were of the same order than those found in P. zopfii. These results would indicate that the sucrose metabolizing enzymes are not related to the algae ability to carry on photosynthesis. 相似文献
1000.
Illuminated intact pea chloroplasts in the presence of O-acetylserine (OAS) catalysed incorporation of SeO32- and SO32- into selenocysteine and cysteine at rates of ca 0.36 and 6 μmol/mg Chl per hr respectively. Sonicated chloroplasts catalysed SeO32- and SO32- incorporation at ca 3.9 and 32% respectively of the rates of intact chloroplasts. Addition of GSH and NADPH increased the rates to ca 91 and 98% of the intact rates, but SeO32- incorporation under these conditions was essentially light-independent. In the absence of OAS, intact chloroplasts catalysed reduction of SO32- to S2- at rates of ca 5.8 μmol/mg Chl per hr. In the presence of OAS, S2- did not accumulate. Glutathione (GSH) reductase was purified from peas and was inhibited by ZnCl2. This enzyme, in the presence of purified clover cysteine synthase, OAS, GSH and NADPH, catalysed incorporation of SeO32- into selenocysteine (but not SO32- into cysteine). The reaction was inhibited by ZnCl2. Incorporation of SeO32- into selenocysteine by illuminated intact chloroplasts and sonicated chloroplasts (with NADPH and GSH) was also inhibited by ZnCl2 but not by KCN. Conversely, incorporation of SO32- into cysteine was inhibited by KCN but not by ZnCl2. It was concluded that SeO32- and SO32- are reduced in chloroplasts by independent light-requiring mechanisms. It is proposed that SeO32- is reduced by light-coupled GSH reductase and that the Se2- produced is incorporated into selenocysteine by cysteine synthase. 相似文献