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1.
A O Adekunle C E Arboleda P H Zervos G L Gerton C Teuscher 《Biology of reproduction》1987,37(1):201-210
Guinea pig (GP) acrosin was purified following acid extraction of testicular acetone powder, pH precipitation of the soluble extract, gel filtration on Sephadex G-100, ion-exchange chromatography on SP-Sephadex, and affinity chromatography on Concanavalin A-Sepharose. Final purification was achieved by re-chromatography on Sephadex G-100. Enzymatic activity was detected by following the hydrolysis of N-benzyloxycarbonylarginyl amide of 7-amino-4-trifluoromethylcoumarin at 37 degrees C, pH 8.0, before and after activation. GP testicular acrosin exhibited a molecular weight of 48,000 by gel filtration and 34,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following SDS-PAGE in gels containing 0.1% gelatin, protease activity was observed to comigrate with the major protein detected by silver staining. The purified GP acrosin showed cross-reactivity with a monospecific polyclonal rabbit antiserum directed against boar sperm acrosin and exhibited reversible pH-dependent activation. The physiochemical characteristics of the purified protein, including the amino acid composition, resemble those reported for acrosins from other species. 相似文献
2.
Chymopapain (EC 3.4.22.6) possesses an essential and a non-essential thiol group, but enzyme preparations produced hitherto always contained less than two thiol groups. Starting from commercial chymopapain or from papaya latex, we have prepared pure enzyme having two thiol groups, which is demanded by mechanistic investigations. The purification was performed by covalent chromatography on activated thiol-Sepharose column, which specifically binds thiol-containing proteins. Elution was effcted with cysteine in a stepwise manner, first at pH 5 then at pH 8. The pure enzyme had a molecular mass of24 700 as estimated by sodium dodecyl sulfate polyacrylamide gel-electrophoresis. Titration of the pure enzyme with 2,2'-dipyridyl disulfide at pH 9 exhibited a biphasic curve. This indicated that under the conditions employed the nonessential thiol group is more reactive than the essential one, which is a characteristic feature of chymopapain B. On the other hand, the magnitude of the rate constant of the same reaction at pH 4, as well as its pH-dependence, was characteristic of chymopapain A. The enzyme with the hybrid kinetic properties was denoted as chymopapain S. We conclude from the above findings that various forms of chymopapain can be classified by their reaction with 2,2'-dipyridyl disulfide. 相似文献
3.
Serine proteinase inhibitors in the Compositae: distribution, polymorphism and properties 总被引:2,自引:0,他引:2
Konarev AV Anisimova IN Gavrilova VA Vachrusheva TE Konechnaya GY Lewis M Shewry PR 《Phytochemistry》2002,59(3):279-291
Multiple molecular forms of inhibitors of trypsin (TI) and chymotrypsin (CI), which are typical digestive enzymes of insects, mammals and micro-organisms, and subtilisin (SI), a proteinase of many bacteria and phytopathogenic fungi, were identified in seeds and vegetative organs of the majority of 128 wild and cultivated species representing 65 genera of three of the subfamilies of the Compositae. Inhibitors with M(r) ranging from 7450 to 7800 and combining activities towards subtilisin and trypsin and/or chymotrypsin (T/C/SI) had the widest distribution and may be involved in plant defense mechanisms. They were found in many species of the subfamilies Carduoideae (genera Carthamus, Centaurea, Cirsium), Cichorioideae (Lactuca, Taraxacum) and Asteroideae (Helianthus, Cosmos, Bidens). Partial amino acid sequencing showed that the safflower (Carthamus tinctorius) T/C/SI and Cosmos bipinnatus T/C/SI, T/SI and C/SI belonged to the potato I inhibitor family. The most active, variable and heterogeneous inhibitors were found in species of the tribe Heliantheae, which is placed in the evolutionary advanced subfamily Asteroideae. Seeds of Helianthus species, Eclipta prostrata, Gailardia aristata, Zinnia elegans and Silphium perfoliatum contained various TI with M(r) ranging from 1500 to 14,750, with some also containing SI. H. annuus seeds contain a unique cyclic TI of M(r) 1514 and similar TI were also present in other Helianthus spp. and the related species Tithonia diversifolia. Zinnia elegans contained a TI with M(r) 11,350 which appeared to represent a novel type of inhibitor distantly related to the cereal subgroup of Bowman-Birk inhibitors. TI and T/SI varied widely in H. annuus lines and wild Helianthus species in their presence or absence and composition. Similar T/SI components were found in the cultivated diploid H. annuus and annual diploid species with the B genome but not in perennials with the A genome. Some T/SI, SI and TI were detected in vegetative organs of sunflower and other Compositae. Studies of the polymorphism and distribution of proteinase inhibitors are relevant to the evolution of protective protein systems and the mechanisms of resistance to pathogenic organisms in the Compositae and other plants. 相似文献
4.
Two trypsin inhibitors (TI-1, TI-2) were isolated from guinea pig plasma and purified to homogeneity. In amino-acid composition as well as molecular masses, TI-1 (Mr 58,000) and TI-2 (Mr 57,000) are similar to each other and to human and mouse alpha 1-proteinase inhibitors, and mouse con-trapsin. The two inhibitors form equimolar complexes with proteinases. The effectiveness of the inhibitors was characterized by association rate constants under second-order rate conditions. The inhibitory action of TI-1 was rapid for bovine trypsin, porcine pancreatic elastase and guinea pig plasma kallikrein, but slow for bovine thrombin and guinea pig plasmin and not detectable for bovine chymotrypsin and porcine pancreatic kallikrein. The inhibitory action of TI-2 was rapid for trypsin and chymotrypsin, but slow for guinea pig plasma kallikrein and not detectable for other proteinases. These results show that TI-1 and TI-2 are physicochemically similar but functionally distinct from each other and from human alpha 1-proteinase inhibitor that inhibits trypsin, chymotrypsin and elastase. 相似文献
5.
Legume proteinase inhibitors (PI/s) abrogate proteolytic activity of proteins and cause adverse effects on growth. In this study, two legume PIs – Archidendron ellipticum Trypsin Inhibitor (AeTI) and Derris trifoliata Trypsin Chymotrypsin Inhibitor (DtTCI) were purified and used along with standard, Soybean Trypsin Inhibitor (STI), either singly or in combination, as antiplasmodial agents against two Plasmodium forms (Pf 3D7/Pf FCR3). Both AeTI/DtTCI were purified to homogeneity by HPLC and showed over 98% trypsin/chymotrypsin inhibition, respectively. DtTCI severely arrested growth of both the Plasmodium forms (IC50 of 9.59 μM and 16.86 μM, respectively). In addition, combination of DtTCI/AeTI had synergistic effect against both Pf 3D7 (FIC – 0.19) and Pf FCR3 (FIC − 0.23). Combinations of DtTCI/STI and AeTI/STI showed additive effects for the parasite forms. Time-course studies indicated DtTCI and combination of DtTCI/AeTI, to be potent inhibitor of schizont rupture. Antiproteolytic action of the PIs on Pf Apical Merozoite Antigen 1 (AMA1) protein revealed that none of the inhibitors (singly/combinations) affected the primary proteolysis of PfAMA1 protein. Notably however, the normal secondary proteolysis of PfAMA1 was abrogated by both DtTCI (singly/combinations) and AeTI. Incidentally, the proteolytic processing of PfAMA1 remained unaffected following STI treatment. 相似文献
6.
D. Gordon Brown 《Oecologia》1988,76(3):467-470
Summary The cost of producing inducible proteinase inhibitors was investigated by manipulating their production with chitin injections in genetically homogeneous tomato plants grown at different nitrogen levels. Proteinase inhibitor production had no detectable effect on fitness-related characters, probably because it represented only a small portion of the nitrogen budget. Nitrogen availability did not influence inhibitor production or its impact on plant fitness. 相似文献
7.
Helicoverpa armigera, a highly polyphagous pest, has a broad host spectrum, causes significant levels of yield loss in many agriculturally important
crops. Serine primarily responsible for most of the proteolytic activity in the larval gut of lepidopteron insects. Neonate
larvae were reared on artificial diet and chickpea seeds smeared with Subabul Trypsin Inhibitor. Larvae fed with artificial
diet showed reduction in larval weight up to 21% (HSTI) and 43% (LSTI). However, larvae fed on seeds showed significant reduction
in weight, 52.4% (HSTI) and 60.3% (LSTI), suggesting that the diet also plays a vital role on the effectiveness of the inhibitors
on larval growth and development. HSTI and LSTI inhibited the gut proteinases from larvae fed on artificial diet significantly
(41.40% and 64.36%) compared to the gut proteinases (27.80% and 38.90%) from larvae fed on chickpea seeds. Seeds smeared with
10,000 TIU resulted in complete mortality of larvae while there was no mortality observed in artificial diet. The results
reveal that LSTI is a stronger inhibitor of insect gut proteinases and for larvae fed with poor nutrition in the natural ecosystems,
low level expression of inhibitor would be enough to affect the growth and development.
Handling editor: Chen-Zhu Wang 相似文献
8.
Paolo Rovero Stefano Pegoraro Stefania Viganò Cristina Amato Lucia Vaccari Ettore Balestreri Romano Felicioli 《Letters in Peptide Science》1995,2(1):27-32
Summary The synthesis and biological activity of a photochromic compound, in which two Lys2 dipeptides are linked through their N-terminal amino groups by an azobenzene moiety, are reported. The synthesis was achieved by a novel solid-phase dimerization strategy, based on the reaction of 4,4-azobenzene dibenzoyl chloride with two N-terminal amino groups of distinct Lys2 dipeptides directly on the resin. Both the trans form and the photostationary state mixture (59% cis and 41% trans) inhibit a plant proteinase which is known to be sensitive to polycationic compounds. 相似文献
9.
10.
Morphine 6-dehydrogenase, which catalyzes the dehydrogenation of morphine to morphinone, has been purified about 440-fold from the soluble fraction of guinea pig liver with a yield of 38%. The purified enzyme was a homogeneous protein on polyacrylamide gel disc electrophoresis and isoelectric focusing. The molecular weight and isoelectric point of the enzyme were 29,000 and 7.6, respectively. The enzyme utilizes both NAD and NADP as a cofactor, and the Km values were 0.12 mM for NAD and 0.42 mM for NADP. The Vmax values for morphine were 588 milliunits/mg of protein (with NAD) and 1600 milliunits/mg of protein (with NADP). The Km values for morphine were 0.12 mM (with NAD) and 0.49 mM (with NADP). The enzyme also exhibited activity for morphine-related compounds: nalorphine, normorphine, codeine, and ethylmorphine; however, 7,8-saturated congeners such as dihydromorphine and dihydrocodeine were poor substrates. The enzyme was inactivated by removal of 2-mercaptoethanol from the enzyme solution. The inactivated enzyme was rapidly recovered by the addition of 2-mercaptoethanol. Phenylarsine oxide and CdCl2 (dithiol modifiers) inhibited competitively toward cofactor binding and noncompetitively toward morphine binding. These results suggest that the enzyme possesses the essential thiol groups, probably vicinal dithiol, at or near the cofactor-binding site. Using the partially purified enzyme, 8-(2-hydroxyethylthio)dihydromorphinone was isolated as the product and identified by UV, mass, and NMR spectra. It was confirmed that morphinone proposed as the dehydrogenation product was nonenzymatically and covalently bound to 2-mercaptoethanol. Accordingly, the isolated morphinone-2-mercaptoethanol conjugate must be formed by two steps: enzymatic production of morphinone from morphine and then nonenzymatic binding of 2-mercaptoethanol to morphinone. 相似文献
11.
Four types of glutathione S-transferase were purified to homogeneity from guinea pig liver by DEAE-cellulose, Sephadex G-75, CM-cellulose, and affinity chromatography. These isozymes were named a, b, c, and d based on the reverse order of elution from a CM-cellulose column, and had specific activities of 89.6, 92.2, 99.0, and 44.0 units/mg, respectively, when assayed with 1 mM each of 1-chloro-2,4-dinitrobenzene and reduced glutathione. All four transferases of guinea pig liver were homodimers. The transferases b, c, and d had a similar molecular weight of 50,000 and their subunit sizes were 25,000, but the corresponding values for transferase a were 45,000 and 23,500, respectively. Transferase a was notably different in the activities towards organic hydroperoxides and 1,2-dichloro-4-nitrobenzene from the other isozymes. Transferases a and b, the major forms in guinea pig liver, were studied with respect to their biochemical properties, including kinetic parameters, absorption and fluorescence spectra, and bilirubin binding. Glutathione peroxidase activity of the transferase a was about 100 times higher than that of other isozymes. In guinea pig liver, it is estimated that transferase a is the major glutathione peroxidase, accounting for about 75% of the total organic hydroperoxide reduction. 相似文献
12.
Hiroaki Kataoka Kazuki Nabeshima Naoto Komada Masashi Koono 《Virchows Archiv. B, Cell pathology including molecular pathology》1989,57(1):157-165
Two new human cell lines, RCM-1 and CoCM-1, have been established from primary colorectal adenocarcinomas. Both cell lines
were unique in that the cultures secreted trypsin inhibitors in vitro. The activities of these inhibitors were accumulated
in serum-free media of both cell lines over a period of several days. Two inhibitors (PI-1 and PI-2) were isolated from serum-free
conditioned medium in which RCM-1 was grown by anion-exchange and gel filtration high-performance liquid chromatography. PI-1
inhibited trypsin and chymotrypsin strongly, and pancreatic elastase weakly. Its molecular weight was about 57 kilodaltons
(Kd) as determined by gel filtration chromatography. It cross-reacted with the antiserum elicited against human α1-antitrypsin in double immunodiffusion. PI-1 corresponding to α1
- antitrypsin was also demonstrated immunohistochemically in both cell lines. PI-2 inhibited trypsin strongly, and chymotrypsin,
kallikrein and plasmin weakly. It had higher molecular weight (200–300 Kd) than that of PI-1, and did not crossreact with
antisera against human α1-antitrypsin, α2-macroglobulin, α1-antichymotrypsin, α2-plasmin inhibitor, inter-α-trypsin inhibitor and urinary trypsin inhibitor. RCM-1 and CoCM-1 are the first colorectal adenocarcinoma
cell lines that secrete functionally active trypsin inhibitors, including α1-antitrypsin in vitro, and are useful for the study of tumor-cell derived proteinase inhibitors. 相似文献
13.
Kredics L Terecskei K Antal Z Szekeres A Hatvani L Manczinger L Vágvölgyi C 《Acta biologica Hungarica》2008,59(2):259-268
Eleven cold-tolerant Trichoderma isolates were screened for the production of proteolytic activities at 10 degrees C. Based on the activity profiles determined with paranitroanilide substrates at 5 degrees C, strain T221 identified as Trichoderma atroviride was selected for further investigations. The culture broth of the strain grown at 10 degrees C in casein-containing culture medium was concentrated by lyophilization and subjected to gel filtration, which was followed by chromatofocusing of the fraction showing the highest activity on N-benzoyl-Phe-Val-Arg-paranitroanilide. The purified enzyme had a molecular weight of 24 kDa, an isoelectric point of 7.3 and a pH optimum of 6.2. The temperature optimum of 25 degrees C and the low thermal stability suggested that it is a true cold-adapted enzyme. Substrate specificity data indicate that the enzyme is a proteinase with a preference for Arg or Lys at the P1 position. The effect of proteinase inhibitors suggests that the enzyme has a binding pocket similar to the one present in trypsin. 相似文献
14.
Al. V. Konarev I. N. Anisimova V. A. Gavrilova V. T. Rozhkova R. Fido A. S. Tatham P. R. Shewry 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,100(1):82-88
A highly sensitive gelatin overlay procedure was used to identify inhibitors of serine proteinases and of the cysteine proteinase
ficin in seeds and leaves of sunflower. One major and two minor groups of trypsin inhibitors were identified in seeds, the
former having a high pI (@10) and also inhibiting chymotrypsin. Three groups of trypsin/subtilisin inhibitors were also present
in seeds, together with three inhibitors of ficin. All groups showed polymorphism between lines of Helianthus annuus, while the trypsin and trypsin/subtilisin inhibitors also varied between wild species of Helianthus, with no apparent relationship to growth type (annual or perennial), genome constitution or ploidy level. Genetic analysis
showed that the major trypsin inhibitor and three groups of trypsin/subtilisin inhibitors are each controlled by single Mendelian
loci, with the three loci for trypsin/subtilisin inhibitors showing recombination values of 0.23–0.40. Purification by RP-HPLC
allowed the M
r
of two trypsin inhibitors to be determined by SDS-PAGE to be about 1,500 and 2,500, while the three trypsin/subtilisin inhibitors
varied in M
r
from about 1,500 to 6,000.
Received: 7 March 1999 / Accepted: 18 March 1999 相似文献
15.
A rapid purification procedure for large scale preparations of yeast proteinase B inhibitors 1 and 2 (IB1 and IB2) is described. By disc gel electrophoresis, amino acid analysis, and end-group determinations, each of the inhibitors is homogeneous. Both inhibitors are polypeptides with molecular weights of 8,500, containing 74 residues. No components other than amino acids could be detected. There is no significant difference in the amino acid compositions of the two inhibitors as analyzed after acid hydrolysis. Both polypeptides are characterized by the total absence of arginine, tryptophan, and sulfur-containing amino acid residues. The proteinase B inhibitors of yeast, therefore, differ fundamentally from proteinase inhibitors of many other organisms, which generally contain a large number of disulfide bridges. Both proteinase B inhibitors have threonine as the NH2-terminal residue and -Val-His-Thr-Asn-COO- as the COOH-terminal sequence. Comparison of peptide maps after tryptic digestion reveals that the two inhibitors differ definitely in only a few tryptic peptides. The inhibitors are rapidly inactivated by digestion with carboxypeptidase A from bovine pancreas at pH 8.5. Inactivation occurs stoichiometrically with the release of threonine, the penultimate residue at the COOH-terminal end of both inhibitors. 相似文献
16.
An endogenous inhibitor of calcium activated neutral proteinase has been purified from human placenta. The procedure included
chromatography on DEAE cellulose, Ultrogel AcA 22 and milli calcium activated neutral proteinase-sepharose in succession.
Endogenous calcium activated neutral proteinase inhibitor was a tetramer with identical subunits of molecular weight 68 kDa.
It was specific for milli calcium activated neutral proteinase (Calpain II) which is inhibited by the formation of an inactive
enzyme-inhibitor complex and not by sequestering Ca2+ from the medium. Although micro calcium activated neutral proteinase (Calpain I) was not inhibited by endogenous calcium
activated neutral proteinase inhibitor, it was protected from autolysis in the presence of the inhibitor. The placental endogenous
calcium activated neutral proteinase inhibitor thus regulates Ca2+ activated proteolysis by ensuring micro calcium activated neutral proteinase activity, while inhibiting milli calcium activated
neutral proteinase. 相似文献
17.
Isolation and characterization of two proteinase inhibitors from the male reproductive tract of mice
Low molecular weight, acid-stable proteinase inhibitors from epididymal and seminal vesicle homogenates were isolated and characterized. The isolation procedure consisted of gel filtration, trypsin affinity, and ion exchange chromatography. The inhibitor from seminal vesicle homogenates has a molecular weight of approximately 6,200, and that of the epididymal inhibitor was estimated at 4,000. Antiserum directed against the seminal vesicle inhibitor did not react with epididymal components. The epididymal inhibitor shows competitive, whereas the seminal vesicle inhibitor shows noncompetitive inhibition against trypsin on double reciprocal plots. Both inhibitors are effective against trypsin and acrosin but not against chymotrypsin, kallikrein, thrombin, or plasmin. To verify site of origin and to investigate androgen dependency of the epididymal inhibitor, mice were efferentiectomized, orchiectomized, or orchiectomized with androgen supplementation. Gel filtration profiles of acid-treated epididymal homogenates from normal and efferentiectomized animals show inhibitor peaks in the same regions. The concentration of acid-stable inhibitor from epididymal homogenates decreased with orchiectomy but returned to normal values when exogenous androgen was supplied. These observations suggest that the low molecular weight inhibitor in the epididymal homogenates is distinct from that in the seminal vesicles. Furthermore, the inhibitor associated with epididymal homogenates is androgen-dependent, and the epididymis is the site of origin of this inhibitor. 相似文献
18.
Benjakul S Visessanguan W Leelapongwattana K 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2003,134(4):579-591
Heat-stable alkaline proteinase was purified from bigeye snapper (Priacanthus macracanthus) ordinary muscle by heat-treatment and a series of chromatographies including Phenyl-Sepharose 6 Fast Flow, Source 15Q and Superose 12 HR 10/30. It was purified to 5180-fold with a yield of 0.8%. The molecular weight of purified proteinase was estimated to be 72 kDa by gel filtration. The proteinase appeared as two proteinase activity bands with molecular weights of 66 and 13.7 kDa on non-reducing SDS-substrate gel. Accordingly, it was found to consist of two different subunits. The optimum pH and temperature for casein hydrolysis were 8.5 and 60 °C, respectively. The proteolytic activity was strongly inhibited by soybean trypsin inhibitor and partially inhibited by ethylenediaminetetraacetic acid, while pepstatin A and E-64 showed no inhibition. Purified proteinase was able to hydrolyze Boc-Phe-Ser-Arg-MCA, but rarely hydrolyzed Z-Phe-Arg-MCA and Z-Arg-Arg-MCA. In addition, it mainly degraded myosin heavy chain, not actin. These results suggest that purified proteinase was serine proteinase, which is probably involved in gel weakening of bigeye snapper surimi. 相似文献
19.
Mariko Shimamura Tadahiko Hazato Takashi Katayama 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,798(1):8-13
An enkaphalin-degrading aminopeptidase using Leu-enkephalin as a substrate was purified about 4100-fold from guinea pig serum. The purified preparation was apparently homogenous, showing on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was approx. 92 000. The amino-peptidase had a pH optimum of 7.0 with Km values of 0.12 mM and 0.18 mM for Leu- and Met-enkephalin, respectively. The enzyme hydrolyzed neutral, basic and aromatic amino acid β-naphthylamides, but did not the acidic one. The enzyme was inhibited strongly by metal-chelating agents, bestatin and amastatin and weakly by puromycin. Among several biologically active peptides, angiotensin III and substance P strongly inhibited the enzyme. 相似文献
20.
Purification and partial characterization of adherence-inhibiting factor (AIF) of platelet-granule fraction in guinea pig were studied. When freshly prepared platelet-granule fraction was subjected to a gel filtration, two neutrophil adherence-inhibiting peaks, designated AIF-I (2,800 kDa) and AIF-II (12 kDa), appeared. AIF-I was sensitive to diisopropylfluorophosphate (DFP) and originated from lysosomes, whereas AIF-II was insensitive to DEP and localized in alpha-granules. Both AIFs were released from platelets by a thrombin stimulation. As the total activity of AIF-I was about 5-fold higher than that of AIF-II, AIF-I was purified and characterized. When purified AIF-I was analyzed on SDS-polyacrylamide gel electrophoresis, the 340 kDa protein band and the other large protein band were observed. Under reducing condition, AIF-I was separated into three components (340, 190 and 165 kDa). AIF-I significantly inhibited neutrophil adherence to artificial substrata and to type IV collagen-coated plastic surface, but not to fibronectin- or plasma-coated plastic surfaces, suggesting that AIF-I inhibits neutrophil adherence not only via nonspecific adsorption sites but also via type IV collagen receptors. 相似文献