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1.
Purification and characterization of two trypsin inhibitors from the hemolymph of Manduca sexta larvae 总被引:1,自引:0,他引:1
Trypsin inhibitory activity from the hemolymph of the tobacco hornworm (Manduca sexta) was purified by affinity chromatography on immobilized trypsin and resolved into two fractions with molecular weights of 14,000 (M. sexta hemolymph trypsin inhibitor (HLTI) A) and 8,000 (HLTI B) by molecular sieve chromatography on Sephadex G-75. Electrophoresis of these inhibitors under reducing conditions on polyacrylamide gels gave molecular weight estimates of 8,300 for HLTI A and 9,100 for HLTI B, suggesting that HLTI A is a dimer and HLTI B is a monomer. Isoelectrofocusing on polyacrylamide gels focused HLTI A as a single band with pI 5.7, whereas HLTI B was resolved into two components with pI values of 5.3 and 7.1. Both inhibitors were stable at 100 degrees C and pH 1.0 for at least 30 min. HLTIs A and B inhibited serine proteases such as trypsin, chymotrypsin, and plasmin, but did not inhibit elastase, papain, pepsin, subtilisin BPN', and thermolysin. In fact, subtilisin BPN' completely inactivated both inhibitors. Both inhibitors formed low-dissociation complexes with trypsin in a 1:1 molar ratio. The inhibition constant for trypsin inhibition by HLTI A was estimated to be 1.45 x 10(-8) M. The HLTI A-chymotrypsin complex did not inhibit trypsin; similarly, the HLTI A-trypsin complex did not inhibit chymotrypsin, indicating that HLTI A has a common binding site for both trypsin and chymotrypsin. The amino-terminal amino acid sequences of HLTIs A and B revealed that both these inhibitors are homologous to bovine pancreatic trypsin inhibitor (Kunitz). 相似文献
2.
Characterization of human placental neuraminidases. Stability, substrate specificity and molecular weight.
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D McNamara G Beauregard H V Nguyen D L Yan M Bélisle M Potier 《The Biochemical journal》1982,205(2):345-351
1. At least two components of neuraminidase can be distinguished on the basis of thermolability and sedimentability by using the artificial fluorogenic substrate 4-methylumbelliferyl N-acetyl-alpha-D-neuraminate. 2. In crude homogenates, thermodenaturation at 25 degrees C showed a biphasic curve corresponding to component A (half-life, 21 min) and B (half-life, 85 min). The two components were partially resolved by centrifugation. A being soluble and B sedimentable. Both had similar pH-activity curves (pH optimum, 4.4), Km values (A, 0.10 mM; B, 0.06 mM) and molecular weight as determined by radiation inactivation (A, 67000; B, 63000). 3. The soluble A form was still aggregated or bound to membranous debris since almost all neuraminidase activity was eluted near or at the void volume of a Sephacryl S-300 column. 4. Both soluble and sedimentable fractions of placenta hydrolysed the GD1A ganglioside and N-acetyl-neuraminyl-D-lactose linearly for 12 h but no fetuin hydrolysis was detected. 5. The neuraminidase activity with the artificial fluorogenic substrate was inhibited by N-acetylneuraminyl-D-lactose but not by the GD1A ganglioside. These preliminary results suggest that there exist two closely related enzymes hydrolysing both the artificial substrate and N-acetylneuraminyl-D-lactose and a third one hydrolysing the GD1A ganglioside exclusively. 相似文献
3.
Two forms of alpha-galactosidase (alpha-d-galactoside galactohydrolase, E.C. 3.2.1.22) which differed in molecular weight were resolved from Cucumis sativus L. leaves. The enzymes were partially purified using ammonium sulfate fractionation, Sephadex gel filtration, and diethylaminoethyl-Sephadex chromatography. The molecular weights of the two forms, by gel filtration, were 50,000 and 25,000. The 50,000-dalton form comprised approximately 84% of the total alpha-galactosidase activity in crude extracts from mature leaves and was purified 132-fold. The partially purified 25,000-molecular weight form rapidly lost activity unless stabilized with 0.2% albumin and accounted for 16% of the total alpha-galactosidase activity in the crude extract. The smaller molecular weight form was not found in older leaves.The two forms were similar in several ways including their pH optima which were 5.2 and 5.5 for the 50,000- and 25,000-dalton form, respectively, and activation energies, which were 15.4 and 18.9 kilocalories per mole for the larger and smaller forms. Both enzymes were inhibited by galactose as well as by excess concentrations of p-nitrophenyl-alpha-d-galactoside sub-strate. K(m) values with this substrate and with raffinose and melibiose were different for each substrate, but similar for both forms of the enzyme. With stachyose, K(m) values were 10 and 30 millimolar for the 50,000- and 25,000- molecular weight forms, respectively. 相似文献
4.
Separation and purification of lymphocyte chemotactic factor (LCF) and interleukin 2 produced by human peripheral blood mononuclear cells 总被引:1,自引:0,他引:1
Two T-cell chemotactic factors, lymphocyte chemotactic factor (LCF) and interleukin 2 (IL-2), were separated and characterized from culture supernatants of concanavalin A-stimulated human peripheral blood mononuclear cells. LCF was purified approximately 7800-fold to homogeneity from culture supernatant using gel filtration and high-performance liquid chromatography (HPLC). LCF was found to be distinct from both IL-2 and interleukin-1. Sephadex G-100 gel filtration of crude supernatants from concanavalin A-stimulated mononuclear cells showed two molecular weight regions of T lymphocyte chemotactic activity. A 10,000- to 25,000-Da region contained both IL-2 and LCF and a 45,000- to 75,000-Da region contained only a high molecular weight form of LCF. Both high and low molecular weight species of LCF eluted with 40-44% acetonitrile from a reversed-phase C18 HPLC column. IL-2 present only in the low molecular weight region eluted from the C18 column with 65-75% acetonitrile. The migration of T lymphocytes to IL-2 was totally inhibited by anti-interleukin 2 receptor antibody while the response of T cells to LCF was unaffected. LCF eluting off the C18 column was purified to homogeneity by two subsequent cycles of gel filtration HPLC. The resultant protein showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular weight of 10,500. The data presented here demonstrate that IL-2 and LCF are distinct lymphocyte chemotactic factors and although they are not readily separable from crude supernatants by molecular sieve chromatography, they can easily be distinguished by reversed-phase HPLC. 相似文献
5.
1. The Virginia opossum (Didelphis virginiana) possessed an arylsulfatase which had a relative molecular weight of 130 +/- 12 kDa, displayed anomalous kinetics, hydrolysed AA2S, and exhibited other properties of arylsulfatase A. No arylsulfatase B was found. 2. The arylsulfatase present in the gray short-tailed opossum (Monodelphis domestica) had a relative molecular weight of 56 +/- 4 kDa, exhibited linear kinetics, was inhibited by chloride, and possessed other characteristics of arylsulfatase B. No arylsulfatase A was found. 3. Arylsulfatases from both species occurred as multiple isozymes which were unaffected by neuraminidase or alkaline phosphatase treatment. 相似文献
6.
K K Yeung A J Owen J A Dain 《Comparative biochemistry and physiology. B, Comparative biochemistry》1979,63(3):329-334
1. beta-N-acetylhexosaminidase isoenzymes from the gastropod, T. cornutus, were purified and their properties studied. 2. The two isoenzymes, designated A and B were separated by DEAE-Sephadex column chromatography and further purified by CM-cellulose, Concanavalin-A-Sepharose-4B and Sephadex G-200 column chromatography. 3. beta-N-Acetylhexosaminidase A and B were purified 416 and 208 fold, with yields of 10.6 and 5.1%, respectively. 4. The two isoenzymes appear homogeneous on polyacrylamide gel electrophoresis, with the A form migrating faster towards the anode than the B form. 5. The purified isoenzymes are virtually free of all other common glycosidase contaminations. 6. The apparent molecular weight of both beta-N-acetylhexosaminidase A and B is about 100,000 when estimated with gel filtration column chromatography and the pH optimum for both is 4.0. 7. Both beta-N-acetylhexosaminidase isoenzyme activities are stimulated by Cl-, Br-, F-, I- and NO3-, and inhibited by Hg+, Ag+, Fe3+, N-acetylglucosamine and N-acetylgalactosamine. 8. The Km values of beta-N-acetylhexosaminidase A and B for the substrate p-nitrophenyl-beta-2-acetamide-2-deoxy-D-glucopyranoside were 2.9 and 3.2 mM, respectively. 相似文献
7.
Klomklao S Benjakul S Visessanguan W Kishimura H Simpson BK Saeki H 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2006,144(1):47-56
Two anionic trypsins (A and B) were purified to homogeneity from yellowfin tuna (Thunnus albacores) spleen by a series of column chromatographies including Sephacryl S-200, Sephadex G-50 and DEAE-cellulose. Purity was increased to 70.6- and 91.5-fold with approximately 2.8% and 15.6% yield for trypsin A and B, respectively. The apparent molecular weight of both trypsins was estimated to be 24 kDa by size exclusion chromatography and SDS-PAGE. Both trypsin A and B appeared as a single band on native-PAGE. Trypsin A and B exhibited the maximal activity at 55 and 65 degrees C, respectively, and had the same optimal pH at 8.5 using TAME as a substrate. Both trypsins were stable to heat treatment up to 50 degrees C and in the pH range of 6.0 to 11.0. Both trypsin A and B were stabilized by calcium ion. The activities were inhibited effectively by soybean trypsin inhibitor, TLCK and partially inhibited by EDTA, but were not inhibited by E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A. Activity of both trypsins continuously decreased with increasing NaCl concentration (0-30%). Apparent Km and Kcat of trypsin A and B for TAME were 0.2-0.33 mM and 66.7-80 S(-1), respectively. The N-terminal amino acid sequences of trypsin A, IVGGYECQAHSQPHQVSLNA, and trypsin B, IVGGYECQAHSQPPQVSLNA, indicated the high homology between both enzymes. 相似文献
8.
125I-Labeled human growth hormone is isolated in high molecular weight (Mr) (300,000, 220,000, and 130,000) and low molecular weight complexes on rat hepatocytes after affinity labeling. The time-dependent formation of low molecular weight complexes occurred at the expense of the higher molecular weight species and was inhibited by low temperature or inhibitors of serine proteinases. Exposure to reducing conditions induced loss of Mr 300,000 and 220,000 species and augmented the amount of Mr 130,000 complexes. The molecular weight of growth hormone (22,000) suggests that binding had occurred with species of Mr 280,000, 200,000, and 100,000. Two-dimensional gel electrophoresis demonstrated that the 100,000-dalton receptor subunit is contained in both the 280,000- and 200,000-dalton species. Reduction of interchain disulfide bonds in the growth hormone receptor did not alter its elution from gel filtration columns, but intact, high molecular weight receptor constituents were separated from lower molecular weight degradation products. Digestion of affinity-labeled growth hormone-receptor complexes with neuraminidase increased the mobility of receptor constituents on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These observations show that the growth hormone receptor is degraded by hepatic serine proteinases to low molecular weight degradation products which can be separated from intact receptor by gel filtration. Intact hormone-receptor complexes are aggregates of 100,000-dalton sialoglycoprotein subunits held together by interchain disulfide bonds and by noncovalent forces. 相似文献
9.
Eosinophil-directed chemotactic inhibitory factors (ECIF) from T lymphocytes of complete Freund's adjuvant-treated guinea pigs were recovered in two separate molecular weight (MW) fractions: the one is eluted near bovine serum albumin (MW about 70,000), and in pH range between 7.0 and 7.5 by chromatofocusing column, whereas the other is near cytochrome c (MW 12,500), and in pH range between 5.0 and 5.5. It was, however, found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoadsorption that the high molecular ECIF is probably an aggregated form of the low molecular ECIF. It was subsequently confirmed that both the ECIF had similar physicochemical properties: sensitive to heating at 56 or 80 degrees C sensitive to enzyme treatment with trypsin and neuraminidase, sensitive in acid but not alkaline condition, and bind to peanut agglutinin-or Limulus polyphemus agglutinin-coupled agarose beads. The inhibitory activity of ECIF was suppressed by previous treatment of eosinophils with D-galactose and sialic acid. ECIF activity was specifically absorbed by eosinophils; the absorption was inhibited by pretreatment of eosinophils with D-galactose and sialic acid. Previous treatment of eosinophils with beta-galactosidase and neuraminidase led the cells not to respond to both ECIF. It was thus suggested that the inhibition by ECIF is receptor-mediating phenomenon, and that ECIF and ECIF receptors on eosinophils contain galactose and sialic acid residues which may play an essential role for the biological activity. 相似文献
10.
Cultured bovine endothelial cells produce both urokinase and tissue-type plasminogen activators 总被引:18,自引:6,他引:12
Cell extracts and conditioned media (CM) from cultured bovine aortic endothelial cells (BAEs) were fractionated by PAGE in the presence SDS, and plasminogen activator (PA) activity was localized by fibrin autography. Multiple molecular weight forms of PA were detected in both preparations. Cell-associated PAs had Mr of 48,000, 74,000, and 100,000 while secreted PAs showed Mr of 52,000, 74,000, and 100,000. A broad zone of activity (Mr 80,000-100,000) also was present in both cellular fractions. In addition, PAs of Mr 41,000 and 30,000 appeared upon prolonged incubation or repeated freezing and thawing of the samples, and probably represent degradation products of higher molecular weight forms. This complex lysis pattern was not observed when CM was subjected to isoelectric focusing. Instead, only two classes of activator were resolved, one at pH 8.5, the other at 7.6. Analysis of focused samples by SDS PAGE revealed that the activity at pH 8.5 resulted exclusively from the Mr 52,000 form; all other forms were recovered at pH 7.6. The activity of the Mr 52,000 form was neutralized by anti-urokinase IgG but was not affected by antitissue activator IgG indicating that it is a urokinaselike PA. The activities of the Mr 74,000-100,000 forms were not affected by anti-urokinase. They were blocked by antitissue activator suggesting that all the forms in this group were tissue-type PAs. The multiple forms of PA were differentially sensitive to inactivation by diisopropylfluorophosphate (DFP). Treatment of CM with 10 mM DFP for 2 h at 37 degrees C only partially inhibited the 52,000-dalton form. However, it completely inactivated the 74,000-dalton partially inhibited the 52,000-dalton form. However, it completely inactivated the 74,000-dalton PA. The activity of the Mr 100,000 form was not affected by this treatment, or by treatment with 40 mM DFP. Thus, cultured BAEs produce multiple, immunologically distinct forms of PA which differ in size, charge, and sensitivity to DFP. These forms include both urokinaselike and tissue-activator-like PAs. The possibility that one of these forms is a zymogen is discussed. 相似文献
11.
A product of histamine-stimulated human lymphocytes, histamine-induced suppressor factor or HSF, was characterized by enzyme treatment, sensitivity to reduction and alkylation, by molecular sieve chromatography, and by polyacrylamide gel electrophoresis. HSF was found to have a wide pH stability (pH 3-10), sensitivity to temperatures greater than 80 degrees C, and to have the properties of a glycoprotein by virtue of its sensitivity to chymotrypsin, trypsin, sodium periodate, and neuraminidase. HSF did not appear to have a serine group(s) in its "active" site since its biologic activity remained intact following treatment with an irreversible serine esterase inhibitor (phenylmethylsulfonyl fluoride). Further, HSF did not appear to have inter- or intra-molecular disulfide linkages because treatment with denaturing and/or reducing agents, followed by alkylation, did not significantly alter its activity. Molecular sieve chromatography employing Sephadex G-100 revealed an apparent molecular weight for HSF of 25-40,000. Electrophoresis of HSF in polyacrylamide gels at pH 8.7 under nonreducing conditions revealed two regions of activity, one region migrating with albumin and the other region anodal to albumin. In addition to suppressing lymphocyte proliferation, the 25-40,000 Mr Sephadex G-100 fractions also inhibited the production of leukocyte inhibitory factor. Of particular interest, gel filtration of supernatants generated by stimulating mononuclear cells with either histamine, dimaprit (but not 2-pyridylethylamine), concanavalin A, or candida albicans resulted in similar elution profiles with regard to inhibition of lymphocyte proliferation. That is, 25-40,000 Mr fractions of supernatants generated by each stimulant suppressed lymphocyte proliferation to a similar degree. The latter findings provide indirect evidence that T lymphocytes, triggered in response to antigen-specific and nonspecific stimuli, elaborate suppressor molecules capable of modulating T-cell function that share certain similarities. 相似文献
12.
Mechanism of action of inter-alpha-trypsin inhibitor 总被引:1,自引:0,他引:1
Inter-alpha-trypsin inhibitor (I alpha I) is a unique proteinase inhibitor that can be proteolyzed by the same enzymes that are inhibited, to generate smaller inhibitors. This study examines the reactions of I alpha I with trypsin, chymotrypsin, plasmin, and leukocyte elastase. Complexes of I alpha I and proteinase were demonstrated by gel filtration chromatography. Complete digestion of I alpha I by each proteinase was not accompanied by a comparable loss of inhibition of that enzyme or a different enzyme. Following proteolysis, inhibitory activity was identified in I alpha I fragments of molecular weight 50,000-100,000 and less than 40,000. Addition of a second proteinase inhibitor prevented proteolysis. Both I alpha I and its complex with proteinase were susceptible to degradation. Kinetic parameters for both the inhibition and proteolysis reactions of I alpha I with four proteinases were measured under physiological conditions. On the basis of these results, a model for the mechanism of action of I alpha I is proposed: Proteinase can react with either of two independent sites on I alpha I to form an inhibitory complex or a complex that leads to proteolysis. Both reactions occur simultaneously, but the inhibitory capacity of I alpha I is not significantly affected by proteolysis since the product of proteolysis is also an inhibitor. For a given proteinase, the inhibition equilibrium constant and the Michaelis constant for proteolysis describe the relative stability of the inhibition and proteolysis complexes; the second-order rate constants for inhibition and proteolysis indicate the likelihood of either reaction. The incidence of inhibition or proteolysis reactions involving I alpha I in vivo cannot be assessed without knowledge of the exact concentrations of inhibitor and proteinases; however, analysis of inhibition rate constants suggests that I alpha I might be involved in plasmin inhibition. 相似文献
13.
Tryptase in rat mast cells: properties and inhibition by various inhibitors in comparison with chymase 总被引:3,自引:0,他引:3
Previous studies with trans-4-(guanidinomethyl)cyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPhBut), a trypsin inhibitor, strongly suggested the involvement of a trypsin-like protease in histamine release from mast cells induced by various secretagogues (Takei, M., Matumoto, T., Endo, K. & Muramatu, M. (1988) Agents and Actions, in press; Takei, M., Matumoto, T., Ito, T., Endo, K. & Muramatu, M.; Takei, M., Matumoto, T., Endo, K. & Muramatu, M. and Takei, M., Matumoto, T., Urashima, H., Endo, K. & Muramatu, M., unpublished results). Two serine proteases, chymase (Benditt, E.F. & Arase, M. (1959) J. Exp. Med. 110, 451-460) and tryptase Kido, H., Fukusen, N. & Katunuma, N. (1985) Arch. Biochem. Biophys. 239, 436-443) were demonstrated in rat peritoneal mast cells. Both enzymes were purified and the effects of inhibitors for trypsin and chymotrypsin on these proteases were examined. The trypsin-like protease was found in saline extract and purified by successive chromatographies on Sephadex G-100 and DEAE-cellulose columns. The molecular mass of this protease was apparently 120,000 Da. This protease showed maximal activity at pH 7.1 and was named pH 7 tryptase. Chymase was obtained from 1.5M NaCl extract. pH 7 Tryptase markedly hydrolysed Boc-Phe-Ser-Arg-NH-Mec and Boc-Val-Pro-Arg-NH-Mec among the various substrates containing arginyl and lysyl bonds but did not cleave Tos-Arg-OMe. Tos-Lys-CH2Cl and diisopropylfluorophosphate strongly inhibited this protease. Various inhibitors for trypsin inhibited pH 7 tryptase, and those for chymotrypsin inhibited chymase. Among the esters of GMCHA examined, GMCHA-OPhBut most strongly and competitively inhibited pH 7 tryptase but it had no effect on chymase. 相似文献
14.
U Ikeda T Toyo-Oka S Hosoda 《Biochemical and biophysical research communications》1986,136(2):773-777
We have identified and isolated two new calcium-activated neutral hydrolases from human ventricular muscles. The one is an esterase, of which molecular weight was 300,000, required millimolar concentration of Ca2+, hydrolyzed Ac-Tyr-OEt X H2O, optiaml pH at 7.0. The other is an amidase, of which molecular weight was 70,000, also required millimolar concentration of Ca2+, hydrolyzed a synthetic substrate for chymotrypsin, Suc-Leu-Leu-Val-Tyr-MCA, with optimal pH at 7.2. Both enzymes did not degrade casein or contractile proteins (myosin, actin, troponin and tropomyosin). Their activities were not inhibited by exogenous protease inhibitors, leupeptin, antipain, monoiodoacetic acid and chymostatin, while the amidase activity was blocked by the endogenous inhibitor against calcium-activated neutral protease (CANP). Thus, their characters are different from chymotrypsin or CANP and they seems to be new hydrolases in the human heart. 相似文献
15.
1. A trypsin and chymotrypsin inhibitor was isolated by extraction of chick-pea meal at pH8.3, followed by (NH4)2SO4 precipitation and successive column chromatography on CM-cellulose and calcium phosphate (hydroxyapatite). 2. The inhibitor was pure by polyacrylamide-gel and cellulose acetate electrophoresis and by isoelectric focusing in polyacrylamide gels. 3. The inhibitor had a molecular weight of approx. 10000 as determined by ultracentrifugation and by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. A molecular weight of 8300 was resolved from its amino acid composition. 4. The inhibitor formed complexes with trypsin and chymotrypsin at molar ratios of 1:1. 5. Limited proteolysis of the inhibitor with trypsin at pH3.75 resulted in hydrolysis of a single-Lys-X-bond and in consequent loss of 85% of the trypsin inhibitory activity and 60% of the chymotrypsin inhibitory activity. Limited proteolysis of the inhibitor with chymotrypsin at pH3.75 resulted in hydrolysis of a single-Tyr-X-bond and in consequent loss of 70% of the trypsin inhibitory activity and in complete loss of the chymotrypsin inhibitory activity. 6. Cleavage of the inhibitor with CNBr followed by pepsin and consequent separation of the products on a Bio Gel P-10 column, yielded two active fragments, A and B. Fragment A inhibited trypsin but not chymotrypsin, and fragment B inhibited chymotrypsin but not trypsin. The specific trypsin inhibitory activity, on a molar ratio, of fragment A was twice that of the native inhibitor, suggesting the unmasking of another trypsin inhibitory site as a result of the cleavage. On the other hand, the specific chymotrypsin inhibitory activity of fragment B was about one-half of that of the native inhibitor, indicating the occurrence of a possible conformational change. 相似文献
16.
Proteins of the human erythrocyte membrane as modified by pronase 总被引:25,自引:0,他引:25
Pronase degrades proteins on the outer surface of the human erythrocyte membrane which run in polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate at a molecular weight of approximately 125,000. Carbohydrate and sialic acid are removed, but fragments of molecular weight 50,000 to 100,000 remain attached to the membrane. The most prominent fragment, one of molecular weight about 73,000, can be labeled with a membrane-impermeable reagent (sulfanilic acid diazonium salt), so it is still accessible from the outside of the cell. Pronase rapidly inactivates membrane-bound acetylcholinesterase, but it has relatively little effect on the facilitated diffusion of glucose; both are inhibited by the diazonium salt. Extensive digestion leads to potassium loss and osmotic lysis. Ghosts prepared in 15 mosm-Tris (pH 7.6) are extensively degraded by pronase: essentially all the protein shifts to low molecular weight. Pronase is even more potent in 3% sodium dodecyl sulfate. Ghosts prepared from intact cells which have been treated with the enzyme hydrolyze when dissolved in the detergent unless steps are taken to inhibit proteolysis. 相似文献
17.
1. Two forms of cytosol neuraminidase (EC 3.2.1.18) (neuraminidase A and neuraminidase B) were isolated and purified from pig brain homogenate, by proceeding through the following steps: centrifugation of brain homogenate at 105 000 X g (1h); ammonium sulphate fractionation (35-55% saturated fraction); column chromatography on Biogel A 5 m; column chromatography on hydroxy apatite/cellulose gel; affinity chromatography on Affinose-tyrosyl-p-nitrophenyloxamic acid. The separation of the two forms of neuraminidase was provided by chromatography on hydroxylapatite/cellulose gel. Neuraminidase A was purified about 500-fold; neuraminidase B about 400-fold. 2. The pH optima and the maximum activities in various buffers were different for neuraminidase A and B (for instance the pH optimum was in sodium acetate/acetic acid buffer, 4.7 for neuraminidase A and 4.9 for neuraminidase B). Ions affected in a different way the two enzymes: K+ activated neuraminidase A but not neuraminidase B; Na+ and Li+ inhibited neuraminidase A at a higher degree than neuraminidase B. Neuraminidase B seemed to be moderately activated by some bivalent cations (Ca2+; Mg2+; Zn2+); neuraminidase A did not. The Km values for sialyllactose were different: 2.2-10(-3) M for neuramindase A; 0.46-10(-3) M for neuraminidase B. 相似文献
18.
Some further aspects of B-lymphocyte-promotor factor (B-LPF) activity have been studied. This activity was present in the supernatants of certain helper-T-cell lines, and it induced polyclonal activation of Ig+ B cells into Ig-secreting cells. It was found that B-LPF induced polyclonal, terminal B-cell differentiation (1) in T-cell- and macrophage-depleted spleen cell populations, (2) in both Lyb-5- and Lyb-5+ cells as well as in small and blast-like splenic B cells, and (3) in normal rather than memory B cells. B-LPF function was neither restricted to major histocompatibility complex gene products nor to immunoglobulin allotypes. B-LPF-like activity was also produced by some B-cell lymphomas/hybrids and by the P388-D1 macrophage line. B-LPF activity was found in three MW fractions: (I) greater than 180,000 (pI greater than 7.0 and 4.5-5.5); (II) 50,000-70,000 (pI greater than 7.0; 6.0-6.5, and 4.5-5.5); and (III) 10,000-15,000 (pI greater than 7.0). All three MW forms of B-LPF activity carried antiserum 6036-defined and AB-1.9.3 monoclonal antibody-defined determinants, and they reacted with chicken gammaglobulin (CGG)-Sepharose but not with human serum albumin-Sepharose. These data indicate that the three MW forms of B-LPF activity are associated/dissociated forms of a 10,000-15,000 MW form (subunit) rather than three different molecular species with B-LPF activity. A comparative study between antigen-specific helper factors and B-LPF was hampered by the finding that the helper-T-cell hybridomas used (e.g., T85-109-45/1) only produced B-LPF in our hands. Previously, it has been described that these helper-T-cell hybrids produced CGG-specific, I-Ak-restricted helper factors. However, one surprising observation was that B-LPF produced by both T85 hybrid cells and L12 T lymphoma cells was absorbed and could be eluted from CGG-Sepharose columns. The relationship of B-LPF to other nonspecific and apparently specific T-helper-cell products is discussed in particular in the light of the observations that many immunologically active molecules are built up from 10,000-12,000 molecular weight domain-like polypeptide structures. 相似文献
19.
Purification and characterization of human colony-stimulating factor 1 from human pancreatic carcinoma (MIA PaCa-2) cells 总被引:3,自引:0,他引:3
Colony-stimulating factor 1 (CSF-1) was purified from the serum-free conditioned medium of a human pancreatic carcinoma cell line (MIA PaCa-2) by a combination of conventional chromatography and high-performance liquid chromatography. The purity of human CSF-1 was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a diffuse single band of Mr 42,000-50,000 and by N-terminal amino acid analysis of glutamate residue. The CSF-1 was stable at 50 degrees C for 30 min. It is sensitive to treatment with trypsin, chymotrypsin, and subtilisin but less sensitive to papain digestion. Treatment of CSF-1 with different glycosidases did not affect the biological activity. Sulfhydryl reagents such as dithiothreitol (DTT), iodoacetic acid, and N-ethylmaleimide did not affect the biological activity at the concentration of 1 mM. However, CSF-1 activity was inhibited totally by the combination of 10 mM DTT and 1 mM SDS. Under denaturing and reducing conditions, CSF-1 appeared on SDS-PAGE as a single protein band of Mr 21,000-25,000 and concurrently lost its activity, indicating that human CSF-1 possibly consists of two similar subunits and that the intact quaternary structure is essential for the biological activity. When treated with neuraminidase and endo-beta-D-N-acetylglucosaminidase D, the molecular weight of CSF-1 was reduced to 36,000-40,000, and to 18,000-20,000 in the presence of mercaptoethanol. Because of the specificity of endo-beta-D-N-acetylglucosaminidase D, it is suggested that the carbohydrate moieties are Asn-linked "complex-type" units. 相似文献
20.
Demetrios G. Mappouras J. Stiakakis Emmanuel G. Fragoulis 《Molecular and cellular biochemistry》1990,94(2):147-156
L-DOPA decarboxylase has been purified to homogeneity from post mortem removed human kidneys. Homogeneity was examined by polyacrylamide gel electrophoresis (PAGE) analysis both in the presence and absence of SDS. The enzyme has a molecular weight of 100,000 daltons estimated by gel filtration and 50,000 daltons determined after SDS-PAGE. Human L-DOPA decarboxylase therefore is a dimer. Polyclonal antibodies produced against human L-DOPA decarboxylase react with the 50,000 daltons enzyme subunit after immuno-blotting and also precipitates enzyme activity. Activity against L-DOPA is partially inhibited by 5-hydroxytryptophan (5-HTP). The effect of various cations on L-DOPA decarboxylase activity has also been tested. 相似文献