Extracellular exo-polygalacturonase secreted from carrot cell cultures. Its purification and involvement in pectic polymer degradation

Exo-polygalacturonase (exo-PGase, EC 3.2.1.67) activity has been detected in a culture filtrate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular exo-PGase was purified to electrophoretic homogeneity using DEAE-Sephadex A-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and preparative polyacrylamide gel electrophoresis (PAGE). The molecular mass of the purified enzyme was calculated to be 48 kDa from Sephadex G-200 gel filtration, and 50 kDa from sodium dodecyl sulfate (SDS)-PAGE after treatment with SDS and 2-mercaptoethanol. The isoelectric point was at pH 6.2. The K_m and V_max values for polygalacturonate (degree of polymerization: 52) were 14.4 μ M and 25.6 μmol (mg protein)⁻¹ h⁻¹, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.6. The enzyme activity was inhibited by Ba²⁺, Cu²⁺, Mn²⁺ and Hg²⁺. The enzyme was involved in ca 15% hydrolysis of the acidic polymer purified from carrot pectic polysaccharides, and connected with the release of galacturonic acid. Even after an exhaustive reaction the enzyme had, however, little or no effect on cell walls from carrot cell cultures.     

N 5, N10-Methylenetetrahydromethanopterin reductase from Methanosarcina barkeri

N ⁵ N ¹⁰-Methylenetetrahydromethanopterin reductase was purified 13-fold to apparent homogeneity from methanol grown Methanosarcina barkeri . The colourless enzyme was found to be composed of four identical subunits of apparent molecular mass 36 kDa. It catalysed the reduction of methylenetetrahydromethanopterin ( K _m=15 μM) to methyltetrahydromethanopterin with reduced coenzyme F₄₂₀ ( K _m=12 μM) at a specific rate ( V _max) of 2200 μmol min⁻¹· mg protein⁻¹ ( K _cat=1320 s⁻¹). With respect to coenzyme specificity, molecular properties and catalytic mechanism the enzyme was found to be similar to CH₂=H₄MPT reductase of Methanobacterium thermoautotrophicum which phylogenetically is only distantly related to M. barkeri .     

Purification and characterization of an endoamylase from tulip (Tulipa gesneriana) bulbs

Amylase activity extracted from tulip ( Tulipa gesneriana L. cv. Apeldoorn) bulbs that had been stored for 6 weeks at 4°C was resolved to 3 peaks by anion-exchange chromatography on diethylaminoethyl-Sephacel. These 3 amylases exhibited different relative mobilities during non-denaturing polyacrylamide gel electrophoresis (PAGE). The most abundant amylase form (amylase I) was purified to apparent homogeneity using hydrophobic interaction chromatography, gel filtration and chromatofocusing. The apparent molecular mass of the purified amylase was estimated to be 51 kDa by sodium dodecyl sulfate-PAGE and 45 kDa by gel filtration chromatography. The purified amylase was determined to be an endoamylase (EC 3.2.1.1) based on substrate specificity and end-product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55°C. The apparent K_m value with soluble starch (potato) was 1.28 mg ml⁻¹. The presence of Ca²⁺ increased the activity and thermal stability of the enzyme. The presence of dithiothreitol enhanced the activity, while β -mercaptoethanol and reduced glutathione had no significant effect. When pre-incubated in the absence of the substrate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) partially inhibited the enzyme. α -cyclodextrins or β -cyclodextrins had no effect on enzyme activity up to 10 m M . In addition to CaCl₂, CoCl₂ slightly enhanced activity, while MgCl₂ and MnCl₂ had no significant effect at a concentration of 2 m M . ZnCl₂, CuSO₄, AgNO₃ and EDTA partially inhibited enzyme activity, while AgNO₃ and HgCl₂ completely inhibited it at 2.0 m M .     

α-Galactosidase from the yeast Torulaspora delbrueckii IFO 1255

The yeast Torulaspora delbrueckii IFO 1255 was selected as the strain fermenting melibiose from 35 strains of Torulaspora species. The strain IFO 1255 produced extracellular and cell-associated forms of α-galactosidase when grown on either melibiose or galactose as the sole carbon source. Most of the enzyme was located outside of the cell membrane: the periplasmic space, or cell walls, or both. α-Galactosidase was purified to homogeneity from the cell-free extract of the strain IFO 1255 by acid treatment and column chromatography on DEAE-Toyopearl 650M and Butyl-Toyopearl 650M. The molecular weight of the purified enzyme was estimated to be 88 000 by SDS-polyacrylamide gel electrophoresis and 530 000 by gel filtration. The enzyme contained 50% of its molecular weight as carbohydrate. Optimum pH and temperature were 4.5–5.5 and 55°C, respectively. The enzyme was inhibited strongly by Ag²⁺, Hg²⁺ and Cu²⁺ each at 1 mmol 1^-1. The K _m (μmol 1^-1) for p -, o -, m -nitrophenyl α-D-galactopyranoside, melibiose, raffinose and stachyose were 2.8, 1.3, 2.8, 4.2, 170 and 230, respectively, and V _max (μmol min^-1 mg protein^-1) for those substrates were 310, 140, 21, 22, 30 and 44, respectively. The properties of α-galactosidase from T. delbrueckii IFO 1255 were similar to those from the related species, Saccharomyces cerevisiae.     

Purification and properties of a neutral invertase from the roots of Cichorium intybus

Multiple activity peaks of neutral invertase (EC 3.2.1.26) were found in chicory roots ( Cichorium intybus L. var. foliosum cv. Flash). The main activity peak was purified by a combination of anion-exchange chromatography, hydrophobic interaction chromatography, chromatofocusing and gel filtration. This protocol produced a 77-fold purification and a specific activity of 1.6 μmol (mg protein)⁻¹ min⁻¹. The mass of the enzyme was 260 kDa as estimated by gel filtration and 65 kDa on SDS-PAGE. Optimal activity was found between pH 7 and 7.5. The purified enzyme exhibited hyperbolic saturation kinetics with a K_m between 10 and 20 mM for sucrose. No other products than glucose and fructose could be detected. Raffinose was hydrolyzed at a rate of 2.4% relative to sucrose whereas the enzyme did not hydrolyze maltose, cellobiose, trehalose, 1-kestose, 1.1-nystose or inulin. Neutral invertase activity was completely inhibited by HgCl₂ and AgNO₃ and partially inhibited by CoCl₂, and ZnSO₄ (1 mM). Pyridoxal phosphate (K_i≅ 500 μ M ), Tris (K_i≅ 1.2 m M ), glucose and fructose (K_i≅ 16 m M ) were strong inhibitors of the enzyme. Fructose and Tris behaved as competitive inhibitors. A possible role for the enzyme's activity in vivo is discussed.     

Purification and kinetic properties of a castor bean seed acid phosphatase containing sulfhydryl groups

An acid phosphatase (EC 3.1.3.2) has been identified and purified from castor bean ( Ricinus communis L., IAC-80 ) seed through sulphopropyl (SP)-Sephadex, diethylaminoethyl (DEAE)-Sephadex, Sephacryl S-200, and Concanavalin A-Sepharose chromatography. The enzyme was purified 2 000-fold to homogeneity, with a final specific activity of 3.8 μkat mg⁻¹ protein. The purified enzyme revealed a single diffuse band with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis, at pH 8.3. The relative molecular mass, determined by high-performance liquid chromatography (HPLC), was found to be 60 kDa. The acid phosphatase had a pH optimum of 5.5 and an akpparent K_m value for p -nitrophenylphosphate of 0.52 m M . The enzyme-catalyzed reaction was inhibited by inorganic phosphate, fluoride, vanadate, molybdate, p -chloromercuribenzoate ( p CMB), Cu²⁺ and Zn²⁺. The strong inhibition by p CMB, Cu²⁺ and vanadate suggests the presence of sulfhydryl groups essential for catalysis. The castor bean enzyme also recognized tyrosine-phosphate and inorganic pyrophosphate (KPP_i) as substrate. The highest specificity constant (V_max/K_m) was observed with KPP_i, making it a potential physiological substrate.     

Purification of an α-L-arabinofuranosidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation

Konno, H., Yamasaki, Y. and Katoh, K. 1987. Purification of an α-L-arabinofurano-sidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation.
An α-L-arabinofuranosidase (α-L-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55) was isolated from a homogenate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The buffer-soluble enzyme was purified to homogeneity by a procedure involving ammonium sulfate fractionation, chromatography on DEAE-Sephadex A-50, Sephadex G-150, Con A-Sepharose 4B and CM-Sephadex C-50, and preparative polyacrylamide gel electrophoresis. The size of this enzyme as determined by polyacrylamide gel electrophoresis in the presence of sodium laurylsulfate and by Sephadex G-200 gel filtration was 94 and 110 kDa, respectively. The isoelectric point was at pH 4.7. The K_m and V_max values for p-nitrophenyl α-L-arabinofuranoside were 1.33 mM and 20.2 μimol (mg protein)^-1 h^-1, respectively. The optimal activity occurred at pH 4.2 with Mcllvaine buffer. The enzyme was stimulated by Ca²⁺ and Zn²⁺, whereas it was strongly inhibited by Cu²⁺, Ag²⁺, Hg²⁺, p-chloromercuri-benzoate and L-arabono-l,4-lactone. The enzyme acted on beet arabinan in an exo-fashion. Furthermore, the enzyme was partially involved in the hydrolysis of the ara-binogalactan and pectic polymer purified from carrot cell walls.     

Citrate synthases from germinating castor bean seeds – I. Purification and properties

The mitochondrial and glyoxysomal citrate synthase (EC 4.1.3.7) from the endosperm of germinating castor beans ( Ricinus communis L., type Sanzibaricnsis) were purified to a final specific activity of 76 and 78 U (mg protein)⁻¹, respectively. Both citrate synthases could be bound to ATP-Sepharose. However, only the mitochondrial enzyme could be eluted by either 100 μ M oxaloacetate or 100 μ M coenzyme A (indicative of affinity chromatography), while the glyoxysomal enzyme was only eluted by 0.5 M KCI (indicative of ion-exchange chromatography). Many properties of the two isoenzymes were similar including the pH dependence and temperature dependence of activity, the pH stability, and the inactivation of the enzyme at elevated temperatures. The most pronounced differences between the two citrate synthases were the isolelectric points of pH 5.9 for the mitochondrial and of pH 9.1 for the glyoxysomal enzyme. Both citrate synthases are dimers in the native form with a molecular weight of 95000 each, as determined by gel filtration on Sepharose CL-6B and by polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate. However, the glyoxysomal citrate synthase existed also as a tetramer with a molecular weight of 200000 in the presence of 10 m M MgCl₂.     

Chloroplast glutathione reductase: Purification and properties

Glutathione reductase was partially purified from isolated pea chloroplasts ( Pisum sativum L. cv. Progress #9). A 1600-fold purification was obtained and the purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)⁻¹ min⁻¹. The enzyme had a native molecular weight of approximately 156 kdalton and consisted of two each of two subunits of about 41 and 42 kdalton. The K_m for oxidized glutathione was 11 μ M and the K_m for NADPH was 1.7 μ M . Enzyme activity was affected by the ionic strength of the assay medium, and maximum activity was observed at an ionic strength of between 60 and 100 m M . The enzyme was inactivated by sulfhydryl modifying reagents and the presence of either oxidized glutathione or NADPH affected the extent of inactivation. Chloroplast glutathione reductase probably serves in the removal of photosynthetically derived H₂O₂ by reducing dehydroascorbate for ascorbate-linked reduction of H₂O₂. Intermediates of this reaction sequence, dehydroascorbate, ascorbate, reduced glutathione, and NADPH had no effect on enzymic activity.     

Purification and characterization of adenine phosphoribosyltransferase from Arabidopsis thaliana

Adenine phosphoribosyltransferase (APRT; EC 2. 4,2. 7) from Arabidopsis thaliana was purified approximately 3800-fold, to apparent homogeneity. The purification procedure involved subjecting a leaf extract to heat denaturation, (NH₄)₂SO₄ precipitation, Sephadex G-25 salt separation, ultracentrifugation and liquid chromatography on Diethylaminoethyl Sephacel, Phenyl Sepharose CL-4B, Blue Sepharose CL-6B and adenosine 5'-monophosphate-Agarose. The purified APRT was a homodimer of approximately 54 kDa and it had a specific activity of approximately 300 μmol (mg total protein)^-1 min^-1. Under standard assay conditions, the temperature optimum for APRT activity was 65°C and the pH optimum was temperature dependent. High enzyme activity was dependent upon the presence of divalent cations (Mn²⁺ or Mg²⁺). In the presence of MnCl₂₊ other divalent cations (Mg²⁺, Ca²⁺, Ba²⁺, Hg²⁺ and Cd²⁺) inhibited the APRT reaction. Kinetic studies indicated that 5-phosphoribose-1-pyrophosphate (PRPP) caused substrate inhibition whereas adenine did not. The K_m for adenine was 4.5±1.5 μ M , the K_m for PRPP was 0.29±0.06 m M and the K_i for PRPP was 1.96±0.45 m M . Assays using radiolabelled cytokinins showed that purified APRT can also catalyze the phosphoribosylation of isopentenyladenine and benzyladenine. The K_m for benzyladenine was approximately 0.73±0.06 m M     

NADH-dependent nitrate reductase from two-row barley roots: Purification, characteristics and comparison with leaf enzyme

NADH-nitrate reductase (EC 1.6.6.1) was purified 800-fold from roots of two-row barley ( Hordeum vulgare L. cv. Daisen-gold) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on TSK-gel (G3000SW). The specific activity of the purified enzyme was 6.2 μmol nitrite produced (mg protein)⁻¹ min⁻¹ at 30°C.
Besides the reduction of nitrate by NADH, the root enzyme, like leaf nitrate reductase, also catalyzed the partial activities NADH-cytochrome c reductase, NADH-ferricyanide reductase, reduced methyl viologen nitrate reductase and FMNH₂-nitrate reductase. Its molecular weight was estimated to be about 200 kDa, which is somewhat smaller than that for the leaf enzyme. A comparison of root and leaf nitrate reductases shows physiologically similar or identical properties with respect to pH optimum, requirements of electron donor, acceptor, and FAD, apparent K_m for nitrate, NADH and FAD, pH tolerance, thermal stability and response to inorganic orthophosphate. Phosphate activated root nitrate reductase at high concentration of nitrate, but was inhibitory at low concentrations, resulting in increases in apparent K_m for nitrate as well as V_max whereas it did not alter the K_m for NADH.     

Ammonium assimilation in bryophytes. l-glutamine synthetase from Sphagnum fallax

Cytosolic and plastidic l -glutamine synthetase (EC 6.3.1.2) isoenzymes from Sphagnum fallax Klinggr. (Klinggr. clone 1) were separated by size-exclusion and ion exchange chromatography. The cytosolic enzyme (GS₁) was purified to apparent electrophoretic homogeneity. The native enzyme had a molecular mass of 390 ± 20 kDa as estimated by gel filtration and was apparently composed of 8 subunits with molecular masses of 48 kDa. GS₁ activity could be measured from pH 6.8 to 8.6 in 50 m M imidazole buffer, with a broad optimum between pH 7.2 and 8.0. The K_m values were 2.5 m M , 0.5 m M and 0.5 m M for l -glutamate, ammonium and ATP, respectively. The enzyme was inhibited by more than 10 m M ammonium or glutamate. The incorporation of ¹⁵NH₄⁺ into amino acids was observed in vivo using ¹⁵ NMR. Label from ammonium was first detected in the amide N of glutamine, and only subsequently in the amino N of glutamate. Moreover, no assimilation was detected in the presence of the specific GS inhibitor methionine sulfoximine. These observations are consistent with a dominant role for GS in the assimilation of ammonium in Sphagnum .     

ISOLATION, PURIFICATION, AND CHARACTERIZATION OF DMSP LYASE (DIMETHYLPROPIOTHETIN DETHIOMETHYLASE (4.4.1.3)) FROM THE RED ALGA POLYSIPHONIA PANICULATA1

Polysiphonia paniculata Montagne is an intertidal red alga known to produce large amounts of the compound dimethylsulfoniopropionate (DMSP). Conversion of this substrate into dimethylsulfide is accomplished in P, paniculata by an enzyme called DMSP lyase (dimethylpropiothetin dethiomethyla.se (4.4.1.3)). DMSP lyase has been purified and characterized from P. paniculata. Enzymie activity is found in two different proteins: the larger with a molecular weight of 9.26 ± 10⁴ daltons and the smaller with a molecular weight of 3.65 ± 10⁴ daltons. Specific activity of the enzyme is 526 μmols min⁻¹mg⁻¹ for the smaller protein a nd 263 μmols min ⁻¹ mg⁻¹ for the la rger protein. The Michaelis-Menten constant (K_m) is 72.8 μM ± 17.15 and the v_max is 1.62 μmols min⁻¹± 0.928 for the 92.6-kDa protein. The p1 of the larger protein is 5.8 and 5.9 for the smaller protein. Interaction with cysteine protease inhibitors L-trans-epoxysuccinyl-leucylamido (4-guanidino)-butane, dithiobis-(2-nitrobenzoate), or N -ethylmaleimide inactivated enzyme activity. The presence of either magnesium or calcium with DMSP lyase enhanced activity al concentrations between 20 and 40 μM but had little effect above these levels. Addition of the divalent chelators ethylenebis(oxyethylenenitrilo) tetraacetic acid and ethylenediaminetetraacetate decreased activity of the enzyme, but activity was restored when either chelator was removed and magnesium or calcium was added to the enzyme .     

Purification and characterization of phosphoglucomutase from peas

Phosphoglucomutase (EC 2.7.5.1) was isolated from pea seeds ( Pisum sativum L. cv. Grenadier) and purified to homogeneity as determined by sodium dodecylsulfate - polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The enzyme was purified by utilizing 25% polyethylene glycol 4000 precipitation, followed by Fractogel-diethyla-minoethyl (DEAE) 650. Fractogel-TSK HW-55(s). and high pressure liquid chroma-tography (HPLC)-(PEI) column chrornatography. The resulting enzyme had a specific activity of 157 units (mg protein)-1. a 152-fold increase over that of the crude plant extract. The molecular weight of the enzyme was 128 to 136 kDa. as determined by native-PAGE and column chromatography, and when it was subjected to SDS-PAGE analysis, it was found to be composed of two subunits having molecular weights ranging from 59 to 64 kDa. Upon SDS-PAGE analysis of a sample purified through HPLC-PEI chromatography. two bands of protein were found: one having a molecular weight of 64 kDa and the other 68 kDa. A pH optimum of 8.6 was found for the enzyme while it was also found that cysleine. Mg²⁺ and glucose 1.6-bisphosphate were necessary for optimal activity Histidine and imidazole only partially fulfilled the cysteine requirement. A 20-min preincubation period in the absence of glucose 1-phosphate was necessary for optimal activity of the enzyme. Without a preincubation period, there was a pronounced lag preceding the linear portion of the reaction as well as a reduction in the V_max. An analysis of the kinetics of the reaction showed K_m values ot 3.6 × 10⁻⁵ and 1.45 × 10⁻⁵ M for glucose 1-phosphate and glucose 1.6-bisphosphate. respectively. A K., of 7.3 × 10⁻⁵ M was obtained for MgCl₂.     

Purification and characterization of an extracellular amylase from Lactobacillus plantarum strain A6

Extracellular amylase from Lactobacillus plantarum A6 was purified by fractionated precipitation with ammonium sulphate and by anion exchange chromatography. The homogeneity of the purified fraction was tested by polyacrylamide gel electrophoresis and showed multiple amylase forms. A major form had an estimated molecular weight of 50 kDa. It was identified as an α-amylase, with an optimum pH of 5.5, an optimum temperature of 65°C and K _m value of 2.38 g l^-1 with soluble starch substrate. The enzyme was inhibited by N -bromosuccinimide, iodine and acetic acid. The enzyme activation energy was 30.9 kJ mol^-1.     

An extracellular β-galactosidase secreted from cell suspension cultures of carrot. Its purification and involvement in cell wall-polysaccharide hydrolysis

β-Galactosidase (β-Galase, EC 3.2.1.23) activity has been detected in a culture medium of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular β-Galase (β-Galase-II) was purified to electrophoretic homogeneity from the concentrated medium using ammonium sulfate precipitation, chromatography on CM-Sephadex C-50. DEAE-Sepharose CL-6B and Sephacryl S-200HR, and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 65 kDa by Sephacryl S-200HR gel-permeation, and 60 kDa by SDS-PAGE after treatment with SDS and 2-mercaptoethanol. The pI was 6.5. The K_m and V_max values for p -nitrophenyl (PNP)-β-D-galactopyranoside were 0.17 m M and 31.9 μmol (mg protein)^-1, h^-1, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.0–4.4. The enzyme activity was inhibited by Co²⁴, Cu²⁺, Hg^2-, p -chloromercuribenzoate (PCMB) and D-galactono-1,4-lactone. The enzyme acted on citrus galactan and larchwood arabinogalactan in an exo-fashion, and was slightly involved in the hydrolysis of an acidic pectic polymer containing arabinosyl and galactosyl residues and in the breakdown of cell walls isolated from carrot cell cultures.     

Properties and significance of an α-amylase produced by Myxococcus coralloides D

M.E.FÁREZ-VIDAL, A. FERNÁNDEZ-VIVAS, F. GONZÁLEZ AND J.M. ARIAS. 1995. The extracellular amylase activity from Myxococcus coralloides D was purified by Sephacryl S-200 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-25. The molecular weight was estimated by SDS-PAGE and by gel filtration as 22.5 kDa. The optimum temperature was 45°C. The pH range of high activity was between 6.5 and 8.5, with an optimum at pH 8.0. Activity was strongly inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Ag⁺, Pb²⁺, Fe²⁺ and Fe³⁺, EDTA and glutardialdehyde, but was less affected by Ni²⁺ and Cd²⁺. Li⁺, Mg²⁺, Ba²⁺, Ca²⁺, N -ethylmaleimide, carbodiimide and phenyl methyl sulphonyl fluoride had almost no affect. The K _m (45°C, pH 8) for starch hydrolysis was 2.0 times 10^-3 gl^-1. Comparison of the blue value-reducing curves with the time of appearance of maltose identified the enzyme produced by M. coralloides D as an α-amylase.     

A β-N-acetylhexosaminidase from Penicillium oxalicum implicated in its cell-wall degradation

High β- N -acetylhexosaminidase (EC.3.2.1.52) activity was detected during autolysis of Penicillium oxalicum . Purification of the enzyme to homogeneity yielded an enzyme with a molecular weight of 132 000 Da by gel filtration and 71 900 Da by SDS polyacrylamide gel electrophoresis, suggesting a dimeric structure. The enzyme is an acidic protein with a pl of 5.0. Optimal activity was at pH 4.0 and 40°C, with a K _m of 0.80 mmol 1^-1 for p -nitrophenyl-β- N -acetylglucosaminide and 1.03 mmol 1^-1 for p -nitrophenyl-β- N -acetylgalactosaminide. The K _i with the competitive inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino- N -phenylcarbamate was 1 μmol 1^-1. Hg²⁺, Ag⁺ and Fe³⁺ were effective inhibitors. β- N -acetylhexosaminidase hydrolysed chitobiose, chitotriose, chitotetrose and chitopentose to monomer to an extent of 92, 74, 44 and 17% respectively in 40 min. This enzyme, in conjunction with a purified endochitinase from P. oxalicum , hydrolysed a cell-wall chitin fraction isolated from this fungus, with the production of N -acetylglucosamine.     

Purification and characterization of glutathione reductase from corn mesophyll chloroplasts

Differences in the apparent molecular weights of the subunits of glutathione reductase (EC 1.6.4.2) from pea chloroplasts and corn mesophyll chloroplasts have been recently reported. In order to more fully describe the differences between the enzymes from these two sources, glutathione reductase from the mesophyll chloroplasts of corn seedlings ( Zea mays L. cv. G-4507) has been purified 200-fold by affinity chromatography using adenosine 2',5'-disphosphate agarose. The purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)^-1 min^-1. The native enzyme had a relative molecular weight of 190 ± 30 kDa and exhibited polypeptides of 65, 63, 34, and 32 kDa when separated on sodium dodecylsulfate-polyacrylamide gels. Comparisons of the results from electroblotting, native molecular weight and subunit molecular weight analyses suggest that the enzyme exists as a heterotetramer. Optimal enzyme activity was obtained at pH 8 in N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES-NaOH) buffer. The sulfhydryl reagent, n -ethylmaleimide, inhibited enzymatic activity when incubated in the presence of NADPH while no inhibition was detected with oxidized glutathione in the incubation mixture. Reduced glutathione (5 m M ) inactivated the enzyme by 50%. This inactivation followed first order kinetics with a rate constant of 0.0028 s^-1. The enzyme was also inactivated by NADPH. The inactivation reached ca 90% within 30 min and followed first order kinetics with a rate constant of 0.0015 s^-1.     

Purification and Characterization of a Ca2+- and Calmodulin-Dependent Protein Kinase from Rat Brain

Abstract: A Ca²⁺- and calmodulin-dependent protein kinase was purified from rat brain cytosol fraction to apparent homogeneity at approximately 800-fold and with a 5% yield. The purified enzyme had a molecular weight of 640,000 as determined by gel filtration analysis on Sephacryl S-300 and a sedimentation coefficient of 15.3 S by sucrose density gradient centrifugation, and resulted in a single protein band of MW 49,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that the native enzyme has a large molecular weight and consists of 11 to 14 identical subunits. The purified enzyme exhibited K _m values of 109 and 30 μM for ATP and chicken gizzard myosin light chain, respectively, and K _a values of 12 n M and 1.9 μM for brain calmodulin and Ca²⁺, respectively. In addition to myosin light chain, myelin basic protein, casein, arginine-rich histone, microtubule protein, and synaptosomal proteins were phosphorylated by the enzyme in a Ca²⁺- and calmodulin-dependent manner. The purified enzyme was phosphorylated without the addition of the catalytic subunit of cyclic AMP-dependent protein kinase. Our findings indicate that there is a multifunctional Ca²⁺- and calmodulin-dependent protein kinase in the brain and that this enzyme may regulate the reactions of various endogenous proteins.