Two Phenolic Amides in the Seed Balls of Sugar Beet (Beta vulgaris L. var. saccharifera Alefeld)

Ferredoxin-dependent sulfite reductase (Fd-SiR) (EC 1.8.7.1) was purified about 1136-fold, with a yield of 11%, from fresh thalli of Porphyra yezoensis by a procedure involving ammonium sulfate precipitation, DEAE-cellulose chromatography, Buty 1-Toyopearl chromatography, Sephadex G-100 gel filtration and ferredoxin-Sepharose affinity chromatography. The purified enzyme was apparently homogeneous, as judged on polyacrylamide disc gel electrophoresis, with a specific activity of 100 units/mg of protein. The molecular weight of the enzyme was estimated to be 70 kilodaltons by gel filtration. On subunit analysis by SDS-PAGE, a single band corresponding to molecular weight of 65 kilodaltons appeared. The purified enzyme (Fd-SiR) showed 5-times higher ferredoxin-dependent activity than methyl viologen-linked activity. In the oxidized form, the enzyme exhibited absorption maxima at 278, 390 (Soret band), 586 (a band) and 714 (CT band) nm, indicating that siroheme is involved in the catalysis of sulfite reduction. The absorbance ratios, A₃₉₀: A₂₁₈ and A₅₈₆ :A₃₉₀, were 0.32 and 0.31, respectively. A plot of the substrate (sulfite) and electron donor (ferredoxin) concentrations versus enzymatic (Fd-SiR) activity yielded sigmoidal curves, giving Hill coefficients («) of 2.3 (for sulfite) and 2.7 (for ferredoxin), respectively. Antibody against the isolated enzyme was raised in rabbits. Analysis of the antiserum by immunodiffusion suggested that it was specific against isolated Fd-SiR. Using the antiserum, dot immunoblotting was performed to determine the immunological similarity of Fd-SiRs from Porphyra yezoensis, Spirulina platensis, Brassica chinensis and Spinacia oleracea. The tests revealed that the four forms of assimilatory Fd-SiR have antigenic determinants in common.     

Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. Purification and properties of the enzyme from the electric organ of Electrophorus electricus

An unspecific carboxylesterase was purified 180-fold from acid-precipitated human liver microsomes. The final preparation was homogeneous on disc electrophoresis and polyacrylamide gel electrophoresis in the presence of 6.25 M urea at pH 3.2. A single symmetrical peak was also found on gel filtration and on velocity sedimentation in the analytical ultracentrifuge, whereas slight heterogeneity was observed on isoelectric focusing.The amino acid composition of the purified enzyme is presented. From the results the partial specific volume (0.745 ml × g^?1) and the minimal molecular weight (60,000) could be calculated. Fingerprint maps of tryptic peptides from the carboxymethylated enzyme are shown.The molecular weight as determined by gel filtration, disc electrophoresis, and analytical ultracentrifugation is in the range of 181,000–186,000. For the molecular weight of the subunits a value of 61,500 has been obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The equivalent weight of the enzyme has been estimated to be 62,500 from stoichiometry of its reaction with diethyl-p-nitrophenyl-phosphate. Partial cross-linking of the subunits with dimethyl suberimidate and subsequent sodium dodecylsulfate polyacrylamide gel electrophoresis yielded three bands with molecular weights of 60,000, 120,000, and 180,000.From these results it is concluded that human liver esterase is a trimeric protein. It is composed of three subunits of equal size, and there is one active site per subunit.     

Purification and subunit structure of mouse liver cystathionase

Cystathionase has been purified from mouse liver by ammonium sulfate precipitation, ethanol precipitation, column chromatography on DEAE-cellulose and on hydrox-ylapatite, as well as Sephadex G-200 gel filtration. These procedures yielded a chromatographically homogeneous enzyme which was purified more than 1000-fold relative to whole liver extract. Overall recovery was approximately 4%. The purified enzyme does not contain detectable carbohydrate and migrates as a single protein component on analytical disc gel electrophoresis. A sedimentation coefficient of 8.3 S has been determined for the active enzyme by rate zonal centrifugation in glycerol gradients. This value suggests a molecular weight for the native enzyme of approximately 160,000 g/mol, a value similar to that estimated by gel filtration. Following sodium dodecyl sulfate gel electrophoresis in the presence of reducing agent and at different gel concentrations, a single protein component with a molecular weight of 40,000 g/mol was obtained. Thus, the enzyme appears to consist of four subunits of equal size. The K_m value for cystathionine at pH 8.1, 37 °C, and in the presence of 1 mm dithioerythritol is approximately 1 mm.     

Purification and characterization of putrescine synthase from cucumber seedlings. A multifunctional enzyme involved in putrescine biosynthesis

The multifunctional enzyme, putrescine synthase has been purified fromCucumis sativus and characterized. This enzyme harbours agmatine iminohydrolase, ornithine transcarbamylase, putrescine transcarbamylase and carbamate kinase activities, whose concerted action results in agmatine → putrescine conversion. The enzyme resolved into two aggregation forms, enzyme aggregated and enzyme monomer upon electrophoresis at pH 8.3. Evidence has been provided by two-dimensional gel electrophoresis that both enzyme aggregated and enzyme monomer comprise of identical polypeptide chains. Under non-reducing conditions on sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the protein moves as a single 150 KDa polypeptide; however, in the presence of 2-mercaptoethanol on sodium dodecyl sulphate-polyacrylamide gel elec trophoresis, it migrates as 3 polypeptides of molecular weight 48,000, 44,000 and 15,000. The enzyme undergoes age-dependentin vivo proteolytic degradation from a 66 KDa polypeptide (primary translational product), through 48 KDa polypeptide to 44 KDa species and finally to small molecular weight peptides. Preliminary results of this work were presented at Golden Jubilee and Annual General Body Meetings of Society of Biological Chemists (India) and the Second Congress of Asian and Ocean Biochemists (1980) held at Bangalore, 1981,Indian J. Biochem. Biophys.,18, 113.     

The minor anionic form of arylsulphatase B (arylsulphatase Bm) of monkey brain. Purification and phosphoprotein nature

The anionic form of arylsulphatase B (arylsulphatase Bm) was purified to apparent homogeneity from monkey brain through steps involving chromatography on diethylaminoethyl-cellulose, Blue-Sepharose, Biogel HTP and finally Biogel P-300 gel filtration. The molecular weight of the purified enzyme as deduced by gel filtration on Biogel P-300 and by sodium dodecylsulphate gel electrophoresis was ∼ 30,000.Escherichia coli alkaline phosphatase treatment of arylsulphatase Bm resulted in the conversion of upto 84% of the enzyme into a less charged form of enzyme, that could not bind to diethylaminoethyl cellulose. Potassium phosphate an inhibitor of alkaline phosphatase prevented this conversion. Upon acid hydrolysis the purified enzyme yielded approximately 7.0 mol of inorganic phosphate per mol of protein.Vibrio cholerae neuraminidase treatment did not alter the charge on arylsulphatase Bm.     

Selection of High Ubiquinone 10-Producing Strain of Tobacco Cultured Cells by Cell Cloning Technique

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase, was purified from a cell-free extract of Streptococcus faecalis R ATCC 8043, using N^α-benzyloxycarbonyl glycine as substrate. The enzyme was purified 1300-fold with an activity yield of 8%. The purified enzyme was homogeneous by disc electrophoresis. The molecular weight of the native enzyme is about 220,000 by gel filtration, and a molecular weight of 32,000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point was 4.48. The enzyme was inhibited by p-chloromercuribenzoate. The presence of divalent cations (i.e., Co²⁺ or Zn²⁺) is essential for its activity.     

Molecular and Enzymatic Properties of Pyridoxamine (Pyridoxine) 5′-Phosphate Oxidase from Baker’s Yeast

Pyridoxamine (pyridoxine) 5′-phosphate oxidase purified from baker’s yeast was found to have a molecular weight of ca, 55,000 daltons based on polyacrylamide gel electrophoresis. The size of the enzyme subunit was analyzed by gel electrophoresis in the presence of sodium dodecylsulfate. This showed that the enzyme was composed of two nonidentical subunits with a molecular weight of 27,000 and 25,000 daltons. Fluorescence titration of the apoenzyme with FMN suggested that the holoenzyme contained one mol of FMN per mol of the enzyme. The K_m value of FMN for apoenzyme was calculated to be ca. 16 nm on both activities of pyridoxamine 5′-phosphate oxidase and pyridoxine 5′-phosphate oxidase.     

Alfalfa Root Nodule Carbon Dioxide Fixation : II. Partial Purification and Characterization of Root Nodule Phosphoenolpyruvate Carboxylase

A nonphotosynthetic phosphoenolpyruvate carboxylase (EC 4.1.1.31) was partially purified from the cytosol of root nodules of alfalfa. The enzyme was purified 86-fold by ammonium sulfate fractionation, DEAE-cellulose, hydroxylapatite chromatography, and reactive agarose with a final yield of 32%. The enzyme exhibited a pH optimum of 7.5 with apparent K_m values for phosphoenolpyruvate and magnesium of 210 and 100 micromolar, respectively. Two isozymes were resolved by nondenaturing polyacrylamide disc gel electrophoresis. Subsequent electrophoresis of these isozymes in a second dimension by sodium dodecyl sulfate slab gel electrophoresis yielded identical protein patterns for the isozymes with one major protein band at molecular weight 97,000. Malate and AMP were slightly inhibitory (about 20%) to the partially purified enzyme. Phosphoenolpyruvate carboxylase comprised approximately 1 to 2% of the total soluble protein in actively N₂-fixing alfalfa nodules.     

Isolation and characterization of argininosuccinate synthetase from human liver

This communication describes the purification and characterization of argininosuccinate synthetase from human liver. By numerous criteria including electrophoresis in sodium dodecyl sulfate containing gels, electrophoresis in nondissociating gels, and analytical ultracentrifugation, the protein is homogeneous at a specific activity of 4.2 mumol/(min mg) assayed at 37 degrees C in the direction of argininosuccinate synthesis. The enzyme has a molecular weight of 183,000, as determined by gel filtration. Electrophoresis in the presence of sodium dodecyl sulfate yielded a single band migrating with an Rf corresponding to 43,000 daltons. Thus, the enzyme is considered to contain four subunits of identical molecular weight. The s20,w of the enzyme is 8.2 S. Antibodies were prepared in rabbits directed against the purified protein. These antibodies react specifically with argininosuccinate synthetase, as determined by electrophoretic analysis of the immunoadsorbed product from crude extracts of human liver. The human enzyme has very similar properties to those published for the beef and rat liver enzymes.     

Purification of Glucoamylase from Lactobacillus amylovorus ATCC 33621

An intracellular glucoamylase (E.C. 3.2.1.3) was purified to homogeneity from Lactobacillus amylovorus on a Fast Protein Liquid Chromatography System (FPLC) with a Mono Q ion-exchanger and two Superose 12 gel filtration columns arranged in series. The enzyme activity was quantified with a specific, chromogenic substrate, p-nitrophenyl-β-maltoside. Preparative gel electrophoresis was then used to further purify active enzyme fractions. Native polyacrylamide gel electrophoresis (Native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of molecular weight 47 kDa. Glucoamylase activity of the purified protein was confirmed by its ability to degrade starch on a 0.025% starch-polyacrylamide gel stained with I₂/KI. Glucoamylase exhibited optimum catalytic activity at pH 6.0 and 45°C, and the enzyme had an isoelectric point near 4.39. The glucoamylase contained high levels of hydrophilic amino acids, comparable to fungal glucoamylases. Received: 12 July 1996 / Accepted: 10 September 1996     

Creatine amidinohydrolase of Pseudomonas putida: crystallization and some properties.

Creatine amidinohydrolase (EC 3.5.3.3, creatinase) of Pseudomonas putida var. naraensis C-83 was purified by column chromatography on sarcosine-hexamethylenediamine-Sepharose and Sephadex G-200 and then crystallized in the presence of ammonium sulfate. The purified preparation appeared homogeneous on disc gel electrophoresis and ultracentrifugal analysis. It was most active at pH 8 and showed a K_m value of 1.33 mm for creatine. Estimation of the molecular weight by the meniscus depletion method yielded a value of 94,000. A value of 47,000 was obtained, however, by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the enzyme is composed of two subunits. Inhibition experiments suggested that a sulfhydryl group is closely related to the creatinase activity.     

Isolation and Characterization of Nα-Benzyloxycarbonyl Amino Acid Urethane Hydrolase II from Lactobacillus fermenti 36 ATCC 9338

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase II, was purified from a cell-free extract of Lactobacillus fermenti 36 ATCC 9338. The enzyme catalyzed the stoichiometric hydrolysis of N^α-benzyloxycarbonyl arginine to form benzyl alcohol and arginine. The enzyme was purified 106-fold with an activity yield of 3%. The purified enzyme was homogeneous by disc gel electrophoresis. The molecular weight of the native enzyme is about 200,000 by gel filtration, and a molecular weight of 27,000 was found for the reduced and denaturated enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point of the enzyme was 5.0, it was inhibited by disodium ethylenediamine tetraacetate and p-chloromercuribenzoate, and the presence of a divalent cation, i.e. Co²⁺, is essential for its activity.     

Spectroscopic properties of spinach catalase

Spinach catalase (hydrogen-peroxide: hydrogen-peroxide oxidoreductase, EC 1.11.1.6) has been purified to homogeneity. The purified enzyme has a specific activity of 25 000 units per mg protein. The presence of 2-mercaptoethanol and phenylmethylsulfonyl fluoride (PMSF) were required for high yields of the enzyme. The molecular weight of the enzyme was estimated to be 125 000 by gel filtration. Subunit analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a single peptide with M_r 55 000. The enzyme, which exhibits optical absorbance maxima at 279, 403, 542, 592 and 723 nm and shoulders at 290, 500 and 630 nm, contains 2 mol iron per mol protein. One of the two irons can be attributed to protoheme, while the other iron appears to be present in a novel heme. The oxidized catalase exhibited two sets of high-spin, ferriheme EPR signals.     

Purification and partial characterization of the catalytic subunit of cAMP-dependent protein kinases from reticulocytes.

The catalytic subunit of cyclic AMP-dependent protein kinases from rabbit reticulocytes has been purified to near homogeneity. It has a molecular weight of 43,000 as judged from gel filtration and by polyacrylamide gel electrophoresis in the presence of sodium dodecyi sulfate and appears to be similar in physical properties and substrate specificity to the comparable enzyme isolated from muscle or liver. The enzyme phosphorylates histones, a protein of 40 S ribosomal subunits from reticulocytes and from Artemia salina, and the low molecular weight heat-stable phosphatase inhibitor (G. A. Nimmo and P. Cohen, 1978, Eur. J, Biochem.87, 341–351). No evidence has been obtained for a direct or indirect role of this enzyme in the regulation of protein synthesis.     

Guanidoacetate methyltransferase from rat liver: Purification,properties, and evidence for the involvement of sulfhydryl groups for activity

Guanidoacetate methyltransferase (EC 2.1.1.2) has been purified about 800-fold from rat liver. The purified preparation shows a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the enzyme is estimated to be 25,000 and 26,000 by Sephadex gel molecular-exclusion chromatography and by electrophoresis in polyacrylamide gradient gel, respectively. The sodium dodecyl sulfate-denatured enzyme also has a molecular weight of 26,000; thus, the enzyme is a monomeric protein. Guanidoacetate methyltransferase as isolated is catalytically inactive, but is readily reactivated by incubation with a thiol. The reactivated enzyme, which contains 3 mol of sulfhydryl groups/mol of enzyme, is again inactivated by oxidized glutathione. This inactivation is accompanied by the disappearance of two sulfhydryl residues. The relationship between the loss of enzyme activity and the number of residues disappeared indicates that the integrity of these sulfhydryl residues is critical for activity. The oxidized enzyme fails to bind the substrate S-adenosylmethionine as evidenced by the equilibrium dialysis study. Alkylation of the nonoxidizable sulfhydryl by N-ethylmaleimide shows that this residue is also essential for activity. UV absorption, fluorescence, and CD spectra show no difference between the reduced and oxidized enzymes, but the former is more susceptible to proteolytic attack by trypsin. The enzyme has an isoelectric pH of 5.3, and is most active at pH 9.0. From the CD spectrum, an α helix content of 15% is calculated. The K_m values for guanidoacetate and S-adenosylmethionine are 97.5 and 6.73 μm, respectively, at pH 8.0 and 37 °C.     

Characterization of Endo β-1,3-Glucanase IV from Flavobacterium dormitator var. glucanolyticae FA-5

Endo β-1,3-glucanase IV (E.C. 3.2.1.6, endo-1,3(4)-β-d-glucanase) from Flav. dormitator var. glucanolyticae FA-5 was shown to be a glycoprotein by gel filtration and sodium dodecyl sulfate gel electrophoresis. The carbohydrate moiety was composed of 17 hexose units. The enzyme had an apparent molecular weight of 3.3 x 10⁴, determined by gel filtration, sodium dodecyl sulfate gel electrophoresis and ultracentrifugation. The enzyme showed maximum reactivity at pH 6.0 and 6.5 for living yeast cells and laminaran, respectively. The enzyme predominantly released laminaripen-taose from a variety of linear β-1,3-glucans and showed transglucanosylation activity. The amino acid composition of the enzyme and some of its physicochemical and enzymatic properties are described.     

Rotein Composition Cf Flasmin Preparation “Hcmclysin”

A commercial preparation of human plasmin (Homolysin), capable of catalyzing the transformation of human growth hormone (hGH) into biologically activated species, was analyzed by electrophoresis and electrofocusing on polyacrylamide gel. Each major component of the preparation was characterized with regard to molecular size (retardation coefficient, K_R). molecular net charge (y-intercept on the Ferguson plot, Y₀), apparent isoelectric point (pi') and enzyme activity. The multiple components of Homolysln revealed by staining corresponded to various aggregation states of plasmin and exhibited full serine protease activity. Pclyacrylamlde gel electrophoresis of Homolysln in the presence of sodium dodecylsulfate (SDS) yielded 2 sub units which corresponded in molecular weight to the known plasmin subunits.     

Subunit Structure of Glucoamylase of Saccharomyces diastaticus

Extracellular glucoamylase produced by a starch-fermenting yeast, Saccharomyces diastaticus 5106-9A, was purified. The enzyme was found to be heterogeneous in molecular weight, ranging from approximately 80K to 66K as estimated by gel filtration, and consisted of two subunits, H and Y. The molecular weight of subunit H was heterogeneous and was determined to be approximately 68K, 59K, and 53K by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of subunit Y was 14K, estimated by the same gel. the molecular weight of the deglycosylated form of subunit H was 41K, suggesting that the heterogeneity of the enzyme was due to glycosyl moieties of subunit H. Subunits H and Y were separated by gel filtration in the presence of sodium dodecyl sulfate. Subunit Y seemed to be hydrophobic, since it was insoluble in an aqueous buffer without detergent.     

Import of peroxisomal hydroxypyruvate reductase into glyoxysomes

A new procedure was used to purify the peroxisomal matrix enzyme hydroxypyruvate reductase (HPR) from green leaves of pumpkin (Cucurbita pepo L.) and spinach (Spinacia oleracea L.). Monospecific antibodies were prepared against this enzyme in rabbits. Immunoprecipitation of HPR from watermelon (Citrullus vulgaris Schrad.) yielded a single protein with a subunit molecular weight of 45 kDa. Immunohistochemical labeling of HPR was found exclusively in watermelon microbodies. Isolated polyadenylated mRNA from light-grown watermelon cotyledons was injected into Xenopus laevis oocytes. The heterologous in-vivo translation product of HPR exhibited the same molecular weight as the immunoprecipitate from watermelon cotyledons, indicating the lack of a cleavable extra sequence. The watermelon HPR translated in oocytes was imported into isolated glyoxysomes from castor bean (Ricinus communis L.) endosperm and remained resistant to proteolysis after the addition of proteinase K. The HPR did not change its apparent molecular weight during sequestration; however, it may have changed its conformation.Abbreviations HPR hydroxypyruvate reductase - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Purification and characterization of mandelonitrile lyase from Prunus lyonii

The enzyme mandelonitrile lyase (EC 4.1.2.10) which catalyzes the decomposition of the cyanohydrin of benzaldehyde has been isolated and purified to homogeneity from mature seeds of the California cherry (Prunus lyonii). The purification procedure involved chromatography on DEAE-cellulose and Con-A-Sepharose with a final recovery of 60% of enzyme activity. Purification of only 4.3-fold yielded a nearly homogeneous preparation. The absorption spectrum of this protein shows maxima at 278, 389, and 463 nm, indicative of its flavoprotein character. The native molecular weight for the lyase was found to be 50,000. The subunit molecular weight of 59,000 was estimated by gel electrophoresis in the presence of sodium dodecylsulfate. The isoelectric point was estimated to be 4.75. The enzyme has a narrow pH optimum around 5.5 and is highly stable at 4 degrees C.