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1.
X Tao G Gao H Z Zhang D X Zhu A Boersma G Lamblin K K Han 《Biological chemistry Hoppe-Seyler》1987,368(3):187-194
CSF-1 was isolated from a large volume of human normal urine (10,000 l), using the following 5 stages of purification: concentration by dialysis, silica gel adsorption, hydrophobic chromatography on phenyl-Sepharose CL-6B, fast protein liquid chromatography (FPLC) and finally preparative electrophoresis on polyacrylamide gels. We have isolated 8 mg of purified CSF-1 which migrated as a single band under non-reducing conditions in SDS-PAGE (staining with Coomassie Blue and the sensitive silver techniques). But in the presence of dithiothreitol, the SDS-PAGE pattern revealed a minor second band with a molecular mass of 50,000 Da. CSF-1 was purified 100,000-fold and has a specific activity of 2.16 X 10(7) units/mg protein. Its apparent molecular mass is 57,000 Da with an isoelectric point, pI = 5.8-6.0. The amino-acid composition is reported and compared with that of murine CSF-1. The carbohydrate content (sialic acid, sulphate groups, N-acetylglucosamine, N-acetylgalactosamine) was also determined, and it shows that CSF is a glycoprotein. 相似文献
2.
G A Gao D X Zhu X Tao J Zheng A Scharfman G Lamblin Y W Shing K K Han 《The International journal of biochemistry》1989,21(4):391-399
1. Colony-stimulating factor (CSF-1) was isolated from a large volume of fresh normal human urine by 5 steps of purification and enrichment. 2. The purification factor is 100,000 fold and the purified compound exhibits a 2.16 x 10(7) U/mg of protein sp. act. 3. The isolated CSF-1 is a sialoglycoprotein with 41.5% of carbohydrate. The almost complete removal of this carbohydrate moiety (up to 91%) was achieved by incubation with trifluoromethane sulfonic acid. 4. The deglycosylated CSF-1 (DG-CSF-1) possesses an apparent Mr 38,000 compared to native CSF-1 with an initial Mr 57,000 (Goa et al., 1988). 5. The features of the interaction of radio-iodinated [125I]CSF-1 with single cell suspensions from various human tissues (bone marrow, spleen, blood, peritoneal cavity, alveolar lavage, lymph node and thymus), were studied. 6. The binding activity of peritoneal macrophages was the highest among the cells examined and erythrocytes, thymus and blood granulocytes showed no CSF-1 binding. 7. On incubation with [125I]CSF-1 at 0 degrees C, cellular binding of [125I]CSF-1 reached a stable maximum within 16 hr. This is in contrast to the association behaviour at higher temperature. 8. At 37 degrees C, cellular associated [125I]CSF-1 levels reached, within 90 min, an unstable maximum which was up to 10 times less than that occurring under the same conditions at 0 degree C. From the Scatchard plot analysis, we obtained the affinity constant and the number of receptor(s). 9. The binding site is sensitive to trypsin. 10. The receptor alone, (labelled by cross-linking to [125I]CSF-1 with di-succinylimidyl-suberate), is a polypeptide with an approx. Mr 110,000. 11. Our results showed that the receptor of CSF-1 is a tyrosin-kinase. 相似文献
3.
Fibroblast stimulation in schistosomiasis. V. Egg granuloma macrophages spontaneously secrete a fibroblast-stimulating factor 总被引:3,自引:0,他引:3
D J Wyler M J Stadecker C A Dinarello J F O'Dea 《Journal of immunology (Baltimore, Md. : 1950)》1984,132(6):3142-3148
Isolated intact egg granulomas from the liver of Schistosoma mansoni-infected mice have been previously shown to elaborate factors in vitro that can stimulate fibroblasts for biological functions that are of potential importance in the pathogenesis of hepatic fibrosis in schistosomiasis. We report here that cell cultures obtained from monodispersed granuloma cell suspensions, and specifically enriched for macrophages (95% to 100%) spontaneously elaborated fibroblast proliferation-stimulating activity in vitro. These cells possessed functional and phenotyptic characteristics of activated macrophages. In contrast, control peritoneal macrophages from uninfected mice lacked such phenotypic characteristics, and did not spontaneously elaborate fibrogenic activity in vitro. The granuloma macrophage activity was present, pre-formed within the isolated cells, and was continuously elaborated during 72 hr of incubation. By gel infiltration chromatography (Sephacryl S-200 sf), fibroblast-stimulating activity was identified in two pooled fractions, one with estimated molecular radius (Mr) of 46 kd to 57 kd and the other with Mr of 10 kd to 16 kd. Preparative isoelectric focusing in granular gel of crude macrophage culture supernatants identified peak activity in fractions with pI approximately 5. Two different serine esterase inhibitors had no effect on the ability of crude granuloma macrophage supernatants to stimulate fibroblast proliferation. Whereas crude and chromatographed fractions of granuloma macrophage supernatant were active for fibroblasts, they had minimal or no interleukin 1 (IL 1) activity when tested in a thymocyte proliferation assay. In contrast, resident peritoneal macrophages from the same infected mice spontaneously secreted substantial IL 1 and fibroblast-stimulating activity in vitro. We conclude that egg granuloma macrophages are activated in vivo to secrete fibrogenic molecules functionally distinct from IL 1, which might contribute to the pathogenesis of hepatic fibrosis in schistosomiasis. 相似文献
4.
Purification and partial characterization of an acidic fibroblast growth factor from bovine pituitary 总被引:9,自引:0,他引:9
Isoelectric focusing has allowed us to fractionate pituitary extracts into basic (pI 8-9) and acidic (pI 4-5) fibroblast growth factor. The acidic fibroblast growth factor (a) is stable upon refocusing, (b) migrates as an acidic protein in urea-containing gel electrophoresis; (c) is not cell-specific, being active with fibroblasts, adrenal, and glial cells, and (d) is a heterogeneous protein fraction with active components of different pI values. The component of pI 4.7, purified to or near homogeneity by isoelectric focusing shows a single peak of activity (Mr = 12,000) in gel chromatography and a single protein band of apparent Mr = 15,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximal restimulation of DNA synthesis initiation on serum-deprived 3T3 fibroblasts is achieved at 1-2 ng/ml; activity with rat glial cells (C6-3D) is less pronounced than with 3T3 fibroblasts. 相似文献
5.
1. An NADP+-dependent malic enzyme was purified 7940-fold from the cytosolic fraction of human skeletal muscle with a final yield of 55.8% and a specific activity of 38.91 units/mg of protein. 2. The purification to homogeneity was achieved by ammonium sulfate fractionation, DEAE-Sepharose chromatography, affinity chromatography on NADP+-Agarose, gel filtration on Sephacryl S-300 and rechromatography on the affinity column. 3. Either Mn2+ or Mg2+ was required for activity: the pH optima with Mn2+ and Mg2+ were 8.1 and 7.5, respectively. The enzyme showed Michaelis-Menten kinetics. At pH 7.5 the apparent Km values with Mn2+ and Mg2+ for L-malate and NADP+ were 0.246 mM and 5.8 microM, and 0.304 mM and 5.8 microM, respectively. The Km values with Mn2+ for pyruvate, NADPH and bicarbonate were 8.6 mM, 6.1 microM and 22.2 mM, respectively. 4. The enzyme was also able to decarboxylate malate in the presence of NAD+. At pH 7.5 the reaction rate was approximately 10% of the rate in the presence of NADP+, with a Km value for NAD+ of 13.9 mM. 5. The following physical parameters were established: s0(20.w) = 10.48, Stokes' radius = 5.61 nm, pI = 5.72 Mr of the dissociated enzyme = 61,800. The estimates of the native apparent Mr yielded a value of 313,000 upon gel filtration, and 255,400 with f/fo = 1.33 by combining the chromatographic data with the sedimentation measurements. 6. The electron microscopy analysis of the uranyl acetate-stained enzyme revealed a tetrameric structure. 7. Investigations to detect sugar moieties indicated that the enzyme contains carbohydrate side chains, a property not previously reported for any other malic enzyme. 相似文献
6.
Protein candidates for the attachment of DNA within eukaryotic cell nuclei were identified by isolating nuclear matrix proteins and determining which of those proteins co-sedimented with DNA within a 5.7 M CsCl gradient. The presence of attached nucleic acid was detected after the proteins were subjected to the denaturing conditions of isoelectric focusing/sodium dodecyl sulfate two-dimensional polyacrylamide gel electrophoresis. The attached nucleic acid was detected with silver staining, ethidium bromide, and Amido Black binding. The nucleic acid was identified as DNA based on its ability to be labeled in vitro by terminal deoxynucleotidyltransferase and DNA polymerase I (Klenow). Three proteins were identified as containing attached DNA, one of which was vimentin. The proteins had apparent Mr and pI values of 70,000, 4.3; 70,000, 5.3; and 57,000, 4.8, respectively. We propose that these proteins are within a class of nuclear proteins containing firmly attached DNA and have referred to them as DNA attachment proteins. 相似文献
7.
G Roy L M Villar I Lazaro M Gonzalez A Bootello P Gonzalez-Porque 《The Journal of biological chemistry》1991,266(18):11495-11501
Two phosphatidylinositol-specific phospholipases C (PI-PLC) have been purified from human spleen. PI-PLCm represents the main activity detected in the membrane, while PI-PLCc is the main activity present in the cytoplasm. PI-PLCm can be resolved into two peaks of activity of high Mr (60,000-70,000) and low Mr (16,000-18,000). High salt concentration ((NH4)2SO4, 2M) dissociates the high Mr form yielding the low molecular form and increasing the specific activity. The same effect of dissociation and potentiation of the activity is observed when membranes solubilized by n-octyl glucoside are subjected to the high voltage conditions of an isoelectric focusing run. The purified Pi-PLCm has a Mr of about 18,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel filtration and a basic pI (9.0-9.2). Purified PI-PLCc has a Mr of 57,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel filtration) and a slightly acid pI (6.2). Other characteristics of both enzymes, such as cations dependence, substrate specificity, optimum pH, and kinetic parameters, are also discussed. 相似文献
8.
W R Hudgins B Hampton W H Burgess J F Perdue 《The Journal of biological chemistry》1992,267(12):8153-8160
A procedure that combined ion exchange, gel permeation, and insulin-like growth factor-binding protein 3 (IGF-BP-3) affinity chromatography with chromatofocusing and reversed-phase high pressure liquid chromatography was used to isolate high molecular weight precursors of human insulin-like growth factor II (IGF-II) from acetic acid extracts of Cohn fraction IV1. Two precursors had isoelectric points (pI) of 5.1 and 5.4 and apparent Mr values of 15,000 and 11,500, respectively. An apparent Mr = 16,000 RLPG/Ser29 variant of IGF-II was also identified in the acetic acid extracts. Amino-terminal amino acid sequencing of the major E domain-containing peptide that had been isolated from apparent Mr = 15,000 IGF-II (pI 5.1), following its digestion with the endoprotease Lys-C, indicated the carboxyl terminus of this precursor was near or at Lys88. During the sequencing of this peptide, a sharply reduced yield of derivatized amino acid occurred at cycle 10, indicating that Thr75 had been posttranslationally modified, possibly by O-glycosylation. To evaluate this possibility, the 125I-labeled high molecular weight IGF-IIs and their endoprotease-generated peptides were treated with glycosidases, and their effects were determined from the change in relative mobilities of the polypeptide and peptides during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Neuraminidase treatment of apparent Mr = 15,000 and 11,500 IGF-II reduced their Mr values to a common value of 10,500. When the desialylated precursors of IGF-II were treated with O-glycosidase, but not when treated with N-glycosidase, the Mr values were reduced further to about 10,000. This was the Mr value that would be predicted for an unglycosylated form of precursor IGF-II that had a carboxyl-terminal end at or near Lys88. When the Ser66-Lys88 endoprotease-generated E domain peptides from pI5.1 and 5.4 high Mr IGF-II were treated with the glycosidases, they had relative mobility changes during sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were similar to those of the intact precursors. Finally, the association of O-linked oligosaccharide with the E domain peptide of IGF-II was confirmed by demonstrating the specificity of binding of the Ser66-Lys88 asialoglycopeptide to jackfruit lectin. 相似文献
9.
Penicillium chrysogenum extracellular acid phosphatase: purification and biochemical characterization 总被引:1,自引:0,他引:1
An extracellular acid phosphatase (EC 3.1.3.2) from crude culture filtrate of Penicillium chrysogenum was purified to homogeneity using high-performance ion-exchange chromatography and size-exclusion chromatography. SDS-PAGE of the purified enzyme exhibited a single stained band at an Mr of approx. 57,000. The mobility of the native enzyme indicated the Mr to be 50,000, implying that the active form is a monomer. The isoelectric point of the enzyme was estimated to be 6.2 by isoelectric focusing. Like acid phosphatases from several yeasts and fungi the Penicillium enzyme was a glycoprotein. Removal of carbohydrate resulted in a protein band with an Mr of 50,000 as estimated by SDS-PAGE, suggesting that 12% of the mass of the enzyme was carbohydrate. The enzyme was catalytically active at temperatures ranging from 20 degrees C to 65 degrees C with a maximum activity at 60 degrees C and the pH optimum was at 5.5. The Michaelis constant of the enzyme for p-nitrophenyl phosphate was 0.11 mM and it was inhibited competitively by inorganic phosphate (ki = 0.42 mM). 相似文献
10.
In a previous report we showed that purified bovine placental lactogen (bPL) exists in two isoforms in the 31,000-33,000 Mr range, each with at least five isoelectric variants differing in approximately 2 orders of magnitude in isoelectric points (pI) 4-6. The multiple isoelectric variants are unique to the bovine hormone. In an effort to determine the nature of these variants endo- and exoglycohydrolase digestions were conducted to determine if this hormone was glycosylated. Analysis of peptide/N-glycosidase F and endoglycosidase F digests of radioiodinated bPL on one-dimensional gel electrophoresis showed a Mr decrease from 31,000 to 24,000 and 33,000 to 26,000 for the two isoforms. Digestion with a mixture of neuraminidase plus mixed exoglycosidases resulted in a Mr decrease of 4,000. Digestion with neuraminidase resulted in a Mr decrease of 2,000. Further analysis of peptide/N-glycosidase F- and neuraminidase-treated bPL by two-dimensional gel electrophoresis showed the isoelectric variants shifted from pI 4.4-6.3 to 4.9-8.0. The sialic acid residues on the N-linkage are responsible for the pronounced acidic character of bPL, but do not account for the residual charge heterogeneity as the different isoelectric variants persist after sialic acid removal. The apparent Mr of the protein after removal of N-linked carbohydrate residues is similar to that of PRL and GH. These enzymatic digestion results demonstrate the presence of N-linked complex oligosaccharide residues attached to the beta-amide group of an asparagine residue. Analyses of the sugar content of the molecule were consistent with the presence of one biantennary N-linked and two O-linked carbohydrate chains. 相似文献
11.
Protein C inhibitor. Purification from human plasma and characterization 总被引:16,自引:0,他引:16
Protein C inhibitor was isolated from human plasma using conventional chromatographic technique consisting of barium citrate adsorption, polyethylene glycol fractionation, DEAE-Sepharose CL-6B treatment, ammonium sulfate fractionation, dextran sulfate-agarose chromatography, gel filtration on ACA-44, and DEAE-Sephacel chromatography. The purified protein C inhibitor is a single polypeptide chain with an apparent Mr = 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitor is heterogeneous in pI: six pIs exist between pH 7.4 and 8.6. The inhibitor was shown to be different from the already known plasma protease inhibitors by chemical and immunological analyses. It migrates to the late alpha 1-globulin region on agarose gel electrophoresis. The inhibitor reduced the amidolytic activity of activated protein C noncompetitively by forming a 1:1 molar complex with the enzyme, determined by the use of a fluorogenic substrate toward activated protein C (Boc-Leu-Ser-Thr-Arg-4-methylcoumaryl-7-amide). The inhibition constant (Ki) of the inhibitor against activated protein C was 5.8 x 10(-8) M. The inhibitor also blocked the prolongation of activated partial thromboplastin time by activated protein C. The immunoglobulin which was produced by the inhibitor completely removed the inhibitory activity present in normal human plasma against activated protein C. This suggests that the inhibitor which we have isolated is the only inhibitor in plasma against activated protein C. 相似文献
12.
A S Kuznetsov N S Parfenova S L Plavinski? 《Biulleten' eksperimental'no? biologii i meditsiny》1990,110(9):271-274
A few alternatives of the binding of healthy patients plasma low density lipoproteins (LDL) with anion exchanger PBE-94 were revealed. In the first case the main part of LDL did not bind to the gel and the isoelectric points of the minor subfractions were 5.8 and 5.3, and pI 4.1. In the second case about half of lipoproteins did not bind to the gel, and the isoelectric points of subfractions were 5.7 and 5.0; and pI 4.1. In the third case when all lipoproteins bound to the PBE-94, there were much more subfractions and their isoelectric points were 6.2, 5.8, 5.2, 4.9, 4.5 and pI 4.1. All LDL of the patients with ischemic heart disease bound to anion exchanger, and the part of subfraction with pI 4.1 was three or five times as great as the one of the healthy person. Increasing of the LDL subfraction with pI 4.1 was observed at prolonged keeping of the LDL obtained from the healthy person plasma. LDL isoelectric point distribution of the persons with carcinoma uterine cervix did not differ from the one of the healthy persons. Acetylation and hexanol modification resulted in the isoelectric point shift from 5.7 to 4.6 and to 4.3 in the case of LDL subfraction to be obtained preparatively using the chromatofocusing. 相似文献
13.
Effects of glycosidase treatment on the physicochemical properties and biological activity of human interferon-gamma 总被引:4,自引:0,他引:4
Highly purified human interferon (IFN)-gamma was treated with a preparation of mixed glycosidases in order to evaluate the effect of carbohydrate depletion on its biological activity, isoelectric point, and molecular size. Glycosidase treatment did not reduce the antiviral activity of IFN-gamma in cultures of human fibroblasts and in bat lung cells. No antiviral activity was observed before or after treatment with glycosidases in pig, mink, bovine, murine, and monkey cells. The degree of neutralization of IFN-gamma activity with specific antibody was also not significantly affected by glycosidase treatment. Several components of IFN-gamma activity were resolved by nonequilibrium pH gradient electrophoresis, with major peaks of activity at pI 8.5 and 8.7. Glycosidase treatment of IFN-gamma resulted in a reduced charge heterogeneity and a higher pI of 9.3. 125I-labeled IFN-gamma was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two bands with molecular weights of 25,000 and 20,000. Glycosidase treatment reduced the apparent molecular weight of these bands to 18,500 and 16,000, respectively. The results suggest that both the Mr = 25,000 and 20,000 bands, thought to be monomeric forms of IFN-gamma, are glycosylated. 相似文献
14.
The major parvalbumins present in the iliofibularis muscle of Xenopus laevis were identified and the total parvalbumin content of different types of single fibers of this muscle was determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS). The criteria used in the identification of proteins as parvalbumins were: a relative molecular mass (Mr) between 10,000 and 14,000, an isoelectric point (pI) between 4.0 and 5.0, and a Ca2+-dependent mobility when run on a polyacrylamide gel in the absence of SDS. Four proteins were thus identified as parvalbumins: PA1, Mr 14,000, pI 4.90; PA2, Mr 11,000, pI 4.90; PA3, Mr 11,000, pI 4.95; and PA4, Mr 11,000, pI 4.25. An ultraviolet absorbance spectrum characteristic of parvalbumins was recorded for a purified preparation of these four proteins. Because the apparent Mr of rabbit parvalbumin in the gel system used was 14,000, whereas the true value is 12,100, it is not excluded that the Mr of component PA1 of 14,000 is an overestimation. The total parvalbumin content of muscles and single muscle fibers was determined using the supernatant obtained after centrifugation of tissue homogenates. Analysis of the protein pattern after electrophoresis in the presence of SDS of this fraction indicated that the Mr 14,000 and 11,000 protein bands contained virtually only parvalbumin. Quantification of the total parvalbumin content of relatively fast (type 1) and slow (type 2) contracting and relaxing single muscle fibers, using laser densitometric analysis of minigels, yielded mean values (mg protein/g wet wt., +/- S.D.) of 5.2 +/- 0.8 for nine type 1 fibers, and 1.9 +/- 1.0 for five type 2 fibers. Both fiber types contained about 2.5-times as much of the Mr 14,000 isoform relative to the combined Mr 11,000 isoforms. 相似文献
15.
Transforming growth factor beta 1 treatment of AKR-2B cells is coupled through a pertussis-toxin-sensitive G-protein(s). 总被引:1,自引:0,他引:1
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Spermidine synthase was purified to apparent homogeneity from human spleens (8700-fold) by affinity chromatography. The native enzyme was composed of two subunits of identical Mr (35,000) and showed an apparent Mr of 62,000 in pore-gradient gel electrophoresis. Its pI was 5.1, Spermine synthase was purified to apparent homogeneity from placenta (5300-fold) and from kidney (4600-fold). The native enzyme was composed of two subunits of identical Mr (45,000) and showed an apparent Mr of 78,000 in pore-gradient gel electrophoresis. In isoelectric focusing it revealed two bands, with pI values of 4.9 and 5.0. Both synthases were present in all human tissues studied, but revealed a clear tissue-specific pattern. Mouse antisera against spermidine synthase revealed only one band, of Mr 35,000, in all purified enzyme preparations and in crude human tissue extracts in immunoblotting. Antisera against spermine synthase showed an immunoreactive band corresponding to the Mr of the subunit of spermine synthase. These antisera did not indicate any cross-reactivity in immunoblotting. Thus spermine synthase and spermidine synthase do not share homologous antigenic sites and are totally different proteins. 相似文献
16.
Mouse splenocytes are induced by pokeweed mitogen to secrete a factor that stimulates mouse hemopoetic (spelling per Nomina Histologica in the Nomina Anatomica, 5th edition, 1983, Williams and Wilkins, Baltimore) progenitor cells to undergo proliferation and differentiation into granulocytes and macrophages in a semi-solid culture system. The granulocyte and macrophage colony-stimulating factor (GM-CSF) was purified with a four-step procedure that includes ultrafiltration, chromatography on DEAE-agarose, Sephacryl S-200, and chromatofocusing gel. The isoelectric point (pI) of 4.2 of the GM-CSF was determined by analytical isoelectrofocusing gel electrophoresis. The sensitivity of the biological activity of GM-CSF to digestion by trypsin and neuraminidase suggests that GM-CSF is a glycoprotein with its sugar moieties at the active site. The GM-CSF is also sensitive to heat denaturation at 60 degrees C or higher suggesting that a three-dimensional conformation is required for its biological activity. The molecular weight of GM-CSF is approximately 57,000 Daltons as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. 相似文献
17.
The regulatory subunit of neural cAMP-dependent protein kinase II represents a unique gene product 总被引:5,自引:0,他引:5
Although the major form of soluble cAMP-dependent protein kinase in bovine cerebral cortex can be classified as a type II kinase, the regulatory subunit (RII) can be distinguished from RII found in other tissues such as heart. Heart and brain RII were distinguished qualitatively by autophosphorylation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mobility of dephosphorylated heart RII shifted from an apparent Mr of 55,000 to 57,000 following autophosphorylation. In contrast, when RII purified from brain was autophosphorylated with [gamma-32P]ATP, two radiolabeled bands were visualized, a minor band (less than or equal to 20%) which migrated with an Mr of 57,000 similar to the heart protein and a band with Mr = 55,000 which did not shift its mobility in response to autophosphorylation. Brain RII was further distinguished from heart RII on the basis of cAMP binding. Millipore filtration and equilibrium dialysis indicated that 2 mol of cAMP bound/mol of RII in contrast to 4 mol/mol with heart RII. Immunological differences were also apparent. Radioimmunoassays using monoclonal antibodies to RII showed that the brain protein had less than 4% of the cross-reactivity of heart RII. Both immunoblotting and immunoprecipitation using monoclonal as well as serum antibodies established that the cross-reactivity in phosphorylated brain RII was associated exclusively with the 57,000 component that behaved like heart RII. The lack of cross-reactivity of neural RII with two different monoclonal antibodies targeted the hinge region of RII as an area where structural differences might be anticipated, and comparative sequence analysis of this region definitively established that the major form of RII in brain is a unique gene product from the RII expressed in heart. 相似文献
18.
Purification of RNA-core induced streptolysin S, and isolation and haemolytic characteristics of the carrier-free toxin 总被引:2,自引:0,他引:2
RNA-core (RNAase-resistant fraction of yeast RNA) induced streptolysin S (SLS) was purified (40% recovery) to apparent electrophoretic homogeneity by hydroxylapatite chromatography followed by gel filtration on Sephadex G-100 in the presence of 6 M-guanidine. HCl. The specific activity of the purified toxin was 3 X 10(6) haemolytic units (mg protein)-1. The Mr of the toxin was below 4000 on the basis of SDS-PAGE and 20 000 by gel filtration in guanidine. HCl. High-voltage isoelectric focusing of the purified toxin allowed the isolation of the carrier-free SLS peptide for the first time. This peptide was basic (pI 9.2) as compared to native SLS (pI 3.6). The native toxin and the peptide had similar haemolytic properties except for the high lability of the peptide, which was stabilized by RNA-core. The Mr of the denatured peptide was about 1800, as estimated by gel filtration. 相似文献
19.
Transplantable human oat cell carcinoma cells of the lung with ectopic vasopressin production were incubated with labeled amino acids and immunoreactive neurophysins in cell extracts were analyzed by isoelectric focusing. When the cells were incubated with L-(35S)-cysteine for 20 h, one major peak (isoelectric point; pI=5.3) and several minor peaks (pI=6.1, 5.7, 5.1, 4.9 and 4.7) of labeled proteins were observed. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the relative molecular mass (Mr) of the pI 5.7 protein was estimated to be 20,000 and that of the pI 6.1 species to be 19,000, while the remainder had a Mr of approximately 10,000. The result of the pulse-labeling experiment has clearly shown that the pI 5.7 and 6.1 proteins, which have affinity for concanavalin A, are biosynthetic precursors for the smaller form of neurophysin with a pI 5.3. When subjected to limited proteolysis with trypsin, the pI 5.7 protein generated a Mr 10,000 protein and a smaller peptide. The Mr 10,000 protein thus produced was identified as neurophysin on the basis of its pH-dependent affinity for vasopressin and the migration pattern on isoelectric focusing. The smaller peptide coeluted with synthetic arginine vasopressin and bound to neurophysin suggesting that it possesses a cysteine-tyrosyl sequence at its N-terminus. Similarly, the pI 6.1 protein liberated neurophysin and vasopressin-like peptide after incubation with trypsin. These results suggests that the glycosylated protein with a pI of 5.7 and a Mr of 20,000 is the common precursor to vasopressin and neurophysin in human oat cell carcinoma of the lung with ectopic vasopressin production. The pI 6.1 protein may be an intermediate in the conversion of the precursor to vasopressin and neurophysin. 相似文献
20.
A rapid four column purification of 2-deoxy-D-glucoside-2-sulphamate sulphohydrolase from human liver 总被引:2,自引:0,他引:2
2-Deoxy-D-glucoside-2-sulphamate sulphohydrolase was extracted from human liver and purified 40 000-fold by a simple four column procedure. The purification was followed using a specific substrate isolated from an acid hydrolysate of heparin, O-(alpha-2-sulphamino-2-deoxy-D-glucopyranosyl)-(1 leads to 3)-L-[6,3H]idonic acid. Only one form of the enzyme was seen on either ion exchange chromatography or isoelectric focussing, with a pI of 6.8. The apparent Mr of the holoenzyme as determined by gel filtration was 190 000 +/- 20 000. Two other larger Mr protein peaks observed on gel filtration appear to be an inactive dimer of the 190 000 dalton peak and a larger aggregate near the exclusion limit of the column. On polyacrylamide disc gel electrophoresis in sodium dodecyl sulphate, with or without prior reduction, each protein peak from the gel filtration column electrophoresed as a single major band with an apparent Mr corresponding to 55 000 +/- 6000. 相似文献