Purification Of Protease Research Articles

Purification of heat-resistant protease from a psychotroph using an affinity column

Purification of heat-resistant protease from a psychotroph using an affinity column

Purification of human platelet calcium-activated protease. Effect on platelet and endothelial function.

Calcium-activated protease (CAP) was purified from the cytosol fraction of homogenized human platelet concentrates using a combination of gel filtration chromatography and affinity chromatography on antipain aminohexyl-Sepharose and activated thiol-Sepharose 4B. Purified CAP is composed of two different polypeptides of Mr = 80,000 and 27,000. Half-maximal protease activity was observed at 0.52 mM Ca2+, and all activity was inhibited by antipain, leupeptin, and N-ethylmaleimide. Activated CAP showed a time-dependent inactivation in the presence of 1 mM Ca2+ with only 5% of the control protease activity remaining after a 1-h exposure to calcium. Preincubation of washed platelets with varying amounts of CAP (0.2-0.4 units) significantly interfered with thrombin-induced platelet aggregation. In addition, ristocetin-induced platelet agglutination in the presence of von Willebrand factor was completely inhibited by 0.4 units of CAP. Concomitant with these protease-induced changes in platelet function, a decrease was observed in a major glycoprotein band of Mr = 150,000 present in platelet membranes and presumed to be glycoprotein Ib. In addition to these effects on platelets, CAP inhibited thrombin-induced production of prostacyclin by cultured human endothelial cells in a dose-dependent manner when the cells were pretreated with CAP. Thus platelet CAP can modulate membrane functions in both platelets and endothelial cells and may thus contribute to the regulation of hemostasis.

Purification and characterization of protease So, a cytoplasmic serine protease in Escherichia coli.

A new cytoplasmic endoprotease, named protease So, was purified to homogeneity from Escherichia coli by conventional procedures with casein as the substrate. Its molecular weight was 140,000 when determined by gel filtration on Sephadex G-200 and 77,000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Thus, it appears to be composed of two identical subunits. Protease So had an isoelectric point of 6.4 and a K(m) of 1.4 muM for casein. In addition to casein, it hydrolyzed globin, glucagon, and denatured bovine serum albumin to acid-soluble peptides but did not degrade insulin, native bovine serum albumin, or the "auto alpha" fragment of beta-galactosidase. A variety of commonly used peptide substrates for endoproteases were not hydrolyzed by protease So. It had a broad pH optimum of 6.5 to 8.0. This enzyme is a serine protease, since it was inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. Although it was not inhibited by chelating agents, divalent cations (e.g., Mg(2+)) stabilized its activity. Protease So was sensitive to inhibition by N-tosyl-l-phenylalanine chloromethyl ketone but not by N-tosyl-l-lysine chloromethyl ketone. Neither ATP nor 5'-diphosphate-guanosine-3'-diphosphate affected the rate of casein hydrolysis. Protease So was distinct from the other soluble endoproteases in E. coli (including proteases Do, Re, Mi, Fa, La, Ci, and Pi) in its physical and chemical properties and also differed from the membrane-associated proteases, protease IV and V, and from two amino acid esterases, originally named protease I and II. The physiological function of protease So is presently unknown.

32] Structure and biosynthesis of aminopeptidases

This chapter describes the structure and biosynthesis of aminopeptidases. Aminopeptidases are integral membrane proteins that can be solubilized without loss of activity either by limited proteolysis or by neutral detergent extraction. The two aminopeptidases—aminopeptidase N and aminopeptidase A—are active on neutral and acidic N-terminal amino acids in peptides and synthetic derivatives, such as β-naphthylamides and p-nitroanilides. The chapter also discusses structure of aminopeptidases regarding their mode of integration in the brush-border membrane; purification of protease and detergent forms of rabbit aminopeptidase N; molecular weight determination and detergent binding of aminopeptidases and their hydrophilic and hydrophobic parts; localization of hydrophilic part on the membrane surface by a quantitative immunochemical technique; relationship between the mode of integration of aminopeptidases in the membrane and their biosynthesis; precipitation of the rabbit enterocyte basolateral membranes by specific anti-aminopeptidase N antibodies; and kinetics of incorporation of newly synthesized aminopeptidase in the basolateral and brush-border membrane fractions.

Partial purification and properties of protease D from larvae of Artemia salina

1. 1. Four alkaline proteases, A, B, C and D were previously detected in Artemia larval extracts. In this paper the purification and properties of protease D are presented. 2. 2. Protease D is active on casein and azocasein. None of the following synthetic compounds were substrates for protease D: benzyloxycarbonyl-β-alanine p-nitrophenyl ester; α-N- benzoyl- dl-arginine p-nitroanilide; benzyloxycarbonyl-glycine p-nitrophenyl ester; benzyloxycarbonyl- l-leucine p-nitrophenyl ester; benzyloxycarbonyl- l-proline p-nitrophenyl ester and benzyloxycarbonyl- l-valine p-nitrophenyl ester. 3. 3. The enzyme is maximally active at pH around 8, it does not require metal ions for activity and it is 50% inhibited in the presence of 0.7 mM dithioerythritol. Its mol. wt was 36,000.

Purification and characterization of alkaline protease and neutral protease from chromatin of rats

It was previously reported that, in addition to a known chymotrypsin-like protease capable of hydrolyzing histones with an optimum pH of 8 (neutral protease), another protease is bound to the chromatin of various rat tissues and in situ hydrolyzes casein more quickly than histones with an optimum pH of 10 (alkaline protease). In the present study, the alkaline protease was purified 14 00-fold to approx. 75% purity from the chromatin of Rhodamine sarcoma. This tumor contains both proteases at higher levels than normal tissues. For purification, affinity columns of Sepharose with bound soybean trypsin inhibitor, casein and histones were successively used. Also, the neutral protease was purified 920-fold to an apparently homogeneous state by affinity chromatography on a Sepharose column with bound soybean trypsin inhibitor under conditions, in which an excess amount of the enzyme was applied on the column so that part of the enzyme would pass through the column without adsorption and the enzyme thus adsorbed was then eluted. The purified alkaline and neutral proteases had molecular weights of approx. 18 000 and 27 000, respectively, and isoelectric points of approx. 11. The former enzyme hydrolyzed casein (100) in preference to histones (18) with an optimum pH of 9.5, whereas the latter enzyme preferred histones (100) to casein (32) with an optimum pH of 8. Their actions against other proteins and synthetic substrates were also studied.

The purification of protease IV of E.coli and the demonstration that it is an endoproteolytic enzyme

A strong proteolytic activity is unmasked and solubilized when E. coli outer membrane fragments are preincubated with 0.083% sodium dodecyl sulfate. This proteolytic activity cleaves αS1 casein into the same degradation products as protease IV, a recently described protease of E.coli located in the outer membrane (Ph. Régnier, preceding paper), it is concluded that sodium dodecyl sulfate solubilizes the same protease. Protease IV has been purified 11,200 fold, probably to homogenetiy, by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by elution of the protein from gel slices. The purified enzyme is fully active, its molecular weight, determined from its migration in denaturating gels is 23,500. αS1 casein is cleaved by protease IV into two large polypeptides which are not further degraded and some small peptides of about 5,000 daltons. The production of discrete polypeptide species suggests that protease IV is an endoproteolytic enzyme.

ラット肝ライソゾーム中の蛋白分解酵素の精製と諸性質

ラット肝ライソゾーム中の蛋白分解酵素の精製と諸性質

Purification and characterization of yeast protease B.

Protease B [EC 3.4.22.9] was purified from baker's yeast by plasmolysis of yeast, acid activation, acid precipitation, and column chromatographies on QAE-Sephadex, SP-Sephadex, D-tryptophan methyl ester-Sepharose 4B and Sephadex G-100. The purified enzyme was inhibited by phenylmethylsulfonyl fluoride and sulfhydryl-blocking reagents. Chymostatin and antipain at extremely low concentrations (1 micro M) inhibited the protease B. The effects of the enzyme on various yeast enzymes were examined by measuring their inactivation. The enzyme inactivated 6-phosphogluconate dehydrogenase [EC 1.1.1.44] and uricase [EC 1.7.3.3], but not malate dehydrogenase [EC 1.1.1.37], alcohol dehydrogenase [EC 1.1.1.1], glutamate dehydrogenase [EC 1.4.1.3], glucose-6-phosphate dehydrogenase [EC 1.1.1.49] or hexokinase [EC 2.7.1.1].

Purification and characterization of protease III from Escherichia coli.

An endoproteolytic enzyme of Escherichia coli, designated protease III, has been purified about 9,600-fold to homogeneity with a 6% yield. The purified enzyme consists of a single polypeptide chain of Mr 110,000 and is most active at pH 7.4. Protease III is very sensitive to metal-chelating agents and reducing agents. The EDTA-inactivated enzyme can be reactivated by Zn2+, Co2+ or Mn2+. Protease III is devoid of activity toward aminopeptidase, carboxypeptidase, or esterase substrates but rapidly degrades small proteins. When fragments of beta-galactosidase are used as substrates for protease III, the enzyme preferentially degrades proteins with molecular weights of less than 7,000. Protease III cleaves the oxidized insulin B chain at two sites with an initial rapid cleavage at Tyr-Leu (16-17) and a second slower cut at Phe-Tyr (25-26).

Purification, characterization and localization of serine protease of Morris hepatoma 8999.

1. A serine protease of hepatoma 8999, isolated in the mitochondrial fraction, was purified and crystallized. The purified enzyme was apparently homogeneous on ultracentrifugal analysis and polyacrylamide disc gel electrophoresis. The ratio of absorbance at 280 nm and 260 nm, A280/A260, was 1.90 and its absorption coefficient, A280 1% was 10.5 cm-1 estimated from dry weight measurements. Its S20, w value was 2.23 S and its molecular weight was estimated to be 24000 +/- 1000. The enzyme contained twice as much lysine, arginine and histidine as chymotrypsinogen did, but had a very similar amino acid composition to serine protease from skeletal muscle. Its isoelectric point was pH 10.6. 2. The substrate specificity of the enzyme was the same as that of chymotrypsin A. Its Km and kcat values for N-acetyl-L-tyrosine ethyl ester, N-acetyl-L-phenylalanine ethyl ester and N-acetyl-L-tryptophan ethyl ester were 0.35 mM and 10.69 s-1, 0.38 mM and 10.7 s-1, and 0.11 mM and 11.8 s-1, respectively. Its activity was completely inhibited by phenylmethylsulfonyl fluoride and partially inhibited with tosylphenylalanine chloromethyl ketone. 3. The enzyme was shown to be located in different granules from the intracellular particules (light and heavy mitochondrial fraction) by sucrose density gradient centrifugation, and it was stained in mast cells of the hepatoma 8999 by the immunofluorescent technique. 4. Serine protease is present in different amounts in various organs of rat and the enzyme from hepatoma 8999 gave a single band that fused completely with those of the enzymes from skeletal muscle, heart, liver and kidney, respectively, on Ouchterlony double-diffusion analysis using antiserum to the crystalline enzyme of hepatoma 8999, but the enzyme from small intestine did not react with the antiserum.

Studies on intestinal protease: Isolation, purification and effect of dietary proteins on alkaline protease activity of the air-breathing fish, Clarias batrachus (Linn.)

1. The effect of dietary protein levels on the proteolytic activity in the intestines of the air-breathing fish, Clarias batrachus (Linn.) has been studied 2. Activity of proteolytic enzymes increased significantly in fishes maintained with a 50% protein diet from those maintained with a 25% protein diet; still higher dietary protein percentage showed no further stimulation of enzyme activity. 3. In a study on the determination of sub-cellular localisation, it has been found that protease activity is more prominent in lysosomes than in other organelles of the cell. 4. A sixty fold purification of alkaline protease from the intestine of Clarias batrachus has been achieved by ion exchange chromatography on DEAE cellulose which has been further checked by polyacrylamide gel electrophoresis.

A new proteolytic enzyme isolated from Mytilus galloprovincialis: Mytilidase. Purification, crystallization and partial characterization

1. 1. A procedure for the advanced purification of acid protease(s) obtained from the hepatopancreas of the sea mussel, Mytilus galloprovincialis, is described. 2. 2. Proteolytic activities at pH 1.6 and 2.6 could not be separated, although presenting different optimal action parameters. 3. 3. The molecular weight of mytilidase, determined by gel filtration on a Sephadex G-200 column, was 36,000 ± 600.

Production and Purification of Thermostable Amylase and Protease of Thermomonospora viridis

Maximum yields of amylase were produced by the thermophilic actinomycete Thermomonospora viridis in a modified Simpson and McCoy medium containing 1.5% corn starch and 0.5% mycological peptone with an initial pH 7.0. Best yields of amylase were obtained after incubation for 48 h, when the pH of the medium had risen to 8.2. Amylase was purified 313-fold by precipitation with n-propyl alcohol, dialysis against tap water, adsorption on Ca(3)(PO(4))(2), and fractionation on Sephadex G-100. Protease was produced in nutrient broth containing 0.5% starch and 1.0% corn steep liquor and at an initial pH 7.0. Maximum yields of protease were produced after 42 h. The protease was purified 54-fold by precipitation with n-propyl alcohol, dialysis against tap water, adsorption on Ca(3)(PO(4))(2), and fractionation on Sephadex G-200.

Purification and properties of blood-coagulating protease from Cephalosporium sp.

Blood-coagulating protease produced extraceîlularly by Cephalosporium sp. was purified. The purified enzyme moved homogeneously with a sedimentation constant, S20, w of 2.90 s by ultracentrifugation and gave a single protein band on the polyacrylamide gel electrophoresis. The enzyme was an EDTA-sensítive alkaline protease and Ca2+ stabilized its activity. Molecular weight of the enzyme was estimated as 39,000 by Svedberg’s method and 124,000 by Andrews’ method. The enzyme was considered to activate Factor X to Xa. The specific blood-coagulating activity of the enzyme was 1/5 of that of bovine thrombin.

Isolation and purification of acid protease from mouse fibroblasts L-929

Isolation and purification of acid protease from mouse fibroblasts L-929

Purification of Protease II from Escherichia coli by Affinity Chromatography and Separation of Two Enzyme Species from Cells Harvested at Late Log Phase

N-Acetyl-D-arginine linked to an agarose matrix has been used to purify protease II from Escherichia coli by affinity chromatography. The specific adsorption of protease II to this absorbent was achieved in 220 mM potassium phosphate buffer pH 7.6, and the enzyme was eluted with L-arginine. Enzyme preparations from cells harvested at late log phase have been resolved into two molecular species which differ in specific activity, kinetic constants and carbohydrate content. Both species appeared homogeneous by electrophoresis in conventional buffers and also in the presence of sodium dodecyl sulfate. Only one enzyme species was obtained by the same procedure using bacteria harvested at the middle of exponential growth.

Production and purification of acid protease from the thermophilic fungus, Penicillium duponti K1014.

A thermophilic fungus, Penicillium duponti K1014, produced an acid protease in a submerged culture. Maximum enzyme production, in a 30-liter fermentor at a preselected optimum growth temperature of 50 C, occurred after 3 days. The productivity of the enzyme was associated with changes of viscosity and pH of culture broth during the growth. The acid protease was isolated from the culture filtrates and was purified ninefold by alcohol precipitation, column chromatography on O-(diethylaminoethyl)cellulose, batchwise treatment with O-(carboxymethyl)cellulose, and by gel filtration. The purified enzyme was homogeneous on disk electrophoresis and sedimentation analysis. The sedimentation constant s(20.w), calculated at a concentration of 0.64%, was 3.98S.

Separation and partial purification of extracellular amylase and protease from Bacillus caldolyticus.

The extracellular amylase and protease from Bacillus caldolyticus can be concentrated by ammonium sulfate precipitation after growth on either solid or in liquid media containing starch, glucose, and brain-heart infusion. Using the Diaflo ultrafiltration system with membranes of various permeability, the enzymes could be separated from each other by extensive flushing with buffer. Best results were obtained with the 50–70% ammonium sulfate fraction as starting material, yielding 72% of the total amylase activity in the low molecular weight fraction (UM-10 fraction: 10000–30000), while 54 and 25% respectively of the protease were retained in the two high molecular weight fractions (50000–100000, and more than 100000). Similar results were obtained with the 20–50% ammonium sulfate fraction, while the fraction of 0–20% saturation contained a low molecular weight protease. The native amylase seems to consist of a number of sub-units, which after extensive flushing accumulate in the fraction with an approximate molecular weight between 10000 and 30000. The enzyme could also be precipitated from cell-free liquid media with ammonium sulfate, followed by separation and purification on ultra-filtration cells. According to the specific activity of the UM-10 fractions a 400-fold purification was obtained compared to the amylase activity of the cell-free medium.

Production and Purification of Acid Protease from the Thermophilic Fungus, Penicillium duponti K1014

A thermophilic fungus, Penicillium duponti K1014, produced an acid protease in a submerged culture. Maximum enzyme production, in a 30-liter fermentor at a preselected optimum growth temperature of 50 C, occurred after 3 days. The productivity of the enzyme was associated with changes of viscosity and pH of culture broth during the growth. The acid protease was isolated from the culture filtrates and was purified ninefold by alcohol precipitation, column chromatography on O-(diethylaminoethyl)cellulose, batchwise treatment with O-(carboxymethyl)cellulose, and by gel filtration. The purified enzyme was homogeneous on disk electrophoresis and sedimentation analysis. The sedimentation constant s(20.w), calculated at a concentration of 0.64%, was 3.98S.