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41.
The efflux of K+ and Na+ from sea urchin eggs during Ca2+ ionophore A23187-induced parthenogenesis was studied in a K+ and Na+-free artificial seawater using extracellular ion-specific electrodes. We have probed this model system with monovalent cation-specific ionophores to determine if they affect K+ efflux in the unfertilized egg and whether any changes in ionophore sensitivity are observed during egg activation. In 500 mM choline chloride, 10 mM CaCl2, 50 mM MgCl2, 10 mM Tris-Cl pH 8.0, A23187 induced a rapid efflux of K+ and Na+ from the eggs after a short lag time (10–15 seconds). After the burst, the rate of K+ efflux remained higher than the pre-activation rate, but was lower than during the burst phase, while the rate of Na+ efflux became nearly zero. Monovalent cation-specific ionophores (valinomycin, gramicidin and nigericin) had no effect on K+ efflux from the unfertilized eggs in our model system. However, once the egg was activated by A23187, each of the above ionophores caused a prolongation of the burst phase for many minutes. These results show that the unfertilized egg plasma membrane (using our artificial conditions) is not susceptible to the monovalent cation-specific antibiotics and suggest that either the inserted cortical granule membrane or the developing fertilization envelope interacts with these ionophores to cause the change in rate-limiting step for K+ efflux observed egg activation. 相似文献
42.
苜蓿二磷酸核酮糖(RuBP)羧化酶体内活化作用的调节 总被引:6,自引:0,他引:6
苜蓿RuBP羧化酶的初活性和活化作用在不饱和光强下与光合速率一样随光强增加而增加。缺硫培养苜蓿叶片的光合速率和RuBP羧化酶的含量、初活性及总活性均比对照有不同程度的降低,其中酶的初活性与光合速率两者减少的趋势比较接近,说明RuBP羧化酶的初活性可能在光合CO_2固定作用中具有决定作用。然而,缺硫植株中酶的活化作用比对照明显增高。酶的活化作用与叶片中的叶绿素,6-PG,NADPH及ATP相对酶含量的比值成正比,与体内的酶量成反比。 相似文献
43.
CNBr cleavage of unreduced proenzyme Clr yielded fragment CP2b, isolated by gel filtration and highpressure gel permeation chromatography. This fragment (˜ Mτ 55000) comprised at least 4 disulphidelinked peptides, which were separated by gel filtration after reduction and alkylation. Peptide CP2bRA4, overlapping the A- and B-chain regions in proenzyme Clr was digested by V8 staphylococcal protease, and the digest separated by reversed-phase HPLC. N-terminal sequence analysis of peptide CP2bRA4SP9 established that Clr activation involves the cleavage of a single Arg-Ile bond, located in the sequence: Gln-Arg-Gln-Arg-Ile-Ile-Gly-Gly 相似文献
44.
The light energy requirements for photoactivation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were studied in a reconstituted chloroplast system. This system comprised isolated pea thylakoids, ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) thioredoxinm and f (Tdm, Tdf) and the photoactivatable enzyme. Light-saturation curves of the photoactivation process were established with once washed thylakoids which did not require the addition of Td for light activation. They exhibited a plateau at 10 W·m–2 under nitrogen and 50 W·m–2 under air, while NADP photoreduction was saturated at 240 W·m–2. Cyclic and pseudocyclic phosphorylations saturated at identical levels as enzyme photoactivations. All these observations suggested that the shift of the light saturation plateau towards higher values under air was due to competing oxygen-dependent reactions. With twice washed thylakoids, which required Td for enzyme light-activation, photophosphorylation was stimulated under N2 by the addition of the components of the photoactivation system. Its rate increased with increasing Td concentrations, just as did the enzyme photoactivation rate, while varying the target enzyme concentration had only a weak effect. Considering that Td concentrations were in a large excess over target enzyme concentrations, it may be assumed that the observed ATP synthesis was essentially dependent on the rate of Td reduction.Under air, Fd-dependent pseudo-cyclic photophosphorylation was not stimulated by the addition of the other enzyme photoactivation components, suggesting that an important site of action of O2 was located at the level of Fd.Abbreviations Fd
ferredoxin
- FBPase
fructose-1,6-bisphosphatase
- FTR
ferredoxin-thioredoxin reductase
- LEM
light effect mediator
- NADP-MDH
NADP-malate dehydrogenase
- Td
thioredoxin 相似文献
45.
46.
The metabolism of N-methyl substituted 7H-dibenzo[c,g]carbazole (N-Me DBC) was investigated in vitro using liver microsomes from 3-methylcholanthrene (MC)-, benzo[c]carbazole (BC) and Arochlor-pretreated mice and rats. N-Me DBC is a potent sarcomatogen devoid of hepatotoxicity and liver carcinogenic activity. The ethyl acetate-extractable metabolites were separated by high performance liquid chromatography (HPLC) and most of them were identified by proton magnetic resonance (PMR), mass spectrometry (MS) and comparison with synthetically prepared specimens. Mouse and rat microsomes gave rise to the same metabolites. The major metabolites were 5-OH-N-Me DBC (50%), N-hydroxymethyl (HMe) DBC (25-30%) and 3-OH-N-Me DBC (10%). Addition of 1,1,1-trichloropropene-2,3-oxide (TCPO) to the standard incubation medium permitted the identification of two dihydrodiols among the minor metabolites. No metabolite of DBC was observed after incubation of N-Me DBC, or its major metabolite N-HMe DBC, with either mouse or rat microsomes, but the possibility of a slight demethylation cannot be totally excluded. The lack of biotransformation at the nitrogen atom site may explain the lack of hepatotoxicity and liver carcinogenic activity of N-Me DBC. The modulation of metabolism by epoxide hydrolase, cytosol and glutathione was also investigated. The results are discussed in the light of data previously obtained with hepatotoxic and hepatocarcinogenic DBC. 相似文献
47.
Electrophoretic analysis of polyphenoloxidase isoenzymes from a variety of angiosperms and from mushroom revealed that the enzymes remain active in the presence of 0.1 % sodium dodecylsulfate. Electrophoresis in the presence of sodium dodecylsulfate allows the detection of latent enzyme forms of polyphenoloxidase, and can also convert slower migrating enzyme forms to faster migrating forms. Electrophoresis in the absence of sodium dodecylsulfate followed by incubation in the presence of sodium dodecylsulfate can also be used to detect latent forms of polyphenoloxidase. Together, these approaches provide a method for screening latent enzymes and give some insight into the mechanism of activation by sodium dodecylsulfate. 相似文献
48.
Bryan B. Fuller 《In vitro cellular & developmental biology. Plant》1987,23(9):633-640
Summary Tyrosinase activity increased in Cloudman S-91 mouse melanoma cell homogenates incubated at 37°C for a minimum of 8 h. Enzyme
activity continued to increase for 48h at which time the maximal level of activation was observed. Activation did not occur
at 4°C and did not occur in the cytosol fraction of the cell, suggesting that the response was localized to melanosomes. The
activated enzyme was resistant to solubilization with the nonionic detergent, Triton X-100, and preparation of homogenates
in this detergent did not inhibit the temperature-dependent activation of the melanosomal fraction of the cell. The activation
process increased the V
Max
of tyrosinase 10-fold and lowered the K
M
by a factor of 2 as determined by the tyrosine hydroxylase assay. The increase in tyrosinase activity was detectable by three
assay methods: tyrosine hydroxylation, melanin synthesis, and by tyrosine decarboxylation. The formation of melanin, however,
was found to be 1/20 that of either tyrosine hydroxylation or decarboxylation, a finding which suggests that the melanin pathway
may be blocked at 5,6-dihydroxyindole. The “self-activation” response could not be mimicked by incubating cell homogenates
with cyclic AMP-dependent protein kinase. Activated tyrosinase could be inhibited by the addition of fresh cell extracts,
a finding which suggests that tyrosinase inhibitors may be present in these cells.
This investigation was supported by Public Health Service grants CA41425 and CA30393 awarded by the National Cancer Institute,
Bethesda, MD and by a research grant from the Proctor and Gamble Company. 相似文献
49.
J. R. Evans 《Planta》1986,167(3):351-358
Photosynthesis in two cultivars of Triticum aestivum was compared with photosynthesis in two lines having the same nuclear genomes but with cytoplasms derived from T. boeoticum. The in-vitro specific activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) isolated from lines with T. boeoticum cytoplasm was only 71% of that of normal T. aestivum. By contrast, the RuBPCase activities calculated from the CO2-assimilation rate at low partial pressures of CO2, p(CO2), were the same for all lines for a given RuBPCase content. This indicates that both types of RuBPCase have the same turnover numbers in-vivo of 27.5 mol CO2·(mol enzyme)–1·s–1 (23°). The rate of CO2 assimilation measured at normal p(CO2), p
a
=340 bar, and high irradiance could be quantitatively predicted from the amount of RuBPCase protein. The maximum rate of RuBP regeneration could also predict the rate of CO2 assimilation at normal ambient conditions. Therefore, the maximum capacities for RuBP carboxylation and RuBP regeneration appear to be well-balanced for normal ambient conditions. As photosynthetic capacity declined with increasing leaf age, the capacities for RuBP carboxylation and RuBP regeneration declined in parallel.Abbreviations PAR
photosynthetically active radiation
- RuBP(Case)
ribulose-1,5-bisphosphate (carboxylase) 相似文献
50.
Lipid activation data for (Ottolenghi, P. (1979) Eur. J. Biochem. 99, 113–131) have been subjected to a regression and fitting analysis based on a recent kinetic model (Sandermann, H. (1982) Eur. J. Biochem, 127, 123–128). The observed kinetic cooperativity could be generated from strictly non-cooperative binding events involving the known number of 30 boundary lipid-binding sites per ATPase monomer. Apparent lipid dissociation equilibrium constants of between 0.3 and 5 μM were obtained, enzyme activity being associated only with the fully lipid-substituted enzyme and enzyme-lipid complexes with less than six unoccupied lipid-binding sites. The enzyme appeared to operate close to a maximum of cooperativity. 相似文献