Ca2+/calmodulin dependent protein kinase from Mycobacterium smegmatis ATCC 607

A soluble Ca²⁺/calmodulin dependent protein kinase has been partially purified (~400 fold) from Mycobacterium smegmatis ATCC 607 using several purification steps like ammonium sulphate precipitation (30-60%), Sepharose CL-6B gel filtration, DEAE-cellulose and finally calmodulin-agarose affinity chromatography. On SDS-PAGE, this enzyme preparation showed a major protein band of molecular mass 35 kD and its activity was dependent on calcium, calmodulin and ATP when measured under saturating histone IIs (exogenous substrate) concentration. Phosphorylation of histone IIs was inhibited by W-7 (calmodulin inhibitor) and KN-62 (CaM-kinase inhibitor) with IC₅₀ of 1.5 and 0.25 m respectively, but was not affected by inhibitors of PKA (Sigma P5015) and PKC (H-7). All these results confirm that purified enzyme is Ca²⁺/ calmodulin dependent protein kinase of M. smegmatis. The protein kinase of M. smegmatis demonstrated a narrow substrate specificity for both exogenous as well as endogenous substrates. These results suggest that purified CaM-kinase must be involved in regulating specific function(s) in this organism.     

Purification of a bifunctional amylase/protease inhibitor from ragi (Eleusine coracana) by chromatography and its use as an affinity ligand

An ammonium sulphate fraction (20–60%) of bifunctional amylase/protease inhibitor from ragi (Eleusine coracana) was purified by affinity chromatography to give 6.59-fold purity with 81.48% yield. The same ammonium sulphate fraction was also subjected to ion exchange chromatography and was purified 4.28-fold with 75.95% yield. The ion exchange fraction was subjected to gel filtration and the inhibitor was purified to 6.67-fold with 67.36% yield. Further sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to check the homogeneity of purified amylase/trypsin inhibitor obtained through affinity, ion exchange and gel chromatography. The molecular weight of the inhibitor was found to be 14 kDa. This purified inhibitor was used as affinity ligand for the purification of a commercial preparation of pancreatic amylase.     

米黑毛霉(M.miehei)天冬氨酸蛋白酶的研究

用硫酸铵分部盐析及离子交换层析技术从米黑毛霉半固体培养物的浸提液中提纯了天冬氨酸蛋白酶,酶活力收率为19.5％,,比活力达3080SU/mg蛋白,提纯8.5倍。用聚丙烯酰胺凝胶电泳、SDS-凝胶电泳、等电聚焦、双向免疫扩散及免疫电泳等方法鉴定该酶均一。本文还报道了关于该酶生化性质、动力学性质及化学修饰的研究结果。该酶以天冬氨酸为其活性的必需基团,系一典型的天冬氨酸蛋白酶。     

Characterisation and inhibition effect of cetrimide on collagenase produced byAspergillus flavus, isolated from mycotic ulcers

Collagenase was found to be the most important enzyme, produced by mycotic keratitis fungi. Therefore,Aspergillus flavus collagenase enzyme has been purified by ammonium sulphate precipitation, Sephadex G25 and DEAE-cellulose chromatography. Electrophoretic analysis for the purified enzyme indicated one subunit of molecular weight of 70–90 KDa when examining on SDS-PAGE. Cetrimide (cetyl trimethyl ammonium bromide) has been tested against the purified collagenase enzyme and indicated reversible competitive inhibitor (kis=0.15 mg/ml) with high promising activity. Cetrimide might be used to inhibit mycotic keratitis fungi.     

Purification and properties of arylsulphatase from the brain of the silkworm Bombyx mori

(1) Arylsulphatase of the silkworm Bombyx mori was partially purified using ammonium sulphate fractionation, ethanol precipitation, Sephadex G-200 gel filtration and Con-A Sepharose chromatography. (2) The purified enzyme preparation was not homogeneous but showed no beta-glucuronidase or beta-galactosidase activities. (3) The kinetic properties of the enzyme indicated that it could be classified under type-2 arylsulphatases of vertebrates. (4) The purified enzyme shows very little activity towards p-nitrophenyl sulphate and none towards cerebroside 3-sulphate.     

Purification and properties of glucose 6-phosphate dehydrogenase from turkey erythrocytes

Glucose 6-phosphate dehydrogenase (G6PD) was purified from turkey erythrocytes by ammonium sulphate precipitation and followed by ADP Sepharose affinity gel chromatography. The yield was 49.71% and specific activity of the enzyme was found to be 44.16 EU/mg protein. By gel filtration the molecular mass was found to be 75 kDa. The enzyme had an optimum pH at 9.0, and optimum temperature at 50 degrees C. Km and Vmax for NADP(+) and glucose 6- phosphate (G6-P) as substrates were also determined and effects of inhibitors such as ATP, NADH and NADPH were examined.     

Lipoxygenase in dark-grown Pisum sativum

Lipid oxidizing activity has been detected in acetone powders from both dark- and light-grown dwarf pea seedlings. This activity has been shown by several methods to be due to lipoxygenase. The enzyme from dark-grown seedlings has been purified 5·7-fold by ammonium sulphate precipitation and gel filtration. CM-cel-lulose chromatography of the purified enzyme yielded four active fractions. The properties of the four lipoxy-genase isoenzymes are described.     

A barley endonuclease specific for apurinic DNA. Isolation and partial characterization

An endonuclease specific for depurinated native DNA was isolated and partially purified from extracts of barley leaves. The procedure included streptomycin sulphate precipitation, ammonium sulphate fractionation, phosphocellulose, hydroxyapatite and Sephadex G-150 chromatography. Purity of the resulting enzyme was determined by gel electrophoresis and gel chromatography and specificity by testing the activity on intact and depurinated bacterial DNAs. At lower concentrations, the enzyme is specific for DNA containing apurinic sites. At higher concentrations, however, it degrades DNA in a non-specific manner. The nuclease has a pH optimum at 7.6, and a molecular weight of about 18000.     

Purification and properties of lactate dehydrogenase from Nocardia asteroides.

The lactate dehydrogenase (LDH) from Nocardia asteroides was purified to homogeneity by ammonium sulphate precipitation, gel filtration on Sephadex G-150 and DEAE-Sepharose column chromatography. The purified enzyme showed a single band in native condition which indicated its homogeneity. SDS-PAGE of the purified enzyme showed the presence of three bands which correspond to molecular weights of 60, 66 and 74 kDa. The pH and temperature optima of the purified enzyme were 9.5 and 50 degrees C, respectively. The metal ions Mn++, Fe++, Co++, Mg++ and Ca++, increased the purified LDH activity. On the other hand, enzyme activity was completely inhibited by CuCl2. Potassium chloride, ammonium sulphate and sodium chloride did not alter the enzyme activity. The purified enzyme exhibited a Km value of 1.6 x 10(-5) M for pyruvate.     

In vitro effects of some antibiotics on glutathione reductase from sheep liver

The effects of gentamicin sulphate, thiamphenicol, ofloxacin, levofloxacin, cefepime, and cefazolin were investigated on the in vitro enzyme activity of glutathione reductase. The enzyme was purified 1,850-fold with a yield 18.76% from sheep liver using ammonium sulphate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE). The enzyme activity was measured spectrophotometrically at 340 nm, according to the method of Carlberg and Mannervik. From these six antibiotics, Ofloxacin, levofloxacin, cefepime, and cefazolin inhibited the activity of the purified enzyme; gentamicin sulphate and thiamphenicol showed little effect on the enzyme activity. The I50 values for these four antibiotics were 0.150 mM, 0.154 mM, 3.395 mM, and 18.629 mM, respectively. The Ki constants were 0.047 +/- 0.034 mM, 0.066 +/- 0.038 mM, 4.885 +/- 3.624 mM, and 6.511 +/- 1.894 mM, respectively and they were competitive inhibitors.     

Inhibitory effects of ofloxacin and cefepime on enzyme activity of 6-phosphogluconate dehydrogenase from chicken liver

In this study, effects of some antibiotics, namely, ofloxacin, cefepime, cefazolin, and ampicillin on the in vitro enzyme activity of 6-phosphogluconate dehydrogenase have been investigated. For this purpose, 6-phosphogluconate dehydrogenase was purified from chicken liver 535-fold with a yield of 18% by using ammonium sulphate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. In order to check the purity of the enzyme, SDS polyacylamide gel electrophoresis (SDS-PAGE) was performed. This analysis revealed a highly pure enzyme band on the gel. Among the antibiotics, ofloxacin and cefepime exhibited inhibitory effects, but cefazolin and ampicillin showed neither important inhibitory nor activatory effects on the enzyme activity. The measured I(50) values by plotting activity percent vs. inhibitor concentration, [I(50)] were 0.1713 mM for ofloxacin and 6.0028 mM for cefepime. Inhibition constants, K(i), for ofloxacin and cefepime were also calculated as 0.2740 +/- 0.1080 mM and 12.869 +/- 16.6540 mM by means of Lineweaver-Burk graphs, and inhibition types of the antibiotics were found out to be non-competitive and competitive, respectively. It has been understood from the calculated inhibitory parameters that the purified chicken enzyme has been quite inhibited by these two antimicrobials.     

Relationship between threonine dehydratase and biosynthesis of tylosin in Streptomyces fradiae.

To elucidate the repression mechanism of ammonium ions on the biosynthesis of tylosin in Streptomyces fradiae NRRL 2702, enzyme activities involved in the metabolism of the aspartate family of amino acids were evaluated in relation to the ammonium ion concentration and tylosin production. It was found that aspartate aminotransferase was essential for both cell growth and tylosin production. However, both threonine dehydratase and valine dehydrogenase were repressed by supplemented ammonium ions at concentrations higher than 50 mM. Threonine dehydratase was purified from cell-free extracts by acetone precipitation, ion-exchange chromatography and gel filtration, and its molecular mass was estimated to be 67,200 Da. The optimum pH and temperature for threonine dehydratase activity were 7.5 and 25 degrees C, respectively, and the Km value for threonine under these optimum conditions was 21 mM. The inhibition pattern of ammonium ions on the activity of threonine dehydratase appeared to be a mixed type.     

Purification and some properties of isocitrate dehydrogenase from Paracoccus denitrificans

NADP-dependent isocitrate dehydrogenase (ICDH) from the bacterium Paracoccus denitrificans was purified to homogeneity. The purification procedure involved ammonium sulphate fractionation, ion exchange chromatography, and gel permeation chromatography. The specific activity of purified ICDH was 801 nkat/mg, the yield of the enzyme 58%. The purity of the enzyme was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. ICDH is a dimer composed of two probably identical subunits of relative molecular weight 90,000. The pH optimum of the enzyme reaction in the direction of substrate oxidation was found to be 5.6; the presence of Mn2+ is essential for enzyme activity. The absorption and fluorescence spectra of the homogeneous enzyme were measured as well.     

Purification and characterization of glutamine synthetase from the unicellular cynabacterium Anacystis nidulans

Glutamine synthetase from the unicellular cynabacterium Anacystis nidulans was found associated with the membrane fraction of cell-free extracts. The enzyme could be solubilized by treatment of the cell membranes with the detergent alkyltrimethylammoniun and was purified to electrophoretical homogeneity by using affinity chromatography on 2′,5′-ADP-Sepharose. The molecular weight of the native enzyme was approx. 575000 but only a single protein band of 47 kDa was detected after sodium dodecyl sulphate gel electrophoresis, which implies a native enzyme complex with twelve identically sized subunits. Values for apparent Michaelis constant of the purified enzyme for ammonium, glutamate and ATP were 20, 5000 and 700 μM, respectively. Alanine behaved as an inhibitor of both activities (transferase and biosynthetic) of glutamine synthetase, whereas aspartate, leucine and lysine inhibited the biosynthetic activity of the enzyme, and glycine and serine only inhibited the transferase activity. Glutamate analogs, such as hydroxylysine, methionine sulfone, methionine sulfoximine and phosphinothricin, which inhibited ammonium uptake in vivo, behaved as potent inhibitors of glutamine synthetase in vitro. A. nidulans glutamine synthetase was inhibited by p-hydroxymercuribenzoate, the effect being reversed by treatment with dithioerythritol, dithiothreitol or mercaptoethanol.     

Ornithine carbamoyltransferase of Streptomyces fradiae: purification and properties

Abstract The enzyme ornithine carbamoyltransferase was purified from Streptomyces fradiae . A 1200-fold increase in specific activity was achieved by ammonium sulphate precipitation, DEAE-cellulose and aminohexyl-agarose chromatography and gel filtration. The purified enzyme has a M _r of 87 000. Its isoelectric point is 5.3 as determined by isoelectric focusing. Apparent K _m values at pH 7.7 for ornithine and carbamoyl phosphate are 1.8 and 1.2 mM, respectively.     

In Vitro Effects of Some Antibiotics on Glutathione Reductase from Sheep Liver

The effects of gentamicin sulphate, thiamphenicol, ofloxacin, levofloxacin, cefepime, and cefazolin were investigated on the in vitro enzyme activity of glutathione reductase. The enzyme was purified 1,850-fold with a yield 18.76% from sheep liver using ammonium sulphate precipitation, 2′, 5′-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE). The enzyme activity was measured spectrophotometrically at 340?nm, according to the method of Carlberg and Mannervik. From these six antibiotics, Ofloxacin, levofloxacin, cefepime, and cefazolin inhibited the activity of the purified enzyme; gentamicin sulphate and thiamphenicol showed little effect on the enzyme activity. The I50 values for these four antibiotics were 0.150?mM, 0.154?mM, 3.395?mM, and 18.629?mM, respectively. The Ki constants were 0.047±0.034?mM, 0.066±0.038?mM, 4.885±3.624?mM, and 6.511±1.894?mM, respectively and they were competitive inhibitors.     

Staphylococcal L-asparaginase: purification and properties of enzymic protein

Staphylococcal L-asparaginase has been purified 400-fold with 40% recovery. The procedure involves ammonium sulphate precipitation and a column chromatography on Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. protein (pI 4.4) with the approximate molecular weight of 125,000 (estimated by Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. The polyacrylamide-SDS gel electrophoresis indicated two subunits with molecular weight 18,000 and 22,000.     

Purification and Characterization of an Endopolygalacturonase Produced by Sclerotinia Sclerotiorum

An endopolygalacturonase (endo-PG), was purified from the culture medium of a local isolate of Sclerotinia sclerotiorum with ammonium sulphate precipitation, cation exchange chromatography and gel filtration. The purified endo-PG had a molecular mass of approximately 18 kDa estimated by gel filtration. The isoelectric point was determined by isoelectric focusing to be approximately 8, suggesting that PG II possesses a net positive charge at physiological pHs. The pH optimum for the enzyme was at pH 4.5. The endo-PG showed essentially the same affinity for pectin and polygalacturonic acid as substrates. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization and partial purification of L-asparaginase from Corynebacterium glutamicum

A high L-asparaginase (L-asparagine amidohydrolase: EC 3.5.1.1) activity was found under conditions of lysine overproduction in cultures of Corynebacterium glutamicum. L-Asparaginase was purified 98-fold by protamine sulphate precipitation. DEAE-Sephacel anion exchange, ammonium sulphate precipitation and Sephacryl S-200 gel filtration. The asparaginase protein was subjected to PAGE under non-denaturing conditions, identified by an in situ reaction and eluted from the gel in an active form. The estimated Mr from gel filtration and SDS-PAGE was 80,000. The L-asparaginase activity was inhibited by the L-asparagine analogue 5-diazo-4-oxo-L-norvaline. Neither D-asparagine nor L-glutamine was a substrate for the enzyme. L-Asparaginase was produced constitutively: its role may be that of an overflow enzyme, converting excess asparagine into aspartic acid, the direct precursor of lysine and threonine.     

Purification and properties of rat brain pyruvate kinase

Rat brain pyruvate kinase was purified to near homogeneity by a three-step process involving ammonium sulfate precipitation and phosphocellulose and Blue-Sepharose CL-6B column chromatography. The enzyme migrated on polyacrylamide gel along with a commercial sample of rabbit muscle pyruvate kinase. The enzyme showed a hyperbolic relationship with phosphoenolpyruvate and ADP, with apparent Km's of 0.18 and 0.42 X 10(-3) M, respectively. The enzyme was inhibited by ATP, the effect being more pronounced at unsaturating concentrations of phosphoenolpyruvate. L-Phenylalanine was found to be a strong inhibitor of the enzyme, with the Ki for inhibitor being 0.11 mM. The inhibition by phenylalanine was more pronounced at pH 7.4 than at pH 7.0, and appeared to be competitive with phosphoenolpyruvate. L-Alanine and fructose 1,6-bisphosphate prevented the inhibition of the enzyme by phenylalanine. Ca2+ was found to be a strong inhibitor of the enzyme, and the inhibition was more marked at saturating phosphoenolpyruvate concentrations. The kinetic properties of the purified brain pyruvate kinase suggest that the enzyme may be distinct from the muscle or liver enzymes.