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1.
The fluorogenic reagent fluorescamine has been used to determine the labeling patterns of Type C spinach chloroplast membrane polypeptides. Membrane polypeptides labeled with fluorescamine were detected by scanning high resolution sodium dodecyl sulfate polyacrylamide gradient slab gels for fluorescence emission. Three membrane polypeptides show a decrease in the extent of labeling when chloroplast membranes are labeled in the light compared to when they are labeled in the dark. These polypeptides have apparent molecular weights 0f 32 000, 23 000 and 15 000. The decrease in labeling observed in the light is abolished or reduced by treatments which inactivate the light-generated transmembrane pH gradient. CF1-depleted chloroplasts show neither a light-activated pH gradient nor a light/dark difference in labeling of these three polypeptides. Both a light-activated pH gradient and light/dark difference in labeling are observed in CF1-depleted chloroplasts which have been treated with N,N'-dicyclohexylcarbodiimide. The same ammonium sulfate fractions of a 2% sodium cholate extract, which are believed to be enriched in the membrane-bound sector of the chloroplast ATPase (CFo) are also found to be enriched in the 32 000, 23 000 and 15 000 molecular weight polypeptides. The three polypeptides are believed to be components of CFo, and the light/dark labeling differences may indicate conformational changes within CFo. Such conformational changes may reflect a mechanism which couples light-generated proton gradients to ATP synthesis. 相似文献
2.
A rapid fluorogenic method for the demonstration of 5′-nucleotide phosphodiesterase in human serum has been developed. This method uses the substrate 4-methylumbelliferyl 5′-thymidylate impregnated in agarose gels or filter paper strips. Zymograms are developed in less than 30 min at 25°C, and the sensitivity of this method has been compared with that of the indigogenic method. 相似文献
3.
Polyacrylamide gels were stained with the sialidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid showing the activity of Vibrio cholerae and Clostridium sordellii sialidases in the gels after electrophoresis. With this fluorogenic method minimum sialidase activities of 5 microU could be determined. The sensitivity of this staining is about 10,000-fold higher compared to protein-staining with Coomassie brilliant blue. For the visualization of other proteins than sialidases the specific sialidase staining could be followed by a protein-staining method in the same gel. 相似文献
4.
C.M. McCullagh R. Gupta A.M. Jamieson J. Blackwell 《International journal of biological macromolecules》1996,18(4):247-253
Rheological measurements have been performed on three molecular weight fractions of purified canine submaxillary mucin (CSM) dissolved in the chaotropic solvent 6 M guanidine hydrochloride (GdnHCI). Solutions of the lower molecular weight fractions are viscoelastic sols, and their dynamic moduli can be scaled with respect to molecular weight and concentration according to linear viscoelasticity theory. In contrast, preparations of the highest molecular weight fraction form viscoelastic gels that exhibit an equilibrium shear modulus, Ge′, which scales with mucin concentration as Ge′ c3. Amino acid and carbohydrate analyses of all three fractions are similar; thus, the differences in rheological behavior are attributed to molecular weight differences, which affect the degree of coil overlap in solutions of a given concentration. These observations demonstrate conclusively that mucin glycoproteins of high molecular weight form gels under conditions in which the mucin chains physically interpenetrate, even when non-covalent intermolecular interactions are extensively disrupted. A comparison of these results with previous studies of purified submaxillary and tracheobronchial mucins indicates that the carbohydrate side-chain length, in addition to molecular weight, is an important determinant of the observed elastic response and the ability to form physical gels 相似文献
5.
A simple method is described for visualizing submicrogram amounts of the cyanogen bromide cleavage products of proteins on sodium dodecyl sulfate-polyacrylamide gels. The peptide fragment mixture is conjugated with the fluorogenic reagent 2-methoxy-2,4,-diphenyl-3-(2H)-furanone prior to electrophoresis. The fluorescent peptide bands are visible under ultraviolet light, thus avoiding the need for fixation and staining. The determination of the structural homology of two immunologically related proteins is presented to illustrate this methodology. 相似文献
6.
The fluorogenic reagent fluorescamine has been used to determine the labeling patterns of Type C spinach chloroplast membrane polypeptides. Membrane polypeptides labeled with fluorescamine were detected by scanning high resolution sodium dodecyl sulfate polyacrylamide gradient slab gels for fluorescence emission.Three membrane polypeptides show a decrease in the extent of labeling when chloroplast membranes are labeled in the light compared to when they are labeled in the dark. These polypeptides have apparent molecular weights of 32 000, 23 000 and 15 000.The decrease in labeling observed in the light is abolished or reduced by treatments which inactivate the light-generated transmembrane pH gradient. CF1-depleted chloroplasts show neither a light-activated pH gradient nor a light/dark difference in labeling of these three polypeptides. Both a light-activated pH gradient and light/dark differences in labeling are observed in CF1-depleted chloroplasts which have been treated with N,N′-dicyclohexylcarbodiimide.The same ammonium sulfate fractions of a 2% sodium cholate extract, which are believed to be enriched in the membrane-bound sector of the chloroplast ATPase (CFo) are also found to be enriched in the 32 000, 23 000 and 15 000 molecular weight polypeptides. The three polypeptides are believed to be components of CFo, and the light/dark labeling differences may indicate conformational changes within CFo. Such conformational changes may reflect a mechanism which couples light-generated proton gradients to ATP synthesis. 相似文献
7.
Aminopeptidases from Plasmodium falciparum, Plasmodium chabaudi chabaudi and Plasmodium berghei 总被引:1,自引:0,他引:1
G. PAUL CURLEY SUSAN M. O'DONOVAN JOHN MCNALLY MARGARET MULLALLY HELEN O'HARA ALICE TROY SUE-ANN O'CALLAGHAN JOHN P. DALTON 《The Journal of eukaryotic microbiology》1994,41(2):119-123
ABSTRACT. Using fluorogenic substrates and polyacrylamide gels we detected in cell-free extracts of Plasmodium falciparum, Plasmodium chabaudi chabaudi and Plasmodium berghei only a single aminopeptidase. A comparative study of the aminopeptidase activity in each extract revealed that the enzymes have similar specificities and kinetics, a near-neutral pH optima of 7.2 and are moderately thermophilic. Each has an apparent molecular weight of 80,000 ± 10,000, determined by high performance liquid chromatography on a calibrated SW500 column. Whilst the P. c. chabaudi and P. berghei activity co-migrate in native polyacrylamide gels, that of P. falciparum migrates more slowly. The three enzymes can be selectively inhibited by ortho -phenanthroline and are thus metallo-aminopeptidases; however, in contrast to other aminopeptidases the metal co-factor does not appear to be Zn2+ . 相似文献
8.
A J Grant P J Russell D Raghavan 《Biochemical and biophysical research communications》1989,162(1):308-315
Elastase activities in intact human bladder cancer cell lines, established from three patients, were measured using a fluorogenic substrate highly specific for elastase, under conditions of physiological pH and ionic strength. This method allowed separation of cell-associated from secreted enzyme activity. As secreted elastase accounted for only 8% of the total, we concluded that the elastases were present at the cell surface. Inhibition studies using extracts of cell-surface elastases showed them to be serine proteinases which were also inhibited by alpha 1-antitrypsin. Partially purified fractions showing the highest specific activity towards the fluorogenic substrate hydrolysed insoluble elastin thus confirming the presence of elastases. This is the first time that elastase activity has been demonstrated in human bladder cancer cells and may represent a mechanism involved in tumour invasion. 相似文献
9.
Michels DA Hu S Schoenherr RM Eggertson MJ Dovichi NJ 《Molecular & cellular proteomics : MCP》2002,1(1):69-74
We report a system for automated protein analysis. In the system, proteins are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde, which reacts with lysine residues and creates a highly fluorescent product. These labeled proteins are analyzed by submicellar capillary electrophoresis at pH 7.5 to perform a first dimension separation. Once the first components migrate from the capillary, a fraction is transferred to a second dimension capillary, where electrophoresis is performed at pH 11.1 to further separate the proteins. Laser-induced fluorescence is used as an ultrasensitive detector of the separated proteins. Successive fractions are transferred from the first dimension capillary to the second dimension capillary for further separation to generate, in serial fashion, a two-dimensional electropherogram. The transfer of fractions is computer-controlled; there is no operator intervention once the sample has been injected. Zeptomoles of labeled proteins are detected, providing exquisite sensitivity. 相似文献
10.
Joseph Schrèvel François Bernard Christèle Maintier Roger Mayer Michel Monsigny 《Biochemical and biophysical research communications》1984,124(3):703-710
By using fluorogenic peptidyl-3-amino-9-ethyl-carbazole a highly selective endopeptidase for the Val-Leu-Gly-Arg sequence was demonstrated in endoerythrocytic stages of . Val-Leu-Gly-Arg-endopeptidase showed a maximum activity in pH range 7.0–8.0; it was completely inhibited by 1 mM leupeptin and 1 mM antipain. A complete inhibition was also obtained by 15 mM chloroquine. This trypsin-like activity was negligible in uninfected red blood cells. The high sensitive fluorogenic procedure could be performed on cell fractions, cell lysates as well as supernatants. 相似文献
11.
W Dubiel G Pratt K Ferrell M Rechsteiner 《The Journal of biological chemistry》1992,267(31):22369-22377
We have identified and purified a protein complex from human red blood cells that activates the multicatalytic protease (MCP). The complex, which we call the regulator, sediments at 11 S and is composed of 30-kDa subunits. The regulator does not hydrolyze fluorogenic peptides, but when multicatalytic protease and regulator are combined, MCP cleaves succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin and Leu-Leu-Glu-p-nitroanilide as much as 60-fold faster. Hydrolysis of several other fluorogenic peptides is stimulated to a lesser extent, and activated MCP does not degrade ubiquitin-lysozyme conjugates, bovine serum albumin, or lysozyme. Latent and activated forms of MCP display similar sensitivity to protease inhibitors, suggesting that activation does not generate new kinds of catalytic sites. In addition, ATP suppresses peptide hydrolysis by activated and latent MCPs to the same extent. Activation involves binding of regulator to MCP, and activated MCP migrates slower on native acrylamide gels. Dissociation of the MCP regulator complex during prolonged sedimentation on glycerol gradients releases active regulator and MCP molecules capable of being reactivated. Moreover, two-dimensional electrophoresis does not reveal changes in MCP or regulator subunits following activation. Thus, activation appears to result from reversible association of regulator subunits with MCP. 相似文献
12.
Localization of chitinolytic enzymes in blood of turbot, Scophthalmus maximus, and their possible roles in defence 总被引:1,自引:0,他引:1
The intracellular localizations ofchitinase and β-N-acetylglucosaminidase were detected in turbot blood smears, using a novel method employing fluorogenic substrates. The two enzymes showed different distributions, with chitinase being more generally distributed and N-acetylglucosaminidase being strongly associated with distinct intracellular bodies, probably lysosomes. The fluorogenic substrates were used to analyse soluble and membrane fractions of homogenates of red and white blood cells prepared on Percoll gradients. In the leucocytes, the chitinase and N-acetylglucosaminidase activities were mostly in the soluble fraction. In the erythrocytes the activities were lower, at about one-hundredth and one-tenth specific activities, respectively, and were distributed between soluble and membrane-bound fractions at about 2 : 1 and 3 : 1, respectively. In contrast, lysozyme had a soluble distribution in leucocytes and was not detected in erythrocytes. Plasma was rich in chitinase and lysozyme activities but had no detectable N-acetylglucosaminidase. Two possible roles for the chitinolytic enzymes are discussed: defence against pathogens and processing of glycoproteins or glucosaminoglycans. Evidence for a defence role for the chitinase and lysozyme is provided by demonstrating that they had inhibitory activity against the chitinous fungus Mucor mucedo . 相似文献
13.
Prabhavathi B. Fernandes Ronald V. Nardi Samuel G. Franklin 《Analytical biochemistry》1978,91(1):101-114
Currently available systems for resolving membrane proteins are based only on size and charge differences. Recently, it has been shown that Triton-urea-acetic acid gels which separate proteins on the basis of charge, size and hydrophobicity are capable of resolving proteins differing only by the substitution of a single neutral amino acid. We have applied this new method to the resolution of bacterial envelope proteins. Conditions for optimal resolution of different bacterial envelope proteins were determined by electrophoresis through transverse urea and Triton X-100 gradient gels. We have also correlated the components resolved in this system with those resolved by classical sodium dodecyl sulfate-gel electrophoresis by using two-dimensional slab gels combining the two systems. Furthermore, envelope protein fractions from different species and strains of bacteria were compared to identify specific proteins. This system appears to be a promising method for investigating envelope proteins which are due to missense mutations. 相似文献
14.
The salivary proteins of Chironomus thummi larvae were separated by SDS gel electrophoresis and characterized by immunological techniques. As a result, five protein fractions (sp-220, sp-180, sp-35, sp-18, and sp-16) with molecular weights Mr=220000, 180000, 35000, 18000 and 16000 were identified in 6%–20% polyacrylamide gradient gels. In addition, three giant proteins fractions (molecular weights exceeding Mr=800000) were detected in composite polyacrylamide-agarose gels. Crossed immunoelectrophoresis allowed us to identify five immunochemically dissimilar organ-specific antigen fractions in the salivary gland secretion. Data were obtained indicating that the protein fractions, sp-220, sp-180, sp-35, sp-18, and sp-16, are immunochemically and structurally similar. The giant secretory proteins and the secretory fractions with low molecular weights were found to be immunochemically unrelated. 相似文献
15.
Immunochemistry of cartilage proteoglycan. Immunodiffusion and gel-electrophoretic studies 总被引:13,自引:8,他引:5
Cartilage proteoglycan is thought to be composed of subunits, core proteins with covalently attached sulphated polysaccharide side chains, which form aggregates by non-covalent association with a link protein. The new technique of non-disruptive extraction followed by fractionation in caesium chloride gradients provides a useful means of preparing relatively pure proteoglycan aggregate, subunit and link fractions. Immunological studies of these fractions led to the identification of an antigen associated with the proteoglycan subunit which was common to several species and to the demonstration of additional species-specific antigens in aggregate and link fractions derived from bovine nasal cartilage. Polyacrylamide-gel electrophoresis with sodium dodecyl sulphate of bovine proteoglycan aggregate and link fractions gave two protein bands in the gels and a protein-polysaccharide band at the origin; subunit fractions gave only the band at the origin. These results are consistent with the current concept of cartilage proteoglycan structure. 相似文献
16.
We have developed a fast and reliable purification protocol to obtain yeast nuclei in intact and pure form and in a reasonable yield. The purified nuclei appear homogeneous at the light and electron microscopic level, are highly enriched in the nuclear marker histone H2B and devoid of mitochondrial, vacuolar and cytosolic marker proteins. On sodium dodecyl sulfate (SDS)-polyacrylamide gels, the nuclear fraction contains unique proteins which distinguishes them from the major yeast subcellular fractions. Yeast nuclei were separated by detergent/salt extraction into soluble, insoluble and membrane fractions. Antibodies raised against subnuclear fractions lead to the identification of an integral nuclear membrane protein and a high-abundance 38-kDa protein which is located in the yeast nucleolus. 相似文献
17.
Clathrin-coated vesicles have been isolated from bovine brain. To allow their further use in biophysical studies, the homogeneity of the preparations has been fully characterized after chromatography on Sephacryl S-1000, which is employed in many studies. It is demonstrated here that clathrin-coated vesicles are not stable on the gels and that their instability is increased in preparations using gels that are not presaturated with phospholipids. In addition, some fractionation occurs during chromatography. It is proposed that the slower migrating fractions contain mainly empty clathrin coats. Changes that occurred during the chromatography step are the result of reversible and irreversible events and are probably related to the assembly/disassembly cycle of clathrin observed in vitro. 相似文献
18.
An electrophoretic analysis of the histones of the house cricket 总被引:1,自引:0,他引:1
The histone complement of the house cricket, an insect, was analyzed by electrophoresis on polyacrylamide gels. Five fractions separated from each other on gels containing 6.25 m urea; their subfractions were resolved in long runs on gels 25 cm long. On comparison with the corresponding fractions in vertebrates, only the F1 fraction of the cricket seems notably different. Its mobility is much lower than that of vertebrate F1's. Further, in contrast to vertebrate F1, which consistently shows some electrophoretic heterogeneity, the F1 from nondividing tissue of cricket appears homogeneous. F1 subfractions were found in testis histone, however, and presumably reflect phosphorylation of F1, as expected of a dividing tissue. The other fractions all display as much heterogeneity as seen in vertebrates, or more. Four subfractions were visible in the F3 fraction, three in F2a1, two in F2a2, and two in F2b. The heterogenity of F2b observed in cricket is of particular interest since F2b subfractions have not been detected in studies of any other organisms. F2b's electrophoretic heterogeneity implies that it, like the other histones, is subject to modification which entails neutralization of basic groups. 相似文献
19.
InbredDrosophila melanogaster stocks were surveyed for α-glucosidases with nondenaturing gel electrophoresis using a fluorogenic substrate to stain the
gels. The glucosidase most active under these conditions is polymorphic. We established that the polymorphism is genetic in
origin and that the glucosidase was not likely to be a previously characterized enzyme. The gene encoding the enzyme was mapped
cytogenetically to 33 A1-2- 33A8-B1, confirming that this is an enzyme not yet reported inD. melanogaster. The enzyme was partially purified by elution from nondenaturing gels, which enabled us to establish that it has optimal
activity at pH 6 and interacts most strongly with α-1–4 glucosides. A developmental and tissue survey suggested that this
enzyme could have a purely digestive role or be involved in carbohydrate metabolism inside the organism. We propose that this
enzyme is involved in either starch digestion or glycogen metabolism. 相似文献
20.
Electrophoretic analysis of liver and testis histones of the frog Rana pipiens 总被引:3,自引:1,他引:2
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《The Journal of cell biology》1975,64(2):389-397
Histones were extracted from frog livers and testes and analyzed by electrophoresis on long polyacrylamide gels and on sodium dodecyl sulfate (SDS)-containing polyacrylamide gels. Frog histones were found to be similar to those of calf thymus except that frog histone fraction F2A2 showed a marked dependence on the temperature at which the long gels were run, and frog histone fraction F3 could be separated from frog F2B on SDS-containing gels. Comparisons between frog liver and frog testis histones indicated that the testis contains as its major F1 component a fast migrating species not found in liver. Testis histones also showed less microheterogeneity of fractions F3 and F2A1 than liver histones. These were the only differences observed between liver and testis histones, even when testis histones were prepared from sperm suspensions that were rich in cells in the late stages of spermiogenesis. Thus it seems that, in Rana, the electrophoretic properties of the basic proteins of sperm differ from those of somatic cells only in the nature of histone F1 and in the degree of microheterogeneity of fractions F2A1 and F3. 相似文献