共查询到20条相似文献,搜索用时 31 毫秒
1.
S H Hixson J L Hurwitz K J Langridge D C Nichols K M Provost A M Wolff 《Biochemical and biophysical research communications》1983,111(2):630-635
Two compounds have been designed to serve as photoaffinity reagents for use with carboxyl proteases. 1,2-Epoxy-3-(4'-azido-2'-nitrophenoxy)propane has been synthesized and shown to react with porcine pepsin in the same fashion as the traditional inhibitor 1,2-epoxy-3-(p-nitrophenoxy)propane, while p-azidophenacyl bromide is similar to other phenacyl bromides in its reaction with pepsin. In combination with p-azido-alpha-diazoacetophenone, previously shown to resemble alpha-diazo carbonyl reagents in its reaction with pepsin, photoaffinity analogs are now available for all three of the widely-used carboxyl protease inhibitors. 相似文献
2.
Tuna pepsinogens and pepsins. Purification, characterization and amino-terminal sequences 总被引:4,自引:0,他引:4
Three pepsinogens (pepsinogens 1, 2, and 3) were purified from the gastric mucosa of the North Pacific bluefin tuna (Thunnus thynuus orientalis). Their molecular masses were determined to be 40.4 kDa, 37.8 kDa, and 40.1 kDa, respectively, by SDS/polyacrylamide gel electrophoresis. They contained relatively large numbers of basic residues when compared with mammalian pepsinogens. Upon activation at pH 2.0, pepsinogens 1 and 2 were converted to the corresponding pepsins, in a stepwise manner through intermediate forms, whereas pepsinogen 3 was converted to pepsin 3 directly. The optimal pH of each pepsin for hemoglobin digestion was around 2.5. N-acetyl-L-phenylalanyl-L-diiodotyrosine was scarcely hydrolyzed be each pepsin. Pepstatin, diazoacetyl-DL-norleucine methyl ester in the presence of Cu2+, 1,2-epoxy-3-(p-nitrophenoxy)propane and p-bromophenacyl bromide inhibited each pepsin, although the extent of inhibition by each reagent differed significantly among the three pepsins. The amino acid sequences of the activation segments of these pepsinogens were determined together with the sequences of the NH2-terminal regions of pepsins. Similarities in the activation segment region among the three tuna pepsinogens were rather low, ranging over 28-56%. A phylogenetic tree for 16 aspartic proteinase zymogens including the three tuna pepsinogens was constructed based on the amino acid sequences of their activation segments. The tree indicates that each tuna pepsinogen diverged from a common ancestor of pepsinogens A and C and prochymosin in the early period of pepsinogen evolution. 相似文献
3.
I Pichová V Kostka 《Comparative biochemistry and physiology. B, Comparative biochemistry》1990,97(1):89-94
1. Two procedures were developed for the preparation of duck pepsinogen, an enzyme from the family of aspartic proteases (EC 3.4.23.1) and its zymogen. 2. The amino acid composition, sugar content and the partial N- and C-terminal sequences of both the enzyme and the zymogen were determined. These sequences are highly homologous with the terminal sequences of chicken pepsin(ogen). 3. Duck pepsinogen and pepsin are unlike other pepsin(ogen)s in being relatively stable in alkaline media: pepsinogen is inactivated at pH 12.1, pepsin at pH 9.6. 4. Duck pepsin is inhibited by diazoacetyl-D,L-norleucine methyl ester (DAN), 1,2-epoxy-3(p-nitrophe-noxy)propane (EPNP), pepstatin and a synthetic pepsin inhibitor Val-D-Leu-Pro-Phe-Phe-Val-D- Leu. The pH-optimum of duck pepsin determined in the presence of synthetic substrate is pH 4. 5. Duck pepsin has a marked milk-clotting activity whereas its proteolytic activity is lower than that of chicken pepsin. 6. The activation of duck pepsinogen is paralleled by two conformational changes. The activation half-life determined in the presence of a synthetic substrate at pH 2 and 14 degrees C is 20 sec. 相似文献
4.
Comparative studies have been made on the effects of diazoacetyl-DL-norleucine methyl ester (DAN), 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and pepstatin on acid proteases, including those from Acrocylindrium sp., Aspergillus niger, Aspergillus saitoi, Mucor pusillus, Paecilomyces varioti, Rhizopus chinensis, and Trametes sanguinea, and also porcine pepsin [EC 3.4.23.1] and calf rennin [EC 3.4.23.4] for comparative purposes. These enzymes were rapidly inactivated at similar rates and in 1:1 stiochiometry by reaction with DAN in the presence of cupric ions. The pH profiles of inactivation of these enzymes were similar and had optima at pH 5.5 to 6. They were also inactivated at similar rates by reaction with EPNP, with concomitant incorporation of nearly 2 EPNP molecules per molecule of enzyme. The pH profiles of inactivation were again similar and maximal inactivation was observed at around pH 3 to 4. Some of the EPNP-inactivated enzymes were treated with DAN and shown still to retain reactivity toward DAN. All these enzymes were inhibited strongly by pepstatin, and the reactions of DAN and EPNP with them were also markedly inhibited by prior treatment with pepstatin. These results indicate that the active sites of these enzymes are quite similar and that they presumably have at least two essential carboxyl groups at the active site in common, one reactive with DAN in the presence of cupric ions and the other reactive with EPNP, as has already been demonstrated for porcine pepsin and calf rennin. Pepstatin appears to bind at least part of the active site of each enzyme in a simmilar manner. 相似文献
5.
D Tsuru S Shimada S Maruta T Yoshimoto K Oda S Murao T Miyata S Iwanaga 《Journal of biochemistry》1986,99(5):1537-1539
Scytalidium lignicolum acid protease B, a pepstatin-insensitive acid protease, was modified by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) with the concomitant loss of its enzyme activity, and an EPNP-labeled peptide was isolated from the thermolysin-digest of the modified enzyme by HPLC. The amino acid sequence of the peptide was determined to be Ile-Leu-Glu-Thr-Gly, which corresponds to the sequence of residue Nos. 51-55 of the enzyme. The results of treatment of the labeled peptide with hydroxylamine suggested that the EPNP moiety is ester-linked to Glu53 of the enzyme. The amino acid sequence around Glu53 of the acid protease B showed high homology with those around the active site Asp residues of calf chymosin and porcine pepsin. These results show that it is highly possible that Glu53 of the acid protease B is one of the amino acid residues involved in its catalytic activity. 相似文献
6.
Two different peptides containing an aspartyl residue reactive with 1, 2-epoxy-3-(p-nitrophenoxy)propane (EPNP) in the acid protease from Rhizopus chinensis were isolated from a peptic digest of the EPNP-modified enzyme. One of the peptides was sequenced as Asp-Thr-Gly-Ser-Asp. The amino acid sequence had very high homology with those around the EPNP-reactive aspartyl residues in rennin (chymosin) [EC 3.4.23.4] and pepsin [EC 3.4.23.1]. The other peptide contained no methionine residue and gave the sequence: Asp-Thr-Gly-Thr-Thr-Leu. The N-terminal aspartyl residue of each peptide was deduced to be the EPNP-reactive site. 相似文献
7.
Extracellular acid and alkaline proteases from Candida olea 总被引:3,自引:0,他引:3
Candida olea 148 secreted a single acid protease when cultured at acidic pH. In unbuffered medium, the culture pH eventually became alkaline and a single alkaline protease was produced. This was the only proteolytic enzyme produced when the organism was grown in buffered medium at alkaline pH. Both proteolytic enzymes were purified to homogeneity (as assessed by SDS-PAGE). The Mr of the acid protease was 30900, the isoelectric point 4.5; optimum activity against haemoglobin was at 42 degrees C and pH 3.3. This enzyme was inactivated at temperatures above 46 degrees C and was inhibited by either pepstatin and diazoacetyl-norleucine methyl ester but was insensitive to inhibition by either 1,2-epoxy-3-(p-nitrophenoxy)-propane or compounds known to inhibit serine, thiol or metallo proteases. The acid protease contained 11% carbohydrate. The alkaline protease had an Mr of 23400 and isoelectric point of 5.4. The activity of this enzyme using azocoll as substrate above 42 degrees C and was inhibited by phenylmethyl-sulphonyl fluoride and irreversible inactivated by EDTA. The enzyme was also partially inhibited by DTT but was insensitive to either pepstatin or p-chloromercuribenzoic acid. 相似文献
8.
A procedure to purify to homogeneity the active form as well as the proenzyme form of the acidic protease of human seminal plasma is described. This involved precipitation with ammonium sulfate, chromatography on diethylaminoethylcellulose, Sephadex G-200, and Sephadex G-100. The molecular weights of the active form and of the proenzyme were determined by electrophoresis and gel filtration to be 35,000 and 42,000, respectively. The proenzyme was more stable than the active form in alkaline solution and can be converted into the active enzyme under acidic conditions. The active form of the acidic protease can hydrolyze hemoglobin, N,N'-dimethylcasein, N-acetyl-L-phenylalanyl-L-diiodotyrosine, and N-benzyloxycarbonyl-L-glutamyl-L-phenylalanine, but cannot hydrolyze bovine serum albumin, ovalbumin, N-benzyloxycarbonyl-L-glutamyl-L-tyrosine. The active form was also inhibited by p-bromophenacyl bromide and 1,2-epoxy-3-(p-nitrophenoxy)propane. 相似文献
9.
J Harris B Coles D J Meyer B Ketterer 《Comparative biochemistry and physiology. B, Comparative biochemistry》1991,98(4):511-515
1. The major glutathione S-transferase (GST) from the common squid Loligo vulgaris has been purified and shown to be a homodimer of subunit molecular mass 24,000 and pI 6.8. 2. It has high activity towards 1-chloro-2,4-dinitrobenzene, p-nitrobenzyl chloride, 4-hydroxynon-2-enal and linoleic acid hydroperoxide, low activity with 1,2-dichloro-4-nitrobenzene and no activity with ethacrynic acid, trans-4-phenyl-3-buten-2-one and 1,2-epoxy-3-(p-nitrophenoxy)propane. 3. The L. vulgaris GST did not cross-react with any of the available polyclonal antibodies raised against mammalian GSTs. 4. Forty amino acids of its N-terminal sequence have been determined. 5. Its activities and primary structure are compared with related proteins from other species. 相似文献
10.
Two kinds of cathepsin D were found in Japanese monkey lung and were named cathepsins D-I and D-II. Cathepsin D-I was partially purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography. It had properties common to other ordinary cathepsins D in terms of the elution position from a DEAE-cellulose column at pH 8.0, the pH-dependence of activity toward acid-denatured hemoglobin, and the molecular weight of 35,000 as determined by Sephadex G-100 gel filtration. On the other hand, cathepsin D-II was purified about 1,000-fold by a combination of ammonium sulfate fractionation and column chromatographies on DEAE-cellulose and Sephadex G-100. It was a very acidic protein as judged from its elution position from a DEAE-cellulose column at pH 8.0, and the high mobility toward the anode on disc gel electrophoresis at pH 8.6. Its molecular weight was determined to be 35,000 by Sephadex G-100 gel filtration and 39,000 by SDS-polyacrylamide gel electrophoresis. It was optimally active at pH 2.8 against acid-denatured hemoglobin as a substrate, showing 80% of the optimal activity at pH 1.0, and almost no activity above pH 4.0. This pH-profile of activity was similar to that of monkey pepsin C (gastricsin). It did not hydrolyze N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine, a synthetic substrate for pepsin, but was inhibited by a series of pepsin inhibitors such as pepstatin, 1,2-epoxy-3-(p-nitrophenoxy)propane, p-bromophenacyl bromide, and diazoacetyl-DL-norleucine methyl ester, although the diazo reagent was a rather weak inhibitor of the enzyme. The amino acid composition of cathepsin D-II was found to be fairly different from those of other cathepsins D. However, it showed a striking resemblance to that of Japanese monkey pepsinogen C, suggesting some evolutionary relationship between them. 相似文献
11.
Pepsinogen C and pepsin C from gastric mucosa of Japanese monkey. Purification and characterization.
A new pepsinogen component, pepsinogen C, was purified from the gastric mucosa of Japanese monkey. The chromatographic behavior of this component on DE-32 cellulose was coincident with that of pepsinogen III-2 previously reported (1), and final purification was performed by large-scale polyacrylamide disc gel electrophoresis. The molecular weight was 35,000 as determined by gel filtration. The ratios of glutamic acid to aspartic acid and of leucine to isoleucine were higher than those of other Japanese monkey pepsinogens. The activated form, pepsin C, had a molecular weight of 27,000 and contained a large number of glutamic acid residues. The optimal pH for hemoglobin digestion was 3.0. Pepsin C could scarcely hydrolyze the synthetic substrate, N-acetyl-L-phenylalanyl-3, 5-diiodo-L-tyrosine (APDT). 1, 2-Epoxy-3-(p-nitrophenoxy)propane (EPNP), p-bromophenacyl bromide, and diazoacetyl-DL-norleucine methyl ester (DAN) inhibited pepsin C [EC 3.4.23.3] in the same way as pepsin III-3 of Japanese monkey. The susceptibility to pepstatin of pepsin C was lower than that of pepsin III-3, and 500 times more pepstatin was required for the same inhibitory effect. The classification and nomenclature of Japanese monkey pepsinogens and pepsins are discussed. 相似文献
12.
GSH peroxidase, GSSG reductase, GSH S-transferase, and gamma-glutamyltranspeptidase activities were measured in the supernatant of 13 human early pregnancy placenta homogenates. From measurements of GSH peroxidase activity with both H2O2 and cumene hydroperoxide as second substrate it was deduced that immature placenta contains only the Se-dependent form. All the specimens investigated exhibited GSSG reductase and gamma-glutamyltranspeptidase activities. GSH S-transferase activity was noted only using 1-chloro-2,4-dinitrobenzene as electrophilic substrate, while no detectable activity was found with 1,2-dichloro-4-nitrobenzene, 1,2-epoxy-3-(p-nitrophenoxy) propane, and p-nitrobenzylchloride. It is concluded that human placenta is equipped, from early pregnancy, with the enzymatic systems which are involved in GSH-mediated cellular detoxication and in preserving the integrity of the sulfhydryl status of the cells. 相似文献
13.
The structure of acid protease from Endothia parasitica in strongly cross-linked form is compared with that of the untreated protein at 2.45-a resolution. The only observed conformation change introduced by the cross-linking reaction is at the N terminal. Otherwise the two main chain structures are essentially identical. Approximately 2 molecules of the inhibitor, 1,2-epoxy-3-(p-nitrophenoxy)propane, are found to be incorporated into each protein molecule. They are covalently bound to the two aspartic residues at the active center. 相似文献
14.
Five pepsinogens were purified to homogeneity from the gastric mucosa of Asiatic black bear and termed pepsinogens I-1, I-2, II-1, II-2, and III. Pepsinogen II-1 was the major component and accounted for more than half of the total pepsinogens. Their molecular weights were estimated to be 40,000 for pepsinogens I-1 and I-2, 38,000 for pepsinogens II-1 and II-2, and 42,000 for pepsinogen III. They resembled each other in amino acid composition, except that pepsinogens I-1 and I-2 contained larger numbers of basic residues than the others. Pepsinogen III was a glycoprotein containing about 3.7% carbohydrate. Each was activated to the corresponding pepsin and their enzymatic characteristics were investigated. The optimal pH against hemoglobin was about 2.2 for pepsin I-1, and about 2.5 for pepsins II-1, II-2, and III. Each pepsin was inhibited by pepstatin as well as porcine pepsin and also by diazoacetyl-DL-norleucine methyl ester, 1,2-epoxy-3-(p-nitrophenoxy)-propane, and p-bromophenacyl bromide. Each pepsin could hydrolyze N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine, but the specific activity was much lower than that of porcine pepsin. Activation peptides corresponding to residues 1-43, 1-25, and 26-43 were isolated from an activation mixture of pepsinogen II-1. The amino acid sequences of these peptides and of the NH2-terminal portions of pepsinogen II-1 and pepsin II-1 were determined, resulting in the complete NH2-terminal 60-residue sequence of pepsinogen II-1. 相似文献
15.
1. The Type B acid protease from Aspergillus niger var. macrosporus was inactivated by reaction with diazoacetyl-DL-norleucine methyl ester (DAN), DL-1-diazo-3-tosylamido-2-heptanone (DTH), and L-1-diazo-3-tosylamido-4-phenyl-2-butanone (DTPB) in the presence of cupric ions. The reaction with DAN took place with 1:1 stoichiometry. The enzyme was also inactivated by reaction with 1, 2-epoxy-3-(p-nitrophenoxy)-propane (EPNP) with concomitant incorporation of approximately two EPNP molecules per molecule of protein. Moreover, these reactions of DAN and of EPNP were markedly inhibited by pepstatin. These results seem to indicate that, as in the case of porcine pepsin [EC 3.4.23.1] and related acid proteases, the enzyme has two essential carboxyl groups at the active site, one reactive with DAN and related diazo reagents in the presence of cupric ions and the other reactive with EPNP, and that pepstatin binds in the vicinity of these residues. 2. The Type A acid protease from the same mold, on the other hand, was found to be markedly less sensitive to these specific inhibitors. Under conditions where the Type B enzyme was completely inactivated by DAN and related diazo reagents, only partial inactivation of this enzyme occurred. The effect of prior mixing of DAN and cupric ions on the pH profile of inactivation was also different from that for the Type B enzyme. Moreover, the Type A enzyme was not inactivated by EPNP. These results thus indicate that the nature of the active site of the Type A enzyme is rather different from that of the Type B enzyme and hence that the Type A enzyme belongs to a different class of acid proteases from the Type B enzyme. 相似文献
16.
F Fukai S Yatomi T Morita S Nishizawa T Nagai T Katayama 《Journal of biochemistry》1989,105(6):968-973
Inhibition of the enzyme activity of glutathione S-transferase (GST) by a physiological concentration of bilirubin was studied using various substrates. When rat liver cytosol was used as an unfractionated GST, its GSH-conjugation activity toward 1-chloro-2,4-dinitrobenzene was decreased to one-half by bilirubin, while the activity toward 1,2-dichloro-4-nitrobenzene, p-nitrobenzyl chloride, or 1,2-epoxy-(p-nitrophenoxy)propane and also the non-selenium dependent GSH-peroxidase activity toward cumene hydroperoxide (CHPx activity) were hardly affected under the same conditions. In contrast, bilirubin inhibited each of the purified GST isozymes and no remarkable difference in bilirubin inhibition was observed with any of the substrates tested. From the chromatographic analysis of the cytosol incubated with [3H]bilirubin, it was found that a major part of the added bilirubin binds to subunit 1 (Ya) of GST isozyme, leaving not only the conjugation activity derived from 3-4 type GST but also the CHPx activity of subunit 2 (Yc) quantitatively intact. The bilirubin inhibition of both the conjugation activity of GST 3-4 and the CHPx activity of GST 2-2 was prevented almost completely by addition of a 3-fold molar excess of GST 1-1. From these results, it was assumed that the enzyme activities of both 3-4 type GSTs and subunit 2 (Yc) were protected from the inhibitory action of bilirubin by the scavenger effect of subunit 1 (Ya). 相似文献
17.
Stimulation of mouse liver glutathione S-transferase activity in propylthiouracil-treated mice in vivo by tri-iodothyronine.
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)
Female C57Bl/6J mice were given drinking water containing 0.05% propylthiouracil to induce a hypothyroid condition. Mitochondrial glycerol-3-phosphate dehydrogenase activity, used as an index of hypothyroidism, was 57.1 +/- 4.5 and 29.4 +/- 3.8 nmol/min per mg of protein for control and propylthiouracil-treated animals respectively. Administration of tri-iodothyronine resulted in an approx. 4.5-fold increase in dehydrogenase activity in propylthiouracil-treated animals. A dose-dependent increase in hepatic GSH S-transferase activity in propylthiouracil-treated animals was observed at tri-iodothyronine concentrations ranging from 2 to 200 micrograms/100 g body wt. This increase in transferase activity was seen only when 1,2-epoxy-3-(p-nitrophenoxy)propane was used as substrate for the transferase. Transferase activity with 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrate was decreased by tri-iodothyronine. Administration of actinomycin D (75 micrograms/100 g body wt.) inhibited the tri-iodothyronine induction of transferase activity. Results of these studies strongly suggest that tri-iodothyronine administration markedly affected the activities of GSH S-transferase by inducing a specific isoenzyme of GSH S-transferase and suppressing other isoenzymic activities. 相似文献
18.
Romsicki Y Scapin G Beaulieu-Audy V Patel S Becker JW Kennedy BP Asante-Appiah E 《The Journal of biological chemistry》2003,278(31):29009-29015
We have characterized the C215D active-site mutant of protein-tyrosine phosphatase-1B (PTP-1B) and solved the crystal structure of the catalytic domain of the apoenzyme to a resolution of 1.6 A. The mutant enzyme displayed maximal catalytic activity at pH approximately 4.5, which is significantly lower than the pH optimum of 6 for wild-type PTP-1B. Although both forms of the enzyme exhibited identical Km values for hydrolysis of p-nitrophenyl phosphate at pH 4.5 and 6, the kcat values of C215D were approximately 70- and approximately 7000-fold lower than those of wild-type PTP-1B, respectively. Arrhenius plots revealed that the mutant and wild-type enzymes displayed activation energies of 61 +/- 1 and 18 +/- 2 kJ/mol, respectively, at their pH optima. Unlike wild-type PTP-1B, C215D-mediated p-nitrophenyl phosphate hydrolysis was inactivated by 1,2-epoxy-3-(p-nitrophenoxy)propane, suggesting a direct involvement of Asp215 in catalysis. Increasing solvent microviscosity with sucrose (up to 40% (w/v)) caused a significant decrease in kcat/Km of the wild-type enzyme, but did not alter the catalytic efficiency of the mutant protein. Structurally, the apoenzyme was identical to wild-type PTP-1B, aside from the flexible WPD loop region, which was in both "open" and "closed" conformations. At physiological pH, the C215D mutant of PTP-1B should be an effective substrate-trapping mutant that can be used to identify cellular substrates of PTP-1B. In addition, because of its insensitivity to oxidation, this mutant may be used for screening fermentation broth and other natural products to identify inhibitors of PTP-1B. 相似文献
19.
Ito H Hirono T Morita Y Nemoto Y Kim YT Takahashi K 《Journal of enzyme inhibition and medicinal chemistry》2008,23(3):352-356
1,2-Epoxy-3-(p-nitrophenoxy)propane (EPNP) is known to inhibit pepsin A and other aspartic proteinases by reacting with the active site aspartic acid residue(s). However, the reaction is considerably slow in general, and therefore, it is desirable to develop similar reagents that are capable of inhibiting these enzymes more rapidly. In the present study, we synthesized a series of novel inhibitors which have a reactive epoxide group linked with peptide by a hydrazide bond, with a general structure: Iva-L-Val-L-Val-(L-AA)(n)-N2H2-ES-OEt (n = 0 approximately 2) (Iva, isovaleryl; AA, bulky hydrophobic or aromatic amino acid residue; ES, epoxysuccinyl). These inhibitors were shown to inhibit porcine pepsin A remarkably faster than EPNP. 相似文献
20.
1. Hepatic cytosolic glutathione S-transferase (GST) activities, toward five substrates, were shown to vary markedly among three laboratory rodent species. 2. Basal GST activities for 1-chloro-2,4-dinitrobenzene (hamster greater than mouse greater than rat), 1,2-dichloro-4-nitrobenzene (mouse greater than rat greater than hamster), p-nitrobenzyl chloride (rat = mouse = hamster), bromosulfophthalein (rat greater than mouse greater than hamster) and 1,2-epoxy-3-(p-nitrophenoxy)propane (mouse greater than rat = hamster) differed with respect to magnitude and distribution among species. 3. GST substrate activities in response to phenobarbital, butylated hydroxy-anisole or 5,5'-diphenylhydantoin treatment were increased more often in mouse and rat as compared to the hamster. 4. These results suggest that basal GST activity, as well as inducibility, differ among rodent species. Since GST are involved in detoxication processes, differences in GST properties may underlie variability in species sensitivity to toxicants. 相似文献