Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues.

Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin.     

Toxic Metabolites of an Unidentified Filamentous Fungus Isolated from Zinnia Leaves

Two ribonucleases (RNases) designated RNase I and RNase II were found in Euphausia superba and isolated by (NH₄)₂SO₄ fractionation, 2 cycles of CM-cellulose chromatography and gel filtration on Sephadex G-100. This procedure resulted in a 2,116-fold purification of RNase I and a 130-fold purification of RNase II. The molecular weight of both purified enzymes was estimated by gel filtration to be 31,500. The isoelectric points were 6.0 (RNase I) and 7.0 (RNase II). Each enzyme hydrolyzed poly A-U, poly U but did not degrade poly G, poly C and DNA. Both enzymes were classified as endonuclease from the hydrolysis product of yeast RNA and poly A. The enzymes were located mainly in the cardiac and pyloric portion of the stomach.     

Purification and chemical properties of two 1,3;1,4-beta-glucan endohydrolases from germinating barley

Two 1,3;1,4-beta-glucan endohydrolases have been purified from extracts of germinating barley by ammonium sulphate precipitation, ion-exchange and gel filtration chromatography. Both enzymes are monomeric, basic proteins. Enzyme I has a molecular weight of 28000 and an isoelectric point of 8.5, while enzyme II has a molecular weight of 33000 and an isoelectric point greater than 10. Enzyme II is a glycoprotein containing 3.6% carbohydrate, of which three residues are probable N-acetylglucosamine, but enzyme I contains only traces of associated carbohydrate. The amino acid compositions of the two 1,3;1,4-beta-glucan endohydrolases are similar and the cross-reactivity of antibodies raised against the purified enzymes suggests that they share common antigenic determinants.     

Purification and characterization of two novel arginine aminopeptidases from Streptococcus mitis ATCC 9811

Two novel aminopeptidases (I and II) which have specificity for amino-terminal arginine residues and strong sensitivity to divalent cations were purified from Streptococcus mitis ATCC 9811 by a procedure that involved treatment with a lytic enzyme for bacterial cell walls, followed by a series of chromatographies. Enzyme I was obtained as a homogeneous protein as judged by polyacrylamide gel electrophoresis and had a specific activity of 484.8 units per mg protein using L-arginine-2-naphthylamide as substrate; its Km value was 2.6 X 10(-5) M. The molecular weight was estimated to be 62,000, and its isoelectric point was pH 4.4. Enzyme II was purified to a specific activity of 128.0 units per mg protein and had a Km value of 3.8 X 10(-5) M. The molecular weight was estimated to be 360,000, and its isoelectric point was pH 5.7. The pH optima of enzymes I and II were 8.6 and 7.6, respectively. Both enzymes were inactivated by sulfhydryl reagents and metal ions but were markedly activated by EDTA. The chloride ion had an inhibitory rather than a stimulatory effect on the activity of both enzymes. Substrate specificity studies indicated that both the enzymes specifically hydrolyze N-terminal arginine residues from a-aminoacyl 2-naphthylamides and peptides, but they could not attack the L-arginyl-L-prolyl-peptide.     

Purification and Some Properties of α-Galactosidase from Candida guilliermondii H-404

Candida guilliermondii H-404, isolated from soil, produced thermostable α-galactosidase, but small amounts of other glycosidases (such as β-galactosidase, α-glucosidase, and β-glucosidase). The enzyme was separated into two fractions by DEAE-Toyopearl 650M chromatography, and the two enzymes were designated galactosidase I and II. These two enzymes had the same molecular weight (270,000 by gel filtration, 64,000 by SDS-PAGE). The isoelectric points of α-galactosidase I and II were 6.16 and 6.21, respectively. These two enzymes were different from each other in pH stability, temperature stability, and effects of Fe^{2 +} and Cu^{2 +} ion on α-galactosidase activity. The enzyme had stronger transfer activity and wider acceptor specificity than α-galactosidases which have been reported.     

Isolation of L-dynurenine 3-hydroxylase from the mitochondrial outer membrane of rat liver.

Outer membrane preparations of rat liver mitochondria were isolated, after the mitochondria had been prepared by mild digitonin treatment under isotonic conditions. L-Kynurenine 3-hydroxylase [EC 1.14.13.9] was solubilized on a large scale from outer membrane by mixing with 1% digitonin or 1% Triton X-100, followed by fractionation into a minor fraction I and a major fraction II by DEAE-cellulose column chromatography. The distribution of total L-Dynurenine 3-hydroxylase was roughly 20 and 80% in fraction I and II, respectively. Fraction I consisted of crude enzyme loosely bound to anion exchanger. In the present investigation, fraction I was not used because of its low activity and rapid inactivation. In contrast, fraction II consisted of crude enzyme with high activity, excluded from DEAE-cellulose column chromatography in the presence of 1 M KC1. In addition, fraction II was purified by Sephadex G-200 gel filtration and DEAE-Sephadex A-50 column chromatography with linear gradient elution, adding 1 M KC1 and 1% Triton X-100 to 0.05 M Tris-acetate buffer, pH 8.1. After isoelectric focusing, the purified enzyme preparation was proved to be homogeneous, since the L-kynurenine 3-hydroxylase fraction gave a single band on disc gel electrophoresis. The molecular weight of this enzyme was estimated to be approximately 200,000 or more by SDS-polyacrylamide gel electrophoresis and from the elution pattern on Sephadex G-200 gel filtration. A 16-Fold increase of the enzyme activity was obtained compared with that of the mitochondrial outer membrane. The isoelectric point of the enzyme was determined to be pH 5.4 by Ampholine isoelectric focusing.     

Resolution and characterization of two soluble calcium-dependent protein kinases from silver beet leaves

Two soluble Ca²⁺-dependent protein kinases (enzymes I and II) have been extensively purified from silver beet leaf tissue by means of a protocol involving batch-wise elution from DEAE-cellulose, Ca²⁺-dependent binding to phenyl-Sepharose, gradient elution from DEAE-Sephacel, gel filtration and binding to Cibacron F3GA-Sepharose CL-6B. Protein kinases I and II are resolved on gradient elution from DEAE-Sephacel and are further distinguished by their different K_m values for ATP and large differences in relative rates of phosphorylation of histone H1, casein and bovine serum albumin (the latter two proteins are relatively poor substrates for enzyme II but not enzyme I). Both enzymes have similar molecular weights as determined from gel filtration (56000 ± 2000 and 57000 ± 3000 for enzymes I and II, respectively). Both enzymes are absolutely dependent on free Ca²⁺ for activity with maximal histone H1 kinase activity being obtained at 0.5 μM free Ca²⁺. A millimolar concentration of Mg²⁺ is required in addition to a micromolar concentration Ca²⁺ for maximal activity. Both enzymes specifically phosphorylate serine residues of histone H1, are thiol activated and are inhibited by lanthanides and a range of calmodulin antagonists and inhibitors of protein kinase C.     

Purification and properties of two exo-cellobiohydrolases from a cellulolytic culture of Fusarium lini

Two distinct exo-cellobiohydrolases (1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91) have been isolated from culture filtrates of Fusarium lini by repeated ammonium sulphate fractionation and isoelectric focusing. The purified enzymes were evaluated for physical properties, kinetics and the mechanism of their action. The results of this work were as follows. (1) A two-step enzyme purification procedure was developed, involving isoelectric focusing and ammonium sulphate fractionation. (2) Yields of pure cellobiohydrolases I and II were 45 and 36 mg l^?1 of culture broth, respectively. (3) Both enzymes were found to be homogeneous, as determined by ultracentrifugation, isoelectric focusing, electrophoresis in polyacrylamide gels containing SDS and chromatography on Sephadex. (4) The molecular weights of the two cellobiohydrolases, as determined by gel filtration and SDS gel electrophoresis, were 50 000–57 000. (5) Both cellobiohydrolases had low viscosity-reducing and reducing sugar activity from carboxymethyl cellulose and high activity with Walseth cellulose and Avicel. (6) The enzymes produced only cellobiose as the end product from filter paper and Avicel, indicating that they are true cellobiohydrolases. (7) Cellobiohydrolase I hydrolysed d-xylan whereas cellobiohydrolase II was inactive towards d-xylan. (8) There was a striking synergism in filter paper activity when cellobiohydrolase was supplemented with endo-1,4-β-d-glucanase [cellulase, 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21).     

Purification and characterization of two extracellular beta-glucosidases from Trichoderma reesei.

A major beta-glucosidase I and a minor beta-glucosidase II were purified from culture filtrates of the fungus Trichoderma reesei grown on wheat straw. The enzymes were purified using CM-Sepharose CL-6B cation-exchange and DEAE Bio-Gel A anion-exchange chromatography steps, followed by Sephadex G-75 gel filtration. The isolated enzymes were homogeneous in SDS-polyacrylamide gel electrophoresis and isoelectric focusing. beta-Glucosidase I (71 kDa) was isoelectric at pH 8.7 and contained 0.12% carbohydrate; beta-glucosidase II (114 kDa) was isoelectric at pH 4.8 and contained 9.0% carbohydrate. Both enzymes catalyzed the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (pNPG). The Km and kcat/Km values for cellobiose were 2.10 mM, 2.45.10(4) s-1 M-1 (beta-glucosidase I) and 11.1 mM, 1.68.10(3) s-1 M-1 (beta-glucosidase II). With pNPG as substrate the Km and kcat/Km values were 182 microM, 7.93.10(5) s-1 M-1 (beta-glucosidase I) and 135 microM, 1.02.10(6) s-1 M-1 (beta-glucosidase II). The temperature optimum was 65-70 degrees C for beta-glucosidase I and 60 degrees C for beta-glucosidase II, the pH optimum was 4.6 and 4.0, respectively. Several inhibitors were tested for their action on both enzymes. beta-Glucosidase I and II were competitively inhibited by desoxynojirimycin, gluconolactone and glucose.     

Purification and properties of cellulase enzymes from Robillarda sp. Y-20

One endo-β-1,4-glucanase (EC 3.2.1.4) and two unique β-glucosidases (EC 3.2.1.21) have been isolated from culture filtrates Robillarda sp. Y-20 by combinations of DEAE A-50 column chromatography and isoelectric focusing. These enzymes were homogeneous on gel filtration, isoelectric focusing and polyacrylamide gel electrophoresis with and without sodium dodecyl sulphate (SDS). The molecular weights of endoglucanase, and the two β-glucosidases, I and II by SDS-polyacrylamide gel electrophoresis were 59000, 76000 and 54000, respectively. The pI values were 3.5, 7.5, and 3.8 for endoglucanase, β-glucosidase I and II, respectively. The major β-glucosidase I was a glycoprotein, but the endoglucanase and β-glucosidase II were not. The endoglucanase rapidly reduced the viscosity of carboxymethyl (CM) cellulose with concomitant production of reducing sugar. The enzyme had very low activity with crystalline cellulose such as insoluble acid treated cellulose, Avicel and filter paper. The endoglucanase attacked celloheptaose to cellotetraose more readily than cellotriose, but did not hydrolyze cellobiose. Both β-glucosidases attacked celloheptaose to cellotetraose more readily than cellotriose and cellobiose, but did not hydrolyze CM-cellulose and insoluble acid treated cellulose. Strong synergism was observed for hydrolysis of CM-cellulose by the endoglucanase and β-glucosidases.     

Gas Chromatographic Determination of Optical Purity of a Chiral Derivatization Reagent,N-Acyl-l-prolyl Chloride

Two endo-type cellulases, tentatively called carboxymethyl cellulases (CMCases) I and II, were purified by gel filtration, ion-exchange chromatography, affinity chromatography, and chromato-focusing from a culture supernatant of Penicillium purpurogenum. Their homogeneity was verified by analytical polyacrylamide gel electrophoresis. The molecular weights of CMCases I and II, estimated by gel filtration, were 72,000 and 50,000, respectively. CMCases I and II contained about 12% and 8% carbohydrate, and had isoelectric points of 4.3 and 3.9, respectively. CMCase I produced cellobiose, glucose, and a trace amount of cellotriose from H₃PO₄-swollen cellulose and Avicel (microcrystalline cellulose), while CMCase II produced cellobiose and cellotriose with a small amount of glucose and cellotetraose. The products from reduced cellopentaose by both enzymes were released predominantly in the β-configuration. CMCase II appeared to act in more random fashion than I against carboxymethyl cellulose. These results suggest that both enzymes attack insoluble cellulose randomly, although there are some differences in the mode of hydrolytic action.     

Purification, properties, and evidence for two subtypes of human placenta hexokinase type I

In human placenta 85% of total hexokinase activity (EC 2.7.1.1) was found in a soluble form. Of this, 70% is hexokinase type I while the remaining 30% is hexokinase type II. All the bound hexokinase is type I. Soluble hexokinase I was purified 11,000-fold by a combination of ion-exchange chromatography, affinity chromatography, and dye-ligand chromatography. The specific activity was 190 units/mg protein with a 75% yield. The enzyme shows only one band in nondenaturing polyacrylamide gel electrophoresis that stains for protein and enzymatic activity; however, two components (with Mr 112,000 and 103,000) were constantly seen in sodium dodecyl sulfate-gel electrophoresis. Many attempts were made to separate these two proteins under native conditions; however, only one peak of activity was obtained when the enzyme was submitted to gel filtration (Mr 118,000), preparative isoelectric focusing (pI 5.9), anion-exchange chromatography, hydroxylapatite chromatography, and affinity chromatography on immobilized dyes and immobilized glucosamine. The high and low molecular weight hexokinases show the same isoelectric point under denaturing conditions as determined by two-dimensional gel electrophoresis. Each hexokinase subtype was obtained by preparative sodium dodecyl sulfate electrophoresis followed by electroelution. Monospecific antibodies raised in rabbits against electroeluted high and low molecular weight hexokinases were not able to recognize the native enzymes but each of them detected both hexokinases on immunoblots. Amino acid compositions and peptide mapping by limited proteolysis of the high and low molecular weight hexokinases were also performed and suggested a strong homology between these two subtypes of human hexokinase I.     

Heat-Moisture Treatment of Cereal Starch Observed by X-ray Diffraction

Chitinases I and II were purified from the culture supernatant of Aeromonas sp. 10S-24 by ammonium sulfate precipitation, SP-Sephadex C-50 chromatography, Sephacryl S-200 gel filtration, and chromatofocusing. Both enzymes were most active at pH 4.0 and the optimum temperature for I and II were 50°C and 60°C. Chitinase I was stable at pHs between 4 and 9 and at temperatures below 50°C and chitinase II was stable at pHs between 5 and 7 and at temperatures below 45°C. The molecular weights were estimated by 8D8 polyacrylamide gel electrophoresis to be 112,000 and 115,000 for I and II respectively, while gel filtration showed the molecular weight to be 114,000 for both types of the enzyme. The pIs for I and II were 7.9 and 8.1, respectively. The activities of both enzymes were inhibited by Ag⁺ and iodoacetic acid.     

The isolation and characterization of copper-resistant mutants ofAspergillus nidulans

The presence of aminopeptidases in the cytoplasm, in the cell wall, and in the cytoplasmic membrane fractions ofStreptococcus sanguis 903 was demonstrated by isoelectric focusing in combination with enzyme-staining procedures. The cytoplasm and the cell wall both had two aminopeptidases (pI 4.25 and 4.3) with broad substrate specificities and one enzyme (pI 4.2) specific for arginine substrates. The former enzymes were both stimulated by Co²⁺ ions; the latter enzyme had no metal cofactor. The cytoplasmic membrane aminopeptidase (pI 4.65) was arginine specific and was not stimulated by metal ions.     

Heterogeneity of Rice Glutelin

Three forms of endopolygalacturonase from Saccharomyces fragilis (Kluyveromyces fragilis) were separated by a procedure including adsorption on Amberlite IRC-50, CM Sephadex C-50 column chromatography and repeated preparative disc electrophoresis. Each endo-PG was almost homogenoeus as judged by polyacrylamide gel electrofocusing and disc electrophoresis. The three enzyme were designated as enzymes I, II and III. Enzymes I and II were similar but enzyme HI different from I and II in isoelectric point. The three enzymes resembled one another in eznyme action on pectic acid and other properties. All the three enzymes showed macerating activity toward the potato and carrot tissues.     

Acid phosphatase in rat liver lysosomal membranes: purification and characterization

Acid phosphatase associated with rat liver lysosomal membranes (M-APase) was purified about 4,200-fold over the homogenate with 10% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included; preparation of lysosomal membranes, solubilization of the membranes with 1% Triton X-100, immunoaffinity chromatography, and gel filtration with FPLC equipped with a Sephacryl S-300HR column. The molecular weight, estimated by gel filtration through TSK SW 3000G, was approximately 320K and SDS gel electrophoresis showed that the enzyme is composed of four identical subunits with an apparent molecular weight of 67K. The enzyme contains about 24.3% carbohydrate consisting of mannose, galactose, fucose, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid in a molar ratio of 38:20:5:36:4:11, respectively. In addition, three soluble forms of acid phosphatase (C-APase I, II, and III) in lysosomal contents were separated from rat liver lysosomal contents with DEAE-Sephacel. These three enzymes were also purified using immunoaffinity chromatography followed by gel filtration. C-APase I, II, III, and M-APase have isoelectric points of 7.7-8.2, 6.6-7.0, 5.7-6.7, and 3.4-3.8, respectively. All four APases are sensitive to endo-beta-N-acetylglucosaminidase H. However, only C-APase III and M-APase are digestible with neuraminidase. Susceptibility of M-APase to neuraminidase in intact tritosomes was examined to study the topography of M-APase in tritosomal membranes. Neuraminidase susceptibility of M-APase was not observed in the intact tritosomes until the tritosomes had been disrupted by osmotic shock.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of an extracellular polygalacturonase from Lactobacillus plantarum strain BA 11

Lactobacillus plantarum produced extracellular polygalacturonase in a medium containing 1.5% low methyl-pectin (w/v) and 0.5% glucose (w/v) as inducers. The enzyme was purified (approximately 70-fold) by ammonium sulphate fractionation, Sephadex G-100 gel filtration and DEAE-cellulose ion exchange chromatography. Two peaks (PG I and PG II) of enzymic activity were obtained from the DEAE-cellulose column. The molecular mass of PG I was similar to that of PG II (32 000 Da). The K _m values of PG I and PG II for sodium polypectate were calculated to be 1.63 mg/ml and 1.78 mg/ml respectively. Their isoelectric points were about pH 5.5. The pH optimum was 4.5, while the optimum temperature was 35°C for both PG I and PG II. The two purified enzymes had similar endo modes of action on polygalacturonic acid, as determined by comparison of viscosity reduction and reducing group release.     

Purification and Characterization of 4,5-Dioxovalerate Reductase (NADPH) from Chlorella regularis

Two types of 4,5-dioxovalerate reductases (NADPH) were partiallypurified and characterized from green alga, Chlorella regularis.The enzyme was separated by DEAE-Sephacel chromatography intotwo peaks: type I (first peak) and type II (second peak). Theactivity ratio of the type II to type I enzyme varied between5 to 7 with a starting cell material. Both enzymes had the samepH optimum at 6.0 and pI value of 4.9. The molecular weightestimated by gel filtration was 33,000 for type I and 99,000for type II enzyme. Both enzymes used only NADPH, but were notspecific for 4,5-dioxovaleric acid (DOVA). Type I enzyme reducedglyoxylate 68-fold faster than DOVA, whereas type II enzymeacted more specifically on a variety of aldehydes than DOVA.It is suggested that these enzymes may not function primarilyas NADPH-DOVA reductases in the metabolic pathway of DOVA. (Received June 15, 1985; Accepted October 14, 1985)     

Purification and properties of the extracellular metallo-proteinases of Chromobacterium lividum (NCIB 10926).

Four extracellular proteolytic enzymes (I-IV) (EC 3.4.22.-) were identified in static cultures of Chromobacterium lividum (NCIB 10926) by agar gel electrophoresis and isoelectric focusing. Proteinases I-III were freed of non-enzymic protein by chromatography on TEAE-cellulose and CM-cellulose. The enzyme mixture was then fractionated in a pH gradient by isoelectric focusing. All three enzymes were shown to be heat-labile metallo-enzymes. Optimal activity occurred at pH 5.6 for enzyme I and at pH 6.2 for enzymes II and III. Remazolbrilliant Blue-hide powder was a sensitive substrate for these enzymes. Proteinase I was also shown to degrade haemoglobin and casein effectively, but not myoglobin, ovalbumin or bovine serum albumin. Proteinases I-III exhibited molecular weight values of 75 000, 72 000 and 67 000 by exclusion chromatography and 71 000 and 66 000 by sodium dodecyl sulphate-poly-acrylamide-gel electrophoresis for enzyme I and II, respectively. The amino acid compositions of enzymes I and II were somewhat similar. Proteinase I was inhibited by EDTA, 1,2-di(2-aminoethoxy)ethane-N,N,N',N'-tetraacetic activity. Mg2+ could substitute for Ca2+ or Mn2+ for Co2+. The interrelationship of proteinases I-III is discussed.     

Isolation,Purification, and Properties of Lytic Enzyme from Polysphondylium pallidum Myxamoebae

Two lytic enzymes (enzyme I and enzyme II) that lysed Micrococcus lysodeikticus were isolated from the crude extract of Polysphondylium pallidum myxamoebae grown in the presence of Klebsiella aerogenes by precipitation with protamine sulfate and by chromatography on DEAE-Sepharose CL-6B. Enzyme I was further purified by gel filtration on a Superose12 column, and enzyme II by chromatography on a MonoQ HR 5/5 column and gel filtration on a Superose12 column. Enzyme I was a basic protein, while enzyme II was acidic. The molecular weights of enzyme I and II were about 14,000 and 22,000, respectively by SDS-polyacrylamide gel electrophoresis. Optimum pHs for the activity were 5.0 for enzyme I and between 3.5 and 4.0 for enzyme II. The maximum activity of enzyme I and II was obtained at 65°C and 45°C to 55°C and at ionic strength of 0.0075 to 0.03 and 0.06, respectively. Both enzymes cleaved the glycosidic bond of β(1,4)-N-acetylmuramyl-acetylglucosamine of the cell wall peptidoglycan of Micrococcus lysodeikticus. These results indicate that the two lytic enzymes of Polysphondylium pallidum myxamoebae are N-acetylmuramidases.