共查询到20条相似文献,搜索用时 46 毫秒
1.
Daniel J. McCormick Walter W. Fredricks Thomas H. Tebo Nickolas J. Calvanico 《Journal of Protein Chemistry》1984,3(3):309-325
The hydrolytic enzymes in pigeon dropping extracts (PDE) were separated into basic and acidic components by DEAE-cellulose chromatography. Six distinct hydrolytic activities were isolated from the acidic group of enzymes. Elastase, trypsin, and two forms of collagenase were the proteases identified. An esterase and a phospholipase were also detected. The sera of symptomatic pigeon breeders, analyzed by crossed immunoelectrofocusing techniques, were shown to contain antibodies to the enzymes trypsin, collagenase, and esterase in the acidic fractions of PDE by staining the immune precipitates with specific chromogenic substrates. These enzymes were purified by preparative isoelectric focusing, affinity chromatography, and gel filtration. The purified elastase hydrolyzed elastin and consisted of a single polypeptide chain (M
r
=23,100). Trypsin hydrolyzed a synthetic arginine substrate, but not elastin or collagen. Its size (M
r
=27,500) and subunit structure were similar to those reported here for elastase. Both enzymes were inhibited by -1-antitrypsin,N-p-tosyl-L-lysine chloromethylketone, and phenylmethylsulfonyl fluoride. Two distinct collagenases were found; both cleaved bovine collagen. The high-molecular-weight (HMW) collagenase was a glycoprotein (M
r
=117,500) and consisted of three polypeptide chains (M
r
=37,100); a low-molecular-weight (LMW) collagenase (M
r
=12,500) was also isolated. The HMW collagenase was inhibited byEDTA,p-chloromercuribenzoic acid, and phenylmethylsulfonyl fluoride, but not by -1-antitrypsin. The source of these enzymes as well as their relationship to the basic hydrolytic enzymes of PDE are discussed. 相似文献
2.
Intracellular proteolytic activities ofB. megaterium KM occur soluble in the cytoplasm and periplasm and insoluble in the membrane. Two proteolytic enzymes were found in the
cytoplasmic fraction by gel filtration on Sephadex G 150 and by polyacrylamide gel electrophoresis. The first enzyme called
CI was stable, had a relative molecular mass ofM
r=105000 (M=105 kg/mol) and was inhibited by EDTA and PMSF, whereas the second, designated CII, was labile and had a relative molecular mass ofM
r=46000 (M=46 kg/mol). Because of its lability it could not be characterized in detail. In the “periplasm” only a single proteolytic
enzyme P (M
r=28000;M=28 kg/mol) inhibited by EDTA could be demonstrated. The extracellular enzyme exhibited similar properties. The membrane proteolytic
activity was sensitive to PMSF and EDTA. The membrane enzymes have not yet been solubilized. In cells of the mutant KM 12
that does not produce the extracellular proteinase, only one type of proteinase, in all its properties identical with the
cytoplasmic proteinase CI, could be demonstrated. 相似文献
3.
P Plevani L Capucci G Badaracco D Breviario N Sacchi G Cattoretti E Ginelli 《Biochemical and biophysical research communications》1982,108(3):1196-1203
The polypeptide structure of terminal transferase purified from human lymphoblasts was examined with an immunoblot procedure using rabbit anti-calf thymus terminal transferase antibodies. Two doublets of bands of Mr 58-56,000 and Mr 44-42,000 are the major immunoreactive polypeptides. Only the Mr 44-42,000 polypeptides can be efficiently renatured after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Controlled degradation with trypsin produces fully active enzyme containing the α and β polypeptides typical of the low molecular weight terminal transferase, suggesting that the different forms of purified terminal transferase may arise by proteolysis of the Mr 58,000 polypeptide. 相似文献
4.
Isolation and characterization from potato tubers of two polypeptide inhibitors of serine proteinases 总被引:7,自引:0,他引:7
G Pearce L Sy C Russell C A Ryan G M Hass 《Archives of biochemistry and biophysics》1982,213(2):456-462
Two polypeptides, isolated to electrophoretic homogeneity from Russet Burbank potato tubers, are powerful inhibitors of pancreatic serine proteinases. One of the inhibitors, called polypeptide trypsin inhibitor, PTI, has a molecular weight of 5100, and inhibits bovine trypsin. The inhibitor is devoid of methionine, histidine, and tryptophan and contains eight half-cystine residues as four disulfide bridges. The second inhibitor, polypeptide chymotrypsin inhibitor II, PCI-II, has a molecular weight of 5700 and powerfully inhibits chymotrypsin. This inhibitor is also devoid of methionine and tryptophan but it contains only six of half-cystines as three disulflde bonds. Both polypeptides strongly inhibit pancreatic elastase. In immunological double diffusion assays, polypeptide trypsin inhibitor and polypeptide chymotrypsin inhibitor II exhibit a high degree of immunological identity (a) with each other, (b) with a polypeptide chymotrypsin inhibitor (PCI-I, Mr 5400) previously isolated from potato tubers, and (c) with inhibitor II, a larger (monomer Mr ~ 12,000) inhibitor of both trypsin and chymotrypsin which has also been previously isolated from potato tubers. The four polypeptide proteinase inhibitors now isolated from Russet Burbank potato tubers cumulatively inhibit all five major intestinal digestive endo- and exoproteinases of animals. The inhibitors are thought to be antinutrients that are present as part of the natural chemical defense mechanisms of potato tubers against attacking pests. 相似文献
5.
A. Pellegrini R. Von Fellenberg 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,797(3):336-342
A new and probably unique elastase inhibitor of horse serum was identified, purified to homogeneity and called pre-α2-elastase inhibitor of the horse. Electrophoretically it migrated immediately in front of the α2 position. Its molecular weight was 188 000 by pore limit polyacrylamide gel electrophoresis and 225 000 by Sephadex G-200 gel filtration. The inhibitor was composed of at least two non-identical polypeptide chains of Mr 68 400 and 87 600. A banding pattern of restricted heterogeneity focused between pH 4.9 and 5.2 was revealed by isoelectric focusing. Of 13 animal, microbial and plant proteinases, horse pre-α2-elastase inhibitor inhibited only pancreatic elastase and trypsin efficiently. Chymotrypsin was inhibited only in traces. No analogy between the elastase inhibitor and the known human serum inhibitors could be found with respect to immunological and biochemical criteria. 相似文献
6.
Two proteolytic enzymes, a cysteine proteinase and a carboxypeptidase, responsible for breakdown of the main storage protein, 13S globulin, were purified from buckwheat seedlings (Fagopyrum esculentum Moench) by (NH4)2SO4 fractionation, gel-filtration on Sephadex G-150, ionexchange chromatography on DEAE-Toyopearl 650 M and chromatofocusing. The cysteine proteinase was purified 74-fold. It has a pH optimum of 5.5, a pI of 4.5 and an apparent molecular mass (Mr) of 71000. The carboxypeptidase was purified 128-fold. It has a pH optimum of 5.3, a pI of 5.8 and a Mr of 78500. Cysteine proteinase hydrolyzed the modified 13S globulin only if the reaction products were eliminated from the incubation mixture by dialysis. Storage protein degradation by the proteinase increased in the presence of carboxypeptidase. We suggest that the two enzymes complete the digestion of 13S globulin after its preliminary hydrolysis by the earlier described enzyme, metalloproteinase, present in dry buckwheat seeds.Abbreviations BSA
bovine serum albumin
- DEAE
diethylaminoethyl
- Mr
apparent molecular mass
- PAGE
polyacrylamide gel electrophoresis
- SDS
sodium dodecyl sulfate 相似文献
7.
Medium-chain fatty acid synthesis by goat mammary-gland fatty acid synthetase. The effect of limited proteolysis.
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Fatty acid synthetase from goat mammary gland was subjected to limited proteolysis by trypsin and elastase. Both proteolytic enzymes selectively cleaved the chain-terminating thioester hydrolase component from the enzyme complex, leaving all other partial activities intact in the core peptides. Trypsin, but not elastase, caused extensive degradation of the released thioester hydrolase. The released thioester hydrolase could be purified to homogeneity by gel filtration. The molecular weight was estimated as 29 000 and the enzyme showed only significant hydrolytic activity toward long-chain acyl-CoA esters. The core peptides retained the ability to synthesize medium-chain acyl-CoA esters in the presence of 2,6-di-O-methyl-alpha-cyclodextrin. The results conclusively show that the terminating thioester hydrolase of goat mammary-gland fatty acid synthetase is not involved in termination of medium-chain-length fatty acid synthesis by this enzyme. 相似文献
8.
Chellakere K. Manjunath Gwendolyn E. Goings Ernest Page 《The Journal of membrane biology》1985,85(2):159-168
Summary Gap junctions (GJ) isolated from rat hearts in presence of the protease inhibitor phenylmethylsulfonylfuoride (PMSF) contain a Mr 44,000 to 47.000 major polypeptide and have a urea-resistant layer of fuzz on their cytoplasmic surfaces, whereas junctions isolated without PMSF are proteolyzed to a Mr 29.500 polypeptide by a serine protease and have smooth cytoplasmic surfaces (C.K. Manjunath, G.E. Goings & E. PageAm. J. Physiol.
246:H865–H875, 1984). Rat liver GJ isolated with or without PMSF contain a Mr 28,000 polypeptide and have smooth cytoplasmic surfaces. Here we examine the origin, type and inhibitor sensitivity of the heart protease; why similar proteolysis is absent during isolation of rat liver gap junctions; and whether the Mr 44.000 to 47,000 cardiac GJ polypeptide is the precursor of the Mr 29,500 subunit. We show that the Mr 44,000 to 47,000 polypeptide corresponds to the unproteolyzed connexon subunit; that proteolysis of this polypeptide occurs predominantly during exposure to high ionic strength solution (0.6m KI) which releases serine protease from mast cell granules; that this protease is inhibitable with PMSF and (less completely) soybean trypsin inhibitor and chymostatin; and thatin vivo degranulation of mast cells by injecting rats with compound 48/80 fails to prevent breakdown of cardiac GJ during isolation. The results support the concept that GJ from rat heart and liver differ in protein composition. 相似文献
9.
Two serine-centred proteolytic enzymes containing catalytically essential histidine residues have been purified to homogeneity from the latex of Elaeophorbia drupifera. The high (117 K) Mr, form, euphorbain d1, and the low Mr, (65 K) form, euphorbain d2, are each composed of subunits of weight 30 000. The subunits differ slightly, as is seen by tryptic mapping and in the amino acid compositions reported for the proteases. Both enzymes have five isoelectric forms, and both display two pH maxima for proteolytic activity. Large molar excesses of sulphydryl-blocking reagents produce some activation of euphorbains d1 and d2. 相似文献
10.
Identification of Several Pathogenesis-Related Proteins in Tomato Leaves Inoculated with Cladosporium fulvum (syn. Fulvia fulva) as 1,3-beta-Glucanases and Chitinases 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Inoculation of tomato (Lycopersicon esculentum) leaves with Cladosporium fulvum (Cooke) (syn. Fulvia fulva [Cooke] Cif) results in a marked accumulation of several pathogenesis-related (PR) proteins in the apoplast. Two predominant PR proteins were purified from apoplastic fluid by ion exchange chromatography followed by chromatofocusing. One protein (molecular mass [Mr] 35 kilodaltons [kD], isoelectric point [pI] ~6.4) showed 1,3-β-glucanase activity, while the other one (Mr26 kD, pI ~6.1) showed chitinase activity. Identification of the products that were released upon incubation of the purified enzymes with laminarin or regenerated chitin revealed that both enzymes showed endo-activity. Using antisera raised against these purified enzymes from tomato and against chitinases and 1,3-β-glucanases isolated from other plant species, one additional 1,3-β-glucanase (Mr33 kD) and three additional chitinases (Mr 27, 30, and 32 kD) could be detected in apoplastic fluids or homogenates of tomato leaves inoculated with C. fulvum. Upon inoculation with C. fulvum, chitinase and 1,3-β-glucanase activity in apoplastic fluids increased more rapidly in incompatible interactions than in compatible ones. The role of these hydrolytic enzymes, potentially capable of degrading hyphal walls of C. fulvum, is discussed in relation to active plant defense. 相似文献
11.
Nimi Osamu Norimoto Yu Nomi Ryosaku 《Bioscience, biotechnology, and biochemistry》2013,77(11):1819-1821
The Kunitz-type trypsin inhibitors, ETIa and ETIb, and chymotrypsin inhibitor ECI were isolated from the seeds of Erythrina variegata. The proteins were extracted from a defatted meal of seeds with 10 mM phosphate buffer, pH 7.2, containing 0.15 M NaCl, and purified by DEAE-cellulose and Q-Sepharose column chromatographies. The stoichiometry of trypsin inhibitors with trypsin was estimated to be 1:1, while that of chymotrypsin inhibitor with chymotrypsin was 1:2, judging from the titration patterns of their inhibitory activities.The complete amino acids of the two trypsin inhibitors were sequenced by protein chemical methods. The proteins ETIa and ETIb consist of 172 and 176 amino acid residues and have Mr 19,242 and Mr 19,783, respectively, and share 112 identical amino acid residues, which is 65% identity. They show structural features characteristic of the Kunitz-type trypsin inhibitor (i.e., identical residues at about 45%) with soybean trypsin inhibitor STI). Furthermore, the trypsin inhibitors show a significant homology to the storage proteins, sporamin, in sweet potato and the taste-modifying protein, miraculin, in miracle fruit, having about 30% identical residues. 相似文献
12.
Summary A xylanase gene from Clostridium acetobutylicum P262 was cloned on a recombinant plasmid pHZ300 which enabled Escherichia coli HB101 cells to produce intracellular xylanase activity. The xylanase gene was located on a 2 kb DNA fragment. The cloned xylanase had an apparent M
r of approximately 28 000 and an isoelectric point of approximately 10. Optimum xylanase activity was obtained at pH 6.0 at 37–43° C. Comparison with a xylanase partially purified from the culture medium of C. acetobutylicum P262 showed that the enzymes had similar characteristics and western blot analysis showed cross-reactivity between antibodies raised against the purified cloned enzyme and a polypeptide of the same M
r from C. acetobutylicum P262. 相似文献
13.
We have identified and isolated two forms of insulin receptor from rat-liver plasma membranes. The smaller (M
r= 90k) is a single polypeptide. The same poly-peptide appears to be the insulin-binding site of the largerM
r=280k). Only the larger, multisubunit, receptor shows high-affinity binding of insulin and negative cooperativity in its dissociation kinetics. 相似文献
14.
Purification and characterization of a proteinase inhibitor from field bean, Dolichos lablab perpureus L 总被引:2,自引:0,他引:2
A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS–PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an M
r of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI–trypsin/chymotrypsin complexes by difference spectral studies. Apparent K
a values of complexes of inhibitor with trypsin and chymotrypsin were 2.1 × 107 M–1 and 3.1 × 107 M–1, respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively. 相似文献
15.
J. R. Efremenko 《Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry》2008,2(3):285-287
The effect of ozonated saline on proteolytic enzymes (trypsin, chymotrypsin, elastase, kallikrein, leucine aminopeptidase), inhibitors of proteolysis and lipid peroxidation (LPO) has been investigated. Injection of ozonated saline caused marked response of the proteolytic system. Low ozone doses did not cause activation of proteolytic enzymes, whereas high doses activated proteases, decreased the level of inhibitors of proteolysis (α1-antitrypsin and α2-macroglobulin) and stimulated accumulation of LPO products. Thus, analyses of proteolytic activity can be used as an indicator of effectiveness and safety of ozone therapy and other treatment programs. 相似文献
16.
Barbara A. McCormack Abigail C. E. Gregory Maria E. Kerry Colin Smith G. Paul Bolwell 《Planta》1997,203(2):196-203
Membrane preparations from suspension-cultured cells of French bean (Phaseolus vulgaris L.) contained callose synthase (EC 2.4.1.34) activity which was preserved upon solubilisation. Following elicitor treatment
of cell cultures, increased activity could be extracted and this increase was maintained during purification. The enzyme was
purified by high-pressure liquid chromatography and active fractions showed a variable association of two polypeptides of
relative molecular masses (Mr) 55 000 and 65 000, the latter being in excess. The Mr-65 000 polypeptide was purified to homogeneity and an antibody raised to it. This antibody showed complex effects on callose
synthase activity when incubated with membrane and soluble extracts. In comparison with other systems, the Mr-55 000 subunit is likely to represent the catalytic subunit while the Mr-65 000 polypeptide is a possible regulatory subunit. The Mr-65 000 polypeptide was immunolocated in membranes at sites of callose synthesis in the plant, in cell plates, in sieve plates,
at the plasma membrane-wall interface of wounded cells and in papillae in infected cells.
Received: 18 January 1997 / Accepted: 8 May 1997 相似文献
17.
Ryozo Nakai Sueharu Horinouchi Takeshi Uozumi Teruhiko Beppu 《Bioscience, biotechnology, and biochemistry》2013,77(11):3061-3065
An alkalophilic Streptomyces strain, KSM-9, producing extracellular cellulases was isolated from soil. Three kinds of cellulases that preferentially hydrolyzed carboxymethylcellulose (CMC) were purified from the strain and designated as CMCase I, II and III. The optimum pH of CMCase I (Mr, 32,000) is 8.5 while those of CMCase II (Mr, 32,500) and III (Mr, 92,000) are at around pH 6.0. CMCase I hydrolyzed CMC in a more random fashion than the other two enzymes. 相似文献
18.
Dipeptidase and carboxypeptidase A activities were determined in cells and luminal contents of the fore-, mid-, and hind-midgut of Musca domestica larvae. Dipeptidase activity was found mainly in hind-midgut cells, whereas carboxy-peptidase activity was recovered in major amounts in both cells and in luminal contents of hind-midguts. The subcellular distribution of dipeptidase and part of the carboxypeptidase A activities is similar to that of a plasma membrane enzyme marker (aminopeptidase), suggesting that these activities are bound to the microvillar membranes. Soluble carboxypeptidase A seems to occur both bound to secretory vesicles and trapped in the cell glycocalyx. Based on density-gradient ultracentrifugation and thermal inactivation, there seems to be only one molecular species of each of the following enzymes (soluble in water or solubilized in Triton X-100): membrane-bound dipeptidase (pH optimum 8.0; Km 3.7 mM GlyLeu, Mr 111,000), soluble carboxypeptidase (pH optimum 8.0; Km 1.22 mM N-carbobenzoxy-glycyl-L-phenylalanine (ZGlyPhe), Mr45,000) and membrane-bound carboxypeptidase (pH optimum 7.5, Km 2.3 mM ZGlyPhe, Mr58,000). The results suggest that protein digestion is accomplished sequentially by luminal trypsin and luminal carboxypeptidase, by membrane-bound carboxypeptidase and aminopeptidase, and finally by membrane-bound dipeptidase. 相似文献
19.
S D Killilea R L Mellgren J H Aylward M E Metieh E Y Lee 《Archives of biochemistry and biophysics》1979,193(1):130-139
Several aspects of the properties of phosphorylase phosphatase in crude rat liver extracts were investigated. Treatment of tissue extracts with either trypsin, ethanol, or urea was found to dissociate phosphorylase phosphatase activity to a form of Mr 35,000. The Mr 35,000 enzyme form was derived from three native enzyme forms. The major cytosolic form of phosphorylase phosphatase had a molecular weight of 260,000 as determined by gel filtration and was dissociated to a Mr 35,000 form by treatment with either ethanol or urea. Treatment of the Mr 260,000 form with trypsin led to its conversion to Mr 225,000 and a Mr 35,000 form. A minor cytosolic form of Mr 200,000 was also present. This minor activity was latent until activated by trypsin treatment and was converted to a Mr 35,000 form by such treatment. The third form was found to chromatograph as a form of molecular weight greater than 500,000 on gel filtration and, like the Mr 200,000 form, was only detected after activation with trypsin. Subsequent to this treatment, it too behaved as a Mr 35,000 enzyme. Although a single major enzyme form was present in the cytosol, multiple molecular weight forms could be generated in crude extracts simply by the use of vigorous mechanical homogenization procedures. This suggested that artifactual forms of enzyme may readily be produced, possibly by proteolytic cleavage of the native enzyme. 相似文献
20.
Phaseolin, the major seed storage protein of Phaseolus vulgaris L., is degraded in the cotyledons in the first 7–10 d following seed germination. We assayed cotyledon extracts for protease activity by using [3H]phaseolin as a substrate and then fractionated the digestion mixtures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in order to identify the cleavage products. The cotyledons of 4-d-old seedlings contain an endopeptidase which cleaves the polypeptides of [3H]phaseolin (apparent molecular weights=51 000, 48 000, 46 000 and 43 000) into three discrete clusters of proteolytic fragments (M
rs=27 000, 25 000 and 23 000). Endopeptidase activity is not detected in the cotyledons until the protein content of these organs starts to decline, shortly after the first day of seedling growth. Endopeptidase activity increases to a maximum level in the cotyledons of 5-d-old seedlings and then declines to a minimum value by day 10. The enzyme was purified 335-fold by ammonium-sulfate precipitation, organomercurial-agarose chromatography, gel filtration and ion-exchange chromatography. The endopeptidase constitutes 0.3% of the protein content in the cotyledons of 4-d-old seedlings. It is a cysteine protease with a single polypeptide chain (M
r=30 000). Optimum hydrolysis of [3H]phaseolin occurs at pH 5. The enzyme is irreversibly inactivated at pH values above 7 and at temperatures above 45° C. The endopeptidase attacks only a limited number of peptide bonds in [3H]phaseolin, without causing any appreciable change in the native molecular weight of the storage protein. The endopeptidase is also able to hydrolyze the bean-seed lectin, phytohemagglutinin. Thus, this enzyme may play a general role in degrading cotyledon proteins of P. vulgaris following seed germination.Abbreviations Da
dalton
- DTT
dithiothreitol
-
M
r
apparent molecular weight
- PAGE
polyacrylamide gel electrophoresis
- PHA
phytohemagglutinin
- SDS
sodium dodecyl sulfate 相似文献