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11.
Etiolated maize leaves (Zea mays L.) contain a major isozyme of NADP-dependent malic enzyme (L-malate dehydrogenase, decarboxylating, EC 1.1.1.40) having an isoelectric point of 5.28±0.03, a Km (L-malate) 0.3–0.6 mM at pH 7.45; a broad pH optimum around pH 6.9 under the conditions of assay; a molecular weight of 280,000 (sometimes accompanied by a minor component of 150,000); and an NAD-dependent activity about 1/50 the NADP-dependent activity. This isozyme, resembling the NADP-malic enzyme of vertebrates, is labeled type 1. The dominant isozyme of young green leaves (type 2) has, however, a pI 4.90±0.03, a Km (L-malate) 0.10–0.15 mM, a pH optimum of 8, and a molecular weight of 280,000. It is also more stable and exhibits an appreciable NAD-dependent activity (1/5–1/7 the NADP activity). Both isozymes show linear kinetics, dependence on Mn or Mg ions, similar Km (NADP+), and the typical increase of Km for L-malate with increasing pH values. Type 1 isozyme of maize is assumed to be cytosolic. Type 2 corresponds in each property to the chloroplast enzyme of bundle-sheath cells. It is present at a low level in etiolated leaves and develops to a high specific activity (up to 100 nmol min-1 mg protein-1 by 150 h illumination) during photosynthetic differentiation, replacing the type 1 form.Abbreviation MES
2 (N-morpholino)ethane sulfonic acid
Work supported by grants from the Consiglio Nazionale delle Ricerche for years 1975 and 1976 相似文献
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An archaeal histone gene from the hyperthermophile Pyrococcus furiosus containing four consecutive putative oligo-dT terminator sequences was used as a model system to investigate termination signals and the mechanism of termination in vitro . The archaeal RNA polymerase terminated with high efficiency at the first terminator at 90°C when it contained five to six T residues, at 80°C readthrough was significantly increased. A putative hairpin structure upstream of the first terminator had no effect on termination efficiency. Template competition experiments starting with RNA polymerase molecules engaged in ternary complexes revealed recycling of RNA polymerase from the terminator to the promoter of the same template. This facilitated reinitiation was dependent upon the presence of a terminator sequence suggesting that pausing at the terminator is required for recycling as in the RNA polymerase III system. Replacement of the sequences immediately downstream of the oligo-dT terminator by an AT-rich segment improved termination efficiency. Both AT-rich and GC-rich downstream sequences seemed to impair the facilitated reinitiation pathway. Our data suggest that recycling is dependent on a subtle interplay of pausing of RNA polymerase at the terminator and RNA polymerase translocation beyond the oligo-dT termination signal that is dramatically affected by downstream sequences. 相似文献
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Paola Barraja Virginia Spanò Diana Patrizia Anna Carbone Girolamo Cirrincione Daniela Vedaldi Alessia Salvador Giampietro Viola Francesco Dall’Acqua 《Bioorganic & medicinal chemistry letters》2009,19(6):1711-1714
A convenient synthesis of the pyrano[2,3-e]isoindol-2-one ring system, an heteroanalogue of angelicin, is reported. Our synthetic approach consists of the annelation of the pyran ring on the isoindole moiety using 5-dialkylamino- or 5-hydroxymethylene intermediates as building blocks. The photoantiproliferative activity of the new derivatives was studied. Some of them bearing the benzyl group at the 8 position were active with IC50 in the micromolar range. Cell cytotoxicity involves apoptosis, alteration of cell cycle profile and membrane photodamage. 相似文献
17.
Lobasso S Saponetti MS Polidoro F Lopalco P Urbanija J Kralj-Iglic V Corcelli A 《Chemistry and physics of lipids》2009,157(1):12-20
The dye 10-N-nonyl acridine orange (NAO) is used to label cardiolipin domains in mitochondria and bacteria. The present work represents the first study on the binding of NAO with archaebacterial lipid membranes. By combining absorption and fluorescence spectroscopy with fluorescence microscopy studies, we investigated the interaction of the dye with (a) authentic standards of archaebacterial cardiolipins, phospholipids and sulfoglycolipids; (b) isolated membranes; (c) living cells of a square-shaped extremely halophilic archaeon. Absorption and fluorescence spectroscopy data indicate that the interaction of NAO with archaebacterial cardiolipin analogues is similar to that occurring with diacidic phospholipids and sulfoglycolipids, suggesting as molecular determinants for NAO binding to archaebacterial lipids the presence of two acidic residues or a combination of acidic and carbohydrate residues. In agreement with absorption spectroscopy data, fluorescence data indicate that NAO fluorescence in archaeal membranes cannot be exclusively attributed to bisphosphatidylglycerol and, therefore, different from mitochondria and bacteria, the dye cannot be used as a cardiolipin specific probe in archaeal microorganisms. 相似文献
18.
Raffaele Lombardi Maria Elena Villani Mariasole Di Carli Patrizia Brunetti Eugenio Benvenuto Marcello Donini 《Transgenic research》2010,19(6):1083-1097
It was previously demonstrated that the tumour-targeting antibody mAb H10 can be transiently expressed and purified at high
levels in Nicotiana benthamiana by using a vacuum-agroinfiltration system boosted by the use of a virus silencing suppressor protein. Scope of this work
was to analyse different steps of protein extraction from agroinfiltrated leaves to optimise the purification process of the
secretory mAb H10 providing new insights in the field of large-scale plant production. Two different extraction procedures
(mechanical shearing/homogenisation and recovery of intercellular fluids -IFs-) were evaluated and compared in terms of purified
antibody yields, antibody degradation and total phenolic compounds content. Mechanical grinding from fresh leaf tissues gave
the highest purification yield (75 mg/kg Fresh Weight -75% intact tetrameric IgG-) and total phenolics concentration in the
range of 420 μg/g FW. The second extraction procedure, based on the recovery of IFs, gave purification yields of 15–20 mg/kg
FW (corresponding to 27% of total soluble protein) in which about 40% of purified protein is constituted by fully assembled
IgG with a total phenolic compounds content reduced by one order of magnitude (21 μg/g FW). Despite a higher antibody degradation,
purification from intercellular fluids demonstrated to be very promising since extraction procedures resulted extremely fast
and amenable to scaling-up. Overall data highlight that different extraction procedures can dramatically affect the proteolytic
degradation and quality of antibody purified from agroinfiltrated N. benthamiana leaves. Based on these results, we optimised a pilot-scale purification protocol using a two-step purification procedure
from batches of fresh agroinfiltrated leaves (250 g) allowing purification of milligram quantities (average yield 40 mg/kg
FW) of fully assembled and functional IgG with a 99.4% purity, free of phenolic and alkaloid compounds with low endotoxin
levels (<1 EU/ml). 相似文献
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De novo root formation in thin cell layers of tobacco: changes in free and bound polyamines 总被引:4,自引:0,他引:4
Patrizia Torrigiani Maria Maddalena Altamura Francesca Capitani Donatella Serafini-Fracassini Nello Bagni 《Physiologia plantarum》1989,77(3):294-301
Thin cell layers excised from tobacco ( Nicotiana tabacum L. cv. Samsun) stem internodes, with an appropriate exogenous hormonal balance, were able to form a greater number of roots, and in a larger percentage of the explants (93%) than when they were excised from pedicels (40%). The developmental sequence of root formation and explant growth were followed by histological analysis. Free and bound [trichloroacetic acid (TCA)-soluble and -insoluble] putrescine and spermidine increased in the explants, particularly when root meristemoids appeared. These meristemoids originated in the superficial (day 6 in culture) or deep (days 10–11) layers and inside the newly formed callus (day 25). At those times, TCA-soluble and, to a lesser extent, TCA-insoluble bound putrescine predominated over the other polyamines. Spermine was always present in trace amounts. Polyamines decreased again when root and callus formation was completed (day 30). The involvement of these three classes of polyamines (free, TCA-soluble and -insoluble) in morphogenic processes is discussed. 相似文献