Purification and partial characterization of a gibberellin 2β-hydroxylase fromPhaseolus vulgaris

A gibberellin 2β-hydroxylase has been purified from mature seeds ofPhaseolus vulgaris. The enzyme is of molecular weight 36,000 and has the characteristics of a dioxygenase; the cofactors areα-ketoglu-tarate, Fe²⁺ and ascorbate, and activity is stimulated by catalase. The V_max of the enzyme is 6.86 nmole h^?1 mg^?1, and the Km values for [1,2-³H₂]GA₁ andα-ketoglutarate are 0.085 μM and 21 μM, respectively. The purified enzyme preparation catalyzes hydroxylation of GA₁, GA₄, GA₉, and GA₂₀ but exhibits a marked preference for the 3-hydroxylated gibberellins as substrate.     

Partial purification and characterization of the soluble DNA polymerase (polymerase-α) from seedlings of Pisum sativum L.

Radish seedlings (Raphanus sativus L. Saxa Treib) were grown in the dark with or without added kinetin (2 mg/l=9.29 M). Low-temperature (77°K) fluorescence emission and absorption spectra of etiolated cotyledons were registered at increasing seedling age before and immediately, 30 s and 30 min after one 1-ms flash. Kinetin was found to induce a higher accumulation of the phototransformable protochlorophyll(ide) P_657–650 in the etiolated cotyledons, especially from day 6 to day 10 after germination. The amount of the P_657–650 protochlorophyll(ide) resynthesized during a 30-min dark period after a 1-ms flash decreased with seedling age. It was smaller in cotyledons from kinetin-treated seedlings at day 6 after germination and at that age only. The ability to perform the Shibata shift decreased with increasing seedling age. In cotyledons from 10- and 13-day-old seedlings, the shift was accomplished to a greater extent when the plants were grown in the presence of kinetin.     

The ∈ subunit of the chloroplast ATP synthase of Pisum sativum

In contrast to the well-characterized spinach ( Spinacea oleracea) chloroplast ATP synthase (CF1–CFo), the properties of the chloroplast ATP synthase from pea (Pisum sativum ) have not been as intensively studied. Preliminary data suggested that the regulatory properties of the two enzymes differ. In the absence of activating treatments the ATPase activity of pea thylakoids in the dark was higher than that in spinach thylakoids. When assayed in the presence of sulfite, the MgATPase activity of pea thylakoids was inhibited to a maximum of 67% by tentoxin, indicating that the dark ATPase activity is in part catalyzed by CF1–CFo. The ATPase activity of purified pea CF1 was also higher than that of spinach CF1 in the absence of activating treatments. These differences could result from the different regulatory properties of the pea or subunit or both. The pea subunit was less effective in binding to or inhibiting the ATPase activity of pea o r spinach CF1 deficient in (CF1-). Spinach inhibited the ATPase activity of pea CF1- at lower concentrations than pea . The gene encoding the pea subunit was cloned and over-expressed. Recombinant pea did not restore low proton permeability to spinach thylakoid membranes reconstitituted with spinach CF1-, although pea was effective when tested with pea thylakoids reconstitituted with pea CF1-. These results confirm earlier suggestions that the C-terminal region of is important in -CF1 and -CFo interactions.     

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.     

Purification and partial characterisation of and α-tocopherol-binding protein from rabbit heart cytosol

An -tocopherol-binding protein has been isolated and purified from rabbit heart cytosol. The purified protein had an apparent molecular mass of 14,200, as derived from SDS-PAGE. The content of the protein in rabbit heart was around 11.8 g per g of tissue. The binding of -tocopherol to the purified protein was rapid, reversible, and saturable. Neither nor tocopherol could displace the bound -tocopherol from the protein, suggesting a high specificity for -tocopherol. -Tocopherol-binding protein did not bind oleate. Transfer of -tocopherol from liposomes to mitochodria was stimulated 8-fold in the presence of the binding protein, suggesting that this protein may be involved in the intracellular transport of -tocopherol in the heart.     

β-Galactosidase of Pediococcus species: induction,purification and partial characterization

Summary Six strains of Pediococcus pentosaceus and two of P. acidilactici had intracellular -galactosidase (-gal) activity when grown in the presence of lactose; all but two strains of P. pentosaceus and one of P. acidilactici had such activity when grown in the presence of glucose. Synthesis of -gal by P. pentosaceus ATCC 25745 was inducible with lactose, galactose, melibiose, lactobionic acid and possibly cellobiose but not with glucose, sucrose, maltose, glycerol, fructose or mannose. Lactose, galactose and possibly maltose, melibiose and lactobionic acid but not glucose, sucrose, glycerol, cellobiose, fructose or mannose induced -gal synthesis by P. acidilactici ATCC 25740. Synthesis of -gal was partially inhibited in P. pentosaceus ATCC 25745 and P. acidilactici ATCC 25740 by glucose added to the medium during growth in the presence of galactose or lactose. Isopropyl -d-thiogalactopyranoside failed to induce synthesis of -gal by either strain during growth on glucose. -Gal from P. pentosaceus ATCC 25745 had a molecular weight of 66,000 and activity optima of pH 6.5 and 45° C. Activity of the enzyme was stimulated by reducing agents, Mg²⁺, Mn²⁺, Zn²⁺ and Co²⁺ but not by Ca²⁺, and was markedly inhibited by ethylenediaminetetraacetate (EDTA), HgCl₂, 1,10-phenanthroline, and an oxidizing agent. The K _mvalues of the enzyme for o-nitrophenol--d-galactopyranoside and lactose were 3.07 and 7.0 mM, respectively, suggesting its low affinity for lactose. Offprint requests to: E. H. Marth     

Thromboxane synthase from bovine lung — Solubilization and partial purification

Prostaglandin endoperoxide synthase and thromboxane synthase were both localized mainly in the microsomal fraction of bovine lung. The capacity to convert prostaglanding H₂ into TXB₂ (thromboxane synthase activity) exceeded the capacity to transform arachidonic acid into products. Thromboxane synthase of lung microsomes was solubilized with Triton X-100 and partially purified by DEAE cellulose chromatography. The preparation thus obtained catalyzed the conversion of PGH₂ to a mixture of TXB₂ and HHT, whereas PGH₁ was predominantly converted to HHD.     

β-Galactosidase from Ginkgo biloba seeds active against β-galactose-containing N-glycans: purification and characterization

In this study, we purified an acidic β-galactosidase to homogeneity from Ginkgo biloba seeds (β-Gal’ase Gb-1) with approximately 270-fold purification. A molecular mass of the purified β-Gal’ase Gb-1 was estimated about 35 kDa by gel filtration and 32 kDa by SDS-PAGE under non-reducing condition, respectively. On the other hand, β-Gal’ase Gb-1 produced a single band with a molecular mass of 16 kDa by SDS-PAGE under reducing condition. The N-terminal amino acid sequences of 32 kDa and 16 kDa molecules were the same and identified as H-K-A-N-X-V-T-V-A-F-V-M-T-Q-H-, suggesting that β-Gal’ase Gb-1 may function as a homodimeric structure in vivo. When complex-type N-glycans containing β-galactosyl residues were used as substrates, β-Gal’ase Gb-1 showed substantial activity for β1-4 galactosyl residue and modest activity for β1-3 galactosyl residue with an optimum pH near 5.0. Based on these results, the involvement of β-Gal’ase Gb-1 in the degradation of plant complex-type N-glycans is discussed.     

Separation, purification, and sequence identification of TGF-β1 and TGF-β2 from bovine milk

Cox and Bürk (Eur. J. Biochem., 1991) reported the partial characterization of Milk Growth Factor (MGF) which stimulated the migration of fibroblasts. We have fractionated the partially purified sample by RP-HPLC and obtained the separation of two peaks of activity. The two active components were isolated as pure MGF-a and MGF-b by RP-HPLC and preparative SDS-PAGE. The purified MGF-a, consisting of a single band by gel electrophoresis and a single peak on an HPLC reversed-phase C-4 column, has the same specific activity as TGF-2 in the fibroblast migration assay. MGF-a was digested by endoprotease Asp-N and the cleaved peptides were analyzed by Edman degradation and plasma desorption mass spectrometry (PDMS). The whole sequence of MGF-a determined by automated sequenator and PDMS of S-pyridylethylated protein and selected fragments was found to be identical to that of TGF-2. MGF-b protein mixture separated by SDS-PAGE was electrophoretically transferred onto a Biometra Glassybond membrane, and the blotted MGF-b protein was directly sequenced on an automated sequenator. The identified 29 amino acids sequence of MGF-b was identical to the amino-terminal sequence of TGF-1. Our study demonstrates that MGF is composed of both TGF-1 and TGF-2. TGF-2 (85%) is the predominant form.     

Basic characterization and partial purification of β-glucosidase from cell-free extracts of Oenococcus oeni ST81

Aims: This study was designed to characterize a β‐glucosidase of Oenococcus oeni ST81, a strain isolated from a Spanish wine of the origin appellation Ribeira Sacra. Methods and Results: The β‐glucosidase of O. oeni ST81 seems to have a periplasmic localization into the cells. This activity was strongly inhibited by gluconic acid, partially inhibited by glucose and not inhibited by fructose, lactate, malate, mannitol or sorbitol. Ethanol increased the activity of this enzyme up to 147%. Among the several metal ions assayed, only Fe²⁺ (10 mmol l^?1) and Cu²⁺ (5 mmol l^?1) exhibited a partial inhibitory effect (40%). This enzyme was partially purified using a combination of ammonium sulfate precipitation and chromatographic methods. The single peak because of β‐glucosidase in all chromatographic columns indicates the presence of a single enzyme with an estimated molecular mass of 140 kDa. The calculated K_m and V_max values for 4‐nitrophenyl‐β‐d ‐glucopyranoside were 0·38 mmol l^?1 and 5·21 nmol min^?1, respectively. The enzyme was stable at pH 5·0 with a value of t_1/2 = 50 days for the crude extract. Conclusions: The β‐glucosidase of O. oeni ST81 is substantially different from those characterized from other wine‐related lactic acid bacteria (LAB), such as Lactobacillus plantarum and Lactobacillus brevis; however, it appears to be closely related to a β‐glucosidase from O. oeni ATCC BAA‐1163 cloned into Escherichia coli. The periplasmic localization of the enzyme together with its high tolerance to ethanol and fructose, the low inhibitory effect of some wine‐related compounds on the enzymatic activity and long‐term stability of the enzyme could be of interest for winemaking. Significance and Impact of the Study: Information regarding a β‐glucosidase from O. oeni ST81 is presented. Although the release of aroma compounds by LAB has been demonstrated, little information exists concerning the responsible enzymes. To our knowledge, this study contains the first characterization of a native β‐glucosidase purified from crude extracts of O. oeni ST81.     

The purification and regulatory properties of α-oxoglutarate dehydrogenase from Acinetobacter lwoffi

The alpha-oxoglutarate dehydrogenase multienzyme complex was purified from Acinetobacter lwoffi to a high degree of homogeneity as shown by gel electrophoresis and analytical ultracentrifugation. Sedimentation-velocity analyses gave s(20,w) values which increased with increasing protein concentration, suggesting dissociation of the complex in dilute solution. The maximum s(20,w) value thereby obtained and the value determined by active enzyme centrifugation were both in the range 28-29S. Electron micrographs of the complex indicated a molecular diameter of 20-22nm (200-220A). The overall activity of the complex was inhibited by NADH, and kinetic studies indicated sites of action on the first and third enzyme components. AMP and ADP relieved this inhibition and also stimulated enzyme activity. Assays specific for the first enzyme component showed this to be the site of action of the adenylates. The activity of the complex varied with energy charge in a manner consistent with its role in energy metabolism.     

Effects of galactomannan as carbon source on production of α-galactosidase by Thermomyces lanuginosus: Fermentation,purification and partial characterisation

Thermophilic fungus Thermomyces lanuginosus CBS 395.62/b strain is able to grow and synthesise extracellular α-galactosidase in media containing galactomannan such as locust bean gum (LBG) or guar gum (GG). Production of extracellular α-galactosidase was enhanced from 1.2 U/mL to 4–6 U/mL meaning about 3–5 times increase by optimisation of medium composition. This enzyme was purified to homogeneity by partial precipitation with 2-propanol and different liquid chromatographical steps. The developed purification protocol yielded 22% of enzyme activity with 900 purified fold. Molecular mass of the purified α-galactosidase enzyme was estimated to be 53 kDa. Maximal catalytic activity of the enzyme was obtained in the acidic pH range between pH 4.6 and 4.8 and in the temperature range 60–66 °C. More than 95% of enzyme activity was remaining after 1-day incubation at 70 °C and on pH in the range from 4.0 to 7.0. The enzyme activity was significantly stimulated by Mg²⁺, Mn²⁺ and K⁺ ions, while considerably inhibited by the presence of Ca²⁺, Ag⁺ and Hg²⁺.     

Overexpression,purification and characterisation of homologous α-l-arabinofuranosidase and endo-1,4-β-d-glucanase in Aspergillus vadensis

In the recent past, much research has been applied to the development of Aspergillus, most notably A. niger and A. oryzae, as hosts for recombinant protein production. In this study, the potential of another species, Aspergillus vadensis, was examined. The full length gDNA encoding two plant biomass degrading enzymes, i.e. α-l-arabinofuranosidase (abfB) (GH54) and endo-1,4-β-d-glucanase (eglA) (GH12) from A. vadensis were successfully expressed using the gpdA promoter from A. vadensis. Both enzymes were produced extracellularly in A. vadensis as soluble proteins and successfully purified by affinity chromatography. The effect of culture conditions on the expression of abfB in A. vadensis was examined and optimised to give a yield of 30 mg/L when grown on a complex carbon source such as wheat bran. Characterization of the purified α-l-arabinofuranosidase from A. vadensis showed an optimum pH and temperature of pH 3.5 and 60 °C which concur with those previously reported for A. niger AbfB. Comparative analysis to A. niger AbfA demonstrated interesting differences in temperate optima, pH stability and substrate specificities. The endo-1,4-β-d-glucanase from A. vadensis exhibited a pH and temperature optimum of pH 4.5 and 50 °C, respectively. Comparative biochemical analysis to the orthologous EglA from A. niger presented similar pH and substrate specificity profiles. However, significant differences in temperature optima and stability were noted.     

The purification of TEM-β-lactamase from cell extract using immobilized copper affinity chromatography

Summary TEM--lactamase was purified by immobilized metal affinity chromatography from E. coli cell extracts. It was hypothesized that this protein could be purified in one step due to the abundence of metal binding residues. A pH gradient eluted the cell extract proteins. EDTA treatment released TEM--lactamase. This procedure is much simpler than the current methods employed for TEM--lactamase purification.     

Production and partial purification of γ-linolenic acid and some pigments fromSpirulina platensis

The polyunsaturated fatty acid -linolenic acid (GLA, 18:36) is of potential pharmaceutical value. The cyanobacteriumSpirulina platensis could become an excellent source for this fatty acid, provided that GLA content could be increased and a GLA concentrate could be obtained at a low cost. Increasing the cell concentration inSpirulina platensis enhanced the fatty acid content and thus the GLA content. This effect was used to further enhance the GLA content of GLA-overproducing strains. Separation of the galactolipids and their purification via urea complexes formation, resulted in a GLA concentrate of over 90% purity.