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.     

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.     

Entamoeba histolytica. I. Aerobic metabolism

The respiration of intact trophozoites harvested from axenic cultures of Entamoeba histolytica was studied with the polarographic technique utilizing the Clark oxygen electrode. A typical Q_o2 value for the freshly harvested amebae was 1 μatom oxygen/mg protein/hr.It was conclusively demonstrated that this parasite, a putative anaerobe, not only consumes oxygen when provided, but has a high affinity for the gas.Added glucose, galactose, and ethanol increased the respiratory rates, whereas other carbohydrates were without effect on the endogenous respiration. Intermediates of the tricarboxylic acid cycle, amino and fatty acids did not stimulate the respiration of E. histolytica.Inhibitors of the mammalian respiratory chain (cyanide, antimycin) as well as agents that inhibit enzymes catalyzing the tricarboxylic acid cycle (malonate, fluoropyruvate, fluoroacetate, fluorocitrate) had little effect on the endogenous or glucose-supported respiration. Alkylating agents (iodoacetamide, iodoacetate), cinnamate, and N-ethylymaleimide strongly inhibited the oxygen consumption of E. histolytica. The chemotherapeutic agents, Paromomycin, Emetine and Metronidazole, at concentrations that inhibit growth in vitro, did not restrict the respiration.Storage of the trophozoites at 4 C led to progressive deterioraion of the parasites and loss of endogenous and glucose-supported respiration. The deterioration was paralled by loss of SH-materials from the amebae. Likewise, sonication or lysis with detergents abolished both the endogenous respiration and response to glucose.Exogenous NADH or NADPH evoked only marginal increases in oxygen consumption of the freshly harvested amebae, but were effective respiratory substrates with stored or sonicated organisms. Addition of vitamin K₃ greatly enhanced the endogenous and glucose-supported respiration of the intact amebae, as well as enhancing the response of stored or sonicated amebae to reduced pyridine nucleotides.     

Regulation of biosynthesis of aspartate family amino acids in the photosynthetic bacterium Rhodopseudomonas palustris

The four amino acids of the aspartate family (l-lysine, l-methionine, l-threonine, and l-isoleucine) are produced in bacteria by a branched biosynthetic pathway. Regulation of synthesis of early common intermediates and of carbon flow through distal branches of the pathway requires operation of a number of subtle feedback controls, which are integrated so as to ensure balanced synthesis of the several end products. Earlier studies with nonsulfur purple photosynthetic bacteria were instrumental in revealing the existence of alternative regulatory schemes, and in this communication we report on the control pattern of a representative of this physiological group not previously investigated, Rhodopseudomonas palustris. The results obtained from study of the properties of four key regulatory enzymes of the aspartate family pathway (-aspartokinase, homoserine dehydrogenase, homoserine kinase, and threonine deaminase) and of the effects of exogenous amino acids (i. e., the end products) on growth of the bacterium indicate that the control schema in Rps. palustris differs substantially from the schemes described for other Rhodopseudomonas species, but resembles the regulatory pattern observed in Rhodospirillum rubrum.Abbreviations A absorbancy - AK -aspartokinase - ASA aspartate -semialdehyde - DTT dithiothreitol - HS l-homoserine - HSDH homoserine dehydrogenase - HSK homoserine kinase - I l-isoleucine - KU Klett-Summerson photometer units - L l-lysine - M l-isoleucine - KU Klett-Summerson photometer units - L l-lysine - M l-methionine - ME -mercaptoethanol - PABA p-aminobenzoic acid - T l-threonine - TD threonine deaminase - RCV synthetic growth medium (see text) - YP agar medium containing 0.3% yeast extract, 0.3% peptone, and 1.5% agar - Y2T synthetic growth medium (see text)     

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.

---

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。