Isolation of auxotrophic mutants in support of ammonia assimilation via glutamine synthetase in Methanobacterium ivanovii

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     

Refolding of serine proteinases

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.     

Protein folding kinetics by combined use of rapid mixing techniques and NMR observation of individual amide protons

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.     

The carotenoid and abscisic acid content of viviparous kernels and seedlings ofZea mays L.

Carotenoid and abscisic acid (ABA) levels were determined in endosperm, embryos and seedlings of wild-type and viviparous (vp) mutants ofZea mays L. Carotenoid concentrations were determined by absorption spectrometry following purification by high-performance liquid chromatography and ABA concentrations by combined gas chromatography-mass spectrometry. Lutein and zeaxanthin were the terminal carotenoids in wild-type tissue. The carotenoid profiles ofvp-1 andvp-8 tissue were similar to that of the wild type; invp-2, vp-5, vp-7 andvp-9 carotenogenesis was blocked at early stages so that xanthophylls were absent. Except forvp-1, where the ABA content was similar to the wild type, the ABA content ofvp embryos was substantially reduced, to 6–16% of the corresponding wild type. Thus, the absence of xanthophylls was associated with reduced ABA content, which was in turn correlated with vivipary. Kernels ofvp-8 had a reduced ABA content although xanthophylls were present. Seedlings of carotenoid-deficient mutants rescued from viviparous kernels contained less ABA than did wild-type seedlings grown in the same way. Furthermore, the ABA concentration of such seedlings did not increase in response to water deficit. Conversely,vp-1 seedlings contained normal levels of carotenoids and ABA. Carotenoid-deficient seedlings did not contain appreciable amounts of chlorophyll so that chloroplast development was not normal. Thus ABA-deficiency could be associated with abnormal plastid development rather than the absence of carotenoids per se.Abbreviations ABA abscisic acid - DAP days after pollination - i.d. internal diameter - FW fresh weight - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - MS mass spectrometry - vp viviparous     

Mass-spectrometric evidence for the double-carboxylation pathway of malate synthesis by Crassulacean acid metabolism plants in light

Phyllodia of the Crassulacean acid metabolism (CAM) plant Kalanchoë tubiflora were allowed to fix ¹³CO₂ in light and darkness during phase IV of the diurnal CAM cycle, and during prolongation of the regular light period. After ¹³CO₂ fixation in darkness, only singly labelled [¹³C]malate molecules were found. Fixation of ¹³CO₂ under illumination, however, produced singly labelled malate as well as malate molecules which carried label in two, three or four carbon atoms. When the irradiance during ¹³CO₂ fixation was increased, the proportion of singly labelled malate decreased in favour of plurally labelled malate. The irradiance, however, did not change either the ratio of labelled to unlabelled malate molecules found in the tissue after the ¹³CO₂ application, or the magnitude of malate accumulation during the treatment with label. The ability of the tissue to store malate and the labelling pattern changed throughout the duration of the prolonged light period. The results indicate that malate synthesis by CAM plants in light can proceed via a pathway containing two carboxylation steps, namely ribulose-1,5-bisphosphate-carboxylase/oxygenase (EC 4.1.1.39) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) which operate in series and share common intermediates. It can be concluded that, in light, phosphoenolpyruvate carboxylase can also synthesize malate independently of the proceeding carboxylation step by ribulose-1,5-bisphosphate carboxylase/oxygenase.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - TMS trimethylsilyl     

The supply of reducing power for nitrite reduction in plastids of seedling pea roots (Pisum sativum L.)

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.     

Localization of Glyoxylate Cycle Enzymes in Glyoxysomes in Euglena*

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.     

Das visuelle System von Zonotrichia leucophrys gambelii

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.

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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).     

recA基因通过同源重组途径参与由整合质粒发动的大肠杆菌染色体复制

我们在前文中报道由整合的F'质粒所发动的大肠杆菌染色体的复制依赖于recA基因。本文报道有关recA、recB、recC以及lexA等在染色体复制中的作用,实验结果说明,recA基因通过同源重组途径而不是通过SOS途径参与复制,而且recA基因和Chi热点无关。实验结果还说明,RecBC酶的依赖于ATP的双链DNA外切核酸酶活性和recA基因的作用无关。     

嗜热脂肪芽孢杆菌质粒的分离

从堆肥和污泥中分离到一批抗药性高温细菌,经电泳检查,发现6株高温细菌细胞中有质粒存在。其中,嗜热脂肪芽孢杆菌T653的细胞DNA提取液电泳图谱上,有三条非染色体DNA条带,用电镜直接观察,证明它们是T653细胞中的三个质粒。测得两个较小质粒的分子量分别为3.6×10~6和45×10~6道尔顿。研究了嗜热脂肪芽孢杆的T653的温度生长条件与其细胞中质粒的关系。T653细胞中三个质粒的明确功能有待进一步探讨。