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1.
Oxidase activity in the developing xylem of branches of Sitka spruce [Picea sitchensis] (Bong) Carr. was expressed in synchrony with the deposition of lignin. The activity was closely associated with the cell
wall but it could be extracted by elution with salt solutions such as 1 M NaCl or CaCl2. A number of different oxidase isoforms with isoelectric points in the range 8–5 were present in these cell wall extracts.
These enzymes displayed a marked preference for the oxidation of coniferyl alcohol and efficiently initiated polymerization
of coniferyl alcohol into insoluble, lignin-like polymers. They also had a substrate preference and profile of sensitivity
to inhibitors that was dissimilar to those reported for classical catechol oxidase or laccase-type polyphenol oxidases. A
novel procedure that combines extraction and affinity chromatography on Concanavalin-A to select high-mannose-type glycoproteins
provided oxidase activity at higher purity and yield than previously used methods. A single band of oxidase activity (apparent
Mr approx. 84 kDa) which was capable of oxidizing α-naphthol/N,N,N′N′-tetramethyl p-phenylene diamine in the absence of added hydrogen peroxide was detected in these cell wall extracts using non-denaturing
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The addition of hydrogen peroxide did not intensify the staining
of this band but it confirmed the presence of a true peroxidase band of apparent Mr approx. 40 kDa. The properties of this coniferyl alcohol oxidase are different from those of laccase-type polyphenol oxidases
(EC 1.10.3.2) previously implicated in lignin deposition in tree species, and their possible roles in this process are discussed.
Received: 9 January 1997 / Accepted: 14 March 1997 相似文献
2.
Barbara A. McCormack Abigail C. E. Gregory Maria E. Kerry Colin Smith G. Paul Bolwell 《Planta》1997,203(2):196-203
Membrane preparations from suspension-cultured cells of French bean (Phaseolus vulgaris L.) contained callose synthase (EC 2.4.1.34) activity which was preserved upon solubilisation. Following elicitor treatment
of cell cultures, increased activity could be extracted and this increase was maintained during purification. The enzyme was
purified by high-pressure liquid chromatography and active fractions showed a variable association of two polypeptides of
relative molecular masses (Mr) 55 000 and 65 000, the latter being in excess. The Mr-65 000 polypeptide was purified to homogeneity and an antibody raised to it. This antibody showed complex effects on callose
synthase activity when incubated with membrane and soluble extracts. In comparison with other systems, the Mr-55 000 subunit is likely to represent the catalytic subunit while the Mr-65 000 polypeptide is a possible regulatory subunit. The Mr-65 000 polypeptide was immunolocated in membranes at sites of callose synthesis in the plant, in cell plates, in sieve plates,
at the plasma membrane-wall interface of wounded cells and in papillae in infected cells.
Received: 18 January 1997 / Accepted: 8 May 1997 相似文献
3.
Ermel FF Follet-Gueye ML Cibert C Vian B Morvan C Catesson AM Goldberg R 《Planta》2000,210(5):732-740
The development of pectin structural features during the differentiation of cambial derivatives was investigated in aspen
(Populus tremula L. × P. tremuloides Michx.) using biochemical and immunocytochemical methods. Comparisons were also made between active and resting tissues.
Active tissues, in particular cambial cells and phloem derivatives, were characterized by a high pectin content. Use of antibodies
raised against arabinan side chains of rhamnogalacturonan 1 (LM6), as well as biochemical analysis, revealed an obvious decrease
from the cortex to the differentiating xylem. Galactan side chains, detected with LM5 antibodies, were present mainly in the
cambial zone and enlarging xylem cells. In contrast, they were totally absent from sieve-tube cell walls. Image analysis of
LM5 immunogold labelling in the cambial zone showed a clustered distribution of galactan epitopes in the radial walls, a distribution
which might result from the association of two different periodic processes, namely the exocytosis of galactan and wall expansion.
Cessation of cambial activity was characterized by cell wall thickening accompanied by a sharp decrease in the relative amount
of pectin and a lowering of the degree of methylesterification. The data provide evidence that the walls of phloem and xylem
cells differ in their pectin composition even at a very early stage of commitment. These differences offer useful tools for
identifying the initial cells among their immediate neighbours.
Received: 12 June 1999 / Accepted: 20 October 1999 相似文献
4.
Mycorrhizal and nonmycorrhizal roots of Allium schoenoprasum were tested for activities of α-mannosidase, β-glucosidase and arabinosidase. Mannosidase activity was higher by a factor
of two in mycorrhizal than in nonmycorrhizal root extracts. The apparent molecular weight of the enzyme was 152 kDa and its
KM was 1.25 mM in colonized roots and 1.85 mM in uncolonized roots. α-Mannosidase activity was further characterized by an acid
pH optimum and Zn2+ dependency. No significant differences could be found between mycorrhizal and nonmycorrhizal roots for β-glucosidase and
arabinosidase activities.
Accepted: 28 August 1995 相似文献
5.
Cloning and functional analysis of a cDNA encoding a starch synthase from potato (Solanum tuberosum L.) that is predominantly expressed in leaf tissue 总被引:10,自引:0,他引:10
Jens Kossmann Gernot J. W. Abel Franziska Springer James R. Lloyd Lothar Willmitzer 《Planta》1999,208(4):503-511
Three isoforms of starch synthase (SS) were shown to be present in soluble potato tuber extracts by activity staining after
native gel electrophoresis. A cDNA encoding SSI from rice was used as a probe to clone a corresponding cDNA from potato. The
deduced amino acid sequence identified the protein as an SS from potato with an Mr of 70.6 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SSI. An analysis of
the expression pattern of the gene indicated that SSI is predominantly expressed in sink and source leaves, and, to a lower extent in tubers. In several independent transgenic
potato lines, where the expression of SSI was repressed using the antisense approach, the activity of a specific SS isoform was reduced to non-detectable levels as
determined through activity staining after native gel electrophoresis. The reduction in the amount of this isoform of SS did
not lead to any detectable changes in starch structure, probably due to the fact that this isoform only represents a minor
activity in potato tubers.
Received: 19 August 1998 / Accepted: 17 December 1998 相似文献
6.
G. J. Grobben W. H. M. van Casteren H. A. Schols A. Oosterveld G. Sala M. R. Smith J. Sikkema J. A. M. de Bont 《Applied microbiology and biotechnology》1997,48(4):516-521
The exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in defined medium were investigated. At equal cell densities, the strain produced 95 mg l−1 exopolysaccharides with glucose and 30 mg l−1 with fructose as the carbohydrate source. High-performance size-exclusion chromatography of the exopolysaccharides produced
on glucose showed the presence of two fractions with relative molecular masses (M
r) of 1.7 × 106 and 4 × 104 in almost equal amounts. The exopolysaccharides produced on fructose contained mainly a fraction of low M
r of 4 × 104. The high-M
r fraction of the purified exopolysaccharides produced on glucose appeared to have a sugar composition of galactose, glucose
and rhamnose in the molar ratio of 5:1:1, whereas the low-M
r weight fraction contained galactose, glucose and rhamnose in the molar ratio of approximately 11:1:0.4. The purified exopolysaccharide
fractions produced on fructose showed comparable ratios. The high-molecular-mass fractions contained terminally linked galactose,
1,2,3-linked galactose, 1,3,4-linked galactose, 1,3-linked glucose and terminally linked rhamnose. The low-molecular-mass
fractions contained mainly 1,3-linked galactose and 1,6-linked galactose and lower amounts of other sugar linkages. The production
of the high-M
r fractions appeared to be dependent on the carbohydrate source, whereas the low-M
r fractions were produced more continuously.
Received: 30 April 1997 / Received revision: 11 June 1997 / Accepted: 14 June 1997 相似文献
7.
The proportion of mated females (M f) of the osmund sawfly, Strongylogaster osmundae, and the sex ratio of the eggs they deposited (r, proportion of males) were estimated in the wild by collecting egg masses. The proportion of mated females at oviposition varied from 0 to 1.0. M f was high (often 1.0) among the females that emerged after hibernation, and lower in the subsequent generations. Mated females of the hibernated generation deposited equal numbers of eggs of both sexes. Mated females of the first and subsequent generations produced more female than male eggs. These results qualitatively agreed with the prediction provided by an evolutionarily stable strategy (ESS) model (if M f < 1 then r < 0.5). However, the quantitative prediction provided by the model [M f (1 − r) = 0.5] was not always observed in the wild, especially where the population density and M f were high. The value of r was often lower than the predicted one. The following simple hypothesis was tested by experimentation: “Females that encounter males frequently estimate the proportion of mated females to be high and deposit eggs with a 1:1 sex ratio.” However, results did not support this hypothesis. Females that copulated soon after emergence and were courted by males two or more times did not show a higher offspring sex ratio than those which mated 1 or 2 days after emergence and experienced no other sexual encounter. Another mechanism for determination of r is suggested, and the reason why the population sex ratio of sawflies is often female-biased (r < 0.5) is discussed. 相似文献
8.
A number of nutritional factors influencing growth and glucose oxidase (EC 1.1.3.4) production by a newly isolated strain
of Penicillium pinophilum were investigated. The most important factors for glucose oxidase production were the use of sucrose as the carbon source,
and growth of the fungus at non-optimal pH 6.5. The enzyme was purified to apparent homogeneity with a yield of 74%, including
an efficient extraction step of the mycelium mass at pH 3.0, cation-exchange chromatography and gel filtration. The relative
molecular mass (M
r) of native glucose oxidase was determined to be 154 700 ± 4970, and 77 700 for the denatured subunit. Electron-microscopic
examinations revealed a sandwich-shaped dimeric molecule with subunit dimensions of 5.0 × 8.0 nm. Glucose oxidase is a glycoprotein
that contains tightly bound FAD with an estimated stoichiometry of 1.76 mol/mol enzyme. The enzyme is specific for d-glucose, for which a K
m value of 6.2 mM was determined. The pH optimum was determined in the range pH 4.0–6.0. Glucose oxidase showed high stability
on storage in sodium citrate (pH 5.0) and in potassium phosphate (pH 6.0), each 100 mM. The half-life of the activity was
considerably more than 305 days at 4 °C and 30 °C, and 213 days at 40 °C. The enzyme was unstable at temperatures above 40 °C
in the range pH 2.0–4.0 and at a pH above 7.0.
Received: 18 November 1996 / Received revision: 3 March 1997 / Accepted: 7 March 1997 相似文献
9.
Emmanuel Collec Yves Colin Florence Carbonnet Claude Hattab Olivier Bertrand Jean Pierre Cartron C. Le Van Kim 《Immunogenetics》1999,50(1-2):16-21
The human Kx blood group antigen is carried by a 37 000 M
r apparent molecular mass membrane polypeptide which is deficient in rare individuals with the McLeod syndrome. The X-linked
human XK gene is transcribed in many tissues including adult skeletal muscle and brain, sieges of disorders observed in McLeod syndrome.
We report here the cloning of the orthologous mouse XK mRNA. Comparison of XK from human and mouse revealed 80% sequence similarity at the amino acid level. The mouse XK gene is organized in two exons and is expressed in many tissues, but its expression pattern is slightly different from that
of the human gene. The presence in mouse erythrocyte membrane of a 43 000 M
r Kx-related protein was demonstrated by immunoblotting with a rabbit antiserum directed against the human protein. With non-reduced
samples, a 140 000 M
r species was detected instead of the 43 000 M
r protein, suggesting that, as demonstrated in the Kx polypeptide might be complexed with another protein in mouse red cells,
presumably the homologue of the human Kell protein of 93 000 M
r.
Received: 22 February 1999 / Revised: 8 June 1999 相似文献
10.
Cytokinins induce two specific morphological alterations in mosses: (i) the differentiation of a tip-growing cell into a
three-faced apical cell (the so-called bud), and (ii) the division of chloroplasts. In a developmental mutant of the moss
Physcomitrella patens (Hedw.) B.S.G. (mutant PC22) impeded in both cellular differentiation (bud production) and chloroplast division, addition
of cytokinin (N6-Δ2-isopentenyladenine) led to bud production after 3 d in the wild type and after 7 d in the mutant. Hormone induced a division
of the mutant macrochloroplasts starting within 24 h and ongoing for 72 h. During this period the abundances of several plastid
proteins changed in both genotypes as judged by two-dimensional-protein gel electrophoresis, silver staining and subsequent
quantification with novel computer software. Eight of these polypeptides were isolated independently, subjected to microsequencing
and thus identified, resulting in the first protein sequence data from a moss. Three polypeptides (24 kDa, 22 kDa, 20 kDa)
were found to be homologous to enhancer protein OEE2 of the oxygen-evolving complex, four to represent isoforms of phosphoglycerate
kinase (EC 2.7.2.3), and one was identified as the β-chain of chloroplast ATPase (EC 3.6.1.34). Possible involvement of these
key enzymes of the chloroplast energy-conversion machinery in organelle division and in cellular differentiation is discussed.
Further sequence information was obtained from both subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39).
Amounts of these polypeptides were not appreciably affected by cytokinin in moss chloroplasts.
Received: 4 July 1996 / Accepted: 4 October 1996 相似文献
11.
M. J. Artolozaga E. Kubátová J. Volc H. M. Kalisz 《Applied microbiology and biotechnology》1997,47(5):508-514
Pyranose 2-oxidase (P2O) was purified 43-fold to apparent homogeneity from the basidiomycete Phanerochaete chrysosporium using liquid chromatography on phenyl Sepharose, Mono Q (twice) and phenyl Superose. The native enzyme has a molecular mass
of about 250 kDa (based on native PAGE) and is composed of four identical subunits of 65 kDa. It contains three isoforms of
isoelectric point (pI) 5.0, 5.05 and 5.15 and does not appear to be a glycoprotein. P2O is optimally stable at pH 8.0 and
up to 60 °C. It is active over a broad pH range (5.0–9.0) with maximum activity at pH 8.0–8.5 and at 55 °C, and a broad substrate
specificity. d-Glucose is the preferred substrate, but 1-β-aurothioglucose, 6-deoxy-d-glucose, l-sorbose, d-xylose, 5-thioglucose, d-glucono-1,5-lactone, maltose and 2-deoxy-d-glucose are also oxidised at relatively high rates. A Ping Pong Bi Bi mechanism was demonstrated for the P2O reaction at
pH 8.0, with a catalytic constant (k
cat) of 111.0 s−1 and an affinity constant (K
m) of 1.43 mM for d-glucose and 83.2 μM for oxygen. Whereas the steady-state kinetics for glucose oxidation were unaffected by the medium at
pH ≥ 7.0, at low pH both pH and buffer composition affected the P2O kinetics with the k
cat/K
m value decreasing with decreasing pH. The greatest effect was observed in acetate buffer (0.1 M, pH 4.5), where the k
cat decreased to 60.9 s−1 and the K
m increased to 240 mM. The activity of P2O was completely inhibited by 10 mM HgCl2, AgNO3 and ZnCl2, and 50% by lead acetate, CuCl2 and MnCl2.
Received: 28 August 1996 / Received revision: 25 November 1996 / Accepted: 29 November 1996 相似文献
12.
In order to clearly establish the properties of the enzymes responsible for hexose phosphorylation we have undertaken the
separation and characterization of these enzymes present in tomato fruit (Martinez-Barajas and Randall 1996). This report
describes the partial purification and characterization of glucokinase (EC. 2.7.1.1) from young green tomato fruit. The procedure
yielded a 360-fold enrichment of glucokinase. Tomato fruit glucokinase is a monomer with a molecular mass of 53 kDa. Glucokinase
activity was optimal between pH 7.5 and 8.5, preferred ATP as the phosphate donor (K
m = 0.223 mM) and exhibited low activity with GTP or UTP. The tomato fruit glucokinase showed highest affinity for glucose
(K
m = 65 μM). Activity observed with glucose was 4-fold greater than with mannose and 50-fold greater than with fructose. The tomato
fruit glucokinase was sensitive to product inhibition by ADP (K
i = 36 μM). Little inhibition was observed with glucose 6-phosphate (up to 15 mM) at pH 8.0; however, at pH 7.0 glucokinase
activity was inhibited 30–50% by physiological concentrations of glucose 6-phosphate.
Received: 4 October 1997 / Accepted: 10 January 1998 相似文献
13.
The plant cytoskeleton has been implicated in a variety of morphogenetic events in higher plants. Most of this work, however,
has concentrated on epidermal cells or primary tissues. We have investigated the cortical microtubular (CMT) and microfilament
(MF) components of the cytoskeleton in a secondary tissue – active vascular cambium of Aesculus hippocastanum L. (horse-chestnut) – and followed the changes in these components during the early stages of differentiation of fusiform
cambial derivatives to axial elements of the secondary vascular system. A correlative approach was used employing indirect
immunofluorescence microscopy of α-tubulin on 6 μm sections, and transmission electron microscopy of 60 nm sections. The study
has demonstrated a rearrangement of the CMT cytoskeleton, from random to helical, as fusiform vascular cambial cells begin
to differentiate as secondary phloem vascular tissue. A similar CMT rearrangement is seen as fusiform cambial cells begin
to differentiate as secondary xylem fibres. This rearrangement is interpreted as evidence of determination of cambial derivatives
towards vascular development. Axially-oriented MF bundles are present in fusiform cambial cells and their axial orientation
is retained in the vascular derivatives at early stages of their development even though the CMTs have become rearranged.
Received: 5 August 1996 / Accepted: 23 September 1996 相似文献
14.
Donald J. Huber 《Physiologia plantarum》1992,86(1):25-32
An approach commonly employed to assess the potential role of the enzyme polygalacturonase (PG, EC 3.2.1.15) in tomato fruit cell-wall pectin metabolism includes correlating levels of extractable PG with changes in specific characteristics of cell wall pectins, most notably solubility and molecular weight. Since information on these features of pectins is generally derived from analyses of subfractions of isolated cell wall, assurance of inactivation of the various isoforms of wall-associated PG is imperative. In the present study, cell wall prepared from ripe tomato (Lycopersicon esculentum Mill. cv. Rutgers) fruit was examined for the presence of active PG and for the ability of phenolic solvents to inactivate the enzyme. Using pectin solubility and Mr (relative molecular mass) changes as criteria for the presence of wall-associated PG activity, pectins from phenol-treated and nonphenol-treated (enzymically active) cell wall from ripe fruit incubated in 50 mM Na-acetate, 50 mM cyclohexanetrans-1,2-diamine tetraacetic acid (CDTA), pH 6.5 (outside the catalytic range of PG), were of similar Mr and exhibited no change in size with incubation time. Wall prepared without exposure to the phenolic protein-denaturants exhibited extensive pectin solubilization and depolymerization when incubated in 50 mM Na-acetate, 50 mM CDTA at pH 4.5, indicating the presence of active PG. Based on the changes in the Mr of pectins solubilized in 50 mM Na-acetate, 50 mM CDTA, pH 4.5, active PG was also detected in wall exposed during isolation to phenolacetic acid-water (PAW, 2:1:1, w/v/v), a solvent commonly employed as an enzyme denaturant. Although the depolymerization of pectins in PAW-treated wall was extensive, oligouronides constituted minor reaction products. Interestingly, PAW-treated wall did not exhibit PG-mediated pectin release when incubated under conditions (30 mM Na-acetate, 150 mM NaCl, pH 4.5) in which nonphenol-treated cell wall exhibited high autolytic activity. In an alternative protocol designed to inactivate PG, cell wall was exposed to Tris-buffered phenol (BP). In contrast to pectins released from PAW-treated wall, pectins solubilized from BP-treated wall at pH 4.5 were indistinguishable in Mr from those recovered from BP-treated wall at pH 6.5 Even when incubated at pH 4.5 at 34°C, conditions under which pectins from PAW-treated wall underwent more rapid and extensive depolymerization, pectins from BP-treated wall exhibited no change in Mr, providing evidence that active PG was not present in these wall preparations. The implications of this study in interpreting the solubility and Mr of pectin in cell wall from ripening fruit are discussed. 相似文献
15.
M. Graf A. Brunella M. Kittelmann K. Laumen O. Ghisalba 《Applied microbiology and biotechnology》1997,47(6):650-657
A new amidohydrolase deacetylating several N-acetyl-1-phenylethylamine derivatives (R)-specifically was found in Arthrobacter aurescens AcR5b. The strain was isolated from a wet haystack by enrichment culture with (R)-N-acetyl-1-phenylethylamine as the sole carbon source. (R) and (S )-N-acetyl-1-phenylethylamine do not serve as inducers for acylase formation. By improving the growth conditions the enzyme production
was increased 47-fold. The amidohydrolase was purified to homogeneity leading to a 5.2-fold increase of the specific activity
with a recovery of 67%. A molecular mass of 220 kDa was estimated by gel filtration. Sodium dodecyl sulfate/polyacrylamide
gel electrophorosis shows two subunits with molecular masses of 16 kDa and 89 kDa. The optimum pH and temperature were pH 8
and 50 °C, respectively. The enzyme was stable in the range of pH 7–9 and at temperatures up to 30 °C. The enzyme activity
was inhibited by Cu2+, Co2+, Ni2+, and Zn2+, and this inhibition was reversed by EDTA.M
Received: 20 September 1996 / Received version: 23 December 1996 / Accepted: 30 December 1996 相似文献
16.
Kamila Rzeznicka Sebastian Schätzle Dominique Böttcher Joachim Klein Uwe T. Bornscheuer 《Applied microbiology and biotechnology》2010,85(5):1417-1425
The nitrile hydratase (NHase, EC 4.2.1.84) genes (α and β subunit) and the corresponding activator gene from Rhodococcus equi TG328-2 were cloned and sequenced. This Fe-type NHase consists of 209 amino acids (α subunit, Mr 23 kDa) and 218 amino acids (β subunit, Mr 24 kDa) and the NHase activator of 413 amino acids (Mr 46 kDa). Various combinations of promoter, NHase and activator genes were constructed to produce active NHase enzyme recombinantly
in E. coli. The maximum enzyme activity (844 U/mg crude cell extract towards methacrylonitrile) was achieved when the NHase activator
gene was separately co-expressed with the NHase subunit genes in E. coli BL21 (DE3). The overproduced enzyme was purified with 61% yield after French press, His-tag affinity chromatography, ultrafiltration
and lyophilization and showed typical Fe-type NHase characteristics: besides aromatic and heterocyclic nitriles, aliphatic
ones were hydrated preferentially. The purified enzyme had a specific activity of 6,290 U/mg towards methacrylonitrile. Enantioselectivity
was observed for aromatic compounds only with E values ranging 5–17. The enzyme displayed a broad pH optimum from 6 to 8.5, was most active at 30°C and showed the highest
stability at 4°C in thermal inactivation studies between 4°C and 50°C. 相似文献
17.
A Pessoa Jr I M de Mancilha S Sato 《Journal of industrial microbiology & biotechnology》1997,18(6):360-363
Sugar cane bagasse hemicellulosic fraction submitted to hydrolytic treatment with 100 mg of sulfuric acid per gram of dry
mass, at 140°C for 20 min, was employed as a substrate for microbial protein production. Among the 22 species of microorganisms
evaluated, Candida tropicalis IZ 1824 showed TRS consumption rate of 89.8%, net cell mass of 11.8 g L−1 and yield coefficient (Yx/s) of 0.50 g g−1. The hydrolyzate supplemented with rice bran (20.0 g L−1), P2O5 (2.0 g L−1) and urea (2.0 g L−1) provided a TRS consumption rate of 86.3% and a cell mass of 8.4 g L−1. At pH 4.0 cellular metabolism was inhibited, whereas at pH 6.0 the highest yield was obtained. The presence of furfural
(2.0 g L−1) hydroxymethylfurfural (0.08 g L−1) and acetic acid (3.7 g L−1) in the hydrolyzate did not interfere with cultivation at pH 6.0.
Received 25 October 1996/ Accepted in revised form 10 March 1997 相似文献
18.
Disruption of the phosphate-starvation response of oilseed rape suspension cells by the fungicide phosphonate 总被引:5,自引:0,他引:5
The influence of the anti-fungal agent phosphonate (Phi) on the response of oilseed rape (Brassica napus L. cv. Jet Neuf ) cell suspensions to inorganic phosphate (Pi) starvation was examined. Subculture of the cells for 7 d in
the absence of Pi increased acid phosphatase (APase; EC 3.1.3.2) and pyrophosphate (PPi)-dependent phosphofructokinase (PFP;
EC 2.7.1.90) activities by 4.5- and 2.8-fold, respectively, and led to a 19-fold increase in V
max and a 14-fold decrease in K
m (Pi) for Pi uptake. Addition of 2 mM Phi to the nutrient media caused dramatic reductions in the growth and Pi content of
the Pi-starved, but not Pi-sufficient cells, and largely abolished the Pi-starvation-dependent induction of PFP, APase, and
the high-affinity plasmalemma Pi translocator. Immunoblotting indicated the cells contain three APase isoforms that are synthesized
de novo following Pi stress, and that Phi treatment represses this process. Phosphonate treatment of Pi-starved cells significantly
altered the relative extent of in-vivo 32P-labelling of polypeptides having Mrs of 66, 55, 45 and 40 kDa. However, Phi had no effect on the total adenylate pool of Pi-starved cells which was about 32%
lower than that of Pi-sufficient cells by day 7. Soluble protein levels, and activities of pyruvate kinase (EC 2.7.1.40) and
ATP-dependent phosphofructokinase (EC 2.7.1.11) were unaffected by Pi starvation and/or Phi treatment. The effects of Phi
on the growth, and APase and PFP activities of Pi-starved B. napus seedlings were similar to those observed in the suspension cells. The results are consistent with the hypothesis that a primary
site of Phi action in higher plants is at the level of the signal transduction chain by which plants perceive and respond
to Pi stress at the molecular level.
Received: 30 December 1996 / Accepted: 19 February 1997 相似文献
19.
Sieve-tube exudate protein (STEP) from Ricinus communis L. seedlings consists of a characteristic set of more than 100 different polypeptides, against which a complex antiserum
was raised. This antiserum cross-reacted with dominant protein species (molecular weights 10–30 kDa) present in the sieve-tube
exudate and, to a lesser extent, with proteins in tissue extracts of Ricinus and a wide range of other plant species. For further elucidation of the nature of individual STEPs in the sieve tubes the
anti-STEP serum was used to screen a cDNA expression library constructed from Ricinus cotyledon mRNA. Two clones that differed in the 3′ untranslated region encoded a protein of 11 kDa which showed striking
homology to bacterial and eucaryotic glutaredoxin sequences. Glutaredoxin activity was confirmed for the recombinant protein
after overexpression in Escherichia coli and characterised in detail in sieve-tube exudate. Michaelis Menten constants (K
m) for reduced glutathione and cysteine were 2 mM and 50 μM, respectively. Besides l-cysteine, dehydroascorbate and protein disulphides were also reduced by the activity present in the sieve-tube exudate. Glutathione,
which is the obligate donor of reduced thiols for glutaredoxin, was present in sieve-tube sap in millimolar concentrations
(up to 3 mM) with a ratio of total to oxidised glutathione of 3:1. It is suggested that glutaredoxin and glutathione in sieve
tubes prevent oxidative damage and may be involved in redox regulation of sieve-tube proteins.
Received: 13 December 1996 / Accepted: 28 December 1996 相似文献
20.
Michniewicz A Ullrich R Ledakowicz S Hofrichter M 《Applied microbiology and biotechnology》2006,69(6):682-688
Cerrena unicolor secreted two laccase isoforms with different characteristics during the growth in liquid media. In a synthetic low-nutrient
nitrogen glucose medium (Kirk medium), high amounts of laccase (4,000 U l−1) were produced in response to Cu2+. Highest laccase levels (19,000 U l−1) were obtained in a complex tomato juice medium. The isoforms (Lacc I, Lacc II) were purified to homogeneity with an overall
yield of 22%. Purification involved ultrafiltration and Mono Q separation. Lacc I and II had M
w of 64 and 57 kDa and pI of 3.6 and 3.7, respectively. Both isoforms had an absorption maximum at 608 nm but different pH optima and thermal stability.
Optimum pH ranged from 2.5 to 5.5 depending on the substrate. The pH optima of Lacc II were always higher than those of Lacc
I. Both laccases were stable at pH 7 and 10 but rapidly lost activity at pH 3. Their temperature optimum was around 60°C,
and at 5°C they still reached 30% of the maximum activity. Lacc II was the more thermostable isoform that did not lose any
activity during 6 months storage at 4°C. Kinetic constants (K
m, k
cat) were determined for 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol and syringaldazine. 相似文献