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41.
R A Roubey G D Ross J T Merrill F Walton W Reed R J Winchester J P Buyon 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(10):3557-3562
C receptor CR3 (iC3b-receptor, CD11b/CD18) plays an essential role in several phagocytic and adhesive neutrophil functions. Recent evidence suggests that stimulus-induced phosphorylation of the CR3 beta-chain, CD18, may mediate certain neutrophil functions by transiently converting the molecule to an activated state. Staurosporine, a protein kinase C inhibitor that blocks PMA-induced CD18 phosphorylation, was used to study the functional relevance of this event. Neutrophils adhered to glass were assayed for binding and phagocytosis of iC3b-opsonized sheep E (EC3bi) in the presence or absence of PMA and/or staurosporine. Binding of EC3bi was markedly increased, not only by PMA, but also by staurosporine and by a combination of both agents (three- to sevenfold). The enhancement of rosetting by staurosporine was likely caused by increased surface expression of CR3 via exocytosis of specific granular contents. In contrast, staurosporine alone did not stimulate phagocytosis of EC3bi and markedly inhibited PMA-induced phagocytosis. Staurosporine also inhibited phagocytosis of yeast beta glucan particles, a CR3 ligand that, in contrast to EC3bi, is bound and ingested without additional prior treatment with PMA. beta glucan phagocytosis was associated with a low level of CD18 phosphorylation. Staurosporine did not block phagocytosis in general, because this agent had relatively little effect on FcR-mediated phagocytosis. These data demonstrate that phagocytosis mediated by CR3 requires activation of CR3 via a staurosporine-sensitive pathway. Increased binding of EC3bi, a function of increased surface expression of CR3, does not require activation of CR3 by such a pathway, confirming previous evidence for the independence of these two phenomena. A direct role for CD18 phosphorylation in the activation of CR3 for phagocytosis is consistent with these data. 相似文献
42.
Cloning, sequence analysis, and overexpression of Escherichia coli folK, the gene coding for 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase. 下载免费PDF全文
The gene coding for the Escherichia coli enzyme 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase has been cloned and sequenced. This gene, designated folK, codes for a protein of 159 amino acids, including an amino-terminal methionine. The protein was overexpressed in E. coli MC4100 by cloning the gene behind the lacUV5 promoter in a high-copy-number plasmid. The enzyme was purified to homogeneity. Amino-terminal analysis of the purified protein showed that the amino-terminal methionine had been removed. The compositional molecular mass (17,945 Da) was identical to the molecular mass determined by mass spectrometry. The enzyme was observed to have a large number of proline residues and migrated anomalously in sodium dodecyl sulfate-polyacrylamide gels, with an apparent molecular mass of 23,000 Da. 相似文献
43.
On the nature of the structural change of the colicin E1 channel peptide necessary for its translocation-competent state 总被引:5,自引:0,他引:5
Acidic pH conditions required in vitro for membrane binding and activity of the channel-forming colicin E1 resulted in an increased susceptibility to proteases of the 178-residue thermolytic channel peptide, an increased accessibility to acrylamide of a fluorescence probe linked to cysteine-505 of the peptide, and an increased partition into nonionic detergent. The structural change in the peptide sensed by the fluorescence probe caused by a transition from pH 6.0 to 3.5 occurred in less than 1 s. The presence of low concentrations of detergents (0.001% SDS or 0.44% octyl beta-D-glucoside) or urea (0.2 M) at pH 6 or 4 also increased the susceptibility of the channel peptide to proteases. The increase in protease susceptibility and acrylamide accessibility at low pH, as well as partition of the peptide into nonionic detergent, suggested that acidic pH or the detergents might cause peptide unfolding. However, the hydrodynamic radius of the channel peptide at pH 6, 21-23 A, was not changed at pH 3.5 or by detergents or urea under conditions that increased the susceptibility of the peptide to protease. The activity of the channel peptide at pH 6 measured with liposomes and planar bilayers, which was a factor of 10(3)-10(4) smaller than that at pH 4, was increased by 2-4 orders of magnitude by 0.001% SDS or 0.44% octyl beta-D-glucoside, with an additional small increment of activity on planar bilayers caused by 0.01% SDS. A small increase in Stokes radius of the peptide in the presence of SDS could be detected that was approximately correlated with increased activity. 相似文献
44.
Paul A. Andrews Merrill J. Egorin Matthew E. May Nicholas R. Bachur 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1982,227(1):83-91
6-Thioguanine (6TG) and its metabolites were analyzed in human plasma with a reversed-phase high-performance liquid chromatographic method. 6TG and related compounds were extracted from plasma with an equal volume of 2 N perchloric acid at a 50–100% recovery efficiency. The neutralized extracts were chromatographed on a μBondapak C18 column by two separate isocratic conditions. 6TG, 6-thiouric acid, 6-thioxanthine, 6-thioguanosine, and 6-methylthiouric acid were analyzed with 0.01 M sodium acetate, pH 3.5–10% methanol as the mobile phase and 340 nm for detection. 6-Methylthioguanine and three unknown metabolites were separated with acetate—25% methanol and 310 nm detection. One of the unknowns was identified as 6-methylthioguanosine. External standard calibration was used for quantitation. The 6TG detection limit was 0.8 nmol/ml in plasma. 相似文献
45.
Ronald E. Clawson Merrill J. Egorin Bonnie M. Fox Louis A. Ross Nicholas R. Bachur 《Life sciences》1981,28(10):1133-1137
The ability of rat liver microsomes and liver slices to metabolize the antineoplastic compound cyclophosphamide was studied at 37° and at elevated temperatures comparable to those used for human systemic hyperthermic antineoplastic therapy. Temperatures above 40.5° and 41.8° inhibited cyclophospamide metabolism by microsomes and liver slices respectively. Therefore, cyclophosphamide may not be a suitable drug for combination with systemic hyperthermia in cancer therapy. 相似文献
46.
47.
Flavokinase (ATP: riboflavin 5'-phosphotransferase, EC 2.7.1.26) purified from rat liver by affinity chromatography, has been immobilized by amide linkage to omega-aminoalkyl-agarose beads. The immobilized enzyme differs from the soluble enzyme in having greater stability, slightly higher Km for the substrates, riboflavin and ATP, a broader pH optimum, and a lower energy of activation. These results suggest that the immobilized enzyme is influenced by the microenvironment of the bead and is subject to some degree of internal diffusional limitation. A small (3 ml), continuous, plug-flow reactor prepared with immobilized flavokinase effects 50% conversion of riboflavin to riboflavin 5'-phosphate (FMN) with a flow rate of 0.16 ml/min, which corresponds to an output of 5 nmol FMN/min. Immobilized flavokinase is effective for phosphorylating riboflavin and numerous riboflavin analogs and provides a facile method for preparing exclusively, unlike other synthetic methods, the 5'-phosphates. 相似文献
48.
49.
A 25,000 molecular weight protein constituent of human amyloid fibrils related to amyloid protein AA
Jane B. Lian Merrill D. Benson Martha Skinner Alan S. Cohen 《Archives of biochemistry and biophysics》1975,171(1):197-205
Amyloid fibrils from a patient with diffuse amyloid disease are dissociated in 6 m guanidine hydrochloride and fractionated by gel chromatography. Two major components are separated on Sepharose 6B. Both proteins are characterized by chromatography, immunodiffusion, discontinuous gel electrophoresis, amino acid tryptic peptide mapping and amino acid sequence analysis. The smaller of the two components is typical of the known protein AA by size (8400 daltons), amino acid composition and a 30-residue N-terminal sequence. The larger of the components (25,000 daltons) undergoes electrophoresis as a single band and appears unaffected by thiol reduction. It differs from protein AA in amino acid content and by its tryptic peptide map, although it contains an N-terminal amino acid sequence identical to protein AA when carried to 20 residues. Treatment of this larger component by mild acid hydrolysis results in the release of the 8400-dalton protein AA. Fractionation after guanidine hydrochloride treatment of this particular amyloid fibril preparation is compared to the fractionation of a typical secondary amyloid preparation that contains only protein AA as the major component. The origin and relationship of the 8,400- and 25,000-dalton protein components is discussed. 相似文献
50.