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11.
Peter W. Wilson Katie E. Osterday Aaron F. Heneghan Anthony D. J. Haymet 《The Journal of biological chemistry》2010,285(45):34741-34745
In this study, we examined the effects that antifreeze proteins have on the supercooling and ice-nucleating abilities of aqueous solutions. Very little information on such nucleation currently exists. Using an automated lag time apparatus and a new analysis, we show several dilution series of Type I antifreeze proteins. Our results indicate that, above a concentration of ∼8 mg/ml, ice nucleation is enhanced rather than hindered. We discuss this unexpected result and present a new hypothesis outlining three components of polar fish blood that we believe affect its solution properties in certain situations. 相似文献
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Influence of sequence and distance between two operators on interaction with the lac repressor 总被引:9,自引:0,他引:9
W T Hsieh P A Whitson K S Matthews R D Wells 《The Journal of biological chemistry》1987,262(30):14583-14591
The influence of additional operator or pseudooperator sequences on the lactose repressor-operator interaction has been investigated. Results of kinetic and equilibrium binding measurements suggest an important in vivo role for the Z-gene pseudooperator in repressor-operator binding; the formation of a ternary, looped complex is indicated by the influence of secondary operator sites on binding parameters. Although the binding affinity of the Z-gene pseudooperator [Oz] is only approximately 1/30 that observed for the primary operator [O], the binding affinity to DNA containing both Oz and O is significantly higher than either sequence alone or the sum of the two. This synergistic effect is enhanced further by replacing the pseudooperator sequence [Oz] with the primary operator sequence and results in an even stronger ternary complex in plasmids with duplicate primary sites. The distance between the center of the two primary operators affects the formation of a ternary complex in the linear DNA molecules. Decreased dissociation rate constants were observed with spacing of operator-like sequences between 300 and 500 base pairs (bp). Minimal influence of the second operator on repressor binding is observed when the operators are separated by approximately 4000 or approximately 100 bp. The significant influence of distance on kinetic and equilibrium parameters was demonstrated by measurements on plasmid pRW1511 [Oi-O-PL-Oz] cleaved with restriction enzymes either in the polylinker region to place Oi-O and Oz on opposite ends of the linear plasmid or outside this region to maintain the sites within 500 bp. These results are consistent with the formation of operator-repressor-pseudooperator ternary complex to generate a looped DNA structure. 相似文献
15.
N-Carbamoyl-beta-alanine (NC beta A) amidohydrolase (EC 3.5.1.6) is regulated in opposing fashion by the substrate, NC beta A and the product, beta-alanine. The native enzyme from rat liver has a molecular weight of 235,000 in the absence of ligands. NC beta A and substrate analogs (N-amidino-beta-alanine, N-carbamoyl-glycine) produced association of the enzyme. beta-Alanine and its analog gamma-aminobutyrate caused dissociation of the enzyme and produced inhibition. Negative cooperativity was observed for the binding of all ligands as measured by the change in polymerization of the enzyme, with an average Hill coefficient (napp) of 0.5. Enzyme that had been dissociated by preincubation with beta-alanine had little or no initial activity; only after a lag of 9 s was a steady state progress curve evident. The existence of a regulatory site is proposed as a model to explain physical and kinetic data. The enzyme activity was highest in rat liver and detectable in kidney; activity was not detected in brain, lung, muscle, or spleen of rat, nor in mouse Ehrlich ascites tumor cells. The rat liver enzyme has a pH optimum of 6.8, with a Km of 6.5 microM for NC beta A and a Ki of 1.08 mM for beta-alanine at this pH. 相似文献
16.
The cloning of chromosomal DNA associated with methicillin and other resistances in Staphylococcus aureus 总被引:19,自引:0,他引:19
Competitive hybridization was used to detect the deletion of chromosomal DNA accompanying the loss of resistance to methicillin (and concomitantly, to cadmium, mercury and tetracycline) from a clinical strain of methicillin-resistant Staphylococcus aureus (MRSA). The method was also used to screen a partial plasmid library of chromosomal HindIII fragments from the MRSA strain. Eight recombinant plasmid clones were identified as containing DNA included in the deletion. These clones were used as probes to screen a phage library of the total DNA of the same MRSA strain, resulting in the isolation of overlapping recombinant phage clones carrying 24 kb of the deleted DNA. Two of the cloned HindIII fragments were associated closely with methicillin resistance, as shown by probing DNA from an independent methicillin-sensitive/resistant transduced strain pair and from two MRSA strains following growth in the presence of high concentrations of methicillin. The endonuclease map of the cloned DNA indicates the presence of four copies of a direct repeat less than 1 kb in size. The map is also consistent with the presence in the chromosome of sequences for mercury resistance (mer A mer B) and for tetracycline-resistance plasmid pT181. 相似文献
17.
The modes of binding to thermolysin of two phosphonamidate peptide inhibitors, carbobenzoxy-GlyP-L-Leu-L-Leu (ZGPLL) and carbobenzoxy-L-PheP-L-Leu-L-Ala (ZFPLA), have been determined by X-ray crystallography and refined at high resolution to crystallographic R-values of 17.7% and 17.0%, respectively. (GlyP is used to indicate that the trigonal carbon of the peptide linkage is replaced by the tetrahedral phosphorus of a phosphonamidate group.). These inhibitors were designed to be structural analogues of the presumed catalytic transition state and are potent inhibitors of thermolysin (ZGPLL, Ki = 9.1 nM; ZFPLA, Ki = 0.068 nM) [Bartlett, P. A., & Marlowe, C. K. (1987) Biochemistry (following paper in this issue)]. ZFPLA binds to thermolysin in the manner expected for the transition state and, for the first time, provides direct support for the presumed mode of binding of extended substrates in the S2 subsite. The mode of binding of ZFPLA displays all the interactions that are presumed to stabilize the transition state and supports the postulated mechanism of catalysis [Hangauer, D. G., Monzingo, A. F., & Matthews, B. W. (1984) Biochemistry 23, 5730-5741]. The two oxygens of the phosphonamidate moiety are liganded to the zinc to give overall pentacoordination of the metal. For the second inhibitor the situation is different. Although both ZFPLA and ZGPLL have similar modes of binding in the S1' and S2' subsites, the configurations of the carbobenzoxy-Phe and carbobenzoxy-Gly moieties are different. For ZFPLA the carbonyl group of the carbobenzoxy group is hydrogen bonded directly to the enzyme, whereas in ZGPLL the carbonyl group is rotated 117 degrees, and there is a water molecule interposed between the inhibitor and the enzyme. For ZGPLL only one of the phosphonamidate oxygens is liganded to the zinc. Correlated with the change in inhibitor-zinc ligation from monodentate in ZGPLL to bidentate in ZFPLA there is an increase in the phosphorus-nitrogen bond length of about 0.25 A, strongly suggesting that the phosphonamide nitrogen in ZFPLA is cationic, analogous to the doubly protonated nitrogen of the transition state. The observation that the nitrogen of ZFPLA appears to donate two hydrogen bonds to the protein also indicates that it is cationic. The different configurations adopted by the respective inhibitors are correlated with large differences in their kinetics of binding [Bartlett, P. A., & Marlowe, C. K. (1987) Biochemistry (following paper in this issue)]. These differences in kinetics are not associated with any significant conformational change on the part of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
18.
Nucleotide sequence of 10 kilobases of rat tyrosine aminotransferase gene 5'' flanking region. 总被引:5,自引:3,他引:2
J Oddos T Grange K D Carr B Matthews J Roux H Richard-Foy R Pictet 《Nucleic acids research》1989,17(21):8877-8878
19.
Diffusion of oxygen through aqueous solutions is of great importance in biological systems. In this work, three models for the diffusion of oxygen through aqueous salt solutions are compared. One model uses mole fraction as the driving force (Fick's Law) and another uses chemical potential. The third model uses the gradient in oxygen activity as the driving force. This new model was chosen because of the availability of oxygen electrodes which directly measure oxygen activity in aqueous solution. These models have been used to reevaluate the technique of measuring O(2) diffusivities. We show that Pick's Law diffusion coefficients do not vary strongly with salt concentration as was erroneously reported in the literature. In addition, we compare the predicted O(2) fluxes of the three models over a wide range in O(2) concentrations. For oxygen concentrations of biological interest, the three models give identical predictions of the flux. 相似文献
20.
Neuropeptide Y mobilizes intracellular Ca2+ and increases inositol phosphate production in human erythroleukemia cells 总被引:2,自引:0,他引:2
A J Daniels E R Lazarowski J E Matthews E G Lapetina 《Biochemical and biophysical research communications》1989,165(3):1138-1144
The intracellular concentration of free Ca2+ was monitored by measuring the fluorescence of fura-2 loaded Human Erythroleukemia Cells. Neuropeptide Y (NPY) increased intracellular Ca2+ in a dose-dependent manner and the 50% effective concentration was 2 nM. Chelation of extracellular Ca2+ by EGTA did not reduce the NPY-mediated increase in cytoplasmic Ca2+, indicating that the increase in fluorescence was due to the release of intracellular Ca2+. A second dose of NPY, after intracellular Ca2+ had returned to basal levels, failed to elicit a response, indicating that the NPY receptor had undergone desensitization. In similar experiments, NPY increased the formation of inositol phosphates, suggesting that the mobilization of Ca2+ from intracellular stores in HEL cells was secondary to the generation of inositol phosphates and stimulation of phospholipase C. 相似文献