Purification and properties of an extracellular protease from Myxococcus virescens.

An extracellular protease from Myxococcus virescens was purified by phosphate precipitation, gel exclusion, and ion-exchange chromatography. The enzyme appeared homogeneous upon disc electrophoresis. The molecular weight of the protease was estimated to be 26,000. The enzyme was rapidly inactivated by ethylenediaminetetraacetate, but the activity could be partially restored by divalent cations. Diisopropylphosphorofluoridate inhibited enzyme activity completely. Michaelis-Menten kinetics were obeyed with casein and hemoglobin as substrates. First-order kinetics were obtained with elastin as the substrate, provided trypsin was in excess. Petidolytic activity indicated that the peptide bonds hydrolyzed by the enzyme were mainly those involving amino acids with nonpolar side chains.     

Purification and characterization of a multicatalytic high-molecular-mass proteinase from rat skeletal muscle.

A proteolytic enzyme was purified from the post-myofibrillar fraction of rat skeletal muscle. The purification procedure consisted of fractionation of the muscle extract by (NH4)2SO4, chromatography on DEAE-Sephacel, fast protein liquid chromatography on Mono Q and gel filtration on Sepharose 6B. The enzyme preparation appeared to be homogeneous as judged by disc electrophoresis in polyacrylamide gels and by immunoelectrophoresis. The isoelectric point of the proteinase is at 5.1-5.2. The enzyme has an Mr of about 650 000 and dissociates into eight subunits of Mr 25 000-32 000 when subjected to electrophoresis in sodium dodecyl sulphate/polyacrylamide gels. The proteinase contains hydrolytic activity against N-blocked tripeptide 4-methyl-7-coumarylamide substrates with an arginine or phenylalanine residue adjacent to the leaving group. Maximum activity with the first group of substrates was at pH 10.5, and this activity was inhibited by leupeptin, chymostatin and Ca2+. Maximum activity with the latter group of substrates was at pH 7.5, and was also inhibited by the two microbial inhibitors, but was activated by Ca2+ ions. By using [14C]methylcasein as a substrate, maximum activity was observed at pH9.0, and this proteolytic activity was not affected by leupeptin, was enhanced by chymostatin and inhibited by Ca2+. Similar effects were observed when benzyloxycarbonyl-Leu-Leu-Glu 2-naphthylamide was used as a substrate. These enzymic activities were abolished by p-hydroxymercuribenzenesulphonic acid or mersalyl acid, whereas a small activation was observed with cysteine or dithiothreitol.     

Purification and characterization of repressor of temperate S. aureus phage phi11

To gain insight into the structure and function of repressor proteins of bacteriophages of gram-positive bacteria, repressor of temperate Staphylococcus aureus phage phi11 was undertaken as a model system here and purified as an N-terminal histidine-tagged variant (His-CI) by affinity chromatography. A approximately 19 kDa protein copurified with intact His-CI (approximately 30 kDa) at low level was resulted most possibly due to partial cleavage at its Ala-Gly site. At approximately 10 nM and higher concentrations, His-CI forms significant amount of dimers in solution. There are two repressor binding sites in phi11 cI-cro intergenic region and binding to two sites occurs possibly by a cooperative manner. Two sites dissected by HincII digestion were designated operators O(L) and O(R), respectively. Equilibrium binding studies indicate that His-CI binds to O(R) with a little more strongly than O(L) and binding species is probably dimeric in nature. Interestingly His-CI binding affinity reduces drastically at elevated temperatures (32-42 degrees C). Both O(L) and O(R) harbor a nearly identical inverted repeat and studies show that phi11 repressor binds to each repeat efficiently. Additional analyses indicate that phi11 repressor, like lambda repressor, harbors an N-terminal domain and a C-terminal domain which are separated by a hinge region. Secondary structure of phi11 CI even nearly resembles to that of lambda, phage repressor though they differ at sequence level. The putative N-terminal HTH (helix-turn-helix) motif of phi11 repressor belongs to the HTH -XRE-family of proteins and shows significant identity to the HTH motifs of some proteins of evolutionary distant organisms but not to HTH motifs of most S. aureus phage repressors.     

Purification of recombinant HIV-1 protease.

A method is described to purify recombinant HIV-1 protease from soluble extracts of Escherichia coli. The isolation involves QAE-Sepharose anion exchange chromatography, hexyl agarose hydrophobic interaction chromatography, MonoS cation exchange chromatography, and Superose 6 size exclusion chromatography. Approximately 100 micrograms of protease was obtained from 18 g E. coli paste. The protein was judged to be homogeneous due to the presence of a single band on a silver-stained SDS polyacrylamide gel.     

Purification and partial characterization of an elastolytic serine protease of Prevotella intermedia.

Elastolytic strains of Prevotella intermedia were isolated from pus samples of adult periodontal lesions. Elastase was found to associate with envelope, and it could be solubilized with guanidine-HCl. The enzyme was purified to homogeneity by sequential procedures including ion-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. This elastase was a serine protease, and its mass was 31 kDa. It hydrolyzed elastin powder, but collagen and azodye-conjugated proteins were not degraded by this enzyme. Both synthetic substrates for human pancreatic (glutaryl-L-alanyl-L-alanyl-L-prolyl-L-leucine p-nitroanilide) and leukocyte elastase (methoxy succinyl-L-alanyl-alanyl-L-prolyl-L-valine p-nitroanilide) were hydrolyzed.     

Purification and characterization of a latent form of multicatalytic proteinase from fish muscle.

1. A latent form of multicatalytic proteinase (MCP) was purified to apparent homogeneity from white croaker muscle by DEAE-Sephacel, Mono-Q, Sephacryl S-300 and second Mono-Q chromatographies. 2. The enzyme preparation was electrophoretically and immunologically similar to MCP purified from the same source by a different method (Folco et al., 1988b, Archs Biochem. Biophys. 267, 599-605) but showed much lower chymotrypsin- and trypsin-like activities. 3. These activities responded to sodium dodecyl sulphate (SDS), urea and heat treatments in different ways: SDS stimulated both activities, urea stimulated the former and inhibited the latter and heating stimulated the former and did not affect the latter. 4. The stimulation of chymotrypsin-like activity by the three treatments was irreversible. 5. Exposure of MCP to SDS or urea in the absence of substrate rapidly inactivated it, whereas heat activation took place irrespective of the presence of substrate. 6. The stimulating effect of SDS on chymotrypsin-like activity was lost in the presence of urea. 7. These results suggest that the enzyme may be activated by different mechanisms.     

26S multicatalytic proteinase complexes decrease during the differentiation of murine erythroleukemia cells.

Changes in multicatalytic proteinase activity during differentiation were investigated using Me2SO-induced differentiation of murine erythroleukemia cells as a model. The apparent ATP-dependent multicatalytic proteinase activity decreased in the Me2SO-treated cells with ATP-dependent incorporation of [3H]diisopropyl fluorophosphate decreasing notably after Me2SO-treatment. This decrease in activity does not seem to arise from a cessation of cell-proliferation, because no significant changes in proteinase activity were observed under different culture conditions. Hydroxyapatite column chromatography was employed to analyze the form of multicatalytic proteinase. It was clearly demonstrated that the 26S form of the proteinase decrease in the differentiated cells relative to normal cells. Multicatalytic proteinase-associated proteins that bind to the proteinase in an ATP-dependent manner were purified on an anti-multicatalytic proteinase IgG conjugated column. Only a small amount of protein was recovered from the differentiated cells. These results suggest that the decrease in multicatalytic proteinase-associated proteins that occurs upon cell-differentiation abolishes the ATP-dependent activity of the proteinase.     

Purification and characterization of multicatalytic proteinase from eggs of the ascidian Halocynthia roretzi

A multicatalytic (high-molecular-weight) proteinase has been purified from eggs of the ascidian Halocynthia roretzi by a procedure including column chromatographies on DEAE-cellulose and hydroxylapatite and gel filtration on Sepharose 6B. The purified enzyme seemed to be homogeneous, as judged by disc-polyacrylamide gel electrophoresis, isoelectrofocusing, sedimentation velocity, and gel filtration. The molecular weight of the enzyme was estimated to be 610,000 by gel filtration. The isoelectric point and the sedimentation coefficient (S20,w) were 6.2 and 22.8S, respectively. The enzyme showed several protein bands with molecular weight ranging from 25,000 to 33,000 on SDS-polyacrylamide gel electrophoresis and a cylindrical or ring-like structure composed of several subunits under the electron microscope, indicating that the enzyme exists as a large molecule consisting of several protein components. The enzyme exhibited chymotrypsin-like and trypsin-like activities whose pH optima were both 7.0. Chymostatin and its analog, calpain inhibitor I, and elastatinal inhibited both activities, whereas leupeptin and antipain only inhibited the latter. The former activity was stimulated by a low concentration of SDS or fatty acid, whereas the latter was not. Thus, the properties of the enzyme purified from ascidian eggs are similar to those of multicatalytic proteinases from mammalian tissues.     

Purification and characterization of an elastolytic protease of Vibrio vulnificus

Large amounts of a highly purified, extracellular elastolytic protease of Vibrio vulnificus were obtained by sequential ammonium sulphate precipitation and hydrophobic interaction chromatography with phenyl-Sepharose CL-4B. The protease had an Mr of about 50,500 (estimated by SDS-PAGE), a pI of 5.7, and a temperature optimum range of 55 to 60 degrees C. The pH optimum and the results of inactivation studies suggested that the enzyme was a neutral metalloprotease. The protease had about 429 amino acid residues, and the first 20 amino-terminal amino acid residues were Ala-Gln-Ala-Asn-Gly-Thr-Gly-Pro-Gly-Gly-Asn-Ser-Lys-Thr-Gly-Arg-Tyr-Glu- Phe-Gly . The purified protease was toxic for mice (about 1.5 mg kg-1 and 4.5 mg kg-1, intraperitoneal and intravenous LD50 values, respectively), and subcutaneous injection of the enzyme elicited rapid and extensive dermonecrosis.     

Purification and characterization of an aspartic protease from potato leaves

A protease was isolated from potato ( Solanum tuberosum L. cv. Pampeana) leaves 48 h after detaching, when aspartic protease (AP) activity is markedly increased. Purification was performed by ammonium sulfate precipitation, ion exchange chromatography and affinity chromatography. A size of 40 kDa was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; it is monomeric and its properties are consistent with those of aspartic proteinases (EC 3.4.23): it has a pH optimum of 3 and it is inhibited by pepstatin. Like other plant APs, leaf AP appears to be glycosylated with a complex-type N-glycan. The enzyme has properties different from those of a tuber AP previously described, indicating that they may have different physiological roles.     

Purification and characterization of an extracellular protease from Flavobacterium arborescens

An extracellular protease from Flavobacterium arborescens has been purified to an apparent homogeneity and characterized. The enzyme is most active at pH 8-10.5, requires no metal cofactor, and is inhibited by diisopropyl fluorophosphate. The protease is nonspecific, is active at temperatures up to 60 degrees C, and is completely free of nucleases. The ease of purification and freedom from nucleolytic contaminants make the protease a useful deproteinizing agent in DNA and RNA manipulations.     

Purification and partial characterization of an alpha-chymotrypsin-like protease of rat peritoneal mast cells.

An alpha-chymotrypsin-like enzyme was isolated from mast cells of the rat peritoneal cavity by extraction with 0.8 M potassium phosphate, 2 per cent protamine sulfate followed by affinity chromatography on hen ovoinhibitor-agarose and adsorption on barium sulfate. This procedure yielded over 9 mg of protease from the peritoneal lavage fluid of 100 rats, equivalent to 44 per cent of the initial activity. The purified protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analytical isoelectric focusing, and amino-terminal sequence analysis. The protease contains no covalently bound carbohydrate and has a molecular weight of approximately 26,000. The enzyme molecule is a single polypeptide chain with an amino-terminal sequence homologous to that of the B chain of bovine alpha-chymotrypsin. The kinetic parameters, Km and kcat, for the hydrolysis of N-benzoyl-L-tyrosine ethyl ester were determined at pH 8.0 and 25 degrees C as 1.1 X 10(-3) M and 84 sec-1, respectively. The value of the second-order rate constant for inactivation of mast cell protease by diisopropylphosphofluoridate was 300 times lower than for bovine alpha-chymotrypsin.     

Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16

Alkaline protease (EC 3.4.21.14) activity, suitable for use in detergents, was detected in the alkaline culture medium of Bacillus sp. KSM-K16, which was originally isolated from soil. The enzyme, designated M protease, was purified to homogeneity from the culture broth by column chromatographies. The N-terminal amino acid sequence was Ala-Gln-Ser-Val-Pro-Trp-Gly-Ile-Ser-Arg-Val-Gln-Ala-Pro-Ala-Ala-His-Asn-Arg-Gly-Leu-Thr-Gly. The molecular mass of the protease was 28 kDa, and its isoelectric point was close to pH 10.6. Maximum activity toward casein was observed at 55°C and at pH 12.3 in 50 mM phosphate/NaOH buffer. The activity was inhibited by phenylmethylsulfonyl flouride and chymostatin. The enzyme was very stable in long-term incubation with liquid detergents at 40°C. The enzyme cleaved the oxidized insulin B chain initially at Leu¹⁵-Tyr¹⁶ and efficiently at ten more sites. Among various oligopeptidyl p-nitro-anilides (pNA) tested, N-succinyl-Ala-Ala-Pro-Phe-pNA was efficiently hydrolyzed by M protease. M protease was precipitated in (NH₄)₂SO₄-saturated acetate buffer (pH 5.0) as plank-like cyrstals.     

Degradation of the S. frugiperda peritrophic matrix by an inducible maize cysteine protease

A unique 33-kDa cysteine protease (Mir1-CP) rapidly accumulates at the feeding site in the whorls of maize (Zea mays L.) lines that are resistant to herbivory by Spodoptera frugiperda and other lepidopteran species. When larvae were reared on resistant plants, larval growth was reduced due to impaired nutrient utilization. Scanning electron microscopy (SEM) indicated that the peritrophic matrix (PM) was damaged when larvae fed on resistant plants or transgenic maize callus expressing Mir1-CP. To directly determine the effects of Mir1-CP on the PM in vitro, dissected PMs were treated with purified, recombinant Mir1-CP and the movement of Blue Dextran 2000 across the PM was measured. Mir1-CP completely permeabilized the PM and the time required to reach full permeability was inversely proportional to the concentration of Mir1-CP. Inclusion of E64, a specific cysteine protease inhibitor prevented the damage. The lumen side of the PM was more vulnerable to Mir1-CP attack than the epithelial side. Mir1-CP damaged the PM at pH values as high as 8.5 and more actively permeabilized the PM than equivalent concentrations of the cysteine proteases papain, bromelain and ficin. The effect of Mir1-CP on the PMs of Helicoverpa zea, Danaus plexippus, Ostrinia nubilalis, Periplaneta americana and Tenebrio molitor also was tested, but the greatest effect was on the S. frugiperda PM. These results demonstrate that the insect-inducible Mir1-CP directly damages the PM in vitro and is critical to insect defense in maize.     

Multiple forms of the 20 S multicatalytic and the 26 S ubiquitin/ATP-dependent proteases from rabbit reticulocyte lysate.

We have used native gel electrophoresis followed by fluorogenic peptide overlay to identify multiple forms of rabbit reticulocyte multicatalytic protease (MCP) or 20 S protease, and two forms of rabbit 26 S ubiquitin/ATP-dependent protease. An abundant, fast-migrating 20 S complex (20 SF) possesses modest ability to hydrolyze the fluorogenic peptide succinyl-Leu-Leu-Val-Tyr-4-methyl-coumaryl-7-amide. In contrast, two minor, slower migrating species cleave the peptide at high rates. A unique 30-kDa polypeptide is associated with one of the active MCPs, and a 160-kDa subunit is associated with the other. Two electrophoretically distinct 26 S proteases can also be isolated from rabbit reticulocyte lysate. The faster migrating form, 26 SF, is more resistant to inactivation by ATP depletion. Despite the differential response to nucleotides and the distinctive electrophoretic mobilities of 26 SF and 26 SS, we have not identified any subunit differences between the two enzymes. In addition to active 26 S proteases, we have discovered and purified a proteolytically inactive particle that contains subunits characteristic of the 26 S protease (e.g. molecular masses between 30 and 110 kDa). Incubation of this protein complex with purified MCP and ATP results in the formation of the 26 S proteases.     

Catalytic activities of the 20 S proteasome, a multicatalytic proteinase complex

The proteasome, a multisubunit, multicatalytic proteinase complex, is attracting growing attention as the main intracellular, extralysosomal, proteolytic system involved in ubiquitin-(Ub) dependent and Ub-independent intracellular proteolysis. Its involvement in the mitotic cycle, and control of the half-life of most cellular proteins, functions absolutely necessary for cell growth and viability, make it an attractive target for researchers of intracellular metabolism and an important target for pharmacological intervention. The proteasome belongs to a new mechanistic class of proteases, the N-terminal nucleophile hydrolases, where the N-terminal threonine residue functions as the nucleophile. This minireview focuses on the three classical catalytic activities of the proteasome, designated chymotrypsin-like, trypsin-like, and peptidyl-glutamyl-peptide hydrolyzing in eukaryotes and also the activities of the more simple Archaebacteria and Eubacteria proteasomes. Other catalytic activities of the proteasome and their possible origin are also examined. The specificity of the catalytic components toward synthetic substrates, natural peptides, and proteins and their relationship to the catalytic centers are reviewed. Some unanswered questions and future research directions are suggested.     

Double-headed protease inhibitors from black-eyed peas. I. Purification of two new protease inhibitors and the endogenous protease by affinity chromatography.

Two new double-headed protease inhibitors have been isolated from black-eyed peas. The isoinhibitors can be purified to homogeneity with greater than 90% recovery in a four-step procedure by means of sequential affinity chromatography on trypsin-Sepharose and chymotrypsin-Sepharose affinity columns. The isoinhibitors both have molecular weights near 8,000 and both have the same NH1-terminal residue serine. Black-eyed pea chymotrypsin and trypsin inhibitor (BEPCI) has an isoelectric point of 5.1 and inhibits trypsin and chymotrypsin simultaneously. Black-eyed pea trypsin inhibitor (BEPTI) has an isoelectric point of 6.5 and inhibits 2 molecules of trypsin simultaneously. BEPTI binds to chymotrypsin-Sepharose above pH 6 but does not inhibit chymotrypsin in the standard inhibitor assay with 10-3 M substrate. These new inhibitors are distinct from the Ventura inhibitor isolated from Serido black-eyed peas. An endogenous seed protease has been isolated from black-eyed peas by affinity chromatography on soybean inhibitor-carboxymethylcellulose affinity columns. A protease-BEPCI complex has been isolated by ion exchange chromatography. A dual physiological function of inhibition and protection of the seed protease is suggested as a plausible role of seed protease inhibitors.