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1.
Purification and characterization of a calcium-activated neutral protease from monkey brain and its action on neuropeptides 总被引:3,自引:0,他引:3
A calcium-activated neutral protease was purified from Japanese monkey brain by ammonium sulfate fractionation and sequential column chromatographies monitored by assay of caseinolytic activity. The purified enzyme gave a single protein band on non-denaturing polyacrylamide gel electrophoresis, and consisted of two subunits with molecular weights of 74,000 and 20,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme required millimolar order calcium ions for activation, and was optimally active at pH 7.5-8.0. Upon incubation with various neuropeptides as substrates, the enzyme preferentially cleaved the peptide bonds with Arg, Lys, or Tyr at the P1 position and an amino acid residue with a bulky aliphatic side chain, such as Leu, Val, or Ile, at the P2 position. The hydrolytic activity toward neuropeptides as well as casein was strongly inhibited by various thiol protease inhibitors. These results suggested that the brain calcium-activated neutral protease may participate in the degradation of neuropeptides in vivo. 相似文献
2.
Using the immunoblot technique, we found that an incubation of purified human myelin in 10 mM Tris-HCl buffer at pH 7.5 resulted in the conversion of the myelinassociated glycoprotein (MAG) to a smaller derivative (dMAG). Exogenously added 5 mM CaCl2 accelerated the conversion of MAG. In buffer containing more than 100 M of EGTA, the conversion was inhibited. In addition, the existence of endogenous calcium in purified myelin was confirmed using atomic absorption spectroscopy. The conversion was also inhibited partially by one of the thiol protease inhibitors, E-64 analogue (E-64-a). These observations suggest that the conversion of MAG is mediated by calcium-activated neutral protease (CANP)-like enzyme. 相似文献
3.
Katsuhiko Yanagisawa Shuzo Sato Nobutada Amaya Tadashi Miyatake 《Neurochemical research》1983,8(10):1285-1293
A calcium-activated neutral protease (CANP) was extracted from human brain and partially purified. The activity was measured using alkali-denatured casein (Hammersten) as a substrate. The optimum pH was around 7.0. The activity required the presence of calcium ions, maximum activity was obtained with over 5 mM calcium ions. TheK
m for the casein concentration was about 1.62 mg/ml. The activity of CANP was inhibited by one of the thiol protease inhibitors, E-64 analogue (E-64-a). The rate of inhibition was about 50% at an E-64-a concentration of 10–5M. This CANP degraded selectively basic protein in myelin proteins and the degradation was inhibited by E-64-a or EGTA. The role of the brain CANP in the process of demyelination was suggested by this study. 相似文献
4.
Anissa Haddar Ali Bougatef Rym Agrebi Alya Sellami-Kamoun Moncef Nasri 《Process Biochemistry》2009,44(1):29-35
An extracellular bleach stable protease producing strain was isolated from marine water sample and identified as Bacillus mojavensis A21 on the basis of the 16S rRNA gene sequencing and biochemical properties. The A21 alkaline protease was purified from the culture supernatant to homogeneity using acetone precipitation, Sephadex G-75 gel filtration and CM-Sepharose ion exchange chromatography, with a 6.43-fold increase in specific activity and 16.56% recovery. The molecular weight of the purified enzyme was estimated to be 20 kDa by SDS-PAGE and gel filtration. The enzyme was highly active over a wide range of pH from 7.0 to 13.0, with an optimum at pH 8.5. The relative activities at pH 11.0 and 12.0 were about 80 and 71.7% of that obtained at pH 8.5. The enzyme was extremely stable in the pH range of 7.0–12.0. It exhibited maximal activity at 60 °C. The thermostability of the enzyme was significantly increased by the addition of CaCl2. The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease.The N-terminal amino acid sequence of the first 20 amino acids of the purified protease was DINGGGATLPQKLYQTSGVL. B. mojavensis A21 protease showed low homology with bacterial peptidases, suggesting that the enzyme is a new protease.The alkaline protease showed high stability towards anionic (0.1% SDS) and non-ionic (1 and 5% Tween 80 and 1% Triton X-100) surfactants. In addition, the enzyme was relatively stable towards oxidizing agents, retaining more than 79 and 70% of its initial activity after 1 h incubation in the presence of 1% H2O2 and 0.1% sodium perborate, respectively. The enzyme showed excellent stability with a wide range of commercial solid and liquid detergents at 30 and 40 °C. Considering its promising properties, B. mojavensis A21 may find potential application in laundry detergents. 相似文献
5.
Antonio Nieto Jesús Avila Manuel Martínez Valdivia 《Molecular and cellular biochemistry》1981,37(3):185-189
Summary The presence of the microtubule-associated protein (MAP2) in the brain of several species has been investigated by SDS-gel electrophoresis and by radioimmunoassay. This assay had
a sensitivity of approx. 10 ng and it was capable of measuring the protein either in purified microtubules or in crude brain
extracts. As determined with this radioimmunoassay, MAP2 accounted for about 10% of the porcine brain microtubule protein and 1% of the protein from a brain extract. Taking porcine
MAP2 as a reference, we have detected polypeptides with the same electrophoretic mobility in brain microtubules from cow, sheep,
rat and chicken. Nevertheless, the MAP2 from these species showed a variable degree of immunoreactivity. Bovine MAP2 appeared closely related to the porcine protein whereas the rat antigen showed low cross-reaction and chicken MAP2 appeared immunologically unrelated to porcine MAP2. Our results suggest a higher variability of the MAP2 sequences as compared to that reported by other authors for the brain microtubule protein, tubulin. 相似文献
6.
Ghorbel-Frikha B Sellami-Kamoun A Fakhfakh N Haddar A Manni L Nasri M 《Journal of industrial microbiology & biotechnology》2005,32(5):186-194
The production and purification of a calcium-dependent protease by Bacillus cereus BG1 were studied. The production of the protease was found to depend specifically on the calcium concentration in the culture medium. This suggests that this metal ion is essential for the induction of protease production and/or stabilisation of the enzyme after synthesis. The calcium requirement is highly specific since other metal ions (such as Mg2+ and Ba2+, which both activate the enzyme) are not able to induce protease production. The most appropriate medium for growth and protease production comprises (g L–1) starch 5, CaCl2 2, yeast extract 2, K2HPO4 0.2 and KH2PO4 0.2. The protease of BG1 strain was purified to homogeneity by ultrafiltration, heat treatment, gel filtration on Sephacryl S-200, ion exchange chromatography on DEAE-cellulose and, finally, a second gel filtration on Sephacryl S-200, with a 39-fold increase in specific activity and 23% recovery. The molecular weight was estimated to be 34 kDa on SDS-PAGE. The optimum temperature and pH of the purified enzyme were determined to be 60°C and 8.0, respectively, in 100 mM Tris-HCl buffer + 2 mM CaCl2. 相似文献
7.
A milk coagulating protease was purified ∼10.2-fold to apparent homogeneity from ginger rhizomes in 34.9% recovery using ammonium sulfate fractionation, together with ion exchange and size exclusion chromatographic techniques. The molecular mass of the purified protease was estimated to be ∼36 kDa by SDS-PAGE, and exhibited a pI of 4.3. It is a glycoprotein with 3% carbohydrate content. The purified enzyme showed maximum activity at pH 5.5 and at a temperature of ∼60 °C. Its protease activity was strongly inhibited by iodoacetamide, E-64, PCMB, Hg2+ and Cu2+. Inhibition studies and N-terminal sequence classified the enzyme as a member of the cysteine proteases. The cleavage capability of the isolated enzyme was higher for αs-casein followed by β- and κ-casein. The purified enzyme differed in molecular mass, pI, carbohydrate content, and N-terminal sequence from previously reported ginger proteases. These results indicate that the purified protease may have potential application as a rennet substitute in the dairy industry. 相似文献
8.
《Insect Biochemistry》1991,21(2):165-176
A lysosomal aspartic protease with cathepsin D activity, from the mosquito, Aedes aegypti, was purified and characterized. Its isolation involved ammonium sulfate (30–50%) and acid (pH 2.5) precipitations of protein extracts from whole previtellogenic mosquitoes followed by cation exchange chromatography. Purity of the enzyme was monitored by SDS-PAGE and silver staining of the gels. The native molecular weight of the purified enzyme as determined by polyacrylamide gel electrophoresis under nondenaturing conditions was 80,000. SDS-PAGE resolved the enzyme into a single polypeptide with Mr = 40,000 suggesting that it exists as a homodimer in its non-denatured state. The pI of the purified enzyme was 5.4 as determined by isoelectric focusing gel electrophoresis. The purified enzyme exhibits properties characteristic of cathepsin D. It utilizes hemoglobin as a substrate and its activity is completely inhibited by pepstatin-A and 6M urea but not by 10 mM KCN. Optimal activity of the purified mosquito aspartic protease was obtained at pH 3.0 and 45°C. With hemoglobin as a substrate the enzyme had an apparent Km of 4.2 μ M. Polyclonal antibodies to the purified enzyme were raised in rabbits. The specificity of the antibodies to the enzyme was verified by immunoblot analysis of crude mosquito extracts and the enzyme separated by both non-denaturing and SDS-PAGE. Density gradient centrifugation of organelles followed by enzymatic and immunoblot analyses demonstrated the lysosomal nature of the purified enzyme. The N-terminal amino acid sequence of the purified mosquito lysosomal protease (19 amino acids) has 74% identity with N-terminal amino acid sequence of porcine and human cathepsins D. 相似文献
9.
Protein kinase C prepared from rat brain was used to phosphorylate a calcium-activated neutral protease, purified from bovine cardiac muscle. Attempts to phosphorylate the enzyme in the presence of calcium were unsuccessful, unless the protease inhibitor leupeptin was also present. Phosphorylation of the 74K subunit of the protease was completely inhibited in the absence of phosphatidylserine and diolein, indicating that phosphorylation of the enzyme was catalysed by the calcium and phospholipid-dependent protein kinase C. 相似文献
10.
Calcium-activated neutral protease (CANP), a putative processing enzyme of the neuropeptide, kyotorphin, in the brain 总被引:2,自引:0,他引:2
Y Yoshihara H Ueda S Imajoh H Takagi M Satoh 《Biochemical and biophysical research communications》1988,155(2):546-553
Kyotorphin (Tyr-Arg) accumulation in the dialysed synaptosol from the rat brain in the presence of an inhibitor of kyotorphin-degrading enzyme, was maximal at neutral pH. This accumulation was activated by calcium ions, but was inhibited by leupeptin and SH-blocking agents, a finding which suggests the involvement of calcium-activated neutral protease (CANP or calpain). In addition, the kyotorphin-precursor protein, being processed by purified mu- or m-CANP, was detected at about 160 kDa on Sephacryl S-300 chromatography of the synaptosol. The present findings seem to be the first evidence for the role of CANP as a processing enzyme of neuropeptide-precursor in nerve terminals. 相似文献
11.
Manuel M. Valdivia Jesús Avila Javier Coll Camilo Colaço Ignacio V. Sandoval 《Biochemical and biophysical research communications》1982,105(4):1241-1249
A radioimmunoassay developed for the microtubule associated protein MAP2 shows that this protein, or related polypeptides are present in all the porcine tissues studied. Nervous tissues (brain, 11 μg MAP2/mg protein; cerebellum, 9.7 μg MAP2/mg protein) contain much higher levels of MAP2 than non-nervous tissues (kidney, 104 ng MAP2/mg protein; lung 89 ng MAP2/mg protein; spleen 66 ng MAP2/mg protein; thyroid 21 ng MAP2/mg protein; liver 9.7 ng MAP2/mg protein). A heat resistant protein doublet of 300,000 with the ability to promote microtubule polymerization has been purified from pig kidney cells by affinity chromatography using MAP2 antibodies. Using a similar purification method a protein of 200,000 daltons has been isolated from Hela cells. 相似文献
12.
《Enzyme and microbial technology》2007,40(7):1433-1440
Pseudomonas aeruginosa PD100 capable of producing an extracellular protease was isolated from the soil collected from local area (garbage site) from Shivage market in Pune, India. The purified protease showed a single band on native and SDS-PAGE with a molecular weight of 36 kDa on SDS-PAGE. The optimum pH value and temperature range were found to be 8 and 55–60 °C, respectively. The enzyme exhibited broad range of substrate specificity with higher activity for collagen. The enzyme was inhibited with low concentration of Ag2+, Ni2+, and Cu2+. β-Mercaptoethanol was able to inactivate the enzyme at 2.5 mM, suggesting that disulfide bond(s) play a critical role in the enzyme activity. Studies with inhibitors showed that different classes of protease inhibitors, known to inhibit specific proteases, could not inhibit the activity of this protease. Amino acid modification studies data and pKa values showed that Cys, His and Trp were involved in the protease activity. P. aeruginosa PD100 produces one form of protease with some different properties as compared to other reported proteases from P. aeruginosa strains. With respect to properties of the purified protease such as pH optimum, temperature stability with capability to degrade different proteins, high stability in the presences of detergents and chemicals, and metal ions independency, suggesting that it has great potential for different applications. 相似文献
13.
A purification procedure is described for a highly active endopeptidase produced by a marine bacterium (Vibrio B-30). The purified enzyme was shown to be homogeneous by ion-exchange chromatography, gel filtration, and electrophoresis. A crystalline preparation was obtained. The pH optimum of the enzyme was about 7.0, and it was stable in the pH range of 5.0–8.5. Using hemoglobin as the substrate, a Km of 0.095 mm was obtained. The temperature optimum of the enzyme was 40 ° in the absence of calcium and about 50 ° in the presence of 10−3 m calcium. Calcium both activated and stabilized the enzyme against thermal denaturation. The enzyme was shown to be a serine protease which was irreversibly inhibited by certain metal-complexing agents. Gel filtration studies revealed that Vibrio B-30 endopeptidase had a molecular weight of 49,000 ± 5,000 but it rapidly autolyzed during the second and third passage through a gel column. Removal of a metal ion (probably calcium) resulted in the formation of a high-molecular-weight, enzymatically inactive component and a low-molecular-weight, partially active component. 相似文献
14.
Rim Nasri Assaâd Sila Naourez Ktari Imen Lassoued Ali Bougatef Maha Karra-Chaâbouni Moncef Nasri 《Process Biochemistry》2012,47(12):1957-1964
An alkaline calcium dependent trypsin from the viscera of Goby (Zosterisessor ophiocephalus) was purified to homogeneity with a 16-fold increase in specific activity and 20% recovery. The purified trypsin appeared as a single band on sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) and native-PAGE. The enzyme had an estimated molecular weight of 23.2 kDa.The optimum pH was 9.0, and the enzyme was extremely stable in various pH buffers between pH 7.0 and 11.0. The optimum temperature for enzyme activity was 60 °C, and the activity and stability of trypsin was highly dependent on the presence of calcium ion. At 60 °C, Ca2+ (5 mM) stimulated the protease activity by 220%. The trypsin kinetic constants, Km and kcat, were 0.312 mM and 2.03 s?1.The enzyme showed high stability towards non-ionic surfactants and oxidizing agent. In addition, the enzyme showed excellent stability and compatibility with some commercial solid and liquid detergents. 相似文献
15.
A serine protease was purified 6.7-fold and with 35% recovery from the seeds Solanum dubium Fresen by a simple purification procedure that combined ammonium sulfate fractionation, cation exchange and gel filtration chromatographies. The enzyme, named dubiumin, has a molecular mass of 66 kDa as estimated by gel filtration and SDS-PAGE. Carbohydrate staining established the existence of a carbohydrate moiety attached to the enzyme. Inhibition of enzyme activity by serine protease inhibitors such as PMSF and chymostatin indicated that the enzyme belongs to the chymotrypsin-like serine protease class. Dubiumin is a basic protein with pI value of 9.3, acts optimally at pH 11.0, and is stable over a wide range of pH (3.0-12.0). The enzyme is also thermostable retaining complete activity at 60 °C after 1 h and acts optimally at 70 °C for 30 min. Furthermore, it is highly stable in the presence of various denaturants (2.0% SDS, 7.0 M urea and 3.0 M guanidine hydrochloride) and organic solvents [CH3CN-H2O (1:1, v/v) and MeOH-H2O (1:1, v/v)] when incubated for 1 h. The enzyme showed a high resistance to autodigestion even at low concentrations. 相似文献
16.
《Plant science》1988,55(3):205-211
Potato (Solanum tuberosum L.) leaves were shown to contain a lipolytic enzyme activity which is stimulated by treatment with purified trypsin, pronase, and to a lesser degree by chymotrypsin. This protease-stimulated activity was stable over a wide range of pH values. Lipolytic enzyme activity also appeared to be regulated by pH, with a pronounced stimulation at pH 6.0 ± 0.5 and a subsequent inactivation at pH 8.0–9.0. This pH stimulation was slightly by ethylene diamine tetracetic acid (EDTA), and was inhibited by Ca2+. Although leupeptin slightly inhibited the pH stimulation, two other protease inhibitors, phenylmethylsulfonyl fluoride (PMSF) and soybean trypsin inhibitor showed no effect. While some of the lipolytic enzyme activitiesn potato leaves (those detected by 1-acyl-2-[6-[(7-nitro-2,1,3 benzoxadiazol-4-yl) amino]-caproyl] phosphatidylcholine (C6-NBD-PC) hydrolysis) are stimulated by protease or pH treatment, others (those detected by 4-methylumbelliferyl laurate (4MUL) hydrolysis) are inactivated by them. The possible physiological significance of this apparent proteolytic activation is discussed. 相似文献
17.
We have introduced a novel glucoamylopullulanase from thermostable alkaliphilic Bacillus subtilis DR8806 from a hot mineral spring in Iran. The enzyme was purified by ion-exchange chromatography following to ammonium sulphate precipitation. The molecular weight of the purified enzyme was estimated to be 65.5 kDa using denaturing acrylamide gel electrophoresis. The enzyme showed high activity over a wide pH range, from pH 5.0 to pH 11.0 with the optimum pH 9.5. Our results also indicated an optimum temperature of the enzyme activity at 70 °C. These features justify the characteristics of the alkaliphilic and thermostable bacterial proteins and enzymes. The enzyme did not require calcium and showed extreme stability with regard to surfactants, including SDS and Triton X-100, and oxidizing agents such as H2O2. These features of the enzyme suggest a promising potential for application in laundry industry. Furthermore, the enzyme was active on pulullan by 68% relative to normal activity on starch. Such characteristics have not already been reported for this type of enzyme, hence we propose that this is a new alkalophilic and thermostable enzyme. 相似文献
18.
Hossein Ghafoori Mansoure Askari Sajjad Sarikhan 《Extremophiles : life under extreme conditions》2016,20(2):115-123
This study reports the purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, Bacillus iranensis, strain X5B. The enzyme was purified to homogeneity by acetone precipitation, ultrafiltration and carboxymethyl (CM) cation exchange chromatography, respectively. The purified protease was a monomeric enzyme with a relative molecular mass of 48–50 kDa and it was inhibited by PMSF indicating that it is a serine-protease. The optimum pH, temperature and NaCl concentration were 9.5, 35 °C and 0.98 M, respectively. The enzyme showed a significant tolerance to salt and alkaline pH. It retained approximately 50 % of activity at 2.5 M NaCl and about 70 % of activity at highly alkaline pH of 11.0; therefore, it was a moderately halophilic and also can be activated by metals, especially by Ca2+. The specific activity of the purified protease was measured to be 425.23 μmol of tyrosine/min per mg of protein using casein as a substrate. The apparent K m and V max values were 0.126 mM and 0.523 mM/min, respectively and the accurate value of k cat was obtained as 3.284 × 10?2 s?1. These special and important characteristics make this serine protease as valuable tool for industrial applications. 相似文献
19.
Purification, Characterization, and Functional Role of a Novel Extracellular Protease from Pleurotus ostreatus 总被引:3,自引:0,他引:3
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Gianna Palmieri Carmen Bianco Giovanna Cennamo Paola Giardina Gennaro Marino Maria Monti Giovanni Sannia 《Applied microbiology》2001,67(6):2754-2759
A new extracellular protease (PoSl; Pleurotus ostreatus subtilisin-like protease) from P. ostreatus culture broth has been purified and characterized. PoSl is a monomeric glycoprotein with a molecular mass of 75 kDa, a pI of 4.5, and an optimum pH in the alkaline range. The inhibitory profile indicates that PoSl is a serine protease. The N-terminal and three tryptic peptide sequences of PoSl have been determined. The homology of one internal peptide with conserved sequence around the Asp residue of the catalytic triad in the subtilase family suggests that PoSl is a subtilisin-like protease. This hypothesis is further supported by the finding that PoSl hydrolysis sites of the insulin B chain match those of subtilisin. PoSl activity is positively affected by calcium. A 10-fold decrease in the Km value in the presence of calcium ions can reflect an induced structural change in the substrate recognition site region. Furthermore, Ca2+ binding slows PoSl autolysis, triggering the protein to form a more compact structure. These effects have already been observed for subtilisin and other serine proteases. Moreover, PoSl protease seems to play a key role in the regulation of P. ostreatus laccase activity by degrading and/or activating different isoenzymes. 相似文献
20.
《Process Biochemistry》2007,42(5):791-797
An extracellular bleach stable protease from the fungus Aspergillus clavatus ES1, isolated from wastewater, was purified and characterized. The protease of ES1 strain was purified to homogeneity using acetone precipitation, Sephadex G-100 gel filtration and CM-Sepharose ion exchange chromatography, with a 7.5-fold increase in specific activity and 29% recovery. The molecular mass was estimated to be 32 kDa on SDS-PAGE. The optimum pH and temperature for the proteolytic activity were pH 8.5 and 50 °C, respectively. The enzyme was stable in the pH range of 7.0–9.0. The protease was activated by divalent cations such as Ca2+ and Mg2+.The alkaline protease showed extreme stability towards non-ionic surfactants (5% Tween 80 and 5% Triton X-100). In addition, the enzyme was relatively stable towards oxidizing agents, retaining more than 71 and 53% of its initial activity after 1 h incubation in the presence of 1 and 2% (w/v) sodium perborate, respectively.The N-terminal sequence of the first 15 amino acids of the purified alkaline protease of A. clavatus ES1 showed high similarity with other fungal alkaline proteases. The activity was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine-protease. 相似文献