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1.
H G Hansma 《The Journal of protozoology》1975,22(2):257-259
The immobilization antigen (i-antigen) fraction of Paramecium aurelia syngen 4 is shown to contain a protease that is activated by mercaptoethaneol. After the protease has been heat-inactivated, the molecular weight of the i-antigen (similar to 250,000 daltons) cannot be decreased by mercaptoethanol treatment. It is demonstrated that the i-antigen is a single polypeptide chain. Reasons are also given why low molecular weight subunits were previously reported by other authors. 相似文献
2.
Arash Zibaee 《Biocontrol Science and Technology》2009,19(8):797-808
The extracellular protease from the entomopathogenic fungus, Beauveria bassiana in the presence of Eurygaster integriceps cuticle was isolated, purified and characterized. Isolate B1 of B. bassiana that shows high virulence against E. integriceps was examined for the production of the cuticle-degrading proteases. Results showed that both subtilisin-like (Pr1) and trypsin-like (Pr2) cuticle-degrading proteases were produced and the enzyme kinetic properties showed better activity of Pr1 in comparison with Pr2. The proteases were purified using acetone precipitation, Sephadex G-100 gel filtration and CM-Sepharose ion exchange chromatography, with a 5.09-fold increase in specific activity and 21.86% recovery. The enzyme molecular weight was estimated to be 47 kDa and the optimal pH and temperature were 8 and 45°C, respectively. The purified protease was activated by divalent cations, Ca2 + and Mg2 +, and inhibited by NaCl, KCl and determined as a serine protease by inhibition of its activity due to using PMSF, EDTA, mercaptoethanol and SDS. Studies on the timing of the protease secretion in the presence of cuticular substrates could provide information about the role of the accumulated hydrolytic enzymes during pathogenesis to better understand these processes. 相似文献
3.
Degradation of the Major Storage Protein of Phaseolus vulgaris during Germination : Role of Endogenous Proteases and Protease Inhibitors 总被引:2,自引:2,他引:0
Cotyledons from Phaseolus vulgaris L. (var. Improved Tendergreen) were tested for their activity on α-N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) and azocasein during a germination periood of 10 days. Both activities increased throughout germination when activity was expressed on the basis of dry weight or protein. That these two activities were most likely due to the action of different enzymes was indicated by the fact that (a) optimal pH for the hydrolysis of BAPNA and azocasein was 8.2 and 5.5, respectively, and (b) the digestion of azocasein was considerably enhanced by mercaptoethanol and partially inhibited by thiol protease inhibitors, N-ethylmaleimide, and E-64, whereas these same regents caused little change in activity toward BAPNA. The three subunits of the major storage protein, G1, disappeared during germination and were accompanied by the accumulation of lower molecular weight products. The breakdown of G1 by extracts of the germinated beans could be demonstrated in vitro at pH 5 to 6. This activity was enhanced by mercaptoethanol and completely abolished by N-ethylmalemide, leupeptin, and E-64. It is concluded that a thiol protease with an acid pH optimum is primarily responsible for the disappearance of the major storage protein during germination. Although an inhibitor of the plant thiol protease, papain, is present in the mature bean and decreases during germination, its role in the control of the breakdown of the storage protein remains to be elucidated. 相似文献
4.
Miroslav Stýblo 《Biological trace element research》1992,34(1):11-18
The molecular structure of plasma and erythrocyte selenium-dependent glutatione peroxidase (GSH-Px) was studied in rats drinking
water containing [75Se]selenious acid, 1.3 mg Se/L. Substantial differences were found using three-step fractionation, including gel filtration
of crude plasma and erythrocyte lysate, gel filtration of75Se-GSH-Px treated by mercaptoethanol, and SDS-electrophoresis. Native plasma75Se-GSH-Px, which exhibited a molecular weight (M
r) of approx 700,000, could be destroyed by mercaptoethanol action, resulting in disintegration of enzyme into several different75Se-protein fragments and release of part of low-mol-wt75Se. Native erythrocyte75Se-GSH-PxM
r, value was found to be 113,000; two75Se-protein fragments arose after mercaptoethanol treatment without75Se release from the enzyme. The75Se-subunits of 22,500 and 21,900 were isolated from plasma and erythrocyte75Se-GSH-Px, respectively. Another minor75Se-GSH-Px was identified in erythrocyte lysate (M
r, 214,000, subunit 22,100), which was considered to be a dimer of the above-mentioned erythrocyte enzyme. It can be assumed,
based on these data, that native plasma GSH-Px, in contrast to erythrocyte enzyme, represents a high-molecular wt complex
composed of several tetramers linked with S—S bonds. A certain part of selenium present in this complex is probably not selenocysteine
and may be released with the mercaptoethanol treatment. 相似文献
5.
We have studied four strains of Tetrahymena thermophila, each of which expresses a different allele of the SerH gene and produces a distinctive surface protein of the immobilization antigen (i-antigen) class. Following exposure of the strains to [3H]ethanolamine or [3H]myristic acid, a protein corresponding in molecular mass to the characteristic i-antigen for that strain became highly labeled, as determined by mobility in sodium dodecylsulfate-polyacrylamide electrophoresis gels. Furthermore, antibodies raised to the i-antigens of the T. thermophila strains selectively immunoprecipitated radioactive proteins having molecular mass identical to that of the i-antigen characteristic for that particular strain. The lipid moieties labeled by [3H]myristate were not susceptible to hydrolysis by exogenous phosphatidylinositol-specific phospholipase C from bacteria. However, when protein extraction was carried out in the absence of phospholipase C inhibitors, radioactive fatty acids derived from [3H]myristate were rapidly cleaved from the putative i-antigens. On the basis of available data, it was concluded that T. thermophila i-antigens contain covalently-linked glycosyl-phosphatidylinositol anchors. 相似文献
6.
F. PAUL DOERDER MUHITTIN ARSLANYOLU YASSER SAAD MICHELLE KACZMAREK MANUEL MENDOZA BARRY MITA 《The Journal of eukaryotic microbiology》1996,43(2):95-100
Until recently, Tetrahymena thermophila has rarely been isolated from nature. With improved sampling procedures, T. thermophila has been found in ponds in many northeastern states. The availability of resident populations makes possible both population and ecological genetic studies. All seven known mating types have been recovered; no eighth mating type has been found. Crosses among whole-genome homozygotes derived from Pennsylvania isolates reveal a spectrum genotypes with mating type alleles resembling traditional A (IV- and VII-) and B(I-) categories. The genotypes differ significantly with respect to mating type frequency, both among themselves and from previously described genotypes. One A-category genotype appears to lack mating type II, while one A-category and all B-category genotypes have low frequencies of mating type III, thus accounting for the low frequency of III in the pond. The low frequency of III in all five B-category genotypes examined suggests that the founding allele in this region was low for III. These and other differences are discussed both in terms of mating type frequencies in the pond and in terms of the possible molecular structure of mat alleles. By contrast, numerous variants of the cell surface immobilization antigen are found in addition to the previously described i-antigens. Variants of the known SerH alleles include those with restriction fragment length polymorphisms and temperature sensitivity as well as alleles with new antigenic specificity. Multiple alleles are present in single ponds. Genes exhibiting serially dominant epistasis over SerH genes also are found. In two instances (K and C), families of antigenically similar polypeptides are expressed in place of H i-antigen. Molecular weight differences suggest that these paralogous i-antigen genes evolve by gene duplication and unequal crossing over within central repeats. The existence of complex patterns of epistasis together with seasonal changes in i-ag frequencies suggest that i-ag play an important, but as yet unknown, ecological role related to the occurrence of frequent conjugation. 相似文献
7.
We have studied four strains of Tetrahymena thermophila , each of which expresses a different allele of the SerH gene and produces a distinctive surface protein of the immobilization antigen (i-antigen) class. Following exposure of the strains to [3 H]ethanolamine or [3 H]myristic acid, a protein corresponding in molecular mass to the characteristic i-antigen for that strain became highly labeled, as determined by mobility in sodium dodecylsulfate-polyacrylamide eiectrophoresis gels. Furthermore, antibodies raised to the i-antigens of the T. thermophila strains selectively immunoprecipitated radioactive proteins having molecular mass identical to that of the i-antigen characteristic for that particular strain. The lipid moieties labeled by [3 H]myristate were not susceptible to hydrolysis by exogenous phosphatidylinositol-specific phospholipase C from bacteria. However, when protein extraction was carried out in the absence of phospholipase C inhibitors, radioactive fatty acids derived from [3 H]myristate were rapidly cleaved from the putative i-antigens. On the basis of available data, it was concluded that T. thermophila i-antigens contain covalently-Iinked glycosyl-phospha-tidylinositol anchors. 相似文献
8.
Bovine liver thiosulfate sulfurtransferase (rhodanese) (EC 2.8.1.1) HAS BEEN REPORTED TO EXIST IN SOLUTION IN A RAPID, PH-dependent equilibrium between monomeric and dimeric forms of molecular weights 18 500 and 37 000 (Volini, M., DeToma, F. and Westley, J. (1967), J. Biol. Chem. 242, 5220). We have reinvestigated the proposed dissociation using sodium dodecylsulfate-polyacrylamide gel electrophoresis. The smallest rhodanese species observed has a molecular weight around 35 000, which is not reduced by severe denaturing conditions, including alkylation in 8 M guanidine-HCl or dialysis against 2% sodium dodecylsulfate and 5% mercaptoethanol. After limited CNBr cleavage, intermediate products of greater than 18 500 molecular weight are formed. The apparent molecular weight of these intermediate fragments is not changed by addition of mercaptoethanol. The total apparent molecular weights of the CNBr fragments after exhaustive cleavage is approx. 45 000 plus or minus 15 000. These results are not consistent with a monomer molecular weight of approx. 18 500 for thiosulfate sulfurtransferase. 相似文献
9.
N. Hajela A. H. Pande S. Sharma D. N. Rao K. Hajela 《Journal of plant biochemistry and biotechnology.》1999,8(1):57-60
A doubleheaded protease inhibitor showing inhibition of bovine pancreatic trypsin and α-chymotrypsin was isolated and purified from the seeds of Phaseolus mungo. The molecular weight of the protease inhibitor was found to be 14.2 kD by SDS-PAGE analysis and gel filtration. The native inhibitor inhibited trypsin and α-chymotrypsin stoichiometrically at the molar ratio 1:1 and 2:1 respectively. The Ki app for trypsin was found to be 0.35 nM and for α-chymotrypsin to be 2.4 nM. Bovine pepsin was not inhibited by the inhibitor. However, the pepsin treated inhibitor was still able to inhibit trypsin and α-chymotrypsin. The inhibitor was stable in 8M urea. Addition of 0.2 M mercaptoethanol resulted in significant loss of inhibitory activity. The inhibitor was extremely heat stable with only 50% loss of inhibitory activity after heating for 100°C for 20 min. Thus, the Phaseolus mungo trypsin/chymotrypsin inhibitor resembles other Bowman-Birk protease inhibitors. 相似文献
10.
Mendelian and non-mendelian mutations affecting surface antigen expression in Paramecium tetraurelia. 总被引:15,自引:9,他引:6
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A screening procedure was devised for the isolation of X-ray-induced mutations affecting the expression of the A immobilization antigen (i-antigen) in Paramecium tetraurelia. Two of the mutations isolated by this procedure proved to be in modifier genes. The two genes are unlinked to each other and unlinked to the structural A i-antigen gene. These are the first modifier genes identified in a Paramecium sp. that affect surface antigen expression. Another mutation was found to be a deletion of sequences just downstream from the A i-antigen gene. In cells carrying this mutation, the A i-antigen gene lies in close proximity to the end of a macronuclear chromosome. The expression of the A i-antigen is not affected in these cells, demonstrating that downstream sequences are not important for the regulation and expression of the A i-antigen gene. A stable cell line was also recovered which shows non-Mendelian inheritance of a macronuclear deletion of the A i-antigen gene. This mutant does not contain the gene in its macronucleus, but contains a complete copy of the gene in its micronucleus. In the cytoplasm of wild-type animals, the micronuclear gene is included in the developing macronucleus; in the cytoplasm of the mutant, the incorporation of the A i-antigen gene into the macronucleus is inhibited. This is the first evidence that a mechanism is available in ciliates to control the expression of a gene by regulating its incorporation into developing macronuclei. 相似文献
11.
Protease inhibitory activity in jackfruit seed (Artocarpus integrifolia) could be separated into 5 fractions by chromatography on DEAE-cellulose at pH 7.6. A minor fraction (I) that did not bind
to the matrix, had antitryptic, antichymotryptic and antielastase activity in the ratio 24:1.9:1.0. Fraction II bound least
tightly to the ion exchanger eluting with 0.05 M NaCl and could be resolved into an elastase/chymotrypsin inhibitor and a
chymotrypsin/trypsin inhibitor by chromatography on either immobilized trypsin or phenyl Sepharose CL-4B. Fractions III and
IV eluted successively with 0.10 M NaCl and 0.15 M NaCl from DEAE-cellulose, inhibited elastase, chymotrypsin and trypsin
in the ratio 1.0: 0.53:0.55 and 1.0:8.9:9.8 respectively. Fraction V, most strongly bound to the matrix eluting with 0.3 M
NaCl and was a trypsin/chymotrypsin inhibitor accounting for 74% of total antitryptic activity. This inhibitor was purified
further. The inhibitor with a molecular weight of 26 kd was found to be a glycoprotein. Galactose, glucose, mannose, fucose,
xylose, glucosamine and uronic acid were identified as constitutent units of the inhibitor. Dansylation and electrophoresis
in the presence of mercaptoethanol indicated that the inhibitor is made up of more than one polypeptide chain. The inhibitor
combined with bovine trypsin and bovine α-chymotrypsin in a stoichiometric manner as indicated by gel chromatography. It had
very poor action on subtilisin BPN′, porcine elastase, pronase,Streptomyces caespitosus protease andAspergillus oryzae protease. It powerfully inhibited the caseinolytic activities of rabbit and horse pancreatic preparations and was least effective
on human and pig pancreatic extracts. Modification of amino groups, guanido groups and sulphydryl groups of the inhibitor
resulted in loss of inhibitory activity. Reduction of disulphide bridges, reduction with sodium borohydride and periodate
oxidation also decreased the inhibitory activity. 相似文献
12.
A B Schneider K Kowalski L M Sherwood 《Biochemical and biophysical research communications》1975,64(2):717-724
“Big” human placental lactogen has been purified by affinity chromatography. On SDS1-polyacrylamide disc gel electrophoresis, “big” hPL had a molecular weight of about 45,000. Following reduction with mercaptoethanol, a single band with a molecular weight of 23,000 was noted. These observations suggest that “big” hPL consists of two peptide chains linked by a disulfide bond. 相似文献
13.
Shuichi Kaminogawa Kunio Yamauchi 《Bioscience, biotechnology, and biochemistry》2013,77(12):2343-2348
A γ-casein-like fraction (FIV) was isolated from the β-casein A hydrolyzate by milk protease and compared with γ-casein. The mobility in polyacrylamide gel electrophoresis, sedimentation coefficient, molecular weight, amino acid composition and terminal amino acids of FIV were almost coincided with those of γ-casein. It is suggested that γ-casein is possibly a product of β-casein hydrolysis by milk protease. 相似文献
14.
Conformation of mucous glycoproteins in aqueous solvents 总被引:5,自引:0,他引:5
Light-scattering techniques have been used to measure the z-average radius of gyration Rg z-average translational diffusion coefficient Dt and weight–average molecular weight Mw of porcine submaxillary mucin (PSM) in solution. PSM isolated at low shear in the presence of protease inhibitors has a Mw about twice as large as a sample prepared without these precautions. The former sample has a Mw of 17 × 106 in 0.1M NaCl, which decreases to 8 × 106 in 6M guanidine hydrochloride (GdnHCl) and then to 2 × 106 on addition of 0.1M mercaptoethanol to the 6M GdnHCl solution. The Rg or D values obtained for PSM in this work superimpose with those of other authors for different mucin glycoproteins, leading to linear log–log relationships to the molecular weight of the protein core. Comparison of these results with those in the literature for denatured proteins suggest that mucins are linear random coils in which the protein core is stiffened by the presence of the oligosaccharide side chains. The length of the oligosaccharides and the nature of the solvent have little effect on the extension of the protein core. This suggests that the stiffness of the protein core is maintained by steric repulsion of the residues at the beginning of the oligosaccharide chains. 相似文献
15.
Nack-Shick Choi Jong Hyun Choi Jung-Hoon Yoon Seung-Goo Lee Jae Jun Song 《Biotechnology letters》2009,31(7):975-978
A protease was purified from Bacillus sp. DJ isolated from Doenjang, a traditional Korean fermented food. Its molecular weight (MW) and isoelectric point (pI) were 18-19 kDa and 6.0–6.5 using 1- or 2-D fibrin zymography, respectively. The protease was optimally active at pH 9 and
55°C. Activity was inhibited by 1 mM PMSF, but not by EDTA, EGTA, aprotinin, or leupeptin, indicating that the protease is
a serine protease. By using a new electrophoretic technique, multiple loading of O’Farrell-type isoelectric focusing (IEF)
slab gel, the first amino acid residues of the N-terminal sequence of the protease were determined as HPLVLVDPIL, which is 80% identical with serine proteases of the subtilase
family. 相似文献
16.
17.
《Bioscience, biotechnology, and biochemistry》2013,77(11):2020-2023
Mixed rumen ciliate protozoa (mainly Entodiniinae) from goats have two kinds of protease; one has a pH optimum of 3.0, the other is active at neutral or alkaline pH. The protease active at neutral or alkaline pH was partially purified from the supernatant after centrifugation of sonicated mixed rumen ciliate protozoa. The supernatant was chromatographed on Bio-Gel A-1.5m and a partially purified protease was obtained. This protease had a molecular weight of more than 400,000. When the sonicated protozoa were heated at 55°C for 15min, the active peak from the Bio-Gel A-1.5m column was shifted to a lower molecular weight, 27,000. The high molecular weight protease was strongly activated by high temprature and SDS, and inhibited by E-64 c. The protease degraded many proteins including those found in rumen bacteria. These findings suggest that rumen ciliate protozoa have high molecular weight protease that plays a role in the digestion of feed and bacterial protein. 相似文献
18.
Two different procedures are described to isolate and purify the reaction center complex from Rhodopseudomonas viridis NHTC 133 by means of the non-ionic detergent dodecyldimethylamine oxide. Both reaction center particles thus obtained were active, as shown by a photobleaching centered at 975 nm.The reaction center also contained, in addition to bacteriochlorophyll, bacteriopheophytin. Other components were also found in this particle: cytochromes C553 and C558 and a menaquinone-like substance.The SDS gel electrophoresis of reaction centers is shown. The molecular weights of the subunits forming the reaction center in 0.5% sodium dodecyl sulfate and 1% mercaptoethanol were calculated as being: 45±1.5 and 37±1.5 kdalton, 29±1.5 and 23±1.5 kdalton.The molecular weight of the complex determined by means of gel filtration (Sepharose 6-B and Bio-Gel P-300) gives a value of approximately 240 kdalton.The minimum molecular weight of the complex calculated by disc gel electrophoresis was 231 kdalton. 相似文献
19.
MARGARETA WELANDER 《Physiologia plantarum》1978,43(3):242-246
The effect of mercaptoethanol at different concentrations on enzyme activity was investigated in leaves from Urtica dioica and Spinacia oleracea. The interference of mercaptoethanol with enzyme activity is dependent on the type of plant, the configuration of the enzyme and the concentration of mercaptoethanol. A stimulation of GDH (glutamate-dehydrogenase) was obtained in both species, while inhibition of GOT (glutamate-oxaloacetate transaminase) and GPT (glutamate-pyruvate transaminase) was demonstrated in Spinacia. The stimulation of GOT and GPT activity in Urtica was probably due to inhibition of phenol oxidase. Conclusions concerning the effect of mercaptoethanol on NR (nitrate reductase) activity were difficult to draw, since mercaptoethanol itself reduced nitrite and interfered with NR determination in tests in vitro. In Urtica. no activity could be obtained at all with the in vitro method, probably because an inhibitor of NR was liberated during the extraction procedure, Activity of NR could however be obtained in both species when using the in vivo method. Addition of protective agents to the extraction medium has been supposed to influence the protein extractability. In conformity with this increasing amount of fresh matter to the same volume of extraction medium resulted in decreased protein extractability. This led to differences in enzyme activity when expressed on a fresh weight basis but the specific activity remained constant. 相似文献
20.
Nobumaro Kawashima Harui Fukushima Ayako Imai Einosuke Tamaki 《Bioscience, biotechnology, and biochemistry》2013,77(9):1141-1145
A leaf protease of tobacco whose activity was enhanced during curing was purified about 60 times with ammonium sulfate fractionation, ethanol precipitation, calcium phosphate gel treatment and Sephadex G-200 column chromatography, and some properties of the protease were examined. The purified enzyme showed the optimum pH at 5.5 and the optimum temperature at 60°C. The protease activity was stable between pH 4.5 and 5.5 at 50°G or at pH 5.5 below 40°C for 1 hr, but completely destroyed at 70°C during 1 hr. The protease activity was greatly activated by reducing agents such as cysteine, glutathione or mercaptoethanol and inhibited by p-chloromercuribenzoate, phenyl- mercuric acetate or silver ions. Metal ions except for silver ion and ethylenediamine tetraacetic acid did not affect the protease activity so far examined. 相似文献