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1.
The search for an unusual cyclic nucleotide-dependent protein kinase in nematodes represented an attempt to gain some insight into the proposed homology of the cAMP and cGMP-dependent protein kinases. Two species of protein kinase were found in high speed supernatants of the mycophagous nematode Aphelenchusavenae. One of the two, bound to DEAE cellulose and was eluted from it in a manner characteristic of the type I cAMP kinase. The enzyme had high affinity for cAMP and dissociated upon binding to the cyclic nucleotide, as judged by the fact that catalytic activity did not bind to a cAMP affinity column. The second enzyme did not bind to DEAE. Unexpectedly, it too had high affinity for cAMP and much lower affinity for cGMP (unlike the cAMPcGMP kinase from insects). The holoenzyme bound tightly to the cAMP affinity column and required a high concentration of the cyclic nucleotide for elution. This latter enzyme is the only example of a cAMP-dependent protein kinase that does not dissociate upon activation.  相似文献   

2.
Purification of thiamine-binding protein from Escherichia coli   总被引:2,自引:0,他引:2  
An affinity column coupled with thiamine pyrophosphate quantitatively absorbs the thiamine-binding activity from a partially purified preparation of Escherichiacoli. The thiamine-binding protein can be eluted from the affinity column in high yield by use of 8 M urea-containing buffer. Approximately 90-fold purification occurs by affinity chromatography yielding a preparation which appears to be homogenous.  相似文献   

3.
The cell surface glycoproteins of hamster NIL cells, labeled with galactose oxidase and NaB3H4, were selectively solubilized by sequential extraction with Tris buffer containing 1) sucrose-ATP-EDTA, 2) zwitterionic detergent (Empigen BB), and 3) 8 M urea. The previously reported “galactoprotein b” (Gap b) and “galactoprotein a” (Gap a or LETS) were isolated by affinity chromatography on insoluble Ricinus communis lectin colums (RCA column) from extracts 2) and 3), respectively. The affinity-purified Gap a contained an actin-like protein, whereas the other affinity-purified galactoproteins did not contain the actin-like protein. The isolated Gap b was heterogeneous, and an additional glycoprotein, specific for NILpy cells was copurified on RCA-column with Gap b.  相似文献   

4.
Trypsin treatment of cultured normal human skin fibroblasts or Hela cells releases material which is retained on a low density lipoprotein (LDL)-Sepharose affinity column, may be eluted from it with 2.5 M KI and, after dialysis, agglutinates LDL or apo-B-coated formocells. Such agglutination is prevented by preincubation of the receptor with LDL in solution or with arginine-rich protamine. Trypsin treatment of “receptor defective” or “receptor negative” mutant fibroblasts releases material which is retained on LDL-Sepharose column but fails to agglutinate LDL-coated formocells. The receptor may be labeled with 6-[3H]-glucosamine·HCl and [3H]-leucine, it is inactivated by heating at 80°C for 10 min and may be obtained from normal fibroblasts or Hela cells, whether they were cultured in presence or in absence of lipoprotein-containing fetal calf serum.  相似文献   

5.
The preparation of monovalent concanavalin A was achieved by incubating metal-free concanavalin A with trypsin at room temperature for 50 hrs. The digest was subjected to affinity chromatography on a column of ovomucoid-agarose to remove the trypsin and subsequent chromatography on Bio-Gel P-150 to resolve the monovalent fragment. Monovalent concanavalin A bound Mn2+, Ca2+ and p-nitrophenyl-α-D-mannopyranoside as determined by ultraviolet difference spectroscopy. The modified protein would not precipitate glycogen or agglutinate Bacillus subtilis cells. The fragment did, however, prevent the agglutination of B. subtilis by native concanavalin A. Preparation of monovalent concanavalin A could not be achieved unless metals were first removed from the native protein.  相似文献   

6.
Studies of the localization of the Na+-dependent sugar transport in monolayers of LLC PK1 cells show that the uptake of a methyl α-d-glucoside, a nonmetabolizable sugar which shares the glucose-galactose transport system, occurs mainly from the apical side of the monolayer. Kinetics of [3H]phlorizin binding to monolayers of LLC PK1 cells were also measured. These studies demonstrate the presence of two distinct classes of receptor sites. The class comprising high affinity binding sites had a dissociation constant (Kd) of 1.2 μM and a concentration of high affinity receptors of 0.30 μmol binding sites per g DNA. The other class involving low affinity sites had a Kd of 240 μM with the number of binding sites equal to 12 μmol/g DNA. Phlorizin binding at high affinity binding sites is a Na+-dependent process. Binding at the low affinity sites on the contrary is Na+-independent. The mode of action of Na+ on the high affinity binding sites was to increase the dissociation constant without modifying the number of binding sites. The Na+ dependence and the matching of Kd for high affinity binding sites with the Ki of phlorizin for the inhibition of methyl α-d-glucoside strongly suggest that the high affinity phlorizin binding site is, or is part of the methyl α-d-glucoside transport system. Binding studies from either side of the monolayer also show that the binding of phlorizin at the Na+ dependent high affinity binding sites occurs mainly from the apical rather than the basolateral side. The specific location of the Na+-dependent sugar transport system in the apical membrane of LLC PK1 cells is, therefore, another expression of the functional polarization of epithelial cells that is retained under tissue culture condition. In addition, since this sugar transport almost disappears after the cells are brought into suspension, it can be used as a marker to study the development of the apical membrane in this cell line.  相似文献   

7.
We have investigated the interaction of VIP and secretin with two human lung carcinoma cell lines in cultures, SW-900 and Calu-1. 125I-labeled VIP binds to and is inactivated by SW-900 and Calu-1 cells in a time- and temperature-dependent manner. The rates of binding and of inactivation were higher at 30°C than at 15°C. At equilibrium, native VIP competitively inhibited the binding of 125I-VIP in the 10?10?10?7M range, half-maximal inhibition being observed at 1.2 nM in SW-900 cells and at 1.1 nM VIP in Calu-1 cells. Scatchard analysis indicated two classes of binding sites with similar characteristics in both cell lines. SW-900 cells have 27 600 sites with a high affinity (Kd = 0.34 nM) and 1062 000 sites with a low affinity (Kd = 61.4 nM). Calu-1 cells have 36 300 sites with a high affinity (Kd = 0.33 nM) and 1148 000 sites with a low affinity (Kd = 78.6 nM). Secretin inhibited tracer binding but with a 5000 times lower potency than native VIP in both cell lines.  相似文献   

8.
Three forms of endo-(1→3)-β-g-glucanases lysing yeast cell walls from Rhizoctonia solani were separated by precipitation with ammonium sulfate and by successive chromatographies on CM Bio-Gel A and Bio-Gel P-60 or P-30, and were finally purified by substrate affinity chromatography on short-chain pachyman-AH-Sepharose CL 6B column. Each preparation was found to be homogeneous on gel filtration and by electrophoresis on acrylamide gel with sodium dodecyl sulfate. They exhibit high activity against insoluble pachyman, but only restricted activity against soluble short-chain pachyman. In the affinity chromatography, three enzymes were found to be strongly absorbed on the column, so that they could be easily eluted with substrate solution using biospecific counter-ligand. It was thus revealed that covalent binding of such a soluble glucan to aminohexyl-Sepharose provides a useful carrier for separation of endo-(1→3)-β-D-glucanases lysing yeast cell walls.  相似文献   

9.
Two valine-sensitive acetohydroxy acid synthase activities were separable from Escherichiacoli K-12 cells by virtue of their different affinities for DEAE-cellulose eluted with a KC1 gradient. These activities appeared to be independent from a valine-resistant cryptic component expressed only in ilvO regulatory mutants. The properties of the first and second activity were coincident to those of extracts of ilvB and ilvHI mutants, respectively. These data prove that the ilvB and ilvHI gene products exist in the cell as physically distinct acetohydroxy acid synthase isoenzymes.  相似文献   

10.
A laboratory isolate of Bacillusbrevis could grow and sporulate on an amino acid, viz., alanine or glutamate or aspartate as single source of carbon and nitrogen. It failed to sporulate if the amino acid was replaced by the corresponding keto acid and ammonium sulphate in the medium, although, normal growth was observed. One of the key enzymes in nitrogen assimilation, the glutamine synthetase, has been purified by DE-52 and affinity column chromatography from both alanine and pyruvate grown cells. The kinetic and other properties of both of these enzymes were studied. The enzyme isolated from alanine grown cells differed significantly from that isolated from pyruvate grown cells (viz.,pH optima, response to Mg++ and other effectors). A possible role of glutamine synthetase in the initiation of bacterial sporulation is discussed.  相似文献   

11.
Changes in neutral amino acid transport activity caused by addition of phytohaemagglutinin-P to quiescent peripheral pig lymphocytes have been evaluated by measurements of 14C-labelled neutral and analogue amino acids under conditions approaching initial entry rates. Utilizing methylaminoisobutyric acid, the best model substrate of System A, we confirmed our previous report (Borghetti, A.F., Kay, J.E. and Wheeler, K.P. (1979) Biochem. J. 182, 27–32) on the absence of this transport system in quiescent cells and its emergence following stimulation. Furthermore, we demonstrated the presence in quiescent cells of an Na+-dependent transport system for neutral amino acids that has been characterized as System ASC by several criteria including intolerance to methylaminoisobutyric acid, strict Na+-dependence, the property of transtimulation and specificity for pertinent substrates such as alanine, serine, cysteine and threonine. Analysis of the relationship between influx and substrate concentration revealed that two independent saturable components contribute to entry of alanine in quiescent cells: a low affinity (Km = ≈4 mM) and a high affinity (Km = ≈0.2 mM) component. The high affinity component could be inhibited in a competitive way by serine, cysteine and threonine, but methylaminoisobutyric acid did not change appreciably its constants. The enhanced activity of alanine transport through the ASC system observed in activated cells resulted from a large increase in the capacity (V) of the high affinity component without any substantial change in the apparent affinity constant (Km).  相似文献   

12.
An affinity column for renin   总被引:1,自引:0,他引:1  
An affinity column for renin was prepared making use of the strong affinity of pepstatin for renin. Pepstatin is an N-acylated pentapeptide from Actinomycetes with the following structure: isovaleryl-L-valyl-L-valyl-4-amino-3-hydroxy-6-methylheptanoyl-L-alanyl-4-amino-3-hydroxy-6-methylheptanoic acid. This peptide was coupled to aminoethylated polyacrylamide gel either directly with the water-soluble carbodiimide, 1-ethyl-3-(dimethylaminopropyl) carbodiimide, or through the N-hydroxy succinimide ester. Submaxillary renin was selectively retained by a small column of the gel and was eluted by a salt gradient to produce a highly pure material. This column was also effective for the purification of renal renin.  相似文献   

13.
Unlike other enzymes of the aromatic multienzyme system, chorismate synthase and the aromatic complex of Neurospora crassa were found to bind to a column of cellulose phosphate and to elute at a relatively high concentration of phosphate (~ 0.2 M). The fact that other enzymes with similar ionic properties failed to bind to phosphocellulose suggests that the binding of the former two enzyme systems is due to a specific affinity for phosphate. This conclusion is not only supported by the fact that these same enzymes did not bind to a column of carboxymethyl cellulose, but also is consistent with the nature of the catalytic reactions of the enzymes. Both the shikimate kinase enzyme of the aromatic complex and chorismate synthase would be expected to have active sites which accomodate a phosphate moiety. We anticipate that other enzymes which involve phospho-substrates will also be amendable to this procedure.  相似文献   

14.
J. Fuska  J. Prousek  J. Rosazza 《Steroids》1982,40(2):157-169
Microbial transformation experiments were conducted with the antitumor lactone withaferin-A. Cunninghamella elegans NRRL 1393 transformed withaferin-A (1a) to 15β-hydroxywithaferin-A (2a) and 12β-hydroxywithaferin-A (3a). The hydroxylated metabolites were isolated by solvent extraction and were purified by column and thin-layer chromatography. Structures of the hydroxylated metabolites were determined by protonand carbon-13 NMR, IR and mass spectral analyses, and by the preparation of acylated derivatives. Compounds 2a and 3a inhibited the growth and biochemical functions of in vitro grown P-388 lymphocytic leukemic cells.  相似文献   

15.
(1) Alkyl sugar inhibition of d-allose uptake into adipocytes has been used to explore the spatial requirements of the external sugar transport site in insulin-treated cells. α-methyl and β-methyl glucosides show low affinity indicating very little space around C-1. The high affinity of d-glucosamine (Ki = 9.05 ± 0.66 mM) is lost by N-acetylation. N-Acetyl-d-glucosamine shows no detectable affinity, indicating that a bulky group at C-2 is not accepted. Similarly 2,3-di-O-methyl-d-glucose (Ki = 42.1 ± 7.5 mM) has lower affinity than 3-O-methyl-d-glucose (Ki = 5.14 ± 0.32 mM) indicating very little space around C-2 but much more around C-3. A reduction in affinity does occur if a propyl group is introduced into the C-3 position. The Ki for 3-O-propyl-d-glucose is 11.26 ± 2.12 mM. 6-O-Methyl-d-galactose (Ki = 87.2 ± 17.9 mM) and 6-O-propyl-d-glucose (Ki = 78.07 ± 12.6 mM) show low affinity compared with d-galactose and d-glucose, indicating steric constraints around C-6. High affinity is restored in 6-O-pentyl-d-galactose (Ki = 4.66 ± 0.23 mM) possibly indicating a hydrophobic binding site around C-6). (2) In insulin treated cells 4,6-O-ethylidene-d-glucose (Ki = 6.11 ± 0.5 mM) and maltose (Ki = 23.5 ± 2.1 mM) are well accommodated by the site but trehalose shows no detectable inhibition. These results indicate that the site requires a specific orientation of the sugar as it approaches the transporter from the external solution. C-1 faces the inside while C-4 faces the external solution. (3) To determine the spatial and hydrogen bonding requirements for basal cells 40 μM 3-O-methyl-d-glucose was used as the substrate. Poor hydrogen bonding analogues and analogues with sterically hindering alkyl groups showed similar Ki values to those determined for insulin-treated cells. These results indicate that insulin does not change the specificity of the adipocyte transport system.  相似文献   

16.
Thiamine pyrophosphokinase (E.C. 2.7.6.2.) from Saccharomyces cerevisiae was found to require the presence of a non-protein, non-metal compound for its activity. myo-Inositol was found capable of stimulating the kinase activity in the presumably resolved but otherwise crude sample of the enzyme. The hexytol was also found capable of inducing the enzyme in growing yeast cells. The cultured yeast cells, in which the kinase had been induced, were used as source of the enzyme for its purification. The compound that had been left adsorbed to the final column of DEAE-Sephadex was proved to have a coenzyme activity towards the enzyme and tentatively identified with myo-inositol 1-pyrophosphate. A sample of synthetic myo-inositol 1-pyrophosphate was made and its coenzyme activity was observed.  相似文献   

17.
The natural affinity of various bacterial glycopeptides and lipopolysaccharides for mammalian cell membranes was estimated quantitatively by comparison with the adsorption of lipopolysaccharide from Escherichia coli NCTC 8623 to erythrocytes, thymocytes, bone marrow cells, spleen cells, peritoneal lymphocytes and macrophages. Immunopotentiating activity was estimated by measuring the ability of the bacterial fractions to stimulate a humoral response to ovalbumin in HAM/1CR mice. When the affinity for mammalian cell membranes was compared with the stimulation of the antibody response, it was found that a negative correlation for peritoneal macrophages (rs=?0.94, P<0.0005) and a positive correlation for peritoneal lymphocytes (rs=+0.97,P<0.0005) and spleen cells (rs=+0.76,P<0.005) existed.  相似文献   

18.
Commerical heparin, 135 USP units/mg, was fractionated by human α-thrombin-agarose affinity chromatography. Heparin was applied to an α-thrombin-agarose column equilibrated with 0.01 M Tris HCl (pH 7.4). Unbound heparin was washed from the column with the equilibration buffer. Bound heparin could be eluted with buffer containing 0.025 M NaCl. The specific activity of bound heparin was as great as 500 USP units/mg. Gel filtration was used to fractionate the heparin into molecular size classes. Low molecular weight heparin, with an average specific activity of 100 USP units/mg, was applied to the α-thrombin-agarose column. Gel filtration of the unbound heparin indicated that larger heparin molecules been selectively removed by the α-thrombin-agarose column. Bound heparin had a specific activity of 270 units/mg. Kinetic results of N-α-tosyl-L-glycyl-L-prolyl-L-arginine-p-nitroanilide hydrolysis by α-thrombin in the presence of heparin correlated with the anticoagulant activity.  相似文献   

19.
A number of β-carboline analogs have been obtained or synthesized, and their in vitro receptor affinities and in vivo antagonist activities determined. The choice of analogs was made in order to explore the importance of the N9-H, the aromatic nitrogen and the C3-ester moiety for high-receptor affinity and antagonist activity of this class of benzodiazepine antagonist. Among the analogs investigated, we describe the properties of 3-cyano-β-carboline (lh), the first potent β-carboline antagonist without a carbonyl at the C3-position.The results obtained indicate: (1) Specific interactions of the C3-substituent with key cationic receptor sites rather than electron-withdrawing properties are important for high-receptor affinity and antagonist activity. (2) Specific in-plane interactions of the atomatic nitrogen with a cationic receptor site, rather than stacking with neutral aromatic residues of the receptor are also important for high affinity and antagonist activity. (3) While the presence of an N9H enhances receptor affinity, interaction with an anionic receptor site does not appear essential for antagonist activity.  相似文献   

20.
The interaction between chick embryo fibroblasts and A1-specific blood group Dolichos biflorus lectin has been studied at various stages of embryo development. The site number ((0.26±0.03) · 106sites/cell) remains the same during development whereas the affinity constant apparently decreases from 8-day cells onwards. The effects of cell number, temperature and time course on the Dolichos binding to fibroblasts were not age dependent. Competitive binding experiments revealed that Dolichos receptor sites were distinct from binding sites of Robina pseudoacacia lectin and concanavalin A, but partially related to binding sites of Ricinus lectin. Thymidine incorporation by fibroblasts in the presence of Dolichos lectin was age dependent. It was inhibited in 6-day cells and weakly stimulated in 16-day cells, but not modified in 12-day cells. Dolichos lectin effects on embryo fibroblasts were very specific because both binding to cells and effect on thymidine incorporation were blocked by N-acetylgalactosamine, the determinant of Dolichos lectin, as well as by Dolichos antiserum.  相似文献   

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