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131.
C A Libreros-Minotta J P Pardo G Mendoza-Hernández J L Rendón 《Archives of biochemistry and biophysics》1992,298(1):247-253
Glutathione reductase (NAD(P)H:GSSG oxidoreductase EC 1.6.4.2.) was purified 1160-fold to homogeneity from the nonsulfurous purple bacteria Rhodospirillum rubrum (wild type). Specific activity of the pure preparation was 102 U/mg. The enzyme displayed a typical flavoprotein absorption spectrum with maxima at 274,365, and 459 nm and an absorbance ratio A280/A459 of 7.6. The amino acid analysis revealed an unusually high content of glycine and arginine residues. Titration of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) showed a total of two free thiol groups per subunit, one of which is made accessible only under denaturing conditions. An isoelectric point of 5.2 was found for the native enzyme. Km values, determined at pH 7.5, were 6.1 and 90 microM for NADPH and GSSG, respectively. NADH was about 2% as active as NADPH as an electron donor. The enzyme's second choice in disulfide substrate was the mixed disulfide of coenzyme A and glutathione, for which the specific activity and Km values were 5.1 U/mg and 3.4 mM, respectively. A native molecular weight of 118,000 was found, while denaturing electrophoresis gave a value of 54,400 per subunit, thus suggesting that R. rubrum glutathione reductase exists as a dimeric protein. Other physicochemical constants of the enzyme, such as Stokes radius (4.2 nm) and sedimentation coefficient (5.71 S), were also consistent with a particle of 110,000. 相似文献
132.
The irreversible thermal unfolding of the class A beta-lactamase I from Bacillus cereus has been investigated at pH 7.0, using differential scanning calorimetry (DSC) and inactivation kinetic techniques. DSC transitions showed a single peak with a denaturation enthalpy of 646 kJ.mol-1 and were moderately scan rate dependent, suggesting that the process was partially kinetically controlled. The inactivation kinetics at constant temperature showed that the irreversible denaturation of the enzyme occurs as the sum of two exponential terms whose amplitudes are strongly temperature dependent within the transition range so that, at the lowest temperatures within this interval, irreversible inactivation would proceed mainly through the slow phase. The fraction of irreversibly denatured enzyme (D) as a function of temperature for a given scanning rate was calculated by numerical integration of the kinetic equation with temperature, using previously determined kinetic parameters. This D form was the most populated of the unfolded states only at temperatures well above the maximum in the calorimetric transition. Combination of the results of kinetic and DSC experiments has allowed us to separate the contribution of the final D state to the excess enthalpy change from the contribution arising from the reversibly denatured forms of the enzyme (I(i), i = 1,..., n), with the resulting conclusion that the scan rate dependence of the calorimetric traces was the result of two different dynamic effects, viz., the irreversible step and a slow relaxation process during formation of the reversibly denatured intermediate states. Finally, the problems of using results obtained at a single scan rate to validate the two-state kinetic model are commented on. 相似文献
133.
Quantitation of molybdopterin oxidation product in wild-type and molybdenum cofactor deficient mutants of Chlamydomonas reinhardtii. 总被引:1,自引:0,他引:1
A simple and reliable procedure of oxidation of molybdenum cofactor (MoCo) from molybdoenzymes by autoclaving samples at 120 degrees C for 20 min yielded a single predominant fluorescent species that could be quantitatively determined by reverse phase high performance liquid chromatography. This method allowed detection and quantitation of molybdopterin in cell-free extracts of the green alga Chlamydomonas reinhardtii. The MoCo oxidation product from C. reinhardtii has the same chromatographic and spectral properties as that of milk xanthine oxidase and chicken liver sulfite oxidase. The oxidized species was also detected in molybdenum cofactor mutants of Chlamydomonas reinhardtii defective at the nit-3, nit-4, nit-5/nit-6 and nit-7 loci, which strongly suggests that active molybdenum cofactor itself is not directly involved in the control of its own biosynthetic pathway. 相似文献
134.
The influence of retinol and retinoic acid, two retinoids of major interest, on the main gel to liquid-crystalline phase transition of different phospholipid membranes has been studied by means of differential scanning calorimetry. Both compounds exerted perturbing effects on the phase transition of membranes composed of dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylethanolamine. At concentrations up to 42.5 mol% of retinoid in the membrane, the delta H was not much affected with respect to the pure phospholipid, indicating a rather slight interaction. As the concentration of retinol was increased the Tc transition temperature decreased. A fluid-phase immiscibility was observed for the system DPPC/retinol at concentrations between 0 and 33 mol%. Almost ideal phase diagrams were obtained for the mixture DPPE/retinol. At concentrations of 33 mol% and higher retinol was able to induce phase separations in DPPC membranes, but not in DPPE. The effect of retinoic acid was much weaker, the Tc and delta H remaining almost unaltered and equal to that of the pure phospholipid up to concentrations of 30 mol%, at neutral pH. Retinoic acid exerted a pH-dependent effect. As the pH decreased, and therefore increased the extent of protonation of retinoic acid, the pertubation of the membrane induced by this compound was less. A strong effect, both on Tc and delta H, was observed at pH 10, where the retinoic acid moiety will be mainly unprotonated and the negative charge will generate repulsive forces thus destabilizing the membrane. The mixture DPPC/retinoic acid presents a region of fluid-phase immiscibility. At low pH, when the retinoic acid moiety was fully protonated, this fluid-immiscibility region extended from 0 to 36 mol% of retinoic acid, but its size decreased with increasing pH, and at pH 10 it was only found from 0 to 3 mol%. These results are discussed in terms of the possible retinoid/phospholipid interactions and the disposition of the retinoid moiety in the bilayer. 相似文献
135.
In unispecific plant stands, the logarithm of mean individual weight (w) depends on the logarithm of density (d) by the -3/4 power law (a slope of -1.5 and an intercept ranging from 2.3 to 5.0). The analysis of the w and d relationships in whole cohorts of two seaweed species from the Strait of Gibraltar shows deviations from the canonical equation. The kelp Phyllariopsis purpurascens (C. Agardh) Henry et South (Phaeophyta) growing at a 30-m depth has the lowest intercept value (0.6) recorded for any plant species and a slope not significantly different from -1.5. The slope value is in accordance with those found in species whose growth is not stopped by reproduction. Irradiance under a single layer of blades was lower than the photosynthetic light compensation point, and this could be due to overdispersion of the shoots and, in consequence, to the low intercept value of the self-thinning equation. The w to d relationships in Asparagopsis armata Harvey (Rhodophyta) show two different components: no dependence between these two variables (slope not significantly different from 0) at densities < 500 shoots·m?2, and a slope more negative (-2.1) than proposed by the -3/2 power law at densities > 500 shoots·m?2. The pattern at high densities could be due to intraspecific competition for light, whereas the slope ~0 at low densities could be related to inhibition of growth by reproduction (cystocarp and carpospore production). Therefore, rather than being considered exceptional, we suggest that a gradient of variability could be expected in the dependence of w on d when specific growth patterns and reproduction are considered. 相似文献
136.
Changes in the species composition, photosynthesis, calcification and size-fractionated carbon metabolism by natural phytoplankton assemblages were monitored in three mesocosms under different nutrient conditions during May 1993. In the 3 enclosures, the decline of the diatom-dominated assemblages was followed by the development of a bloom of the coccolithoporid Emiliania huxleyi. Highest growth of E. huxleyi was observed in the mesocosm with a high N : P ratio, suggesting this species is a good competitor at low phosphate concentrations. The transition from diatom- to E. huxleyi-dominated assemblages brought about a sharp reduction of the phytoplankton standing stock and carbon-specific photosynthetic rate. The relative contribution of the smaller size fraction to total photosynthesis increased as the succession progressed. Calcification rate and E. huxleyi cell-specified calcite production were highest during the early stages of development of the E. huxleyi bloom. Distinct changes in the patterns of 14C allocation into biomolecules were noticed during the diatom-E. huxleyi succession. The diatom-dominated assemblage showed high relative 14C incorporation into low molecular weight metabolites (LMWM), whereas proteins and, specially, lipids accounted for the largest proportion of carbon incorporation in the E. huxleyi bloom. The patterns of photoassimilated carbon metabolism proved to be strongly dependent on cellular size, as protein relative synthesis was significantly higher in the smaller than in the larger size fraction, irrespective of the nutrient regime and the successional stage. These results are discussed in relation to the ecological and physiological features of small phytoplankton. 相似文献
137.
Antonio Bode Benita Casas Emilio Fernández Emilio Marañón Pablo Serret Manuel Varela 《Hydrobiologia》1996,341(3):225-234
Summary Chlorophyll-a and primary production on the euphotic zone of the N-NW Spanish shelf were studied at 125 stations between 1984 and 1992. Three geographic areas (Cantabrian Sea, Rías Altas and Was Baixas), three bathymetric ranges (20 to 60 m, 60 to 150 m and stations deeper than 200 m), and four oceanographic stages (spring and autumn blooms, summer upwelling, summer stratification and winter mixing) were considered. One of the major sources of variability of chlorophyll and production data was season. Bloom and summer upwelling stages have equivalent mean and maximum values. Average chlorophyll-a concentrations approximately doubled in every step of the increasing productivity sequence: winter mixing — summer stratification — high productivity (upwelling and bloom) stages. Average primary production rates increased only 60% in the described sequence. Mean (± sd) values of chlorophyll-a and primary production rates during the high productivity stages were 59.7 ± 39.5 mg Chl-a m–2 and 86.9 ± 44.0 mg C m–2 h–1, respectively. Significant differences in both chlorophyll and primary production resulted between geographic areas in most stages. Only 27 stations showed the effects of the summer upwelling that affected coastal areas in the Cantabrian Sea and Rías Baixas shelf, but also shelf-break stations in the Rías Altas area. The Rías Baixas area had lower chlorophyll than both the Rías Altas and the Cantabrian Sea areas during spring and autumn blooms, but higher during summer upwelling events. On the contrary, primary production rates were higher in the Rías Baixas area during blooms in spring and autumn. Mid-shelf areas showed the highest chlorophyll concentrations during high productivity stages, probably due to the existence of frontal zones in all geographic areas considered. The estimated phytoplankton growth rates were comparable to those of other coastal upwelling systems, with average values lower than the maximum potential growth rates. Doubling rates for upwelling and stratification stages in the northern and Rías Altas shelf areas were equivalent, despite larger biomass accumulations during upwelling events. Low turnover rates of the existing biomass in the Rías Baixas shelf in upwelling stages suggests that the accumulation of phytoplankton was due mainly to the export from the highly productive rías, while the contribution of in situ production to these accumulations was relatively lower. 相似文献
138.
Plasmid DNA carrying either the nitrate reductase (NR) gene or the argininosuccinate lyase gene as selectable markers and the correspondingChlamydomonas reinhardtii mutants as recipient strains have been used to isolate regulatory mutants for nitrate assimilation by insertional mutagenesis. Identification of putative regulatory mutants was based on their chlorate sensitivity in the presence of ammonium. Among 8975 transformants, two mutants, N1 and T1, were obtained. Genetic characterization of these mutants indicated that they carry recessive mutations at two different loci, namedNrg1 andNrg2. The mutation in N1 was shown to be linked to the plasmid insertion. Two copies of the nitrate reductase plasmid, one of them truncated, were inserted in the N1 genome in inverse orientation. In addition to the chlorate sensitivity phenotype in the presence of ammonium, these mutants expressed NR, nitrite reductase and nitrate transport activities in ammonium-nitrate media. Kinetic constants for ammonium (14C-methylammonium) transport, as well as enzymatic activities related to the ammonium-regulated metabolic pathway for xanthine utilization, were not affected in these strains. The data strongly suggest thatNrg1 andNrg2 are regulatory genes which specifically mediate the negative control exerted by ammonium on the nitrate assimilation pathway inC. reinhardtii. 相似文献
139.
María E. Jiménez-Capdeville Tomás A. Reader Eduardo Molina-Holgado Robert W. Dykes 《Neurochemical research》1996,21(1):1-6
This study examined the effects of a nerve transection on monoamine release from primary somatosensory cortex. The technique
of microdialysis was employed to sample extracellular levels of norepinephrine (NE), 3,4-dihydroxyphenylacetic acid (DOPAC),
5-hydroxyindole-3-acetic acid (5-HIAA) and homovanillic acid (HVA) in the barrel field of freely moving rats following the
surgical transection of the contralateral infraorbital nerve. Microdialysates obtained 3, 4, and 5 days after deafferentation
were analyzed using high-performance liquid chromatography with electrochemical detection. We found a significant increase
in the release of the dopamine metabolites, DOPAC and HVA from the deafferented cortex. Three days after deafferentation the
release of DOPAC was three-fold higher in the deafferented than in the control animals, and remained about 100% higher in
the next two days in this group of animals. The release of HVA showed a gradual increase following the deafferentation procedure,
since a 92% larger value on day 3 increased to a 338% difference on day 5. On the other hand, the release rate of NE and the
levels of the serotonin metabolite 5-HIAA were not significantly affected by the deafferentation procedure. These results
are discussed in the context of the possible participation of dopamine in the reorganization of the deafferented somatosensory
cortex. 相似文献
140.
Ma Rosario de la Torre Angela Casado Ma Encarnación López-Fernández Diana Carrascosa Ma Concepción Casado Domenico Venarucci Vincenzo Venarucci 《Neurochemical research》1996,21(8):885-888
In order to investigate the role of two free radical detoxificant enzymes in patients with aging brain disorders, superoxide
dismutase (SOD) and catalase (CAT) activities have been measured in blood from male and female human patients of different
ages with several types of aging brain disorders. When compared with activities in the normal population, we have detected:
1) SOD and CAT activities are decreased in patients with Parkinson disease. 2) SOD activity seems to be normal and CAT activity
is decreased in patients with dementia. 3) In the patients with stroke, SOD activity is normal, while CAT activity is decreased.
SOD activity was measured in red blood cells using the Minami and Yoshikawa method. CAT activity was measured in hemolysates
by the method of Aebi. We can conclude that SOD and CAT activities in patients with Parkinson disease are decreased. 相似文献