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921.
V J Chen A M Orville M R Harpel C A Frolik K K Surerus E Münck J D Lipscomb 《The Journal of biological chemistry》1989,264(36):21677-21681
The nonheme iron oxidase isopenicillin N synthase catalyzes the formation of two new internal bonds in the tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form the beta-lactam and thiazolidine rings of isopenicillin N. Concomitantly, O2 is reduced to 2 H2O. The recombinant enzyme from Cephalosporium acremonium (Mr = 38,400), expressed as an apoenzyme in Escherichia coli, binds 1 g atom of Fe2+/mol of enzyme to reconstitute full activity. M?ssbauer spectra of the 57Fe-enriched enzyme exhibit parameters (delta = 1.30 mm/s, delta EQ = 2.70 mm/s) which unambiguously show that the active site iron is high spin Fe2+. Anaerobic binding of ACV causes a substantial decrease in the isomer shift parameter delta (delta = 1.10 mm/s, delta EQ = 3.40 mm/s) showing that the substrate perturbs the iron site and makes its coordination environment much more covalent. Nitric oxide (NO) binds to the EPR silent active site iron to give an EPR active species (g = 4.09, 3.95, 2.0; S = 3/2) similar to those of the nitrosyl complexes of many other mononuclear Fe2+-containing enzymes. The rhombicity of the EPR spectrum is increased (g = 4.22, 3.81, 1.99) by anaerobic addition of ACV suggesting that the substrate binds to or near the iron without displacing NO. Interestingly, the enzyme.ACV.NO complex displays an optical spectrum similar to that of ferric rubredoxin in which the iron has only thiol coordination. This suggests that the Fe2+ of the enzyme.ACV.NO complex acquires Fe3+ character and that the cysteinyl thiol moiety of ACV coordinates to the iron. Similar substrate thiol coordination to the iron of the enzyme.ACV complex is the most probable explanation for the large decrease in isomer shift observed. These results provide the first evidence for the direct involvement of iron in this unique O2-dependent reaction and suggest novel roles for iron and oxygen in biological catalysis. 相似文献
922.
Visualization of the polarity of isolated titin molecules: a single globular head on a long thin rod as the M band anchoring domain? 总被引:14,自引:8,他引:6
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TII, the extractable form of titin, was purified from myofibrils and separated by high resolution gel permeation chromatography into two fractions (TIIA and TIIB). Novel specimen orientation methods used before metal shadowing and EM result in striking pictures of the two forms. Molecules layered on mica become uniformly oriented when subjected to centrifugation. TIIB comprises a very homogeneous fraction. All molecules reveal a single globular head at one end on a long and very thin rod of uniform diameter. The lengths of the rods have a very narrow distribution (900 +/- 50 nm). TIIA molecules seem lateral oligomers of TIIB, attached to each other via the head regions. While dimers are the predominant species, trimers and some higher oligomers can also be discerned. Mild proteolysis destroys the heads and converts TIIA and TIIB into TIIB-like rods. Similar molecules also result from titin purified from myofibrils by certain established purification schemes. Headless titin molecules show in gel electrophoresis only the TII band, while head bearing molecules give rise to two additional polypeptides at 165 and 190 kD. Immunoelectron microscopy of myofibrils identifies both titin-associated proteins as M band constituents. We speculate that in the polar images of TII the globular head region corresponds to the M band end of the titin molecules. This hypothesis is supported by immunoelectron micrographs of TIIB molecules using titin antibodies of known epitope location in the half sarcomere. This proposal complements our previous immunoelectron microscopic data on myofibrils. They showed that epitopes present only on the nonextractable TI species locate to the Z line and its immediately adjacent region (Fürst, D. O., M. Osborn, R. Nave, and K. Weber. 1988. J. Cell Biol. 106:1563-1572). Thus, the two distinct ends of the titin molecule attach to Z and M band material respectively. 相似文献
923.
Plasmid pBP11 contains a sequence homologous to Tn21-like element Tn2410 encoding dihydropteroate synthetase and beta-lactamase OXA-2. The nucleotide sequence of a 1.5 kb segment of this region has been determined including the bla gene. It reveals strong sequence homology with the OXA-2 operon of plasmid R46. The implications of an additional 319 bp segment in pBP11 for the different evolution of R46/pKM101 and pBP11 are discussed. 相似文献
924.
925.
Uwe-G. Maier Klaus D. Grasser Michael M. Haa? Günter Feix 《Molecular & general genetics : MGG》1990,221(2):164-170
Summary A 216 by promoter fragment of the 19 kDa protein zein gene pMS1, containing the CCAAT and TATA boxes, was analysed by a variety of techniques for in vitro interactions with nuclear proteins from endosperm tissue. HMG proteins were found to form stable complexes with these A/T-rich promoter sequences and several specific DNA-binding proteins appear to be involved in the formation of DNA-protein complexes with this fragment. A 29 bp region spanning the two CCAAT boxes was protected from DNase I digestion in footprinting experiments. 相似文献
926.
Estimation of the effect of photoinhibition on the carbon gain in leaves of a willow canopy 总被引:21,自引:0,他引:21
The occurrence of photoinhibition of photosynthesis in leaves of a willow canopy was examined by measuring the chlorophyll-a fluorescence ratio of F
V/F
M (FM is the maximum fluorescence level of the induction curve, and FV is the variable fluorescence, F
V=F
M–F
0, where F0 is the minimal fluorescence). The majority of the leaves situated on the upper parts of peripheral shoots showed an afternoon inhibition of this ratio on clear days. This was the consequence of both a decrease in F
M and a rise in F
O. In the same leaves the diurnal variation in intercepted photosynthetic photon flux density (PPFD) was monitored using leaf-mounted sensors. Using the multivariate method, partial least squares in latent variables, it is shown that the dose of PPFD, integrated and linearly weighted over the last 6-h period, best predicts photoinhibition. Photoinhibition occurred even among leaves that did not intercept PPFDs above 1000 mol·m–2·s–1. Exposure of leaves to a standard photoinhibitory treatment demonstrated that the depression in the F
V/F
M ratio was paralleled by an equal depression in the maximal quantum yield of CO2 uptake and a nearly equal depression in the rate of bending (convexity) of the light-response curve of CO2 uptake. As a result, the rate of net photosynthesis is depressed over the whole natural range of PPFD. By simulating the daily course in the rate of net photosynthesis, it is estimated that in the order of one-tenth of the potential carbon gain of peripheral willow shoots is lost on clear days as a result of photoinhibition. This applies to conditions of optimal temperatures. Photoinhibition is even more pronounced at air temperatures below 23° C, as judged from measurements of the FV/FM ratio on clear days: the afternoon inhibition of this ratio increased in a curvilinear manner from 15% to 25% with a temperature decrease from 23° to 14° C.Abbreviations and Symbols FO
minimum fluorescence
- FV
variable fluorescence
- FM
maximum fluorescence
- PLS
partial least squares in latent variables
- PPFD
photosynthetic photon flux density
- VPD
water vapour-pressure deficit
This study was supported by the Swedish Natural Science Research Council. We are indebted to Dr. Jerry Leverenz (Department of Plant Physiology, University of Umeå, Sweden) for guidance with the modelling of the photosynthesis data. 相似文献
927.
λ-Glutamylcysteine synthetase in higher plants: catalytic properties and subcellular localization 总被引:1,自引:0,他引:1
λ-Glutamylcysteine synthetase activity (EC 6.3.2.2) was analysed in Sephacryl S-200 eluents of extracts from cell suspension
cultures ofNicotiana tabacum L. cv. Samsun by determination of λ-glutamylcysteine as its monobromobimane derivative. The enzyme has a relative molecular
mass (Mr) of 60000 and exhibits maximal activity at pH 8 (50% at pH 7.0 and pH9.0) and an absolute requirement for Mg2+. With 0.2mM Cd2+ or Zn2+, enzyme activity was reduced by 35% and 19%, respectively. Treatment with 5 mM dithioerythritol led to a heavy loss of activity
and to dissociation into subunits (Mr 34000). Buthionine sulfoximine andl-methionine-sulfoximine, known as potent inhibitors of λ-glutamylcysteine synthetase from mammalian cells, were found to be
effective inhibitors of the plant enzyme too. The apparent Km values forl-glutamate,l-cysteine, and α-aminobutyrate were, respectively, 10.4mM, 0.19 mM, and 6.36 mM. The enzyme was completely inhibited by glutathione
(Ki=0.42 mM). The data indicate that the rate of glutathione synthesis in vivo may be influenced substantially by the concentration
of cysteine and glutamate and may be further regulated by feedback inhibition of λ-glutamylcysteine synthetase by glutathione
itself. λ-Glutamylcysteine synthetase is, like glutathione synthetase, localized in chloroplasts as well as in the cytoplasm.
Chloroplasts fromPisum sativum L. isolated on a Percoll gradient contained about 72% of the λ-glutamylcysteine synthetase activity in leaf cells and 48%
of the total glutathione synthetase activity. In chloroplasts ofSpinacia oleracea L. about 61% of the total λ-glutamylcysteine synthetase activity of the cells were found and 58% of the total glutathione
synthetase activity. These results indicate that glutathione synthesis can take place in at least two compartments of the
plant cell.
Dedicated to Professor A. Prison on the occasion of his 80th birthday 相似文献
928.
Nitrogen-starved sunflower plants (Helianthus annuus L. cv. Peredovic) cannot absorb NO
3
–
or NO
2
–
upon initial exposure to these anions. Ability of the plants to take up NO
3
–
and NO
2
–
at high rates from the beginning was induced by a pretreatment with NO
3
–
. Nitrite also acted as inducer of the NO
2
–
-uptake system. The presence of cycloheximide during NO
3
–
-pretreatment prevented the subsequent uptake of NO
3
–
and NO
2
–
, indicating that both uptake systems are synthesized de novo when plants are exposed to NO
3
–
. Cycloheximide also suppressed nitrate-reductase (EC 1.6.6.1) and nitrite-reductase (EC 1.7.7.1) activities in the roots. The sulfhydryl-group reagent N-ethylmaleimide greatly inhibited the uptake of NO
3
–
and NO
2
–
. Likewise, N-ethylmaleimide promoted in vivo the inactivation of nitrate reductase without affecting nitrite-reductase activity. Rates of NO
3
–
and NO
2
–
uptake as a function of external anion concentration exhibited saturation kinetics. The calculated Km values for NO
3
–
and NO
2
–
uptake were 45 and 23 M, respectively. Rates of NO
3
–
uptake were four to six times higher than NO
3
–
-reduction rates in roots. In contrast, NO
2
–
-uptake rates, found to be very similar to NO
3
–
-uptake rates, were much lower (about 30 times) than NO
2
–
-reduction rates. Removal of oxygen from the external solution drastically suppressed NO
3
–
and NO
2
–
uptake without affecting their reduction. Uptake and reduction were also differentially affected by pH. The results demonstrate that uptake of NO
3
–
and NO
2
–
into sunflower plants is mediated by energy-dependent inducible-transport systems distinguishable from the respective enzymatic reducing systems.Abbreviations CHI
cycloheximide
- NEM
N-ethylmaleimide
- NiR
nitrite reductase
- NR
nitrate reductase
-
pHME
p-hydroxymercuribenzoate
This research was supported by grant PB86-0232 from the Dirección General de Investigatión Científica y Técnica (Spain). One of us (E.A.) thanks the Consejeria de Educación y Ciencia de la Junta de Andalucia for the tenure of a fellowship. We thank Miss G. Alcalá and Miss C. Santos for their valuable technical and secretarial assistance. 相似文献
929.
A cyclic AMP response element mediates repression of tyrosine aminotransferase gene transcription by the tissue-specific extinguisher locus Tse-1 总被引:30,自引:0,他引:30
Tyrosine aminotransferase (TAT) gene expression is liver specific and inducible by glucocorticoids and via the cAMP signaling pathway. In fibroblasts and other nonliver cells the gene is subject to negative control by the trans-dominant tissue-specific extinguisher locus Tse-1. We identified a hepatocyte-specific enhancer that is repressed by Tse-1. Two distinct sequence motifs are absolutely essential for function of this enhancer: a cAMP response element (CRE), which is the target for repression by Tse-1, and a hepatocyte-specific element. The specificity of the enhancer is generated by the combination of these two essential elements, which are fully interdependent. In vivo footprinting indicates that Tse-1 acts by affecting protein binding at the CRE. A direct antagonism between Tse-1 and the cAMP signaling pathway suggests that Tse-1 plays a role in control of developmental activation of the TAT gene. 相似文献
930.
Different pathways mediate virus inducibility of the human IFN-alpha 1 and IFN-beta genes 总被引:26,自引:0,他引:26
N J MacDonald D Kuhl D Maguire D N?f P Gallant A Goswamy H Hug H Büeler M Chaturvedi J de la Fuente 《Cell》1990,60(5):767-779