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21.
The role of a miniscaffolding protein, miniCipC1, forming part of Clostridium cellulolyticum scaffolding protein CipC in insoluble cellulose degradation was investigated. The parameters of the binding of miniCipC1, which contains a family III cellulose-binding domain (CBD), a hydrophilic domain, and a cohesin domain, to four insoluble celluloses were determined. At saturating concentrations, about 8.2 micromol of protein was bound per g of bacterial microcrystalline cellulose, while Avicel, colloidal Avicel, and phosphoric acid-swollen cellulose bound 0.28, 0.38, and 0.55 micromol of miniCipC1 per g, respectively. The dissociation constants measured varied between 1.3 x 10(-7) and 1.5 x 10(-8) M. These results are discussed with regard to the properties of the various substrates. The synergistic action of miniCipC1 and two forms of endoglucanase CelA (with and without the dockerin domain [CelA2 and CelA3, respectively]) in cellulose degradation was also studied. Although only CelA2 interacted with miniCipC1 (K(d), 7 x 10(-9) M), nonhydrolytic miniCipC1 enhanced the activities of endoglucanases CelA2 and CelA3 with all of the insoluble substrates tested. This finding shows that miniCipC1 plays two roles: it increases the enzyme concentration on the cellulose surface and enhances the accessibility of the enzyme to the substrate by modifying the structure of the cellulose, leading to an increased available cellulose surface area. In addition, the data obtained with a hybrid protein, CelA3-CBD(CipC), which was more active towards all of the insoluble substrates tested confirm that the CBD of the scaffolding protein plays an essential role in cellulose degradation. 相似文献
22.
Summary Periplasmic-leaky (lky) Hfr mutant strains of Escherichia coli K12, grown in low-phosphate Tris medium, excreted alkaline phosphatase (AP) into the extracellular fluid. The lky207 mutation, which proved to induce the highest AP excretion rate, was transferred to an F- host, carrying a phoS, T mutation allowing constitutive AP biosynthesis. Use of high-phosphate LB-rich medium for growing this F-
lky strain improved cell biomass, extracellular AP activity and excretion specificity in favour of the enzyme. Physiological studies helped us to develop a new culture medium (LB 8.3) giving higher enzyme and excretion yields. LB 8.3 medium also increased cell viability of lky mutants stored at 4° C.Using optimized culture conditions, the highest extracellular enzyme activity produced by lky mutant 706 was reached in the late stationary growth phase and was equal to 1,400 U/ml of culture medium (i.e., 6 times the intracellular AP content of wild-type strain, Ga15, developed in derepressed conditions); AP released into the extracellular fluid corresponded to 34% of total excreted proteins and was equivalent to a purified enzyme preparation. 相似文献
23.
Methylation of (R,S)-DOPA with diazomethane gave the trimethyl derivative in which the phenolic hydroxy groups and the carboxy group were methylated. N-Methylated side products were also formed. N-Acylation of the racemic trimethyl derivative with (S)-α-methoxy-α-trifluoromethylphenylacetyl chloride gave two diastereomeric amides which were resolved by gas chromatography, the diastereomer derived from (S)-(−)-DOPA cluting first. The procedure was also applied to α-methyl-DOPA. 相似文献
24.
Summary The addition of serum to quiescent NIH 3T3 mouse cell cultures resulted in a 10- to 20-fold increase of Rb influx which was resistant to ouabain, and only a three- to fourfold activation of ouabain-sensitive Rb influx. Stimulation of the ouabain-resistant Rb influx following serum addition reached its maximum within 2 min. The stimulation of ouabain-resistant Rb influx was a result ofV
m increase while theK
m for Rb was unchanged. Ouabain-resistant Rb influx, after serum addition, was resistant to amiloride and sensitive to ethacrinic acid. Replacing chloride in the medium by NO3
–, CO3
– and CH3COO– resulted in a drastic decrease in the ouabain-resistant Rb influx. It appeared, therefore, that the ouabain-resistant Rb influx in NIH 3T3 cells was Cl–-dependent. 相似文献
25.
The cellular growth ofChlamydomonas reinhardii is modified by the addition of a total exogenous histone fraction. These modifications may be related to chloroplast DNA replication; they are different according to the different classes of histones. The H1 subfraction seems to be responsible for the effect of the total histone fraction. 相似文献
26.
27.
Alan K. Keenan Alma Gal Alexander Levitzki 《Biochemical and biophysical research communications》1982,105(2):615-623
Turkey erythrocyte adenylate cyclase was activated by GppNHp and l-epinephrine to its stable, highly active form. In this form the enzyme could be solubilized by Lubrol-PX and subsequently re-inserted into phospholipid vesicles concomitantly with the removal of up to 99.3% of the Lubrol. The ability of GTP and l-epinephrine to reverse the GppNHp/epinephrine activated state was taken as a measure for the reappearance of hormone sensitivity in the reconstituted vesicles. An incomplete but significant reappearance of hormone sensitivity in the reconstituted adenylate cyclase was achieved. This hormone sensitivity was found to be stereospecific for (?)epinephrine. The 125I-cyanopindolol binding properties of the reconstituted β-receptor depend on the nature of the detergent and the phospholipids used in the reconstitution. 相似文献
28.
In this study we examined the effect of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the bumetanide-sensitive Na+/K+/Cl- transporter in quiescent BALB/c 3T3 cells. We have shown that exposure of quiescent BALB/c 3T3 cultures to phorbol ester did not inhibit the basal bumetanide-sensitive Rb+ influx or efflux. In fact, at high concentration (100 ng/ml), TPA slightly stimulated the bumetanide-sensitive Rb+ influx and efflux. However, when the quiescent cultures were stimulated by serum or by defined growth factors, the stimulated fraction of the bumetanide-sensitive Rb+ influx was drastically inhibited by exposure of the cells to the phorbol ester TPA. Based on the above findings, we propose that activation of protein kinase C by the phorbol ester TPA does not inhibit the Na+/K+/Cl- cotransport activity; however it does suppress only the growth-factors-stimulated fraction of the cotransport in quiescent BALB/c 3T3 cells. These data propose that activation of kinase C has a regulatory feedback effect on the stimulation of the Na+/K+/Cl- cotransport activity by growth factors. 相似文献
29.
We previously identified 3-amino-1-phenylbutane (APB) as an oxidative N-dealkylated, metabolite of the antihypertensive agent labetalol. Labetalol has two asymmetric centers and is used clinically as a mixture of the four possible stereoisomers; APB has one asymmetric center. We now report an enantiospecific gas chromatographic/mass spectrometric assay for APB in urine. After adding the internal standard 1-methyl-2-phenoxyethylamine and alkalinizing, the urine samples were extracted with ether. The extracts were derivatized with the optically active acid chloride prepared from (S)-alpha-methoxy-alpha-trifluoromethylphenylacetic acid. The derivatives were separated by capillary gas chromatography and detected by electron capture negative ion chemical ionization mass spectrometry with selected ion monitoring. The derivative of the R enantiomer eluted first, and the [M--32]- ions were monitored for both the drug and the internal standard. The method was linear in the 0.05-2.5 micrograms enantiomer-1 ml-1 range and had inter-assay and intra-assay coefficients of variation of less than 6%. The assay was used in the analysis of urine samples from a patient in labetalol therapy and no interference was found. Further studies are needed to elucidate the oxidative metabolism of labetalol and its stereochemical aspects. 相似文献
30.
P Galéra D Vivien S Pronost J Bonaventure F Rédini G Loyau J P Pujol 《Journal of cellular physiology》1992,153(3):596-606