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1.
In an electrophoretic survey of lactate dehydrogenase (LDH) isozymes in neotropical cichlid fishes (Perciformes: Cichlidae) several species were discovered in which a cathodal liver-restricted isozyme is expressed along with the highly anodal eye-restricted isozyme (LDH-C4) typically encountered in perciform fishes. Biochemical characterization of these two isozymes from the basketmouth cichlid, Acaronia nassa (Heckel), strongly suggested that they were non-orthologous and challenged the accepted view that eye- and liver-restricted LDH isozymes are alternative expressions of the same (LDH-C *) gene. In this study, antiserum raised against cypriniform (goldfish) liver-restricted LDH-C4 failed to cross-react with the basketmouth liver-restricted analogue while effectively titrating the eye-restricted, anodal isozyme and, at higher titres, the LDH-B4, heart-restricted isozyme from all cichlid species. Anti-serum raised against basketmouth muscle-restricted LDH-A4 failed to titrate any of the eye- and liver-restricted isozymes. These data confirm the orthology of eye- and liver-restricted LDH isozymes in Cypriniform and Perciform fishes as originally proposed, suggest that the liver-restricted isozyme of cichlid fishes is non-orthologous and further raise the question of identity and evolutionary origin of this anomalous LDH activity.  相似文献   

2.
Carbonic anhydrase (CA) isozymes were identified and isolated from three strains of Phaeodactylum tricornutum [University of Texas Culture Collection (UTEX 640), North Eastern Pacific Culture Collection at the University of British Columbia B31 and Culture Collection of Algae and Protozoa 1052/1A]. External (CAext) and internal CA activity was detected by potentiometric assay of intact cells and cell homogenates of air and high CO2-grown cells. CAext was detected only in UTEX 640 grown under CO2-limited conditions and present in trace amounts in cells grown on high CO2. CA isozymes in cells extracts were separated by cellulose acetate electrophoresis and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All three strains had two CA bands in common, while UTEX 640 had a third, faster-running band which was absent from extracts of high CO2-grown cells and thus was the external isozyme. The internal CA isoforms of the UTEX 640 strain were shown to have molecular masses of 28 and 25 kDa, and the external 24 kDa. A fourth CAext isozyme with a molecular weight of 23.5 kDa was later detected using a polyclonal CA antibody. The CA isozymes were low-CO2-inducible proteins because Western blot analysis, using a polyclonal antibody, indicated that CA expression was repressed in high CO2-grown cells. CA localization, using both immunofluorescence and immunogold techniques, with air-grown cells indicated that the CAext was located in the periplasmic space and on the cell membrane, whereas in high CO2-grown cells only internal CA was detected.  相似文献   

3.
Abstract: Highly purified casein kinase II (CK II) isozymes from bovine brain gray matter (BBGM) were obtained by means of a new purification procedure consisting of one phosphocellulose and three Mono-Q steps. The phosphocellulose eluate showed two BBGM-CK II activities. The first minor component (BBGM-CK IIa) was eluted with 0.9 M NaCl and the major component was eluted at 1.1 M NaCl (BBGM-CK IIb). The protein complexes responsible for these two activities were comprised of three subunits, i.e., α (40 kDa), α' (38 kDa), and β (28 kDa), with various subunit ratios. The two isozymes displayed the same behavior on Superose 12 fast protein liquid chromatographic gel filtration and sucrose density centrifugation. BBGM-CK IIa and b showed chromatographic and biochemical differences including differing K m for ATP and GTP and K i for heparin and 2,3-bisphosphoglycerate. The properties of the main peak (BBGM-CK IIb) were studied in detail. The stimulatory effect of Mg2+, Mn2+, and Co2+ was highly dependent both on the nature of the substrate and on ionic type and concentration. It is surprising that with phosvitin as substrate, BBGM-CK IIb was fully active even in the absence of Mg2+ and NaCl. The inhibitory effect of heparin and the stimulatory effects of NaCl, KCl, spermine, and polylysine were highly dependent on the ionic strength, buffer type, and substrate. BBGM-CK II isozymes phosphorylated stathmine in the presence of polylysine, but the requirement for polybasic compounds was not absolute, as is the case with calmodulin and clathrin β-light chain. The unusual chromatographic behavior and biochemical properties of these BBGM-CK II isozymes, compared with the classical CK II, could be explained at least in part by their subunit ratios.  相似文献   

4.
Abstract: We have investigated the isozymes of a phosphoinositide-specific phospholipase C (PLC) in bovine retina using several monoclonal antisera to PLCβ1, γ1, and δ1. Immunoblot analysis showed that all three isozymes were present in the retina. Immunocytochemical localization in frozen bovine retina sections showed that PLCγ1 was present in the photoreceptor cell layer, outer plexiform cell layer, inner plexiform cell layer, and ganglion cell layer. Immunoreaction within the photoreceptor cell layer was dependent on dark/light adaptation state of retinas. Immunoblot analysis of rod outer segments (ROS) with monoclonal or polyclonal antibodies to PLCγ1 showed the presence of an immunoreactive band of 140 kDa. ROS prepared from retinas light-adapted in vitro had more PLCγ1 on immunoblots than ROS from dark-adapted retinas. PLC enzyme activity in ROS from light-adapted retinas was 69 and 46% higher than ROS from dark-adapted retinas, when assayed in the presence and absence of ATP, respectively. This increase in enzyme activity was observed at [Ca2+]free between 0.32 and 100 µ M . These results demonstrate the presence of PLCγ1 in bovine ROS and show that ROS prepared from light-adapted retinas are enriched in this isozyme, suggesting that light may promote the binding of this isozyme to bleached ROS membranes.  相似文献   

5.
The mediation of tentoxin-induced chlorosis through inhibition of chloroplast coupling factor 1 (CF1) ATPase activity was investigated through an examination of the effects of tentoxin on electrophoretically-separated CF1 ATPases from sensitive and insensitive Nicotiana species. Sensitive species exhibited three major ATPases, only one of which was inhibited at some concentrations of tentoxin. Insensitive Nicotiana species showed the same three "isozymes"upon electrophoresis but none of the isozymes were tentoxin sensitive. CF1 isolated from Zea mays L. cv. Pioneer 3541, which is insensitive to tentoxin in vivo based on lack of chlorosis, exhibited two ATPases, one of which was sensitive to tentoxin. The concentration/activity relationships between tentoxin and ATPase inhibition of the sensitive isozyme did not correlate well with the chlorosis induced at similar levels of tentoxin in vivo. Both Oenothera hookeri Torr. & Gray and the CF1-deficient I iota mutant derived from it are sensitive to tentoxin as determined by loss of chlorophyll and ultrastructural changes typical of the tentoxin syndrome. These results support a mechanism of action different from inhibition of CF1 for tentoxin-induced chlorosis.  相似文献   

6.
Although there are many indications that N cycling in grassland ecosystems changes under elevated atmospheric CO2 partial pressure (pCO2), most information has been obtained in short‐term studies. Thus, N budgets were established for four years under ambient and 60 Pa pCO2 at two levels of N fertilization in two contrasting model ecosystems: Trifolium repens L. (white clover) and Lolium perenne L. (perennial ryegrass) were planted in soil in boxes in the Swiss FACE experiment. While T. repens showed an 80% increase in harvested biomass with no change in biomass allocation under elevated atmospheric pCO2 compared to ambient conditions, L. perenne showed an increase only in the biomass of the roots. During the four years of the experiment, the systems gained N both from N retained in the soil and from stubble/stolon and roots left after the final harvest; in total between 11 and 86 gN m−2. Nitrogen retention in the soil was between 4 and 64 g m2. The L. perenne system gained the most N and retained the most N in the soil at high N fertilization and elevated atmospheric pCO2. The input of new C and N into the soil correlated well in the L. perenne systems but not in the T. repens systems. Elevated atmospheric pCO2 led neither to an increase in N retention in the soil nor did it reduce the loss of N from the soil. In the L. perenne systems, N fertilization played the main role in both the retention of N and the sequestration of C, while in the T. repens systems symbiotic N2 fixation may have controlled N retention in the soil.  相似文献   

7.
1. The photosynthetic response to elevated CO2 and nutrient stress was investigated in Agrostis capillaris, Lolium perenne and Trifolium repens grown in an open-top chamber facility for 2 years under two nutrient regimes. Acclimation was evaluated by measuring the response of light-saturated photosynthesis to changes in the substomatal CO2 concentration.
2. Growth at elevated CO2 resulted in reductions in apparent Rubisco activity in vivo in all three species, which were associated with reductions of total leaf nitrogen content on a unit area basis for A. capillaris and L. perenne . Despite this acclimation, photosynthesis was significantly higher at elevated CO2 for T. repens and A. capillaris , the latter exhibiting the greatest increase of carbon uptake at the lowest nutrient supply.
3. The photosynthetic nitrogen-use efficiency (the rate of carbon assimilation per unit leaf nitrogen) increased at elevated CO2, not purely owing to higher values of photosynthesis at elevated CO2, but also as a result of lower leaf nitrogen contents.
4. Contrary to most previous studies, this investigation indicates that elevated CO2 can stimulate photosynthesis under a severely limited nutrient supply. Changes in photosynthetic nitrogen-use efficiency may be a critical determinant of competition within low nutrient ecosystems and low input agricultural systems.  相似文献   

8.
Supernatant malate dehydrogenase (MDH) isozymes (as visualized by starch gel electrophoresis) are encoded by two distinct gene loci in both the largemouth and smallmouth bass. When an interspecific F1 hybrid is formed between these two fish, a unique MDH isozyme is generated. The results of freeze-thaw molecular hybridization (which is the first application of this technique to MDH) indicate that this unique isozyme in the F1 hybrid is a heterodimer composed of one subunit of each parental type. The F1 hybrids produced F2 hybrids which in turn formed the F3 hybrid population. The inheritance of alleles at the MDH-B locus is consistent with a single Mendelian autosomal locus. Furthermore, there is no evidence of linkage between the lactate dehydrogenase-E locus and the MDH-B locus.  相似文献   

9.
Abstract: Two cannabinoid receptors belonging to the superfamily of G protein-coupled membrane receptors have been identified and cloned: the neuronal cannabinoid receptor (CB1) and the peripheral cannabinoid receptor (CB2). They have been shown to couple directly to the Gi/o subclass of G proteins and to mediate inhibition of adenylyl cyclase upon binding of a cannabinoid agonist. In several cases, however, cannabinoids have been reported to stimulate adenylyl cyclase activity, although the mechanism by which they did so was unclear. With the cloning of nine adenylyl cyclase isozymes with various properties, including different sensitivities to αs, αi/o, and βγ subunits, it became important to assess the signaling pattern mediated by each cannabinoid receptor via the different adenylyl cyclase isozymes. In this work, we present the results of cotransfection experiments between the two types of cannabinoid receptors and the nine adenylyl cyclase isoforms. We found that independently of the method used to stimulate specific adenylyl cyclase isozymes (e.g., ionomycin, forskolin, constitutively active αs, thyroid-stimulating hormone receptor activation), activation of the cannabinoid receptors CB1 and CB2 inhibited the activity of adenylyl cyclase types I, V, VI, and VIII, whereas types II, IV, and VII were stimulated by cannabinoid receptor activation. The inhibition of adenylyl cyclase type III by cannabinoids was observed only when forskolin was used as stimulant. The activity of adenylyl cyclase type IX was inhibited only marginally by cannabinoids.  相似文献   

10.
The influence of salinity on the activity of nitrate reductase (NR, EC 1.6.6.1) and the level of the molybdenum cofactor (MoCo) as affected by the source and concentration of nitrogen was studied in annual ryegrass ( Lolium multiflorum cv. Westerwoldicum). Plants grown in sand were irrigated with nutrient solution with an electrical conductivity of 2 or 11.2 dS m−1, containing nitrogen (0.5 or 4.5 m M ) in the form of NH4NO3 or NaNO3 Salinity-treated (11.2 dS m−1) plants produced less biomass and more organic nitrogen while accumulating more NO3 than control plants. Increased nitrogen concentration in the irrigation solutions enhanced biomass and organic nitrogen production as well as NO3 accumulation irrespective of the electrical conductivity. Salinity inhibited shoot growth and increased shoot NR activity of plants receiving 4.5 m M NH4NO3 or NaNO3. Similar effects were observed in roots of plants grown in 4.5 m M NaNO3. Nitrate added to a complementation medium containing ryegrass MoCo and the NR apoprotein of Neurospora crassa mutant nit-1 stimulated the activity of the reconstituted NR (NADPH-nitrate reductase, EC 1.6.6.3). Increased salinity and nitrogen in the nutrient solutions caused an increase of MoCo content in roots and shoots. Similar results were observed for NR activity in the shoots. The increase of MoCo in response to salinity was more pronounced than that of NR, especially in the roots. We conclude that the pool size of MoCo in ryegrass is not constant, but varies in response to nutritional and environmental factors.  相似文献   

11.
Abstract Alkali-tolerant Aspergillus fischeri Fxn1 produced two extracellular xylanases. The major xylanase ( M r 31000) was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange chromatography and preparatory PAGE. Xylose was the major hydrolysis product from oat spelt and birch wood xylans. It was completely free of cellulolytic activities. The optimum pH and temperature were 6.0 and 60 °C, respectively. pH stability ranged from 5 to 9.5 and the t1 / 2 at 50 °C was 490 min. It had a K m of 4.88 mg ml−1and a V max of 588 μmol min−1 mg−1. The activity was inhibited (95%) by AlCl3 (10 mM). This enzyme appears to be novel and will be useful for studies on the mechanism of hydrolysis of xylan by xylanolytic enzymes.  相似文献   

12.
Plant respiration draws on substrate pools of different functional/biochemical identity. Little is known about the effect of nitrogen deficiency on those pools' sizes, half-lives and relative contribution to respiration, and consequently, of carbon residence time in respiratory metabolism. Here we studied how nitrogen fertilization affects the respiratory carbon supply system of shoots and roots of Lolium perenne , a perennial grass. Plants grown at two nitrogen supply levels in continuous light were labelled with 13CO2/12CO2 for intervals ranging from 1 h to 1 month. The rate and isotopic composition of shoot, root and plant respiration were measured, and the time-courses of tracer incorporation into respired CO2 were analysed by compartmental modelling. Nitrogen deficiency reduced specific respiration rate by 30%, but increased the size of the respiratory supply system by 30%. In consequence, mean residence time of respiratory carbon increased with nitrogen deficiency (4.6 d at high nitrogen and 9.2 d at low nitrogen supply). To a large extent, this was due to a greater involvement of stores with a long half-life in respiratory carbon metabolism of nitrogen-deficient plants. At both nitrogen supply levels, stores supplying root respiration were primarily located in the shoot, probably in the form of fructans.  相似文献   

13.
The role of the ascorbate-glutathione cycle and AOS detoxification was investigated during leaf growth of defoliated and undefoliated plants of ryegrass ( Lolium perenne L. cv. Bravo). Antioxidants and related enzymatic activities were located in elongating leaf bases (ELBs) of undefoliated plants, following a decreasing gradient from basal (meristem) to distal segments, inverse to H2O2 levels. In the meristematic zone, the intense activity of the ascorbate-glutathione cycle and the supply of reducing power by the oxidative pentose phosphate pathway allowed the maintenance of both antioxidant reduction and H2O2 detoxification. BCNU (1–3 bis(2-chloroethyl)- N -nitrosourea), a glutathione reductase inhibitor, induced an increase in the meristematic zone in both H2O2 and antioxidant levels and a decrease in reduced/oxidized ratios of glutathione and ascorbate. These changes were associated with a reduced foliar regrowth activity. In the absence of BCNU, defoliation did not modify the ratios of reduced/oxidized antioxidants, although it triggered a temporary increase in H2O2 level. The results are discussed on the basis of a possible control of leaf growth by glutathione and ascorbate.  相似文献   

14.
The catalase activity and the isozyme pattern of the metalloenzyme system superoxide dismutase (SOD) have been determined in pea ( Pisum sativum L., cv, Lincoln) leaves of different ages (apical, middle and lower), during several stages of plant development. Pea seedlings were grown in full nutrient solution in a phytotron. Catalase activity was determined polarographically, and superoxide dismutase isozymes (Mn-SOD, Cu, Zn-SOD I and Cu, Zn-SOD II) were separated by acrylamide gel electrophoresis and their relative amounts quantified by densitonietry. The results indicate that the relative amounts of SOD isozymes are slightly different in leaves of different ages during plant growth and, interestingly, each molecular form of SOD shows a clearly distinct pattern during plant development. These changes in the relative percentages of SOD isozymes could be due to the induction of the distinct molecular forms of SOD by the metals Mn, Cu and Zn, translocated to the different leaves as a result of plant development. The relative percentage of the Mn-SOD isozyme showed a similar pattern to that of catalase activity, suggesting a possible link between these two metalloenzymes at subcellular level, both cooperating to remove the toxic effects of O-2 and H2O2.
An additional conclusion is that before a certain metalloenzyme can be used as a marker to assess the plant micronutrient status, it is essential to have a detalled study of its activity pattern in leaves of different age during plant development.  相似文献   

15.
Membrane-bound [NiFe]-hydrogenase from Hydrogenophaga sp. AH-24 was purified to homogeneity. The molecular weight was estimated as 100±10 kDa, consisting of two different subunits (62 and 37 kDa). The optimal pH values for H2 oxidation and evolution were 8.0 and 4.0, respectively, and the activity ratio (H2 oxidation/H2 evolution) was 1.61 × 102 at pH 7.0. The optimal temperature was 75 °C. The enzyme was quite stable under air atmosphere (the half-life of activity was c . 48 h at 4 °C), which should be important to function in the aerobic habitat of the strain. The enzyme showed high thermal stability under anaerobic conditions, which retained full activity for over 5 h at 50 °C. The activity increased up to 2.5-fold during incubation at 50 °C under H2. Using methylene blue as an electron acceptor, the kinetic constants of the purified membrane-bound homogenase (MBH) were V max=336 U mg−1, k cat=560 s−1, and k cat/ K m=2.24 × 107 M−1 s−1. The MBH exhibited prominent electron paramagnetic resonance signals originating from [3Fe–4S]+ and [4Fe–4S]+ clusters. On the other hand, signals originating from Ni of the active center were very weak, as observed in other oxygen-stable hydrogenases from aerobic H2-oxidizing bacteria. This is the first report of catalytic and biochemical characterization of the respiratory MBH from Hydrogenophaga .  相似文献   

16.
The genetic variability of one of the liver isozymes of aldehyde oxidase (AOX-B2 or AOX-2) and the stomach isozyme of alcohol dehydrogenase (ADH-C2) has been examined among strains of mice. Evidence is presented for a fourth allele of Aox-2 and a third allele of Adh-3 . The hybrid allozyme pattern for mouse liver AOX was consistent with a dimeric subunit structure for this enzyme.  相似文献   

17.
Abstract : Altered hypothalamic-pituitary-adrenal (HPA) function (increased plasma cortisol level) has been shown to be associated with mood and behavior. Protein kinase C (PKC), an important component of the phosphatidyl-inositol signal transduction system, plays a major role in mediating various physiological functions. The present study investigates the effects of acute (single) and repeated (10-day) administrations of 0.5 or 1.0 mg/kg doses of dexamethasone (DEX), a synthetic glucocorticoid, on B max and K D of [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding, PKC activity, and protein expression of PKC isozymes, α, β, γ, δ, and ε in the membrane and the cytosolic fractions of rat cortex and hippocampus. It was observed that repeated administration of 1.0 mg/kg DEX for 10 days caused a significant increase in B max of [3H]PDBu binding to PKC, in PKC activity, and in expressed protein levels of the γ and ε isozymes in both the cytosolic and the membrane fractions of the cortex and the hippocampus, whereas a lower dose of DEX (0.5 mg/kg for 10 days) caused these changes only in the hippocampus. On the other hand, a single administration of DEX (0.5 or 1.0 mg/kg) had no significant effect on PKC in the cortex or in the hippocampus. These results suggest that alterations in HPA function from repeated administration of glucocorticoids may modulate PKC-mediated functions.  相似文献   

18.
To clarify the mechanism of aluminum (Al) toxicity and Al tolerance, we isolated a new clone (pAL201) from a tobacco cDNA library. Northern blot hybridization analysis indicated that the expression of pAL201 is induced by Al treatment and phosphate (P1) starvation. The complete cDNA sequence suggested that this clone encodes a moderately anionic peroxidase (EC 1.11.1.7). Analysis by isoelectric focussing indicated that a moderately anionic peroxidase (approximately pI 6.7) and two cationic peroxidases (pI 9.2 and 9.7) in the soluble fraction are activated by Al treatment and P1 starvation, while two moderately anionic isozymes are repressed by these stresses. We suppose that Al ion stress can control the activity of some peroxidase isozymes, one of which is probably induced by enhanced gene expression of pAL201. There is a possibility that some of these isozymes have some functions in Al ion stress.  相似文献   

19.
Abstract. The 15N isotope was used to compare the uptake and the assimilation of NH4+ and NO3 nitrogen in ryegrass ( Lolium perenne L.) during regrowth after cutting. Uptake of nitrate-N, expressed per plant, was at all times greater than ammonium-N uptake and assimilation decreased in roots and stubble while its assimilation was maintained at a high level in leaves. It has been suggested that ammonium assimilation is directly related to the availability of carbohydrates in the sink organ (leaves) resulting from their remobilization from the source organs (roots and stubble). Nitrate reduction decreased in all organs, while the uptake of NO3 was still high. After this first period of regrowth, nitrogen assimilation both from nitrate and ammonium increased in all the plants. Nitrate reduction capacity (expressed in μg NO3-N reduced per g D.W. per d) is 7.5 and 22.5 times greater in leaves than in stubble and roots, respectively. Therefore, nitrogen assimilation in stubble and particularly in roots was mainly dependent on ammonium nitrogen.  相似文献   

20.
The effect of MRS broth on the stability of hydrogen peroxide (H2O2) has been studied. Known concentrations (1–100 μg ml−1) of H2O2 were prepared in distilled water, phosphate buffer (pH 7·0) and MRS broth (pH 6·2 and 3·9). H2O2 was very stable in aqueous and buffer solutions but it was rapidly degraded in MRS broth (pH 3·9). The presence of H2O2 in MRS broth (pH 6·2) could not be detected.  相似文献   

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