PURIFICATION AND CHARACTERIZATION OF THE 20S PROTEASOME FROM THEALGA CHARA CORALLINA (CHAROPHYCEAE)1

We purified the 20S proteasome from the alga Chara corallina Willd with DEAE–ion‐exchange column chromatography and preparative nondenaturing PAGE. The analysis of the purified enzyme bynondenaturing PAGE gave a single band whose molecular mass was estimated to be about 600,000 Da by gel permeation chromatography and whose isoelectric point was at pH 5.5. Two‐dimensional gel electrophoresis gave at least 12 spots with molecular masses from 26,000 to 32,000 Da in a wide range of isoelectric points. The 20S proteasome hydrolyzed three types of artificial substrates used to differentiate chymotrypsin‐like, trypsin‐like, and peptidyl glutamyl peptidase activities. Both the chymotrypsin‐like and the peptidyl glutamyl peptidase activities were enhanced by SDS. In the presence of 0.03% SDS, the optimal pH for both activities was 8.5. Trypsin‐like activity of the 20S proteasome had a broad pH optimum in an alkaline region and was not activated but inhibited by SDS. Its chymotrypsin‐like activity was inhibited by N‐ethylmaleimide, p‐chloromercuribenzoic acid, and chymostatin. In contrast, its peptidyl glutamyl peptidase activity was not inhibited by chymostatin. Moreover, proteasome inhibitors MG 115 and MG 135 were effective against the chymotrypsin‐like activity and less so against the peptidyl glutamyl peptidase activity. These properties were very similar to those of the proteasomes of mammalian, yeast, and spinach cells. The large size of Chara cells will make in vivo manipulations and investigations of the proteasome proteolytic system possible.     

Purification and Some Properties of Carboxylesterase from Arthrobacter globiformis; Stereoselective Hydrolysis of Ethyl Chrysanthemate

An esterase with excellent stereoselectivity for (+)-trans-ethyl chrysanthemate was purified to homogeneity from Arthrobacter globiformis SC-6-98-28. The purified enzyme hydrolyzed a mixture of ethyl chrysanthemate isomers stereoselectively to produce (+)-trans-acid with 100% stereoisomeric purity. The apparent molecular weight of the purified enzyme was 43,000 on SDS–polyacrylamide gel electrophoresis, and 94,000 on gel filtration chromatography. The optimum conditions for the ester hydrolysis were pH 10.0 at 45°C. The purified esterase hydrolyzed short-chain fatty acid esters, but did not have detectable activity on long-chain water-insoluble fatty acid esters. The enzyme activity was inbibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride.     

Further purification and characterization of trehalases from the American cockroach,Periplaneta americana

Summary A soluble trehalase was purified more than 200-fold from the male accessory gland of the American cockroach,Periplaneta americana, by CM-cellulose, hydrophobic chromatography, and Sephacryl S-200 gel filtration. The final preparation was homogeneous as judged by polyacryl-amide gel electrophoresis in the absence and presence of SDS, isoelectric focusing, and immuno-diffusion tests. The purified enzyme was maximally active at pH 5.2, and showed high specificity for trehalose with aK _m of 0.98 mM. The isoelectric point was 4.7. The molecular weight of the enzyme (75,000) was determined by molecular sieve chromatography and SDS-polyacrylamide gel electrophoresis. The amino acid composition was determined and compared with those of trehalases purified from other sources. The trehalase could be stained for carbohydrate with the periodic acid-Schiff's reagent following SDS-polyacrylamide gel electrophoresis, indicating that it was a glycoprotein. Another soluble trehalase and two types of fat body trehalases could be highly purified by the method described. A comparison of the properties of trehalases from the accessory gland and the fat body showed some resemblance.     

Purification and Characterization of an Extracellular Alkaline Serine Protease from Aspergillus terreus (IJIRA 6.2)

An extracellular alkaline serine protease has been purified from Aspergillus terreus (IJIRA 6.2). The purification procedure involved chromatography on DEAE-Sephadex A25, phosphocellulose, hydroxyapatite, casein-Sepharose, gel filtration on Sephacryl-S-300 and by glycerol density gradient centrifugation. The enzyme was further purified to apparent homogeneity through a combination of electrophoresis in polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) with or without protease substrate (gelatin) and subsequent regeneration of its activity in situ by removal of SDS. The active enzyme was visualized in a zymogram or on the basis of protease activity exhibited on an X-ray film. The protein in the unstained segment of the gel was electroeluted. The eluted protein with protease activity exhibited a molecular mass of 37,000-daltons on electrophoresis in SDS-polyacrylamide gel. A sedimentation coefficient of 3.2S was obtained by glycerol density gradient contrifugation. Maximum activity of protease was observed at pH 8.5 and at 37°C. Purified protease was active between pH 5.5 and 9.5 and was found to be stable up to 60°C. With Na-caseinate, the K _m of the purified protease was found to be 0.055 mM. Antipain, phenylmethane sulfonyl fluoride, and chymostatin served as non-competitive inhibitors. Substrate specificity was determined by using a synthetic chromogenic peptide containing N-P-Tosyl-Gly-Pro-Arg-p-nitroanilide. Results showed that the protease cleaved the peptide on the -COOH end of arginine residue. Received: 8 October 1999 / Accepted: 3 November 1999     

Purification, characterization and inhibition of sterol C24-methyltransferase from Candida albicans

Solubilized sterol C24-methyltransferase (24-SMT) was purified to homogeneity from a cell extract of the yeast Candida albicans (Ca) by anion exchange chromatography, gel permeation chromatography and fast performance liquid chromatography using a Mono Q column. The purified enzyme has an apparent molecular mass of 178 kDa on gel permeation chromatography and 43 kDa on SDS/PAGE, indicating that it is composed of four identical subunits. The substrate requirement of the native enzyme has an optimal specificity for zymosterol with associated kinetic constants of K_m 50 μM and k_cat of 0.01 s⁻¹. The product of the enzyme incubated with zymosterol was fecosterol. Inhibition of the catalyst was observed with substrate analogs designed as transition state analogs (25-azalanosterol, K_i = 54 nM and 24 (R,S),25-epiminolanosterol, K_i = 11 nM) or as mechanism-based inactivators (26,27-dehydrozymosterol, K_i 9 μM) and k_inact = 0.03 min⁻¹) of the C24-methylation reaction. Product analogs ergosterol and fecosterol, but neither cholesterol nor sitosterol, inhibited activity affording K_i values of 20 and 72 μM, respectively. Ammonium and thia analogs of the intermediates of the sterol C24-methyl reaction sequence were effective growth inhibitors exhibiting IC₅₀ values that ranged from 3 to 20 μM.     

An activated by cobalt alkaline aminopeptidase from <Emphasis Type="Italic">Bacillus mycoides</Emphasis>

An intracellular arginine—specific aminopeptidase synthesized by Bacillus mycoides was purified and characterized. The purification procedure for studied aminopeptidase consisted of ammonium sulphate precipitation and three chromatographic steps: anion exchange chromatography and gel permeation chromatography. A molecular weight of ∼50 kDa was estimated for the aminopeptidase by gel permeation chromatography and SDS-PAGE. The optimal activity of the enzyme on arginyl-β-naphthylamide as a substrate was at 37°C and pH 9.0. The enzyme showed maximum specificity for basic amino acids: such as Arg and Lys but was also able to hydrolyze aromatic amino acids: Trp, Tyr, and Phe. Co²⁺ ions activated the enzyme, while Zn²⁺, Cu²⁺, Hg²⁺ and Mn²⁺ inhibited it. The enzyme is a metalloaminopeptidase whose activity is inhibited by typical metalloaminopeptidase inhibitors: EDTA and 1,10-phenanthroline. Analysis of fragments of the amino acid sequence of the purified enzyme demonstrated high similarity to Amp S of Bacillus cereus and APII of B. thuringensis.     

Purification and Some Properties of Thiosulfate Dehydrogenase from Acidithiobacillus ferrooxidans

Abstract

Thiosulfate dehydrogenase was purified from Acidithiobacillus ferrooxidans using three purification steps. The purification procedure involved ammonium sulfate fractionation, ion‐exchange chromatography, and gel permeation chromatography. Specific activity of the purified enzyme (after IEC) was 3.26 nkat/mg, and yield of the enzyme was 78%. The purity of the enzyme was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme is a tetramer composed of four probably identical subunits of relative molecular weight 45,000. The pH optimum of the enzyme reaction in the direction of substrate oxidation was found to be 3.0. The isoelectric point of the enzyme was 8.3. Enzyme activity was found to be particularly sensitive to the histidine‐selective reagent diethylpyrocarbonate. Reagents selective for arginine, cysteine, and tryptophane had no effect on enzyme activity.     

Purification and properties of 3-ketovalidoxylamine A C-N lyase from Flavobacterium saccharophilum

3-Ketovalidoxylamine A C-N lyase was purified about 900-fold from the cell-free extract of Flavobacterium saccharophilum by ammonium sulfate fractionation, column chromatography on CM cellulose and gel filtration on Sephacryl S-200. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 36,000 by gel filtration on Sephacryl S-200 and by SDS polyacrylamide gel electrophoresis, indicating that the enzyme is a monomer. The optimum pH was found at 9.0. The enzyme activity was inhibited by EDTA or ethyleneglycol bis(beta-aminoethylether)-N,N'-tetraacetic acid and the inhibition was reversed by Ca2+ ion. The enzyme was able to eliminate p-nitroaniline or p-nitrophenol from p-nitrophenyl-3-ketovalidamine (IV) or p-nitrophenyl-alpha-D-3-ketoglucoside (VI), but not from p-nitrophenyl-1-epi-3-ketovalidamine or p-nitrophenyl-beta-D-3-ketoglucoside. Apparent Km values for IV and VI were 0.24 mM and 0.5 mM, respectively.     

Purification of a -hydantoinase using a laboratory-scale Streamline phenyl column as the initial step

A -hydantoinase from Thermus sp. was overexpressed in Escherichia coli and purified to homogeneity for subsequent crystallization. The purification was performed with hydrophobic interaction chromatography as the capture step followed by anion-exchange chromatography and gel permeation chromatography as intermediate purification and polishing steps, respectively. The hydrophobic interaction step was done in fluidized bed mode in a laboratory-scale Streamline column made from conventional laboratory equipment. The whole purification protocol could be finished within one day. The purified enzyme crystallizes. The crystals are suitable for X-ray protein structure analysis and diffract to at least 2.3 Å resolution. Complete data sets have been measured up to 2.6 Å resolution. The X-ray structure is currently being solved.     

PURIFICATION OF BOVINE PINEAL HYDROXYINDOLE O-methylTRANSFERASE BY IMMUNOADSORPTION CHROMATOGRAPHY

A procedure is described for the use of immunoadsorption chromatography of hydroxyindole O-methyltransferase (HIOMT). HIOMT was purified from bovine pineal extract by affinity chromatography on immunoglobulins (Ig)-Sepharose. The overall purification was about 45-fold; the yield was 84%. This enzyme constitutes about 2.0% of the soluble proteins in the pineal gland. The enzyme represented a single precipitin line on Ouchterlony double diffusion plate and immunoelectrophoresis. Ultracentrifugation analysis indicated the existence of molecular aggregates of enzyme and disc gel electrophoresis showed one main protein band and several minor bands. However sodium dodecyl sulphate (SDS) gel electrophoresis showed a single protein band with subunit molecular weight 38,000 demonstrating bovine pineal HIOMT to be polymer enzyme of a single subunit. The properties of the purified enzyme including disc gel electrophoretic pattern, the effect of pH, chemicals and substrates and immunological properties were identical with those of the crude enzyme.     

Effect of Pepsin Inhibitor (S-PI) on the Growth of Rhodotorula glutinis No. K-24

A tissue-type transglutaminase (TGase) was purified from liver tissue of the red sea bream, Pagrus major, by ion-exchange chromatography and heparin-Sepharose affinity chromatography. Its activity was assessed using a fluorometric assay to measure the incorporation of monodansylcadaverine into N,N′-dimethyl casein. The molecular mass of purified TGase was estimated to be 78kDa by SDS–polyacrylamide gel electrophoresis. The enzyme required Ca²⁺ to express its activity, although 10 mM Sr²⁺ also activated the enzyme fully. TGase activity was maximal at pH 9.0–9.5, and the enzyme was strongly inhibited by sulfhydryl reagents. The purified enzyme catalyzed the cross-linking of myosin heavy chain obtained from Alaska pollack, resulting in gelation of an actomyosin solution. The partial amino acid sequence of this fish TGase showed divisionally significant similarity to TGase from guinea pig liver.     

Purification and characterization of an aminopeptidase from the chloroplast stroma of barley leaves by chromatographic and electrophoretic methods

Aminopeptidases catalyze the cleavage of amino acids from the amino terminus of protein or peptide substrates. Although some aminopeptidase activities have been found in plant chloroplasts, the identity of these proteins remains unclear. In this work, we report the purification to apparent homogeneity of a soluble aminopeptidase from isolated barley chloroplasts which preferentially degraded alanyl-p-nitroanilide (Ala-pNA). After organelle isolation in a density gradient and precipitation of soluble proteins with ammonium sulfate, the proteins were purified in three consecutive steps including hydrophobic interaction, gel permeation and ion-exchange chromatographies. The purified enzyme appeared as a single band with a M_r of 84 000 in sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. The M_r of the native enzyme was estimated to be 93 000 by gel permeation chromatography, suggesting that the protein is a monomer. Mass spectrometry analysis of tryptic digests indicates that the primary structure of the protein has not been reported previously. The enzyme was characterized as a metalloprotease as it could be totally inhibited by 1,10-phenanthroline. Strong inhibition could also be observed using the specific aminopeptidase inhibitors amastatin and bestatin. Besides Ala-pNA, the purified protein could also cleave with decreasing activity glycyl-pNA, leucyl-pNA, lysyl-pNA, methionyl-pNA and arginyl-pNA. The possible physiological role of this enzyme in the chloroplast stroma is discussed.     

Purification and Some Properties of α-Galactosidase from Tubers of Stachys affinis

An α-galactosidase from tubers of S. affinis was purified about 130 fold by ammonium sulfate fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. The purified enzyme showed a single protein band on disc gel electrophoresis. The molecular weight of the enzyme was determined to be approximately 42,000 by gel filtration and 44,000 by SDS disc gel electrophoresis. The optimum reaction pH was 5.2. The enzyme hydrolyzed raffinose more rapidly than planteose. The activation energy of raffinose and planteose by the enzyme was estimated to be 7.89 and 11.4 kcal/mol, respectively. The enzyme activity was inhibited by various galactosides and structural analogs of d-galactose. Besides hydrolytic activity, the enzyme also catalyzed the transfer reaction of d-galactosyl residue from raffinose to methanol.     

Isolation and purification of cell wall-associated arginine carboxypeptidase fromStreptococcus mitis ATCC 15909

An arginine carboxypeptidase was isolated from the cell walls ofStreptococcus mitis ATCC 15909 by mutanolysin extraction of the walls. The enzyme was purified 32-fold by gel filtration on Sephacryl S-300, affinity chromatography on Arginine-Sepharose 4B and by rechromatography on Sephacryl S-300. The molecular mass of the enzyme was calculated to 122 kDa by gel filtration. The enzyme released arginine from the carboxy terminal of hippurylarginine and, at a low rate, lysine from furylacryloylalanyl-lysine and hippuryl-lysine. The carboxypeptidase seemed firmly bound to the cell wall because SDS treatment of the walls did not release measurable amounts of activity.     

Purification and characterization of human placental microsomal aminopeptidase: immunological difference between placental microsomal aminopeptidase and pregnancy serum cystyl-aminopeptidase

Human placental microsomal aminopeptidase (microsomal PAP) was purified 3,880-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100 and also trypsin digestion, and subjected to zinc sulfate fractionation, chromatographies with DE-52, hydroxylapatite, Sephacryl S-300 and lentil lectin-Sepharose 4B, and finally affinity chromatography with bestatin-Sepharose 4B. Microsomal PAP was separated from aminopeptidase A (AAP) by affinity chromatography. The apparent relative molecular mass (Mr) of the enzyme was estimated to be 220,000 by high-performance liquid chromatography with an aqueous gel column. The purified enzyme gave almost a single band with a molecular mass of 140,000 by sodium dodecyl sulfate (SDS) gel electrophoresis. The isoelectric point of the enzyme was 5.2. The purified enzyme was most active at pH 8.0 with L-leucine-p-nitroanilide as substrate; the Km value for this substrate was 1.1 mmol/l. The microsomal PAP was immunologically different from the pregnancy serum cystyl aminopeptidase (serum PAP).     

Properties of genetically overproducedE. coli xylose isomerase

Summary Xylose isomerase was purified from a transformedE. coli strain (LE392-pRK248/pTXI-1) (Lastick et al., 1986) that overproduces the enzyme by induction of the strong lambda P_L promotor. Kinetic data, N-terminal sequence analysis, SDS polyacrylamide gel electrophoresis, size exclusion chromatography and immunodiffusion were used to compare the overproduced enzyme with xylose isomerase purified from xylose induced, non-transformedE. coli LE392 cells; no differences between these purified enzyme preparations were found.     

The predominant form of mammalian DNA topoisomerase Iin vivo has a molecular mass of 100 kDa

DNA topoisomerase I was purified to apparent homogeneity from human HeLa cells as a single polypeptide with a molecular mass of 100 kDa, as assayed by both gel filtration column chromatography and SDS-polyacrylamide gel electrophoresis. No smaller forms of the enzyme were detected in the purified fraction. Therefore, smaller forms, which have been observed by other investigators, are likely to be the result of proteolysis during isolation and are not relevant to thein vivo activity of DNA topoisomerase I.Abbreviations 2-ME 2-Mercaptoethanol - DTT Dithiothreitol - PMSF Phenylmethylsulfonyl Fluoride - SDS Sodium Dodecyl Sulfate     

Purification and characterization of an extracellular prolyl endopeptidase from Agaricus bisporus

Prolyl endopeptidase [EC 3.4.21.26] was purified to homogeneity from the culture filtrate of Agaricus bisporus by a procedure that comprised ammonium sulfate fractionation, anion-exchange chromatographies on DEAE-Toyopearl and DEAE-Sephadex, hydroxylapatite chromatography, and high-performance liquid chromatography (HPLC) on a TSKgel G 2000 SW column. The overall activity recovery was 8.6%. The enzyme was most active at or around pH 7.5 and was stable in the range of pH 5-9 when checked with Z-Gly-Pro-beta-naphthylamide as a substrate. The isoelectric point of the enzyme was about 4.8. The enzyme was a monomeric protein of molecular weight 78,000 +/- 2,000 as judged by gel permeation chromatography on Sephadex G-150 and electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide gel. The enzyme hydrolyzed Pro-X bonds and at least five subsites (S3, S2, S1, S1', and S2') were found to be involved in enzyme-substrate binding. Among them, S2, S1, and S1' subsites of the enzyme showed high stereospecificity. The enzyme was strongly inhibited by diisopropylfluorophosphate (DFP), Z-Gly-Pro-CH2Cl, Z-Pro-prolinal, Z-Pro-pyrrolidine, Z-Thiopro-pyrrolidine, Z-Pro-thiazolidine, Z-Thioprothiazolidine, and p-chloromercuribenzoate (PCMB), while it was not inhibited by phenyl-methylsulfonyl fluoride (PMSF), E-64, iodoacetamide, or metal chelators. Although the A. bisporus enzyme showed no immunological cross reaction with anti-bovine prolyl endopeptidase antiserum, the other characteristics were quite similar to those of mammalian and plant enzymes.     

Purification and characterization of an intracellular heat-stable proteinase (pernilase) from the marine hyperthermophilic archaeon Aeropyrum pernix K1

A novel intracellular serine proteinase from the marine aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) that we designated pernilase was purified by ammonium sulfate precipitation, anionic-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified enzyme was composed of a single polypeptide chain with a molecular mass of 50 kDa as determined by SDS-PAGE. The proteinase had a broad pH profile (pH 5–10) with an optimum pH of 9.0 for peptide hydrolysis. The optimum temperature for enzyme activity was 90°C. The enzyme was strongly inhibited by diisopropyl fluorophosphate (DFP) and phenylmethyl sulfonylfluoride (PMSF), suggesting that it corresponds to a serine proteinase. The enzyme was highly resistant to the reducing agents dithiothreitol and 2-mercaptoethanol but sensitive to the denaturing reagents guanidine-HCl and urea and also to the detergent sodium dodecyl sulfate (SDS). Pernilase showed high substrate specificity for Boc-Leu-Gly-Arg-MCA peptide. Thermostability of this enzyme showed half-lives of 85 min at 100°C and 12 min at 110°C. Received September 24, 1997 / Accepted May 20, 1998     

Improved purification of α-amylase isolated fromRhizomucor pusillus by affinity chromatography

A highly purified extracellular -amylase was isolated fromRhizomucor pusillus with minimum loss of enzymatic activity. The enzyme was purified from the mycelium-free liquid filtrate of the thermophilic moldRhizomucor pusillus. Maximum enzyme yields were attained after 5 days of growth on liquid starch-yeast extract at 45°C and pH 7.0. The crude enzyme preparation was first concentrated 80-fold by ultrafiltration. Purification was recently achieved with high-performance liquid chromatography and Waters Protein Pak 300 SW. Improved purification was then achieved with a dextrin-bound affinity column, with a 59-fold increase in specific activity from the crude enzyme preparation. This final enzyme preparation produced a single band on polyacrylamide gel electrophoresis. The molecular weight determined by SDS gel electrophoresis was 52,000 daltons.