Cell wall proteins from sugar beet cells in suspension culture

Several proteins were extracted from the purified cell walls of suspension-cultured sugar beet cells with 0.5% EDTA (pH 6.8) after prior extraction of the walls with 0.5% deoxycholate and then with 2 molar NaCl. Two abundant proteins (P-I and P-II protein) were separately purified to homogeneity by procedures that included fractionation with ammonium sulfate, column chromatography on DEAE-cellulose and butyl Toyopearl, and preparative polyacrylamide electrophoresis. P-I exists as a dimer of identical subunits, and P-II is composed of four different subunits. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that quite different polypeptides are present in the culture medium and in the NaCl and EDTA extracts of the wall.     

Acid and alkaline invertases in suspension cultures of sugar beet cells

Alkaline invertase was induced during the initiation of suspension cultures of single cells from leaf explants of sugar beets in Murashige-Skoog liquid medium which contained benzyladenine. This activity was barely detectable in the leaves themselves. In suspension cultures, the presence of both acid and alkaline invertases was detected; alkaline invertase was only present in the cytoplasm of the cultured cells, whereas acid invertase was present in the cytoplasm and cell walls, and was also detected in the culture medium. The cell wall contained at least three types of acid invertase; two of these activities were solubilized by saline (saline-released) and EDTA (EDTA-released), respectively, and the third remained tightly associated with the cell wall. Saline-released and EDTA-released invertases from the cell wall showed the significant differences in their properties: the saline-released enzyme had the highest affinity for sucrose among the invertases tested, and was easily bound to cell walls, to DNA, and to a cation exchanger, unlike the EDTA-released enzyme. Sucrose is the source of carbon for plant cells in suspension culture and is probably degraded in the cell wall by the saline-released invertase, which had the highest activity and the highest affinity for sucrose. Hexose products of this degradation would be transported to cytoplasm. Soluble invertase, EDTA-released invertase from the cell wall, and one of two extracellular invertases behaved similarly upon chromatography on DEAE-cellulose. They had similar activity profiles with changing pH, and similar K_m values for sucrose. Thus it appears that they are identical. Two extracellular invertases found in the growth medium of the suspension cultures were probably identical with those in the soluble fraction of callus and seedlings of sugar beets, because they showed similar behaviors during chromatography on DEAE-cellulose, and had similar activity profiles with changing pH and K_m values for sucrose.     

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S -adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S -adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S -adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence ( Bvcycll ).     

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S -adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S -adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S -adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence ( BvcycII ).     

Purification and properties of a nitrate reductase inactivating factor from rice cells in suspension culture

The nitrate reductase inactivating factor in cultured rice cellswas purified 320-fold. The purification procedure involved precipitationwith (NH₄)₂SO₄, fractionation at pH 4.0, adsorption on CM-cellulose,and gel filtration on Sephadex G-200. The molecular weight wasestimated to be 200,000 from the Sephadex G-200 gel filtration. The inactivating factor shows maximal activity at pH 8.0 andappears to be located in the cytoplasm of the cultured ricecells. The inactivating factor was more stable to heat treatmentthan NADH nitrate reductase. The factor inactivated nitratereductase complex except for reduced methylviologen nitratereductase. It had no influence on the activity of nitrite reductase,glutamate dehydrogenase, and NADH diaphorase, but inactivatedxanthine oxidase. The inactivating factor had no protease activitywhen casein, bovine serum albumin, or nitrate reductase fractionwas used as the substrate. The type of inactivation of nitratereductase by the inactivating factor was noncompetitive. Inhibitionof the inactivating factor by o-phenanthroline, EDTA, and p-chloromercuribenzoicacid suggested the involvement of a metal and sulfhydryl groupat its active site. (Received January 28, 1977; )     

Purification and properties of a glucoamylase fraction from the culture filtrate of Rhizopus nodosus

A glucoamylase was isolated from the culture filtrate of Rhizopus nodosus and was separated from the acid lipase by DEAE-cellulose chromatography at pH 8.0 It was purified by Concanavalin A Sepharose 4B affinity chromatography followed by CM-Sephadex chromatography 387 fold with 30.7% yield. The homogeneity of the enzyme were confirmed by polyacrylamide gel electrophoresis and immunological studies. The different physico-chemical properties of the enzyme were studied. The molecular weight of the enzyme was found to be 71,000. Ethylenediaminetetraacetic acid had no effect on the enzyme whereas Hg2+ partially inhibited the enzyme activity. Tryptophan residues were found to be essential for the enzyme activity.     

Transformation of sugar beet cell suspension cultures

Summary A sugar beet transformation method was developed using particle bombardment of short-term suspension cultures of a breeding line FC607. Highly embryogenic suspension cultures derived from leaf callus were bombarded with the uidA (gusA) reporter gene under the control of either the osmotin or proteinase inhibitor II gene promoter, and the npt II selectable marker gene. Transient uidA expression was visualized as 500–4000 blue units per 200 mg of bombarded cells 2 d after bombardment. Stably-transformed calluses were recovered on both kanamycin and paromomycin media. The greatest number of GUS (+) calluses was obtained when 50 or 100 mgl⁻¹ of kanamycin was applied 2 d after transformation for 3–5 wk, followed by either no selection or reduced levels of the antibiotic. PCR analyses of the GUS (+) callus lines revealed the expected size fragment for uidA and npt II genes. Stable incorporation of the uidA gene into the genome was confirmed by Southern blot analyses. Several transformed embryos were detected by histochemical β-glucuronidase (GUS) staining.     

Immunolocalization of PIP aquaporins in protoplasts from the suspension culture of sugar beet mesophyll under isoosmotic conditions and osmotic stress

Protoplasts from the suspension culture of sugar beet (Beta vulgaris L.) mesophyll were found to change their volume in response to short-term osmotic stress. When the sorbitol concentration in the external medium was increased 1.5-fold (from 0.4 to 0.6 M) or decreased from 0.4 to 0.25 M, the volume of protoplasts decreased and increased, respectively, by 55–60%. These changes started immediately after the shift in osmoticum concentration and completed within 1–3 min. In the presence of an endocytosis marker FM1-43, its fluorescence increased conspicuously after replacement of isotonic medium with the hypotonic solution but did not change after the substitution with hypertonic medium. At the same time, the hypertonic shrinkage of protoplasts was accompanied by accumulation of fluorescent material in the periplasmic space. The western blot analysis with the use of immune serum for conservative sequence of PIP-type aquaporins revealed their presence in the plasmalemma and intracellular membranes. This conclusion was confirmed by indirect immunofluorescence microscopy: the membrane-bound secondary antibodies labeled with a fluorescent probe Alexa-Fluor 488 were distributed comparatively uniformly on the boundary between the plasmalemma and the protoplast internal compartment. As evident from micrographs of protoplasts exposed to the hypotonic treatment, the fluorescence was smoothly distributed over the plasmalemma after protoplast swelling but its intensity was not so bright. The protoplast shrinkage during the hypertonic treatment resulted in heterogeneous alternate distribution of fluorescent and transparent plasmalemma regions, the fluorescence of stained regions being very intense. The results are interpreted as the evidence that the short-term osmotic stress activates exo-and endocytosis. The migrating regions of the plasmalemma were depleted of PIP-type aquaporins; hence, the induction of osmotic stress has no effect on the amount of this type aquaporins in the plasma membrane.     

Purification and conservation of infective sugar beet mosaic virus

Sugar beet mosaic virus (SBMV) was precipitated by polyethyleneglycol (PEG) 6000 from the cell sap of infected sugar beet leaves. After centrifugation and addition of dextrane T 10 the virus was lyophilized. Its infectious activity was demonstrated by mechanical transmission toChenopodium quinoa Willd. and to sugar beet. Stability of infectious activity of the lyophilized virus was verified.     

Purification and some properties of five anthraquinone specific glucosyltransferases from Cinchona succirubra cell suspension culture

Five anthraquinone-specific glucosyltransferases were partially purified from Cinchona succirubra cell suspension culture by fractional precipitation with ammonium sulphate, gel filtration and chromatofocusing on a fast protein liquid chromatography system. Five, distinct glucosylating activities were resolved with apparent pI values of 5.3, 4.8, 4.5, 4.3 and 4.1. They accepted emodin, anthrapurpurin, quinizarin, 2,6-dihydroxy anthraquinone and 1,8- dihydroxy anthraquinone as the best substrates, respectively. These enzymes exhibited similar characteristics as to pH optimum (pH 7) in histidine/HCl buffer, M, 50 000, had no cation requirement and were inhibited by various SH-group reagents. The K_m value of the respective anthraquinones for either of the five enzymes was 10 μM. The physiological role of these novel enzymes is discussed in relation to the biosynthesis of anthraquinone glucosides in this tissue.     

Purification and some properties of the membrane-bound hydroxyproline-containing glycoprotein from tobacco cells cultured in suspension

Membrane-bound hydroxyproline-containing glycoprotein from culturedtobacco cells was solubilized with chloroform. The glycoproteinwas homogeneous on acryl-amide gel electrophoresis and in ultracentrifugationanalysis. The glycoprotein was composed of 74% carbohydrateand 26% protein, and the carbohydrate moiety was composed ofgalactose, arabinose and rhamnose. The S₂₀ w value of the glycoproteinwas 5.4. ¹Department of Biochemistry, University of Texas Health ScienceCenter, San Antonio, Texas 78284, U.S.A. (Received December 15, 1979; )     

Thermophilic methanogenesis from sugar beet pulp by a defined mixed bacterial culture

Summary Thermophilic degradation of sugar beet pulp was studied in batch cultures at 55°C by different associations of bacteria, includingClostridium thermocellum,Methanobacterium sp. andMethanosarcina MP.C. thermocellum produced acetate, succinate, methanol, ethanol, H₂ and CO₂. The coculture ofC. thermocellum andMethanobacterium sp. produced trace amounts of ethanol and succinate; acetate concentration was about three times higher than in theC. thermocellum monoculture. The association of this coculture withMethanosarcina MP produced 5.5 mmol CH₄/g dry weight sugar beet pulp.     

Purification and properties of glucoamylase from Endomycopsis sp. 20-9]

Homogenous yeast (Endomycopsis sp. 20-9) glucoamylase was isolated from cultural medium. The homogeneity of the enzyme preparation is demonstrated by means of polyacrylamide gel disc electrophoresis, isofocusing and ultracentrifugation. Amino acid composition, molecular weight (53 000), sedimentation constant (4.3S) and isoelectric point (pI 3.80-3.82) of the enzyme are determined. Glucoamylase is found to be glucoprotein.     

Extraction,characterization and spontaneous emulsifying properties of pectin from sugar beet pulp

The effects of organic acid extractants on the yield and characteristics of pectin from sugar beet pulp were investigated with citric acid, malic acid and lactic acid at different pH (1.5 and 2.0) and time (1 h and 2 h). The results demonstrated that the yields of pectins were directly correlated with the decrease of pH and reaction time, and the optimum yield of 17.2% was obtained at pH 1.5 and 2 h. Furthermore, the acid type also affected the physicochemical characteristics of pectin, especially on the esterification degree (42–71), galacturonic acid content (60.2–77.8%), emulsion activity (35.2–40.1%) and emulsion stability (62.1–79.4%), and a relatively single pectin mainly consisted of homogalacturonan could be obtained under a suitable reaction condition, which was an excellent crude material for the production of emulsion activity.     

Purification and properties of two forms of glucoamylase fromAspergillus niger

A. niger produced α-glucosidase, α-amylase and two forms of glucoamylase when grown in a liquid medium containing raw tapioca starch as the carbon source. The glucoamylases, which formed the dominant components of amylolytic activity manifested by the organism, were purified to homogeneity by ammonium sulfate precipitation, ion-exchange and two cycles of gel filtration chromatography. The purified enzymes, designated GA1 and GA2, a raw starch digesting glucoamylase, were found to have molar masses of 74 and 96 kDa and isoelectric points of 3.8 and 3.95, respectively. The enzymes were found to have pH optimum of 4.2 and 4.5 for GA1 and GA2, respectively, and were both stable in a pH range of 3.5–9.0. Both enzymes were thermophilic in nature with temperature optimum of 60 and 65°C, respectively, and were stable for 1 h at temperatures of up to 60°C. The kinetic parametersK _m andV showed that with both enzymes the branched substrates, starch and amylopectin, were more efficiently hydrolyzed compared to amylose. GA2, the more active of the two glucoamylases produced, was approximately six to thirteen times more active towards raw starches compared to GA1.     

Purification and properties of S-adenosyl-L-homocysteine hydrolase from leaves of spinach beet.

S-Adenosyl-l-homocysteine hydrolase (EC 3.3.1.1) has been isolated from spinach-beet leaves and purified 100-fold. The enzyme catalyzes both the hydrolysis of S-adenosyl-l-homocysteine to adenosine and l-homocysteine and its synthesis from these compounds. The equilibrium constant for the reaction is 1.8 × 10^?6 in relation to hydrolysis. The enzyme shows optimum activity at pH 8.5. Enzyme preparations were stabilized by the addition of bovine serum albumin. The K_m for S-adenosylhomocysteine was 41 μm in the hydrolysis reaction and for adenosine, dl-homocysteine, and l-homocysteine it was 13 μm, 2.2 mm, and 1.2 mm, respectively.The enzyme was inhibited by S-adenosylmethionine, homocysteine, and adenine. These inhibitions and the K_m values determined are discussed in relation to the regulation of the enzyme in vivo and especially its effect on methylation reactions using S-adenosylmethionine as methyl donor.     

Purification and some properties of three forms of glucoamylase from a Rhizopus species.

1. Three forms of glucoamylase [EC 3.2.1.3] were simultaneously purified from a Rhizopus species by (NH4)2SO4 fractionation and successive chromatographies on Sephadex G-75, DEAE-Sephadex, and CM-Sephadex, and were finally separated from each other by means of recycling chromatography on Bio-Gel P-150. The purification achieved was 3--4 fold from crude extract with respect to each glucoamylase; the yields of the three glucoamylases, designated as Gluc1, Gluc2, and Gluc3 in order of content, were 39, 7, and 0.4%, respectively. All the purified enzymes were homogeneous in polyacrylamide gel electrophoresis, isoelectric focusing, and ultracentrifugation. 2. The three glucoamylases were glycoproteins differing in both amino acid composition and carbohydrate content, but showed a common antigenicity in immunodiffusion. The molecular weights of Gluc1, Gluc2, and Gluc3 were estimated to be 74,000, 58,600, and 61,400, respectively, by sedimentation equilibrium and these values were verified by SDS-polyacrylamide gel electrophoresis. The specific activities of the three enzymes toward starch were in the opposite order to their molecular weights. 3. The three glucoamylases had the same broad pH optima in the range pH 4.5--5.0 and shared a common susceptibility to inactivation by heat, extreme pH, and such divalent cations as Hg2+, Pb2+, and Mn2+, indicating close similarity in enzymatic properties.