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1.
W H Massover 《Biochimica et biophysica acta》1979,579(1):169-180
Pure ferritin from male mouse liver produces a single band of monomers (RF = 0.199) with electrophoresis in polyacrylamide gels at pH 9.0. The five sub-bands within this monomeric band appear to represent charge isomers having the same molecular size. Ferritin from BH3 transplantable mouse hepatoma shows two overlapping bands of monomers (RFA = 0.208 and RFB = 0.240); further electrophoretic studies show that these bands represent two subpopulations of molecules differing both in charge and size. Sub-bands are not found in this hepatoma ferritin. The larger tumor ferritin reaches the same end migration position as all liver isoferritins on gradient gels, signifying a very similar or identical molecular size; however, the absence of sub-bands indicates that this hepatoma ferritin differs in charge from the homologous liver proteins. Liver and hepatoma ferritins both produce a single prominent subunit band corresponding to nominal molecular weights of 22 250 and 21 700, with polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. With electrophoresis on polyacrylamide gradient slabs containing sodium dodecyl sulfate and dithiothreitol, both liver and hepatoma ferritins now reveal two subunits bands situated at identical positions. The polypeptides of these two closely spaced bands have a nominal molecular weight difference of less than 1000. Neither the hepatoma nor the liver seems to produce the ferritins found in the other tissue. Nevertheless, all these ferritins are composed of the same two types of subunits, albeit in different relative amounts. Observed distinctions in the ferritins from these normal or neoplastic cells must reflect differences in assembly and processing, as well as in the regulated expression of the same ferritin genes. 相似文献
2.
H J Schneider J Markl W Schartau B Linzen 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1977,358(9):1133-1141
The hemocyanin of the North American tarantula Eurypelma californicum (Dugesiella californica) is dissociated at pH 9.6 into monomers (Mr about 70 000) and dimers (Mr about 140 000), which were separated by gel filtration. The monomer peak was resolved by preparative polyacrylamide gel electrophoresis and yielded 4 protein bands, three of which (1, 3 and 4M) are apparently homogeneous. Band 2 contains two sub-fractions (2I and 2II). The dimer peak contains two dimers (bands 4D and 5). Upon treatment with 5mM cysteine the dimer band 5 is dissociated, yielding only one type of monomer identical with band 3. The other dimer, which was only partially dissociated by 10mM EDTA, is most probably a heterodimer, one component being electrophoretically indistinguishable from band 2II. After treatment of the native hemocyanin with sodium dodecylsulfate and analysis in gradient gel slabs, 6 polypeptide chains were observed (labeled a - f). They correspond to the products of alkaline dissociation as follows: band 1 = e, band 2I = a, band 2II = c, band 3 = f, band 4M = d, band 4D = b plus c, band 5 = f. The molecular weights were determined by dodecylsulfate gel electrophoresis in gradient gels, and by sedimentation equilibrium analysis and found to range between 67 000 and 76 000. The sedimentation coefficients are between 4.4 and 4.7 S for the monomers and 6.6 and 6.7 for the dimers. The isoelectric points range from pH 4.5 to pH 5.4. The findings are discussed with respect to the limitations of molecular weight determination by conventional dodecylsulfate gel electrophoresis, to the structure of the hemocyanin oligomers and to possible biological significance. 相似文献
3.
Molecular size heterogeneity of ferritin in mouse liver 总被引:2,自引:0,他引:2
W H Massover 《Biochimica et biophysica acta》1985,829(3):377-386
As much as 4% of the total protein in pure liver ferritin from mice with short-term parenteral iron overload produces a minor band migrating anodally to the major (alpha) band of holoferritin with non-denaturing polyacrylamide gel electrophoresis. The components in this minor band and the alpha band have been isolated to purity by preparative electrophoretic fractionation. The protein in the minor band is ferritin, since it contains ferric iron and fulfills defining criteria at the level of biochemistry, immunology and ultrastructure. Native polyacrylamide electrophoresis with pore-size-gradient gels shows that the ferritin molecules in the minor band have a slightly smaller diameter than the holoferritin in the alpha band. Isoelectric focusing reveals that the smaller ferritin has an identical number and range of charge isomers (pI 4.9-5.3) as the larger ferritin, but the relative amount of each size class within some isoferritin bands differs. The smaller ferritin molecules are structurally intact and are made from polypeptide subunits with Mr 18 000; the larger ferritin molecules have subunits with Mr 22 000. The minor species of hepatic ferritin thus has a smaller molecular size because it is made mainly from smaller subunits. No minor electrophoretic band can be detected in liver ferritin obtained from mice with normal iron levels. These results demonstrate that siderosis induces the formation of molecular size polymorphism (macroheterogeneity) in mouse liver ferritin. The new smaller hepatic ferritin could serve to redistribute excess iron into the main storage organs during the early response to iron overload, since it appears to be identical to one of the two types of serum ferritin molecules present in these siderotic mice. 相似文献
4.
Purification of RNAase II to electrophoretic homogeneity is described. The exonuclease is activated by K+ and Mg2+ and hydrolyses poly(A) to 5'-AMP, exclusively as described by Nossal and Singer (1968, J. Biol. Chem. 243, 913--922). To separate RNAase II from ribosomes, DEAE-cellulose chromatography was used. Two additional chromatographic steps give a preparation that yields 10 bands after analytical polyacrylamide gel electrophoresis. Preparative polyacrylamide gel electrophoresis resulted in a final preparation which on analytical polyacrylamide gels gives a single band. A molecular weight of 76 000 +/- 4000 was obtained from Sephadex G-200 chromatography, with three bands from sodium dodecyl sulfate (SDS) denaturation and SDS gel electrophoresis. The subunits have a molecular weight of 40 000 +/- 2000, 33 000 +/- 2000, and 26 000 +/- 1000. The enzyme thus appears to consist of three dissimilar subunits. 相似文献
5.
本试验利用聚丙烯酰胺凝胶梯度电泳分步染色法直接对玉米苗期酯酶同工酶和过氧化物酶同工酶各酶带的分子量进行了比较测定。酯酶同工酶 E_1、E_2、E_3~F、E_3~S、a、b、c 各酶带的分子量分别为<20000,35200、33000、38500、29900、28500、34000道尔顿过氧化物酶同工酶 PX_4~F和 PX_4~S酶带的分子量分别为131000和149000道尔顿。根据酶带在均匀胶和梯度胶中的位置变化对各酶带的生化性质作了初步分析,发现 E_3~F和 E_3~S、PX_4~F 和 PX_4~S 在迁移率上的差异主要是分子量的差异。本文为同工酶的分子量测定提供了一个简便的方法。 相似文献
6.
The molecular weights of esterase and peroxidase isozymes of maize seedlings were directly determined by improved polyacrylamide gradient gel electrophoresis. The different isozyme bands developed in polyacrylamide slab gel electrophoresis (uniform gel) were identified in polyacrylamide gradient gel electrophoresis by means of isozyme variants. The molecular weights of esterase isozymes E1, E2, E3F, E3S, a, b, c, named according to isozyme patterns in uniform gel, are <20000, 35200, 33000, 38500, 29900, 28500, 34000 doltons respectively. The molecular weights of peroxidase isozymes PX4F and PX4S are 131000 and 149000 doltons respectively. According to the band location in uniform gel and in gradient gel, some biochemical properties of the isozyme bands and relationships between the isozyme bands were analyzed. The possible errors in the determination of smaller molecular weight isozymes are discussed. 相似文献
7.
Márcio H.L Arndt Marcelo M Santoro 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1998,119(4):667-675
The hemoglobin of Biomphalaria glabrata was purified to homogeneity by gel filtration column followed by anion exchange chromatography. The dissociation products were analyzed by a 5–15% gradient polyacrylamide gel electrophoresis containing sodium dodecyl sulfate (SDS-PAGE) giving a band of 270 kDa and a band of 180 kDa after reduction with β-mercaptoethanol. The same profile was obtained in a 3.5% agarose gel electrophoresis containing SDS (SDS-AGE) but showed additional bands of higher molecular weight. These bands were proposed to be monomers, dimers and trimers, since they showed a good correlation in a plot of Rf versus log Mr. After partial reduction in a two-dimensional SDS-AGE, the proposed monomers and dimers produced two and four bands, respectively, likely indicating one to four chains crosslinked by disulfide bridges. Digestion with four different proteases yielded several equivalent fragments with molecular weights multiples of its minimum molecular weight (17.7 kDa). The circular dichroism spectrum of the protein showed a characteristic high α-helix content (70%). It was proposed that this hemoglobin is a pentamer with a molecular weight of aproximately 1.8×103 kDa, assembled by five 360-kDa subunits, each formed by two 180-kDa chains linked in pairs by disulfide bridges and each of these chains, in turn, comprised by ten heme binding domains linked in tandem. These data are compared to the published information for other planorbid extracellular hemoglobins. 相似文献
8.
《Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis》1978,517(2):419-428
Purification of RNAase II to electrophoretic homogeneity is described. The exonuclease is activated by K+ and Mg2+ and hydrolyses poly(A) to 5′-AMP, exclusively as described by Nossal and Singer (1968, J. Biol. Chem. 243, 913–922). To separate RNAase II from ribosomes, DEAE-cellulose chromatography was used. Two additional chromatographic steps give a preparation that yields 10 bands after analytical polyacrylamide gel electrophoresis. Preparative polyacrylamide gel electrophoresis resulted in a final preparation which on analytical polyacrylamide gels gives a single band. A molecular weight of 76 000 ± 4000 was obtained from Sephadex G-200 chromatography, with three bands from sodium dodecyl sulfate (SDS) denaturation and SDS gel electrophoresis. The subunits have a molecular weight of 40 000 ± 2000, 33 000 ± 2000, and 26 000 ± 1000. The enzyme thus appears to consist of three dissimilar subunits. 相似文献
9.
L Vulimiri N Catsimpoolas A L Griffith M C Linder H N Munro 《Biochimica et biophysica acta》1975,412(1):148-156
Ferritins purified from horse spleen and from rat liver, kidney, heart and hepatoma were analyzed by quantitative polyacrylamide gel electrophoresis. From the migration characteristics of these ferritins at several gel concentrations, Ferguson plots were constructed and the molecular sizes and charges (apparent valences) together with their statistical variability were obtained by applying Rodbard computer programs to the data. Finally, ellipses were drawn describing the 95% confidence limits of these data for size and charge and were used to identify those ferritins that differed in size and/or charge. By these criteria, many of the tissue ferritins were differentiated from one another in terms of their molecular size and/or charge. Among the various tissue ferritin monomers, the molecular sizes were essentially similar (420 000-490 000) except for the two heart ferritins which were larger (530 000 and 626 000, respectively). However, the estimated charges on rat liver, kidney and hepatoma monomers (30-38 net protons per molecule) differed from that of spleen monomer (51 net protons per molecule) while the larger rat heart ferritin also had a greater charge (83 net protons) than the smaller (40 net protons). Apoferritins prepared chemically by removal of iron from the holoferritins had migration properties indistinguishable from the parent holoferritins. The migration properties of minor (dimeric) ferritin bands on the gels were compared with those of the monomer bands. The molecular sizes of the minor bands were larger than those of the major bands, and were not inconsistent with a doubling in size. However, charge differences varied, being either similar for major and minor forms (spleen ferritin), approximately twice for the minor form (rat hepatoma ferritin) or five times greater for the minor form (rat liver ferritin). These differences in behavior were confirmed by using minimally sieving gels, on which the major bands of horse spleen ferritin failed to separate whereas those of rat liver ferritin were readily separable. It is concluded that dimers of ferritins from different tissues may associate in different ways. 相似文献
10.
Organization of a multifunctional protein in pyrimidine biosynthesis. Analyses of active, tryptic fragments 总被引:7,自引:0,他引:7
The multifunctional protein which catalyzes the first three steps of pyrimidine biosynthesis in hamster cells can be cleaved by trypsin into enzymatically active fragments. When the fragments are separated by nondenaturing polyacrylamide gel electrophoresis, three major polypeptide bands appear. Carbamyl phosphate synthetase (EC 2.7.2.9), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3) activities are associated with 129,000-, 660,000-, and 94,000-dalton bands, respectively. Further analysis of these fragments by denaturing polyacrylamide gel electrophoresis has shown that the aspartate transcarbamylase band seen on the nondenaturing gel is actually a large aggregate of 39,000-dalton fragments and the dihydroorotase band is a dimer of 44,000-dalton fragments. The data reported here indicate that (i) this multifunctional protein is composed of three enzymatically independent domains, (ii) the sum of the molecular weights of these three domains (129,000 + 39,000 + 44,000 = 212,000) is similar to that of the undigested monomer (220,000 daltons), and (iii) a site important to the formation of the native multimeric protein is probably near the aspartate transcarbamylase domain. 相似文献
11.
M band protein can be specifically extracted from fresh chicken breast muscle myofibrils suspended in 5 mM Tris-HCl pH 8.0. During discontinuous polyacrylamide gel electrophoresis the isolated protein separates into three bands which can be identified as two separate components (A, B) and a complex of the two. When partially purified fractions of the separated components are combined, an increase in the intensity of the band containing the complex can be shown. The polypeptide chain weights of the two components are 100,000 (A) and 40,000 (B) daltons as estimated by sodium dodecyl sulfate- (SDS-) polyacrylamide gel electrophoresis. Antibody prepared against total M band protein stains only the M band of the myofibril and is completely absorbed by M band protein. M band protein also absorbs the M band staining specifically from antibody which stains both I and M bands. Immunodiffusion data indicate that anti-M band is a mixture of two specific antibodies, one against each component. 相似文献
12.
M C Linder C A Goode R Gonzalez C Gottschling J Gray G M Nagel 《Archives of biochemistry and biophysics》1989,273(1):34-41
Ferritin purified from horse heart and applied to nondenaturing polyacrylamide gel electrophoresis migrated as a single band that stained for both iron and protein. This ferritin contained almost equal amounts of fast- and slow-sedimenting components of 58 S and 3-7 S, which could be separated on sucrose density gradients. Iron removal reduced the sedimentation coefficient of the fast-sedimenting ferritin to 18 S, and sedimentation equilibrium gave a molecular weight 650,000, with some preparations containing ferritin of 500,000 molecular weight as well. Sedimentation rates of the 3 S and 7 S ferritins were not affected by iron removal, and sedimentation equilibrium data were consistent with Mr's 40,000 and 180,000, respectively. Preparations of ferritin extracted from horse spleen contained only 67 S (holo) or 16 S (apo) ferritin and no slow-sedimenting species. When examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all of the ferritins contained the usual H and L subunits (23 and 20 kDa, respectively), but the slow-sedimenting (3 S and 7 S) heart apoferritins also contained appreciable quantities (ca 25%) of three larger subunits of 42, 55, and 65 kDa. All the subunits reacted positively in Western blots to polyclonal antibodies made against specially purified large heart or spleen ferritins containing only 20- and 23-kDa subunits. Similar results were obtained for ferritins from rat heart. The results indicate that mammalian heart tissue is peculiar not just in having an abnormally large iron-rich ferritin but also in having iron-poor ferritins of much lower molecular weight, partly composed of larger subunits. 相似文献
13.
We have reinvestigated the association and dissociation of ferritin and apoferritin in phosphate buffer (pH 7.2, I = 0.05). When oligomer-enriched solutions of horse spleen ferritin were mixed with more concentrated, but unenriched solutions of horse spleen apoferritin, there was dissociation of the ferritin oligomers, as determined by polyacrylamide gel electrophoresis and from iron/protein ratios. Some evidence was also obtained for association of monomers in the mixture of ferritin and apoferritin after pelleting and redissolution of pellets in minimal volumes of the phosphate buffer. Monomer-enriched, biosynthetically labeled rat liver ferritin was pelleted, redissolved in minimal volumes of phosphate buffer, and separated by polyacrylamide gel electrophoresis; the fractions were isolated and counted. The results revealed that an association of monomers of the rat liver ferritin had taken place which doubled the concentration of dimers. However, our results also indicate that association by concentration was limited to a fraction of monomers. 相似文献
14.
Rat liver beta-glucuronidase (EC 3.2.1.31), both from microsomal and lysosomal fractions, were purified about 9500-fold over the homogenate with high yield using affinity chromatography prepared by coupling purified specific immunoglobulin G against rat preputial gland beta-glucuronidase to Sepharose 2B and isoelectric focusing. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of approximately 310000. In dodecylsulfate polyacrylamide gel electrophoresis, the microsomal beta-glucuronidase showed a single band corresponding to a molecular weight of 79000, while the lysosomal beta-glucuronidase had three distinct bands which consisted of one major and two minor bands corresponding to molecular weight of 79000, 74000, and 70000, respectively. A broad pH activity curve with a single optimum at pH 4.4 was observed in both the microsomal and the lysosomal beta-glucuronidases. Immunological gel diffusion technique with rabbit antiserum against rat liver lysosomal beta-glucuronidase revealed that both enzymes had the same or quite similar antigenic determinants. 相似文献
15.
Individual hexokinase isoenzymes (isoHK) are isolated from normal and malignant human stomach mucosa. IsoHK from tumour tissue are found to have KM for glucose 10 times as low as isoHK from normal tissue. Molecular weights of individual isoHK from normal and tumour tissues are similar (at the range of 112,000-125,000). The treatment of protein preparation with 8M urea in the presence of 1% sodium docecyl sulphate resulted in the appearance of a single band with molecular weight of 58,000-60,000 for all the isoHK under polyacrylamide gel electrophoresis. Intensive bands with molecular weight of 60,000 and 96,000 and a number of minor bands were observed under polyacrylamide gel disc elect-ophoresis in the absence of urea. 2-Mercaptoethanol did not affect the results of disc electrophoresis. It is concluded that the molecule of human hexokinase consists of two subunits with molecular weight of 60,000. 相似文献
16.
B Akerstr?m L L?gdberg H Babiker-Mohamed S Lohmander L Rask 《European journal of biochemistry》1987,170(1-2):143-148
Rabbit alpha 1-microglobulin was purified from the urine of sodium-chromate-treated animals by the use of gel chromatography on Sephadex G-100, affinity chromatography on concanavalin-A--Sepharose and ion-exchange chromatography on DEAE-Sephadex. Rabbit alpha 1-microglobulin had a molecular mass of 25.6 kDa on SDS/polyacrylamide gel electrophoresis. Alpha 1-microglobulin has previously been purified from the urine of humans, guinea-pigs and rats by similar methods, and the molecular masses of the four homologues were compared by SDS/polyacrylamide gel electrophoresis and gel chromatography in a denaturing medium. By these two methods the human homologue was 6 kDa and 3 kDa larger, respectively, than the other three proteins. Endoglycosidase F digestion of alpha 1-microglobulin, followed by SDS/polyacrylamide gel electrophoresis, revealed three protein bands in the human alpha 1-microglobulin sample, and only two bands in guinea-pig, rat and rabbit alpha 1-microglobulin, with a gap between each band of 2.6--2.9 kDa. The amino-terminal amino acid sequences of the four homologues were determined and between 72% and 81% homology was seen. The five amino-terminal amino acids present in the other species were missing in guinea-pig alpha 1-microglobulin. Our results indicate that human alpha 1-microglobulin is substituted with two N-linked oligosaccharides, while only one is attached to each of the other alpha 1-microglobulins, and that the extra glycosylamine-linked oligosaccharide in the human protein is attached to asparagine in position 17. Finally it is shown that all four homologues inhibit antigen stimulation of human lymphocytes, a finding which is consistent with our previous suggestion that the N-linked oligosaccharides carry the immunosuppressive activity of alpha 1-microglobulin. 相似文献
17.
Multiple forms of human renin. Purification and characterization. 总被引:10,自引:0,他引:10
F X Galen C Devaux T Guyenne J Menard P Corvol 《The Journal of biological chemistry》1979,254(11):4848-4855
Human renin was purified from a juxtaglomerular cell tumor with a high renin content, 24.2 Goldblatt units/mg of protein. The purification procedure comprised three steps: gel filtration, DEAE-cellulose chromatography, and preparative isoelectric focusing. Five forms of renin amounting to 5.3 mg of enzyme were obtained with isoelectric points of 4.95, 5.10, 5.35, 5.55, and 5.70. They were all glycoproteins. The three major fractions had very similar specific activities, 868, 860, and 809 Goldblatt units/mg of protein. These fractions produced a single band on analytical isoelectric focusing and a single arc on immunoelectrophoresis. On polyacrylamide gel electrophoresis at pH 7.8, each fraction consisted of two renin bands with the same molecular weight, but different net charges. The molecular weight determined by gel filtration and Fergusson plot analysis on polyacrylamide gel was 38,000 to 42,000. The optimum pH determined on N-acetyltetradecapeptide substrate was 6.5, and the Km was 6.8 x 10(-6) M. These parameters were identical with those for standard human kidney renin. Antibodies raised against tumor renin completely inhibited the activity of both tumor and standard renin. Under dissociating conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel electrophoresis in the presence of 6 M urea), part of the purified enzyme dissociated into two smaller fragments (Mr = 20,000 and 25,000) containing renin activity. 相似文献
18.
C Bagnara C Gaudin J P Belaich 《Biochemical and biophysical research communications》1986,140(1):219-229
Avicelase assay of gel slices after non-denaturing polyacrylamide gel electrophoresis of concentrated supernatants from Cellulomonas fermentans revealed four active bands. One of them corresponded to the principal active band on CM-cellulose. Among the three others, at least one did not correspond to any active band on CM-cellulose and might reflect the presence of an exoglucanase (EC 3.2.1.91). The active band on CM-cellulose was composed of two endoglucanases (EC 3.2.1.4), called CFA and CFB, which we purified by the means of DEAE-Trisacryl chromatography and high performance liquid chromatography (anion exchange chromatography and gel chromatography). These two monomeric enzymes differ in their molecular weights (40,000 and 57,000 for CFA and CFB, respectively) and in their catalytic constants in the reaction with CM-cellulose (Km were 1.5 g/l and 59 g/l for CFA and CFB, respectively), but have similar modes of action on this substrate and similar substrate specificities. 相似文献
19.
Purification and Properties of Undecyl Acetate Esterase from Pseudomonas cepacia Grown on 2-Tridecanone 总被引:6,自引:3,他引:3
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Undecyl acetate esterase has been purified from Pseudomonas cepacia grown on the methyl ketone, 2-tridecanone. The K(m) for undecyl acetate was 2.3 x 10(-2) M. Polyacrylamide gel electrophoresis indicated that two esterase bands were being recovered during purification. These bands were separated by preparative polyacrylamide gel electrophoresis. Molecular weights were estimated to be approximately 34,500 by several methods. Molecular sieve polyacrylamide gel electrophoresis indicated that the two esterases had the same molecular weight but different charge, which is indicative of isoenzymes. 相似文献
20.
Theta-Toxin, an oxygen-labile hemolysin produced by Clostridium perfringens, was purified 3300 fold from culture filtrate by successive chromatography on DEAE-Sephadex A-50 and Sephadex G-150. The purified toxin gave two distinct bands in disc electrophoresis, while the same material, after mild reduction with dithiothreitol, yielded a single band, indicating that the purified theta-toxin contained, as well as a reduced, active form, an oxidized, inactive form of toxin. These two forms of the toxin had a similar, if not identical molecular size. The purified preparation gave a single band in a sodium dodecyl sulfate polyacrylamide gel electrophoresis and formed a single precipitin line with National Standard gas gangrene (C. perfringens) antitoxin. By sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular weight of theta-toxin was estimated to be 51 000, the value being in exact accordance with that obtained by amino acid analysis. The amino acid composition of theta-toxin was very close to that of cereolysin, an oxygen-labile hemolysin produced by Bacillus cereus. The amino-terminal residue of theta-toxin was lysine as determined by the Dansyl method. 相似文献