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31.
Various enzymes involved in the initial metabolic pathway for ammonia assimilation by Methanobacterium ivanovii were examined. M. ivanovii showed significant activity of glutamine synthetase (GS). Glutamate synthase (GOGAT) and alanine dehydrogenase (ADH) were present, wheras, glutamate dehydrogenase (GDH) was not detected. When M. ivanovii was grown with different levels of NH
+
4
(i.e. 2, 20 or 200 mM), GS, GOGAT and ADH activities varied in response to NH
+
4
concentration. ADH was not detected at 2 mM level, but its activity increased with increased levels of NH
+
4
in the medium. Both GS and GOGAT activities increased with decreasing concentrations of NH
+
4
and were maximum when ammonia was limiting, suggesting that at low NH
+
4
levels, GS and GOGAT are responsible for ammonia assimilation and at higher NH
+
4
levels, ADH might play a role. Metabolic mutants of M. ivanovii that were auxotrophic for glutamine were obtained and analyzed for GS activity. Results indicate two categories of mutants: i) GS-deficient auxotrophic mutants and ii) GS-impaired auxotrophic mutants.Abbreviations GS
Glutamine synthetase
- GOGAT
glutamate synthase
- GDH
glutamate dehydrogenase
- ADH
alanine dehydrogenase 相似文献
32.
Bovine trypsinogen and chymotrypsinogen were successfully refolded as the mixed disulfide of glutathione using cysteine as the disulfide interchange catalyst. The native structures were regenerated with yields of 40%-50% at pH 8.6 and 4 degrees C, and the half-time for the refolding was approximately 60-75 min. We then refolded threonine-neochymotrypsinogen, which is a two-chain structure held together by disulfide bonds and produced on cleavage of Tyr 146-Thr 147 in native chymotrypsinogen [Duda CT, Light A, J Biol Chem 257 9866-9871, 1982]. Neochymotrypsinogen was denatured and fully reduced, and the thiols were converted to the mixed disulfide of glutathione. The two polypeptide fragments, representing the amino- and carboxyl-terminal domains, were separated on Sephadex G-75. Mixtures of the polypeptide fragments varying in the ratio of their concentration from 1:5 to 5:1 were refolded with yields of 21-28%. The lack of dependence on the concentration of either fragment and the relatively high yields suggest independent folding of the amino- and carboxyl-terminal domains. When the globular structures of the domains formed, they then interacted with one another and produced the native intermolecular disulfide bridge and the proper geometry of the active site. 相似文献
33.
Protein folding kinetics by combined use of rapid mixing techniques and NMR observation of individual amide protons 总被引:9,自引:0,他引:9
A method to be used for experimental studies of protein folding introduced by Schmid and Baldwin (J. Mol. Biol. 135: 199-215, 1979), which is based on the competition between amide hydrogen exchange and protein refolding, was extended by using rapid mixing techniques and 1H NMR to provide site-resolved kinetic information on the early phases of protein structure acquisition. In this method, a protonated solution of the unfolded protein is rapidly mixed with a deuterated buffer solution at conditions assuring protein refolding in the mixture. This simultaneously initiates the exchange of unprotected amide protons with solvent deuterium and the refolding of protein segments which can protect amide groups from further exchange. After variable reaction times the amide proton exchange is quenched while folding to the native form continues to completion. By using 1H NMR, the extent of exchange at individual amide sites is then measured in the refolded protein. Competition experiments at variable reaction times or variable pH indicate the time at which each amide group is protected in the refolding process. This technique was applied to the basic pancreatic trypsin inhibitor, for which sequence-specific assignments of the amide proton NMR lines had previously been obtained. For eight individual amide protons located in the beta-sheet and the C-terminal alpha-helix of this protein, apparent refolding rates in the range from 15 s-1 to 60 s-1 were observed. These rates are on the time scale of the fast folding phase observed with optical probes. 相似文献
34.
Plastids were separated from extracts of pea (Pisum sativum L.) roots by sucrose-density-gradient centrifugation. The incubation of roots of intact pea seedlings in solutions containing 10 mM KNO3 resulted in increased plastid activity of nitrite reductase and to a lesser extent glutamine synthetase. There were also substantial increases in the activity of glucose-6-phosphate and 6-phosphogluconate dehydrogenases. No other plastid-located enzymes of nitrate assimilation or carbohydrate oxidation showed evidence of increased activity in response to the induction of nitrate assimilation. Studies with [1-14C]-and [6-14C]glucose indicated that there was an increased flow of carbon through the plastid-located pentose-phosphate pathway concurrent with the induction of nitrate assimilation. It is suggested that there is a close interaction through the supply and demand for reductant between the pathway of nitrite assimilation and the pentose-phosphate pathway located in the plastid. 相似文献
35.
LYNN B. GRAVES RICHARD N. TRELEASE ALBIN GRILL WAYNE M. BECKER 《The Journal of eukaryotic microbiology》1972,19(3):527-532
SYNOPSIS. We demonstrated previously microbodies in Euglena gracilis grown in the dark on 2-carbon substrates. We have now established in Euglena the particulate nature of enzymes known in other organisms to be localized in microbodies (glyoxysomes and leaf peroxisomes). On a linear sucrose gradient the glyoxylate cycle enzymes band together at a nigner equilibrium density (1.20 g/cm3) than mitochondrial marker enzymes (1.17 g/cm3), establishing the existence in Euglena of glyoxysomes similar to those of higher plants. Glyoxylate (hydroxypyruvate) reductase and, under certain conditions, also glycolate dehydrogenase co-band with the glyoxylate cycle enzymes, suggesting that Euglena glyoxysomes, like those of higher plants, may contain peroxisomal-type enzymes. Catalase, an enzyme characteristic of microbodies from a variety of sources, was not detected in Euglena. 相似文献
36.
H. -G. Hartwig 《Cell and tissue research》1970,106(4):556-583
Zusammenfassung Der Verlauf der Sehbahn und die Lokalisation der optischen Zentren wurden bei Zonotrichia leucophrys gambelii (nordamerikanischer Ammernfink) nach einseitiger Augenexstirpation mit den Techniken von Nauta-Fink-Heimer, Bodian und Bielschowsky erforscht. Die Untersuchungen erstreckten sich über einen Zeitraum von 3 bis zu 120 Tagen nach der Operation. Zonotrichia leucophrys gambelii besitzt ein für Vögel typisches visuelles System. Die Hauptmasse der Optikusfasern endet im Stratum griseum et fibrosum superficiale des Tectum opticum. Weitere zentrale Endgebiete sind: Nucleus geniculatus lateralis, Nucleus lateralis anterior, Nucleus superficialis synencephali, Nucleus externus, tectales Grau und Nucleus ectomamillaris als Kern der basalen optischen Wurzel. Alle Fasern werden im Chiasma opticum total gekreuzt, auch der Tractus isthmo-opticus, ein efferentes Bündel, dessen Ursprung im Nucleus isthmo-opticus zu finden ist. Dieses efferente Fasersystem läßt sich im Stumpf des durchtrennten N. opticus noch 120 Tage nach der Operation gut versilbern. Eine direkte Verbindung von Retina und Hypothalamus war lichtmikroskopisch nicht nachweisbar. Neurosekretorisch aktive Zellen des Hypothalamus können zwar einen engen räumlichen Kontakt mit den optischen Fasern haben, Synapsen sind aber an diesen Stellen nicht zu erkennen. Es werden passagere Opticusfasern beschrieben, die auf dem Weg zum Nucleus lateralis anterior und Nucleus superficialis synencephali den Hypothalamus durchsetzen.
Neurohistological and experimental studies of the visual system in Zonotrichia leucophrys gambelii
Summary The course of the optic pathways and the positions of the optic centers have been investigated with unilaterally enucleated white-crowned sparrows, Zonotrichia leucophrys gambelii, using the techniques of Nauta-Fink-Heimer, Bodian, and Bielachowsky. The investigation involved birds examined 3–120 days after enucleation. The white-crowned sparrow has a typically avian visual system. The major bundles of optic fibers terminate in the stratum griseum et fibrosum superficiale of the tectum opticum. Further terminal areas are the nucleus geniculatus lateralis, nucleus lateralis anterior, nucleus superficialis synencephali, nucleus externus, the tectal gray, and the nucleus ectomamillaris of the basal optic root. There is a complete crossing of all fibers in the chiasma, including those of the tractus isthmo-opticus, an efferent bundle with its origin in the nucleus isthmo-opticus. This efferent fiber system can be well impregnated in the stump of the sectioned optic nerve up to 120 days after the operation. No direct connection between the retina and hypothalamus could be demonstrated by light microscopy. Hypothalamic neurosecretory cells can occur in close contact with optic fibers but no synapses have been recognized. Some optic fibers pass through the hypothalamus enroute to the nucleus lateralis anterior and the nucleus superficialis synencephali.
Mit Unterstützung durch die Deutsche Forschungsgemeinschaft. Herrn Prof. Dr. D.S. Farner, Department of Zoology, University of Washington, Seattle, Wash., danke ich für die Förderung dieser Studien (National Institutes of Health Research Grant No. 5 ROI NB 06187 to Professor D. S. Farner). 相似文献
37.
38.
39.
The relationship between phosphate status and cyanide-resistant respiration in bean roots 总被引:8,自引:0,他引:8
Bean ( Phaseolus vulgaris L.) seedlings were cultured on complete or phosphate-deficient nutrient medium. After 14 days of culture on phosphate-deficient medium the visible symptoms of Pi deficiency were observed only in the shoot, the fresh and dry weights of the roots were slightly higher than in control plants. The decreased Pi content in the roots had little effect on total respiration rate but had an effect on the level of inhibition of respiration by cyanide. The high resistance of respiration to cyanide observed in Pi -deficient roots was the result of the suppression of cytochrome path activity and an increased participation of the alternative, cyanide-resistant pathway. The cytochrome pathway activity increased when inorganic phosphate was supplied to Pi -deficient roots for 1 or 3.5 h. It is speculated that the suppression of cytochrome pathway in Pi -deficient roots may result from restriction of the phosphorylating capacity or a partial inhibition of cytochrome oxidase activity. 相似文献
40.
Howard Griffiths Mark S. J. Broadmeadow Anne M. Borland Clive S. Hetherington 《Planta》1990,181(4):604-610
Short-term measurements of instantaneous carbon-isotope discrimination have been determined from mass-spectrometric analyses of CO2 collected online during gas exchange for the epiphytic bromeliad Tillandsia utriculata L. Using this technique, the isotopic signature of CO2 exchange for each phase of Crassulacean acid metabolism (CAM) has been characterised. During night-time fixation of CO2 (Phase I), discrimination () ranged from 4.4 to 6.6, equivalent to an effective carbon-isotope ratio (13C) of –12.3 to –14.5 versus Pee Dee Belemnite (PDB). These values reflected the gross photosynthetic balance between net CO2 uptake and refixation of respiratory CO2, characteristic of CAM in the Bromeliaceae. When for the relative proportion of external (p
a
) and internal (p
i) CO2 is taken into account, calculated p
i/p
a decreased during the later part of the dark period from 0.68 to 0.48. Measurements of during Phase II, early in the light period, showed the transition between C4 and C3 pathways, with carboxylation being increasingly dominated by ribulose bisphosphate carboxylase (Rubisco) as increased from 10.5 to 21.2 During decarboxylation in the light period (Phase III), CO2 leaked out of the leaf and the inherent discrimination of Rubisco was expressed. The value of calculated from on-line measurements (64.4) showed that the CO2 lost was considerably enriched in 13C, and this was confirmed by direct analysis of the CO2 diffusing out into a CO2-free atmosphere (
13C = + 51.6 versus PDB). Instantaneous discrimination was characteristic of the C3 pathway during Phase IV (late in the light period), but a reduction in showed an increasing contribution from phosphoenolpyruvate carboxylase. The results from this non-invasive technique confirm the observations that double carboxylation involving both phosphoenolpyruvate carboxylase and Rubisco occurs during the transient phases of CAM (II and IV) in the light period.Abbreviations and Symbols CAM
Crassulacean acid metabolism
-
H+
(dawn-dusk) variation in titratable acidity
-
13C
carbonisotope ratio of plant organic material, relative to Pee Dee Belemnite (vs. PDB)
-
discrimination against 13CO2,
-
p
i, p
a
internal, external partial pressures of CO2
- Rubisco
ribulose1,5-bisphosphate carboxylase
- PAR
photosynthetically active radiation
- PEPCase
phosphoenolpyruvate carboxylase
We are grateful for financial support in respect of research grants (GR3/5360, GR3/6419) and a studentship awarded by the Natural Environment Research Council, UK. 相似文献