Identification and characterization of a rhizosphere β-galactosidase from Pisum sativum L.

Plant enzyme activities in the rhizosphere potentially are a resource for improved plant nutrition, soil fertility, bioremediation, and disease resistance. Here we report that a border cell specific β-galactosidase is secreted into the acidic extracellular environment surrounding root tips of pea, as well as bean, alfalfa, barrel medic, sorghum, and maize. No enzyme activity was detected in radish and Arabidopsis, species that do not produce viable border cells. The secreted enzyme activity was inhibited by galactose and 2-phenylethyl 1-thio-β-d-galactopyranoside (PETG) at concentrations that altered root growth without causing cell death. A tomato galactanase encoding gene was used as a probe to isolate a full length pea cDNA clone (BRDgal1) from a root cap-border cell cDNA library. Southern blot analysis using full length BRDgal1 as a probe revealed 1–2 related sequences within the pea genome. BRDgal1 mRNA expression was analysed by whole mount in situ hybridization (WISH) and found to occur in the outermost peripheral layer of the cap and in suspensions of detached border cells. No expression was detected within the body of the root cap. Repeated efforts to develop viable hairy root clones expressing BRDgal1 antisense mRNA under the control of the CaMV35S promoter, whose expression in the root cap is limited to cells at the root cap periphery only during root emergence, were unsuccessful. These data suggest that altered expression of this enzyme is deleterious to early root development. The first two authors contributed equally to the completion of this project.     

α-glucosidase from grape berries: Partial purification and characterization

-Glucosidase (-D-glucoside glucohydrolase EC 3.2.1.20) was purified approximately 30-fold from grape berries (Vitis vinifera var. Riesling). Besides maltose the enzyme preparation hydrolyzes to a lesser extent maltotriose, isomaltose, and starch. It has a pH optimum of 5.1 and a molecular weight of about 100,000. Tris, glycerol, several mono-and disaccharides were tested as inhibitors. The kinetic behavior of ribose, fructose, cellobiose, sucrose, turanose, methylglucopyranoside, Tris, and glycerol was fully investigated. The inhibition studies suggest a Ping-Pong mechanism, with the second substrate concentration being constant, that can be treated as a Uni Bi system. The purified enzyme is stable when stored frozen at-20° C. The grape-berry -glucosidase may exist as multiple forms (pI 7.2 and 8.2 respectively), and it does not require ions for its activity.This work was supported by the consiglio Nazionale delle Ricerche, Roma, Italy     

The partial purification and characterisation of gibberellin 2β-hydroxylases from seeds of Pisum sativum

The gibberellin (GA) 2-hydroxylases in mature and immature seeds of Pisum sativum have been partially purified and characterised. The enzymes are unstable when stored below pH 7.0 or in the absence of a thiol reagent. The optimum assay pH is between 7.4 and 7.8 and activity is dependent upon the presence of -ketoglutarate, Fe²⁺ and ascorbate. The 2-hydroxylase activities for GA₁, GA₄, GA₉ and GA₂₀ are chromatographically inseparable and correspond to a protein of M_r 44000. The rate of GA 2-hydroxylation varies according to substrate and some evidence indicates that the 2-hydroxylase activities for GA₁ and GA₄ and for GA₉ and GA₂₀ may reside in different proteins. During pea seed maturation, the specific activity of the enzyme(s) increases dramatically and reaches a maximum at a time when endogenous GA₉, GA₂₀, GA₂₉ and GA₅₁ are also at their greatest concentration. This correlation is not the result of substrate induction of enzyme activity. Since the GA 2-hydroxylases operate at maximal rate at low substrate concentrations they are incapable of rapidly 2-hydroxylating excessive quantities of (exogenously applied) GA₁ or GA₂₀. On the basis of the kinetic parameters of the GA 2-hydroxylase activities, a generalised model is discussed for the control of the steady-state levels of bioactive hormone under normal physiological conditions.Abbreviations DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - GA_n gibberellin A_n - HPLC high-performance liquid chromatography - HSS high-speed supernatant - LSS low-speed supernatant - PMSF phenylmethane sulphonyl fluoride     

Partial purification of endo- and exo-β-D-glucanase enzymes from Zea mays L. seedlings and their involvement in cell-wall autohydrolysis

The proteins dissociated from isolated Zea seedling cell wall using high-ionic-strength salt solutions have been found to include a number of enzymes which appear to participate in autolytic reactions of the cell wall. These enzymes caused extensive degradation of enzymatically inactive cell wall, liberating as much as 100 g/mg dry weight over a 48-h period. Lithium chloride (3M) was shown to be most effective in yielding protein and wall-degrading activities.Molecular-sieve chromatography of the cell-wall protein resolved endo--D-glucanase and exo--1,3-glucanase (EC 3.2.1.58) activities when Avena glucan and laminarin, respectively, were employed as substrates. The exoenzyme (molecular weight around 60,000) was strongly inhibited by inorganic mercury at a concentration which suppressed the release of monosaccharide from autolytically active cell wall. The endo--D-glucanase (MW around 26,000), which showed a marked preference for substrates of mixed-linkage, exhibited features indicating that it initiates the autolytic solubilization of wall glucan.Cell-wall -D-glucan, recovered as a component of an alkali-soluble cell-wall fraction, served as a substrate for the purified glucanases. Their hydrolysis pattern, assessed using gel exclusion chromatography and product analysis, confirmed that they hydrolyze -D-glucan. The products generated by the endoglucanase were similar in molecular-size distribution to those liberated from autolytically active-wall. Exoglucanase activity was required for extensive hydrolysis of -D-glucan in vitro.During coleoptile development the autolytic activity of the cell wall increased dramatically. This increased activity, however, did not parallel the growth potential of the tissue, but more closely reflected an increase in cell-wall -D-glucan, the primary substrate for autolytic reactions.These results were presented, in part, as papers at the annual meeting of the American Society of Plant Physiologists. Ohio State University, Columbus, in August 1979     

Partial purification and property of pyridoxine (pyridoxamine)-5′-phosphate oxidase isozymes from wheat seedlings

Isozymes of pyridoxine (pyridoxamine)-5′-phosphate oxidase (EC 1.4.3.5) were isolated from the extract of wheat seedlings by column chromatographies. From DEAE-Sephadex A-50, two fractions having pyridoxine-5′-phosphate oxidase activity were separated by eluting with ~0.075 and ~0.125 m phosphate buffers (pH 8.0). These fractions were further fractionated on a Blue-Sepharose CL-6B column, from which again two activities were eluted by 1.0 m KCl solution. One fraction, designated as E-I, used only pyridoxine 5′-phosphate as substrate, whereas the other, designated as E-II, oxidized not only pyridoxine 5′-phosphate but also pyridoxamine 5′-phosphate with approximately equal rates. The mobility on polyacrylamide disc gel electrophoresis and the substrate specificity of these two fractions were different. Therefore, they were concluded to be isozymes.     

Partial purification and kinetics of oestriol 16α-glucuronyltransferase from the cytosol fraction of human liver

An enzyme that conjugates the 16α-hydroxyl group of oestriol with glucuronic acid was found in the cytosol fraction of human liver. The enzymic activity could not be sedimented when the cytosol fraction was centrifuged at 158000g_av. for 120min. The oestriol 16α-glucuronyltransferase was purified 100-fold by 0–30% saturation of the cytosol fraction with ammonium sulphate followed by filtration of the precipitate through Sephadex G-200. The activity was eluted at the void volume. The product of the reaction, oestriol 16α-monoglucuronide, was identified by paper chromatography and by crystallization of radioactive product to constant specific radioactivity. The optimum temperature was 37°C, and the activation energy was calculated to be 11.1kcal/mol. The apparent Michaelis–Menten constants for oestriol and UDP-glucuronic acid were 13.3 and 100μm respectively. Cu²⁺, Zn²⁺ and Hg²⁺ inhibited, whereas Mg²⁺, Mn²⁺ and Fe²⁺ stimulated the enzyme. Substrate-specificity studies indicated that the amount of oestradiol-17β, oestradiol-17α and oestrone conjugated was not more than about 5% of that found for oestriol. Oestriol 16α-monoglucuronide, a product of the reaction, did not inhibit the 16α-oestriol glucuronyltransferase; in contrast, UDP, another product of the reaction, inhibited the enzyme competitively with respect to UDP-glucuronic acid as the substrate, and non-competitively with respect to oestriol as the substrate. ATP and UDP-N-acetylglucosamine did not affect the oestriol 16α-glucuronyltransferase. 17-Epioestriol acted as a competitive inhibitor and 16-epioestriol as a non-competitive inhibitor of the glucuronidation of oestriol. 5α-Pregnane-3α,20α-diol also inhibited the enzyme non-competitively. It is most likely that the oestriol 16α-glucuronyltransferase described here is bound to the membranes of the endoplasmic reticulum.     

Cloning and characterization of a DNA polymerase β gene from Trypanosoma cruzi

A gene coding for a DNA polymerase β from the Trypanosoma cruzi Miranda clone, belonging to the TcI lineage, was cloned (Miranda Tcpolβ), using the information from eight peptides of the T. cruzi β-like DNA polymerase purified previously. The gene encodes for a protein of 403 amino acids which is very similar to the two T. cruzi CL Brener (TcIIe lineage) sequences published, but has three different residues in highly conserved segments. At the amino acid level, the identity of TcI-polβ with mitochondrial polβ and polβ-PAK from other trypanosomatids was between 68–80% and 22–30%, respectively. Miranda Tc-polβ protein has an N-terminal sequence similar to that described in the mitochondrial Crithidia fasciculata polβ, which suggests that the TcI-polβ plays a role in the organelle. Northern and Western analyses showed that this T. cruzi gene is highly expressed both in proliferative and non-proliferative developmental forms. These results suggest that, in addition to replication of kDNA in proliferative cells, this enzyme may have another function in non-proliferative cells, such as DNA repair role similar to that which has extensively been described in a vast spectrum of eukaryotic cells.     

Partial purification and further characterization of the novel endoglucosaminidase from human serum that hydrolyses 4-methylumbelliferyl-N-acetyl-β-d-chitotetraoside (MU-TACT hydrolase)

A novel endoglucosaminidase, originally described by Den Tandt et al. [Int. J. Biochem.20 (1988), 713–719] and bearing the provisional name MU-TACT hydrolase, was purified from human serum 56,000-fold by means of ammonium sulphate precipitation, anion-exchange chromatography, Con A-Sepharose chromatography and gel filtration on Sepharose CL-6B followed by Superose 12HR. Based on the latter technique the native apparent molecular weight of the enzyme appeared to be equal to that of myoglobin, being approx. 17 kD. The enzyme eluted clearly at a different volume than lysozyme. MU-TACT is a commercially available substrate for lysozyme. For unknown reasons two major peptides co-purify that give bands on SDS-PAGE of 55–60 and 31 kD, respectively.     

Partial purification, characterization and nitrogen regulation of the lysine ɛ-aminotransferase of Streptomyces clavuligerus

The L-lysine ɛ-aminotransferase (LAT) of Streptomyces clavuligerus was partially purified and characterized. The 51.3-kDa enzyme exhibited optimal activity at pH 7.0–7.5 and 30°C. It catalyzed transfer of the terminal amino group of L-lysine or L-ornithine to α -ketoglutarate. Oxalacetate and pyruvate were also used as acceptors of the amino group but with very low efficiency. Increasing ammonium concentrations added to chemically-defined medium MM enhanced the formation of LAT and decreased production of cephalosporins by S. clavuligerus. In cultures grown in the absence of lysine, greater enhancement of LAT formation by ammonium and less repression of cephalosporin biosynthesis were observed. In the chemically-defined GSPG medium, ammonium ions decreased cephalosporin production without showing an effect on LAT formation. Received 20 August 1996/ Accepted in revised form 15 November 1996     

Partial purification and characterization of intracellular car☐ypeptidase ofCandida albicans

Intracellular car☐ypeptidase was partially purified from the yeast form ofCandida albicans H-317 by acid dialysis, ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Peptidase activity was measured with an enzyme-coupled colorimetric assay. Fractionation by native polyacrylamide gel electrophoresis and Sephacryl S-200 gel filtration indicated the presence of a single peak of car☐ypeptidase, with the isoelectric point at pH 4.6 and a molecular weight of 100,000. The pH optimum and apparentK_m usingN-carbobenzoxy-l-phenylalanine-l-leucine (N-Cbz-l-Phe-l-Leu) as substrate were 6.5 and 2 × 10⁻⁴M, respectively. The best substrates for the enzyme wereN-Cbz-Ala-X peptides, withN-Cbz-Ala-Leu giving the highest rate of hydrolysis. Substrates that gave 7% or less of the control (N-Cbz-Phe-Leu) rate of hydrolysis were Gly-Leu, Gly-Phe, Ile-Phe, Ile-Met, Glu-Phe, Glu-Tyr, Val-Phe, and Pro-Phe. There was no detectable hydrolysis of the followingN-Cbz peptides; Gly-Met, Gly-Val, Gly-Tyr, Gly-Ile, or Ile-Val. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride,p-chloromercuribenzoate, benzyloxy-carbonyl-l-phenylalanine chloromethyl ketone, and tosyl-l-phenylalanine chloromethyl ketone, but was not affected by EDTA, tosyl-l-lysine chloromethyl ketone, pepstatin A, leupeptin, bestatin, or antipain. Although secretory proteinases are thought to play a role in the pathogenesis of this organism, the role of this intracellular car☐ypeptidase has yet to be determined.     

Partial purification and comparative characterization of α-amylase secreted byLactobacillus amylovorus

An -amylase (E.C. 3.2.1.1.) secreted byLactobacillus amylovorus was partially purified and characterized. This high-molecular-weight enzyme [Imam SH, Burgess-Cassler A, Côté GL, Gordon SH, Baker FL (1991) Curr Microbiol 22:365–370] was quantified with a clinical -amylase assay adapted to a microplate format. It was isolated from concentrated cell-free culture medium by ammonium sulfate precipitation, ion exchange, and hydrophobic interaction chromatographies. The enzyme was not particularly thermostable, but like three other microbial -amylases tested for comparison, was renaturable following treatment with SDS and heat. The pH optimum and pI were 5.5±0.5 and 5.0, respectively; its temperature optimum was 60–65°C, and the molecular weight on SDS gels was 140±10 kDa.     

Partial purification and properties of a β-N-acetylglucosaminidase from the fungus Sclerotinia fructigena

1. As cultures of the fungus Sclerotinia fructigena autolysed, the filtrates contained increasing quantities of a beta-N-acetylglucosaminidase. 2. The enzyme was purified up to 42-fold by a combination of isoelectric focusing and gel filtration. 3. It ran as a single band in cellulose acetate strip electrophoresis and in isoelectric focusing (pI3.76). 4. The enzyme did not readily hydrolyse chitin or a glycopeptide with terminal N-acetylglucosamine residues, but rapidly degraded the N-acetylglucosamine dimer NN'-diacetylchitobiose; the monomer was readily utilized by the fungus as a nitrogen source. The K(m) value for hydrolysis of p-nitrophenyl beta-2-acetamido-2-deoxy-d-glucopyranoside at 37 degrees C was 2.0mm. The Sclerotinia enzyme was generally less susceptible to inhibition by 2-acetamido-2-deoxygluconolactone and other related sugars than the corresponding enzyme from other sources. Inhibition by excess of substrate was observed. 5. The culture filtrate also contained N-acetylgalactosaminidase activity; conflicting evidence was obtained as to whether the same enzyme was responsible for both hexosaminidase activities.     

Resolution of DNA polymerase-α-primase complex and primase free

DNA Polymerase-α from embryonic chicken brain was resolved on DEAE-cellulose into 3 comPonent activities that remained distinct uPon rechromatograPhy. Product formation by each activity required exogenously added temPlate-Primer DNA, all 4 deoxynucleoside triPhosPhates, and a divalent metal cation. Each form incorPorated [³H]-dTTP or [³H]-dCTP into a high molecular weight Product that was identified as DNA by its chromatograPhic behavior and its sensitivity to DNase. High ionic strength, N-ethylmaleimide, and the Polymerase-α-sPecific inhibitor aPhidicolin inhibited each activity; the aPParentK _i value of aPhidicolin was 3.0 μM in each case. Based on these results, the 3 activities were identified as multiPle forms of DNA Polymerase-α . ExPeriments using embryonic chicken brains of various ages indicated that Polymerase-α1, and Polymerase-α3 reached maximal activity in 9-day-old embryos, while Polymerase-α₂ activity was elevated at a slightly later develoPmental stage. Using Poly (dC) as temPlate, high Primase activity was detected in Polymerase-α₁, fractions.     

Partial purification and properties of γ-glutamyltranspeptidase from mycelia of Morchella esculenta

Three -glutamyltranspeptidase (enzymes I, II and III) were partially purified from the cell free extracts of the cultured mycelia of Morchella esculenta Fr. The molecular masses of enzymes were 155,000 (I), 219,000 (II) and 102,000 (III). All of them catalyzed both hydrolysis and transpeptidation of various -glutamyl compounds. -l-Glutamyl-cis-3-amino-l-proline occurring in the cultured mycelia of this fungus was a good substrate for both reactions. K _m values for hydrolysis were in the order of 10^-4 to 10^-5 M, and those for transpeptidation were in the order of 10^-2 to 10^-4 M. The enzymes were inhibited by a -glutamyltranspeptidase inhibitor, l-serine plus borate.Abbreviations -GTP -glutamyltranspeptidase - HPLC High-performance liquid chromatography     

Effect of phosphatidylcholine vesicles on the activity of DNA polymerase-α

Summary Phosphatidylcholine vesicles stimulate the activity of the DNA polymerase- from calf thymus. This effect is dependent upon the way of addition of the Mg ions, and the extent of the ³H-dTTP incorporation is closely related to the concentration of the vesicles. A role of phospholipids on the activity of the DNA-related enzymes is suggested.     

α,α-trehalase from the brine shrimp Artemia salina. purification and properties

• 1.1. Trehalase (α,α-trehalose 1-d-glycohydrolase, E.C. 3.2.1.28) from cryptobiotic Artemia salina embryos was purified and characterized.
• 2.2. Most of the enzyme activity was present in an insoluble form and could be solubilized by deoxycholate treatment at high ionic strength and sonication.
• 3.3. The enzyme had a molecular weight of 75,000 and its isoelectric point was 6.2. It was optimally active at pH 5.6 with a K_m = 4.3 × 10⁻³ M for its natural substrate.
• 4.4. The enzyme was highly specific for trehalose, only lactose and cellobiose being hydrolyzed to a limited extent.