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11.
A process using ligand-coupled particles in aqueous polyethylene glycol-dextran two-phase polymer systems was developed to achieve a highly selective, scaleable biochemical separation process. Product protein is bound to the ligand-coupled particles that quantitatively distribute to the polyethylene glycol-rich upper phase. Other proteins and contaminants partition preferentially to the dextran-rich lower phase.The process offers significant advantages over affinity partitioning here the ligand is coupled to the backbone of a polyethylene glycol polymer. These advantages include a much wider diversity of ligands that can be coupled to particles and more effective confinement of the ligand in the process. Affinity partition with ligands coupled to particles is more amenable to scale-up than is affinity chromatography. A variety of commercially available Sepharose-based particles are suitable for this process. Homogenates from Saccharomyces cerevisiae, which is genetically altered to overproduce pyruvate kinase, and Cibacron blue F3G-A-coupled Sepharose particles are used as a model system for the process. Binding studies with/without aqueous two-phase systems show that the formation of a two-phase system after the adsorption equilibrium is reached does not affect the apparent dissociation constant. Binding of protein to ligand-coupled particles is more rapid in single-phase systems than in the polymer two-phase system. Single-phase binding eliminates the mass transfer resistance associated with redistribution of product protein from the dextran-rich bottom phase to the polyethylene glycol-rich top phase. 相似文献
12.
B Singer F Chavez S J Spengler J T Ku?mierek L Mendelman M F Goodman 《Biochemistry》1989,28(4):1478-1483
The effect of alkyl group size on ability to act as deoxythymidine triphosphate (dTTP) has been studied for the carcinogen products O2-methyl-, O2-ethyl-, and O2-isopropyl-dTTP by using three types of nucleic acids as template and DNA polymerase I (Pol I) or Klenow fragment as the polymerizing enzymes. Apparent Km and relative Vmax values were determined in primer extension on M13 DNA at a single defined site, in poly[d(A-T)], and in nicked DNA. These data are the basis for calculation of the relative rate of insertion opposite A, relative to dTTP. The insertion rate for any O2-alkyl-dTTP is much higher than for a mismatch between unmodified dNTPs. Unexpectedly, O2-isopropyl-dTTP is more efficiently utilized than O2-methyl-dTTP or O2-ethyl-dTTP on each of the templates. O2-isopropyl-dTTP also substitutes for dTTP over extended times of DNA synthesis at a rate only slightly lower than that of dTTP. Parallel experiments using O4-methyl-dTTP under the same conditions show that it is incorporated opposite A more frequently than is O2-methyl-dTTP. Therefore, both the ring position and the size of the alkyl group influence polymerase recognition. Once formed, all O2-alkyl-T.A termini permit elongation, as does O4-methyl-T.A. In contrast to the relative difficulty of incorporating the O-alkyl-dTTPs, formation of the following normal base pair (C.G) occurs rapidly when dGTP is present. This indicates that a single O-alkyl-T.A pair does not confer significant structural distortion recognized by Pol I. 相似文献
13.
14.
Lectin binding as a probe of proliferative and differentiative phases in primary monolayer cultures of cutaneous keratinocytes 总被引:1,自引:0,他引:1
The surface of cells in the cutaneous epidermis of the newborn rat exhibits a discrete change in lectin-binding specificity from Griffonia simplicifolia I-B4 (GS I-B4), specific for alpha-D-galactosyl residues, to Ulex europeus agglutinin I (UEA), specific for alpha-L-fucose, as the cell leaves the basal layer and differentiates. Primary monolayer cultures of rat keratinocytes maintained in low Ca2+ medium (0.08 mM) exhibited a characteristic unimodal pattern in the ratio of bound UEA to bound GS I-B4 (UEA/B4 ratio) over a 7-day culture period as determined by a quantitative fluorometric assay. The UEA/B4 ratio was initially low between Days 1 and 2 (0.56 +/- 0.05), steadily increased to a maximum of 0.84 +/- 0.09 between Days 2 and 4, and then gradually decreased to 0.41 +/- 0.07 between Days 6 and 7. Estimation of DNA synthesis showed (a) a higher [3H]thymidine incorporation when the UEA/B4 ratio was low and (b) a steady but lower incorporation between Days 3 and 4, coincident with the higher UEA/B4 ratio. Autoradiographic results further showed that cells stained intensely with UEA failed to incorporate [3H]thymidine into their nuclei. Electrophoresis of [3H]fucose-labeled material isolated on UEA-Sepharose 4B revealed that the changes in labeling by [3H]fucose, bound UEA, and the UEA/B4 ratio in the monolayer were related in part to variable expression of "96K-associated UEA-bound" radioactivity corresponding to a major class of lectin-specific cell-surface glycoproteins (GP96 fraction) identified in situ. Overall, the results suggest that (a) the increase in the UEA/B4 ratio between Days 2 and 4 reflects the progression of a proportion of the cells in the monolayer to an early spinous cell stage, the ultimate fate of which is desquamation into the medium and (b) the decrease in the UEA/B4 ratio between Days 5 and 7 reflects a consequent proliferative response to this loss of cells. This system should be useful for studying environmental influences on the homeostasis of cell proliferation and differentiation in the cutaneous epidermis. 相似文献
15.
E. -D. Schulze J. Čermák M. Matyssek M. Penka R. Zimmermann F. Vasícek W. Gries J. Kučera 《Oecologia》1985,66(4):475-483
Summary Leaf gas exchange, transpiration, water potential and xylem water flow measurements were used in order to investigate the daily water balance of intact, naturally growing, adult Larix and Picea trees without major injury. The total daily water use of the tree was very similar when measured as xylem water flow at breast height or at the trunk top below the shade branches, or as canopy transpiration by a porometer or gas exchange chamber at different crown positions. The average canopy transpiration is about 12% lower than the transpiration of a single twig in the sun crown of Larix and Picea. Despite the similarity in daily total water flows there are larger differences in the actual daily course. Transpiration started 2 to 3 h earlier than the xylem water flow and decreased at noon before the maximum xylem water flow was reached, and stopped in the evening 2 to 3 h earlier than the water flow though the stem. The daily course of the xylem water flow was very similar at the trunk base and top below the lowest branches with shade needles. The difference in water efflux from the crown via transpiration and the water influx from the trunk is caused by the use of stored water. The specific capacitance of the crown wood was estimated to be 4.7 x 10-8 and 6.3 x 10-8 kg kg-1 Pa-1 and the total amount of available water storage was 17.8 and 8.7 kg, which is 24% and 14% of the total daily transpiration in Larix and Picea respectively. Very little water was used from the main tree trunk. With increasing transpiration and use of stored water from wood in the crown, the water potential in the foliage decreases. Plant water status recovers with the decrease of transpiration and the refilling of the water storage sites. The liquid flow conductance in the trunk was 0.45 x 10-9 and 0.36 x 10-9 mol m-2s-1 Pa-1 in Larix and Picea respectively. The role of stomata and their control by environmental and internal plant factors is discussed. 相似文献
16.
17.
C D Perchonock I Uzinskas T W Ku M E McCarthy W E Bondinell B W Volpe J G Gleason B M Weichman R M Muccitelli J F DeVan 《Prostaglandins》1985,29(1):75-81
A series of desamino-2-nor-leukotriene analogs has been prepared by the reaction of various thiols with several methyl trans-4,5-epoxy-6Z-alkenoates, followed by deprotection. The products were assessed for their ability to antagonize the LTD4-induced contraction of the isolated guinea pig trachea. Several compounds displayed potent leukotriene antagonist activity, i.e., KB values in the sub-micromolar range, while only minimally affecting basal airway tone. The most potent analog, 4-hydroxy-5-(2-carboxyethylthio)-6Z-nonadecenoic acid, antagonized both LTD4- and LTE4-induced contractions of the trachea in an apparently competitive fashion. These agents possess increased potency relative to SK&F 101132, the first leukotriene analog identified as having LT-antagonist activity. Thus, these results demonstrate that deletion of the peptide amino group can produce leukotriene analogs which have minimal intrinsic contractile activity on the isolated guinea pig trachea, yet possess potent leukotriene-antagonistic effects. 相似文献
18.
M. Kučera 《Journal of invertebrate pathology》1984,43(2):190-196
Gel permeation, preparative isoelectric focusing, and affinity chromatography were used to purify three inhibitors of proteolytic activity from perchloric acid extracts of last instar Galleria mellonella larvae. Electrofocusing experiments revealed three isoinhibitors with different isoelectric points: inhibitor I-1 with p1 of pH 5.6, inhibitor I-2, pH 7.7, and inhibitor I-3 (of small inhibitory activity), pH 8.6. By affinity chromatography on trypsin-Sepharose 4B the I-1 was purified 9.7 ×, but 71.1% of inhibitory activity was lost. Molecular mass of the inhibitory complex was 12,600 Da. I-1 and I-2 are relatively stable to heat at several pHs with minor stability at pH 10. I-1 and I-2 inhibit serine proteases about 2.5 times as much as sulfhydryl proteases. In the same ratio protease P-1 and protease P-2 from Metarhizium anisopliae are inhibited. 相似文献
19.
Bonnie J. Reger Maurice S.B. Ku Jeffrey W. Pottert John J. Evans 《Phytochemistry》1983,22(5):1127-1132
The ribulose-1,5-bisphosphate carboxylase/oxygenase purified from maize (a C4 monocot) to homogeneity has a MW of532 000 and sedimentation coeffici 相似文献
20.