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91.
Recent crystallographic studies on the mutant human hemoglobin Ypsilanti (beta 99 Asp-->Tyr) have revealed a previously unknown quaternary structure called "quaternary Y" and suggested that the new structure may represent an important intermediate in the cooperative oxygenation pathway of normal hemoglobin. Here we measure the oxygenation and subunit assembly properties of hemoglobin Ypsilanti and five additional beta 99 mutants (Asp beta 99-->Val, Gly, Asn, Ala, His) to test for consistency between their energetics and those of the intermediate species of normal hemoglobin. Overall regulation of oxygen affinity in hemoglobin Ypsilanti is found to originate entirely from 2.6 kcal of quaternary enhancement, such that the tetramer oxygenation affinity is 85-fold higher than for binding to the dissociated dimers. Equal partitioning of this regulatory energy among the four tetrameric binding steps (0.65 kcal per oxygen) leads to a noncooperative isotherm with extremely high affinity (pmedian = .14 torr). Temperature and pH studies of dimer-tetramer assembly and sulfhydryl reaction kinetics suggest that oxygenation-dependent structural changes in hemoglobin Ypsilanti are small. These properties are quite different from the recently characterized allosteric intermediate, which has two ligands bound on the same side of the alpha 1 beta 2 interface (see ref. 1 for review). The combined results do, however, support the view that quaternary Y may represent the intermediate cooperativity state of normal hemoglobin that binds the last oxygen. 相似文献
92.
A step leading to the formation of the covalent complexes between porcine pancreatic elastase (PPE) and 7-[(alkylcarbamoyl)amino]-4-chloro-3-ethoxyisocoumarins (alkylHNCO-EICs) is the formation of the noncovalent Michaelis complex. No average structures are available for the Michaelis complexes of PPE with alkylHNCO-EICs. We present the results of an initial step in obtaining these structures and have determined kinetic constants as well. The kinetic results indicate that formation of the Michaelis complex is what differentiates the effectiveness of these inhibitors in inactivating PPE. The structural and kinetic results together suggest that the structure of the Michaelis complex is necessary for the design of potent alkylHNCO-EIC inhibitors of PPE. Two novel alkylHNCO-EICs are predicted to be the best inhibitors of this series. An alternate mechanism for serine protease inhibition is also proposed. Evidence for, and studies that may add support to, the hypothesized mechanism are discussed. 相似文献
93.
E. J. Nücken R. B. Henschke F. R. J. Schmidt 《Molecular & general genetics : MGG》1991,229(1):137-146
Summary Tn21-related transposons are widespread among bacteria and carry various resistance determinants at preferential sites, hs1 and hs2. In an in vivo integrative recombination assay it was demonstrated that these hot spots direct the integration of aminoglycoside resistance genes like aadB from Klebsiella pneumoniae and aacAI from Serratia marcescens, in a recA
– background. The maximum required recognition sequence which must be present in both the donor and recipient plasmids is 5 CTAAAACAAAGTTA 3 (hs2). The double-site-specific recombination occurred with a frequency of 10–5–10–6. The resulting structures include not only replicon fusion products but also more complex structures carrying two copies of the donor plasmid or simply the donor gene flanked by hs elements. hs1 and hs2 are thought to act as recognition sites for a trans-acting site-specific recombinase. By the use of Tn21 deletion derivatives, it has been shown that the recombinase is not encoded by Tn21. This new integrative recombination system is involved in the acquisition of new genes by Tn21-related transposons and their spread among bacterial populations. 相似文献
94.
Fabrizio Arigoni P. Alexandre Kaminski Hauke Hennecke Claudine Elmerich 《Molecular & general genetics : MGG》1991,225(3):514-520
Summary The nucleotide sequence of a 4.1 kb DNA fragment containing the fixABC region of Azorhizobium caulinodans was established. The three gene products were very similar to the corresponding polypeptides of Rhizobium meliloti. The C-terminal domains of both fixB products displayed a high degree of similarity with the -subunits of rat and human electron transfer flavoproteins, suggesting a role for the FixB protein in a redox reaction. Two open reading frames (ORF) were found downstream of fixC. The first ORF was identified as fixX on the basis of sequence homology with fixX from several Rhizobium and Bradyrhizobium strains. The second ORF potentially encoded a 69 amino acid product and was found to be homologous to a DNA region in the Rhodobacter capsulatus nif cluster I. Insertion mutagenesis of the A. caulinodans fixX gene conferred a Nif– phenotype to bacteria grown in the free-living state and a Fix– phenotype in symbiotic association with the host plant Sesbania rostrata. A crude extract from the fixX mutant had no nitrogenase activity. Furthermore, data presented in this paper also indicate that the previously identified nifO gene located upstream of fixA was probably a homologue of the nifW gene of Klebsiella pneumoniae and Azotobacter vinelandii. 相似文献
95.
Determination of the chromosomal site for the human radiosensitive ataxia telangiectasia gene by chromosome transfer 总被引:3,自引:0,他引:3
The chromosomal localization of the gene which complements radiation hypersensitivity of AT cells was studied by microcell-mediated chromosome transfer. A 6-thioguanine-resistant derivative of an immortalized AT cell line, AT2KYSVTG, was used as a recipient for microcell-mediated chromosome transfer from 4 strains of mouse A9 cells, 3 of which carried a human X/11 recombinant chromosome containing various regions of chromosome 11, while the other carried an intact X chromosome. HAT-resistant microcell hybrids were isolated and examined for their radiosensitivity and chromosome constitution. The microcell hybrid clones obtained from the transfer of an intact X chromosome or an X/11 chromosome bearing the pter → q13 region of chromosome 11 did not show a difference in radiosensitivity from parental AT cells, while those obtained from the transfer of X/11 chromosomes bearing either the p11 → qter or the pter → q23 region of chromosome 11 exhibited a marked radioresistance which was comparable to normal human fibroblasts. A HAT-resistant but radiosensitive variant was further obtained from the microcell fusion with an A9 cell strain carrying an X/11 chromosome bearing the 11p11 → qter region, in which a deletion at the 11q23 region was found. The results indicate that the gene which complements a radiosensitive phenotype of AT is located at the q23 region of chromosome 11. 相似文献
96.
The effects of the local anesthetic dibucaine on coupling between electron transport and ATP synthesis-hydrolysis by the coupling-factor complex (CF0CF1 ATPase) were investigated in thylakoid membranes from Spinacia oleracea L. cv. Monatol. Evidence is presented that inhibition of ATP synthesis was produced by a specific uncoupling mechanism which was based on dibucaine-membrane surface interactions rather than on the interaction of dibucaine with the ATPase complex. Dibucaine reduced the osmotic space of thylakoid vesicles. At low pH of the medium it stimulated ATP hydrolysis beyond the rates obtained with optimum concentrations of ‘classical’ uncouplers. After addition of dibucaine, there was displacement of membrane-bound Mg2+ and strong thylakoid stacking in the presence of only low Mg2+ concentrations. Inhibition of ATP synthesis and transmembrane pH gradient increased with medium pH. Hydrolysis of ATP by isolated CF1 and the CF0CF1 complex was only slightly affected by dibucaine. The data are discussed assuming the involvement of localized proton channels on the membrane surface in protonic coupling of electron transport and ATP synthesis. A hypothesis for the mechanisms of action of local anesthetics at the thylakoid membrane is presented. 相似文献
97.
The second-order rate constant (k4) for the oxidation of monosubstituted phenols and anilines by lactoperoxidase compound II was examined by Chance's method [B. Chance, Arch. Biochem. Biophys.
71 (1957), 130–136]. When the electronic states of these substrates were calculated by an ab initio molecular orbital method, it was found that the log k4 value correlates well with the highest occupied molecular orbital (HOMO) energy level but not with the net charge or frontier electron density. These results are essentially similar to those reported previously in the case of horseradish peroxidase [J. Sakurada, R. Sekiguchi, K. Sato, and T. Hosoya, Biochemistry
29 (1990), 4093–4098], showing some dissimilar features which are considered to reflect the structural difference between the two enzymes.Abbreviations HOMO
highest occupied molecular orbital
- HRP
horseradish peroxidase
- LPO
lactoperoxidase (EC 1.11.1.7)
- LUMO
lowest unoccupied molecular orbital 相似文献
98.
An experimental test of the eddy correlation technique over a Mediterranean macchia canopy 总被引:4,自引:1,他引:3
R. VALENTINI G. E. SCARASCIA MUGNOZZA P. DE ANGELLS R. BIMBI 《Plant, cell & environment》1991,14(9):987-994
Abstract. Flux densities of water vapour and carbon dioxide were measured for a Mediterranean macchia canopy. Results show good agreement between the measured available energy and the sum of latent sensible and heat flux densities determined with the eddy correlation technique. Joint evaluation of the Bowen ratio, aerodynamic resistance, canopy resistance and the 'omega factor' suggests that the macchia canopy is intermediate in aerodynamic roughness between coniferous and deciduous canopies. Maximum daytime carbon flux densities ranged from -14 to -22(μnol m−2 s−1 on a ground area basis. The ratio of transpiration to assimilation (E/A) was a function of incident photo-synthetic photon flux density below about 400 μmol m−2 s−1 and above it was fairly constant at 272 mol mol−1 (H2 O/CO2 ). The relationship between carbon influx and canopy conductance was linear. Results show promising applications of the eddy correlation technique for evaluating physiological features of canopies, treated as unitary functional systems. 相似文献
99.
The general structure of cytochrome oxidase is reviewed and evidence that the enzyme acts as a redox-linked proton pump outlined. The overall H+/e
– stoichiometry of the pump is discussed and results [Wikström (1989),Nature
338, 293] which suggest that only the final two electrons which reduce the peroxide adduct to water are coupled to protein translocated are considered in terms of the restrictions they place on pump mechanisms. Direct and indirect mechanisms for proton translocation are discussed in the context of evidence for redox-linked conformational changes in the enzyme, the role of subunit III, and the nature of the CuA site. 相似文献
100.
The initial (F0), maximal (FM) and steady-state (FS) levels of chlorophyll fluorescence emitted by intact pea leaves exposed to various light intensities and environmental conditions, were measured with a modulated fluorescence technique and were analysed in the context of a theory for the energy fluxes within the photochemical apparatus of photosynthesis. The theoretically derived expressions of the fluorescence signals contain only three terms, X=J2p2F/(1–G), Y=T/(1–G) and V, where V is the relative variable fluorescence, J2 is the light absorption flux in PS II, p2F is the probability of fluorescence from PS II, G and T are, respectively, the probabilities for energy transfer between PS II units and for energy cycling between the reaction center and the chlorophyll pool: F0=X, FM=X/(1–Y) and FS=X(1+(YV/(1–Y))). It is demonstrated that the amplitudes of the previously defined coefficients of chlorophyll fluorescence quenching, qP and qN, reflect, not just photochemical (qP) or nonphotochemical (qN) events as implied in the definitions, but both photochemical and nonphotochemical processes of PS II deactivation. The coefficient qP is a measure of the ratio between the actual macroscopic quantum yield of photochemistry in PS II (41-1) in a given light state and its maximal value measured when all PS II traps are open (41-2) in that state, with 41-3 and 41-4. When the partial connection between PS II units is taken into consideration, 1-qP is nonlinearily related to the fraction of closed reaction centers and is dependent on the rate constants of all (photochemical as well as nonphotochemical) exciton-consuming processes in PS II. On the other hand, 1-qN equals the (normalized) ratio of the rate constant of photochemistry (k2b) to the combined rate constant (kN) of all the nonphotochemical deactivation processes excluding the rate constant k22 of energy transfer between PS II units. It is demonstrated that additional (qualitative) information on the individual rate constants, kN-k22 and k2b, is provided by the fluorescence ratios 1/FM and (1/F0)–(1/FM), respectively. Although, in theory, 41-5 is determined by the value of both k2b and kN-k22, experimental results presented in this paper show that, under various environmental conditions, 41-6 is modulated largely through changes in k
N, confirming the idea that PS II quantum efficiency is dynamically regulated in vivo by nonphotochemical energy dissipation.Abbreviations Chl
chlorophyll
- F0, FM and FS
initial, maximal and steady-state levels of modulated Chl fluorescence emitted by light-adapted leaves
- PS I and II
photosystem I and II
- qP and qN
(previously defined) photochemical and nonphotochemical components of Chl fluorescence quenching 相似文献