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991.
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
992.
The biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin has been studied with a microsomal preparation obtained from etiolated seedlings of sorghum. The biosynthetic pathway involves tyrosine, N-hydroxytyrosine, and p-hydroxyphenylacetaldehyde oxime as early intermediates (M?ller, B. L. and Conn, E. E. (1980) J. Biol. Chem. 254, 8575-8583). The use of deuterium-labeled tyrosine and mass spectrometric analyses demonstrate that the alpha-hydrogen atom of tyrosine is retained in the conversion of tyrosine to p-hydroxyphenylacetaldehyde oxime. This excludes p-hydroxyphenylpyruvic acid oxime as intermediate in the pathway. A high pressure liquid chromatography method was developed to separate the (E)- and (Z)-isomers of p-hydroxyphenylacetaldehyde oxime. The microsomal enzyme system was found to produce initially the (E)-isomer of p-hydroxyphenylacetaldehyde oxime. An isomerase then converts the (E)-isomer to the (Z)-isomer, which is the isomer preferentially utilized by the microsomal enzyme system in the subsequent biosynthetic reactions. The (E)-isomer produced in situ is more efficiently converted to the (Z)-isomer than exogenously added (E)-isomer and may thus be metabolically channeled. 相似文献
993.
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 下载免费PDF全文
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
994.
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
995.
V gene rearrangement is required to fully activate the hypermutation mechanism in B cells 总被引:10,自引:0,他引:10
J Roes K Hüppi K Rajewsky F Sablitzky 《Journal of immunology (Baltimore, Md. : 1950)》1989,142(3):1022-1026
Nonproductively rearranged H and L chain loci of B cell hybridoma lines expressing heavily mutated antibodies were cloned and partially sequenced. The results confirm earlier data showing that somatic point mutations are as frequent in nonproductively rearranged loci containing a rearranged V gene as in productively rearranged loci. They establish in addition that in nonproductive H chain loci which bear a DJH rearrangement the frequency of somatic mutations is more than 10 times lower (0.2%) than in VDJH loci expressed by the same cells (2.5%). Thus, the hypermutation mechanism operating in B cell differentiation is targeted at V genes rearranged to the J locus and may require nucleotide sequences associated with both V and J elements in order to be fully activated. An inversion of the JH2 segment was detected in one DJH locus. This inversion appears to be the result of a secondary joining event occurring occasionally in the course of B cell development. 相似文献
996.
997.
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
998.
Purified potato tuber (Solanum tuberosum L. cv Bintie) mitochondria contain soluble, highly latent NAD+- and NADP+-isocitrate dehydrogenases, NAD+- and NADP+-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160, 25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP+-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP+-malate dehydrogenase activity is probably due to unspecificity of the NAD+-malate dehydrogenase. NADP+-specific isocitrate dehydrogenase had much lower Kms for NADP+ and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD+-specific enzyme (101 micromolar for NAD+ and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP+-specific isocitrate dehydrogenase whereas the NAD+-specific enzyme had a sharp optimum at pH 7.8. Externally added NADP+ stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where external NADPH oxidation was inhibited. This shows that (a) NADP+ is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP+-reducing activities of malate dehydrogenase and the NADP+-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes. 相似文献
999.
Inhibition of zeaxanthin formation and of rapid changes in radiationless energy dissipation by dithiothreitol in spinach leaves and chloroplasts 总被引:22,自引:11,他引:11 下载免费PDF全文
Demmig-Adams B Adams WW Heber U Neimanis S Winter K Krüger A Czygan FC Bilger W Björkman O 《Plant physiology》1990,92(2):293-301
Dithiothreitol, which completely inhibits the de-epoxidation of violaxanthin to zeaxanthin, was used to obtain evidence for a causal relationship between zeaxanthin and the dissipation of excess excitation energy in the photochemical apparatus in Spinicia oleracea L. In both leaves and chloroplasts, inhibition of zeaxanthin formation by dithiothreitol was accompanied by inhibition of a component of nonphotochemical fluorescence quenching. This component was characterized by a quenching of instantaneous fluorescence (Fo) and a linear relationship between the calculated rate constant for radiationless energy dissipation in the antenna chlorophyll and the zeaxanthin content. In leaves, this zeaxanthin-associated quenching, which relaxed within a few minutes upon darkening, was the major component of nonphotochemical fluorescence quenching determined in the light, i.e. it represented the `high-energy-state' quenching. In isolated chloroplasts, the zeaxanthin-associated quenching was a smaller component of total nonphotochemical quenching and there was a second, rapidly reversible high-energy-state component of fluorescence quenching which occurred in the absence of zeaxanthin and was not accompanied by Fo quenching. Leaves, but not chloroplasts, were capable of maintaining the electron acceptor, Q, of photosystem II in a low reduction state up to high degrees of excessive light and thus high degrees of nonphotochemical fluorescence quenching. When ascorbate, which serves as the reductant for violaxanthin de-epoxidation, was added to chloroplast suspensions, zeaxanthin formation at low photon flux densities was stimulated and the relationship between nonphotochemical fluorescence quenching and the reduction state in chloroplasts then became more similar to that found in leaves. We conclude that the inhibition of zeaxanthin-associated fluorescence quenching by dithiothreitol provides further evidence that there exists a close relationship between zeaxanthin and potentially photoprotective dissipation of excess excitation energy in the antenna chlorophyll. 相似文献
1000.
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