共查询到20条相似文献,搜索用时 31 毫秒
1.
Sacco C. De Vries Marco C. Harmsen Martin T. R. Kuiper Hans J. M. Dons Joseph G. H. Wessels 《Plant molecular biology》1983,2(6):295-303
Summary The molecular cloning of cDNA corresponds to pea seedling mRNA sequences encoding a shoot-specific polypeptide, the small subunit of the ribulose 1,5 biphosphate carboxylase and a component of the light-harvesting chlorophyll a/b complex is described. cDNA prepared from polysomal poly(A)RNA of light-grown shoots was enriched for shoot-specific and light-induced sequences by heterologous liquid hybridization with mercurated polysomal poly(A)RNA of dark-grown roots, followed by sulfhydryl chromatography. Cloned shoot-specific sequences were identified by 2D electrophoretic analysis of hybrid release translation products. The cloned shoot-specific sequence corresponded to a mRNA of 850 nt present both in light-and dark-grown shoots, and produced anin vitro translation product of Mr27 500 and isoelectric point of 4.7. 相似文献
2.
Phytochrome destruction begins immediately following actinic irradiation of 4-day-old, dark-grown oat (Avena sativa L., cv. Garry) shoots grown in open containers. When grown in closed containers, otherwise identical oat shoots exhibit a delay of about 40 minutes between irradiation and the onset of destruction. This delay can be attributed to accumulation of ethylene by several criteria, including elimination of the delay by mercuric perchlorate. These data provide an explanation for otherwise contradictory observations concerning the presence of a delay prior to the onset of destruction. 相似文献
3.
Identification of Ca2+-stimulated polyphosphoinositide phospholipase C in isolated plant plasma membranes 总被引:16,自引:0,他引:16
A polyphosphoinositide phospholipase C has been identified in highly purified plasma membranes from shoots and roots of wheat seedlings. The enzyme preferentially hydrolysed phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate and had a different phosphoinositide substrate profile from soluble phospholipase C. The enzyme activity was lower in plasma membranes isolated from light-grown shoots than from dark-grown ones, whereas no differences in activity between plasma membranes from light- and dark-grown roots were seen. Maximum activity of the membrane-bound enzyme was observed around pH 6. It was activated by micromolar concentrations of Ca2+, but not by GTP or GTP analogues. The enzyme may participate in signal transduction over the plant plasma membrane. 相似文献
4.
Variation of the polypeptide composition of mitochondria isolated from different potato tissues 总被引:8,自引:1,他引:7
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
des Francs-Small CC Ambard-Bretteville F Darpas A Sallantin M Huet JC Pernollet JC Rémy R 《Plant physiology》1992,98(1):273-278
The protein contents of mitochondria from different potato (Solanum tuberosum L.) tissues (tubers, dark-grown shoots, and green leaves) grown in a greenhouse or in vitro were compared by two-dimensional polyacrylamide gel electrophoresis. Two different methods were used: using the method that gave the highest resolution, an average number of 360 polypeptides was revealed on the mitochondrial patterns after silver staining. The mitochondrial protein patterns of etiolated tissues (tubers, dark-grown shoots) are roughly similar but distinct from those of green leaves. The four subunits of the glycine decarboxylase complex (involved in photorespiration) and a few other polypeptides are very abundant in green tissues, compared with nonphotosynthetic tissues. Conversely, some other polypeptides that are abundant in tubers and dark-grown shoots are hardly detectable in green leaf mitochondria. A rabbit antiserum was raised against a 40 kilodalton polypeptide that is among the most characteristic of these nonphotosynthetic tissue-specific polypeptides, and the N-terminal sequence of this polypeptide was determined. No effect of in vitro culture was observed on the protein composition of mitochondria isolated from differentiated tissues. However, the protein patterns of callus and cell suspension mitochondria are distinct from those of any differentiated tissues, although their basic pattern is clearly mitochondrial. 相似文献
5.
Modifications of Sulfhydryl Groups on Phytochrome and Their Influence on Physicochemical Differences between the Red- and Far-Red-Absorbing Forms
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Phytochrome extracted from shoots of dark-grown rye (Secale cereale cv Rymin) and oat (Avena sativa cv Garry) as the far-red-form (Pfr) and/or under conditions conducive to oxidation exhibited a blue shift in the visible absorption maximum of its red-light-absorbing form (Pr) relative to that measured in vivo. This spectral alteration could not be reversed but could be prevented by inclusion of 10 millimolar diethyldithiocarbamate and 140 millimolar 2-mercaptoethanol in homogenization buffers. Similar blue shifts were induced in purified rye phytochrome by addition of the sulfhydryl-modifying reagent, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). In spectrally normal phytochrome (i.e., no detectable blue shift), Pfr had three to four more sulfhydryls available for rapid reaction with DTNB than did Pr. This difference was maintained over a 2.5-hour time course. Phytochrome purified under conditions resulting in a blue-shifted Pr absorption maximum exhibited a decreased short-term reactivity of Pfr to DTNB. Comparison of the binding and elution of altered and unaltered phytochrome from agarose-immobilized Cibacron blue 3GA confirmed that the Pfr form of spectrally normal phytochrome had a greater affinity for the dye than did the Pr form but that spectral alteration of phytochrome was accompanied by a loss of this difference as evidenced by an increased binding of Pr to the dye. It was concluded that phytochrome has highly reactive sulfhydryl residues located on the portion of the protein that undergoes conformational changes on interconversion of Pr and Pfr and that these residues require rigorous protection in order to extract the native form of the protein from plant tissue. 相似文献
6.
Purification and Properties of Amine Oxidase from Epicotyls of Pisum sativum 总被引:4,自引:2,他引:2
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A procedure has been developed for the purification of amine oxidase (E.C. 1.4.3.4) from etiolated pea epicotyls (Pisum sativum cv. Little Marvel). The enzyme is sensitive to copper chelating reagents and carbonyl reagents, but is not inhibited by sulfhydryl reagents. The purified enzyme has a molecular weight of 1.85 × 105, as determined by sedimentation equilibrium centrifugation, and has been shown to be specifically stimulated by phosphate. 相似文献
7.
Correlation between Polyribosome Level and the Ability to Induce Nitrate Reductase in Dark-grown Corn Seedlings 总被引:11,自引:10,他引:1
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Nitrate reductase can be induced in excised shoots of 3-day-old dark-grown Zea mays (var. WF9 × M14) seedlings in the absence of light. In contrast, leaves of 10-day-old dark-grown seedlings require a light treatment in order to induce enzymatic activity. Leaves of 10-day-old dark-grown seedlings contain a very low level of polyribosomes while 3-day-old shoots contain a very high level of polyribosomes. There is a gradual loss of polyribosomes from 3 to 10 days and a gradual loss of in vitro protein synthetic activity of the ribosome preparations. The loss of polyribosomes and decrease in their amino acid-incorporating activity correlate positively with the loss of ability to induce nitrate reducase activity as leaves of dark-grown corn seedlings age. These results corroborate and extend our previous results, in that light is not required for nitrate reductase induction per se in leaves of dark-grown seedlings but is required to reactivate the protein synthetic apparatus of older leaves. 相似文献
8.
Hirofumi Ogawa Yoshihisa Ishiguro Motoji Fujioka 《Archives of biochemistry and biophysics》1983,226(1):265-275
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 Km values for guanidoacetate and S-adenosylmethionine are 97.5 and 6.73 μm, respectively, at pH 8.0 and 37 °C. 相似文献
9.
A growth period from day 4 to day 11 after sowing of dark-grown oat seedlings ( Avena sativa L. cv. Flämingskrone) was studied. Etioplasts were prepared from various stages. By means of one and two dimensional gel eletrophoresis developmental changes in the polypeptide pattern of the prolamellar body-prothylakoid complex of etioplasts were studied. Polypeptides were described by their molecular weights and pI values after isoelectric focusing. The polypeptide map changes continuously with age, with the onset of effects of senescence around day 7. Concomitantly, proto-chlorophyllide and proteins are degraded, with a higher degradation rate for proto-chlorophyllide. An analysis of protochlorophyllide fluorescence displayed a constant ratio (fluorescence emission at 650 nm/fluorescence emission at 630 nm) from day 6 to day 11. It is concluded, that the best stages for greening studies of dark-grown oat seedlings occur between day 4 and day 6 after sowing. 相似文献
10.
Phytochrome Pelletability Induced by Irradiation in Vivo: TEST FOR IN VITRO BINDING OF ADDED [S]PHYTOCHROME
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Pratt LH 《Plant physiology》1980,66(5):903-907
Undegraded, highly purified [35S]phytochrome was immunoaffinity-purified either from dark control oat (cv. Garry) shoots or from etiolated oat shoots that were previously irradiated first with red and then with far-red light so that, if proper extraction conditions had been utilized, about 60% of the total phytochrome would have been pelletable. When [35S]phytochrome was added to extraction buffer immediately prior to homogenization of etiolated oat shoots, pelletability assays indicated that there was no preferential binding of [35S]phytochrome regardless of (a) whether it was purified from dark control or irradiated shoots, (b) whether it was added as phytochrome-red-absorbing form or phytochrome-far-red-absorbing form, or (c) whether it was added to dark control or red-irradiated shoots. Similarly, binding of [35S]phytochrome to resuspended pellets obtained from crude oat extracts was not specific for the source of [35S]phytochrome, for its form, or for the irradiation treatment given to intact shoots used to prepare the resuspended pellets. No evidence was obtained to support the hypothesis that phytochrome binds with specificity to particulate material in vitro under conditions used to assay for light-enhanced, in vivo-induced phytochrome pelletability. 相似文献
11.
Prostaglandin A isomerase has been purified 120-fold from rabbit serum by the use of ammonium sulfate fractionation, isoelectric focusing, and Sephadex G-200 chromatography. The molecular weight of the enzyme was estimated to be 110,000 from the elution volume on Sephadex G-200. Prostaglandin A isomerase is a heterogeneous protein with respect to charge. This has been concluded from the spread of enzymatic activity over 1 pH unit after isoelectric focusing. The enzymatic activity is inhibited by N-ethylmaleimide but not by other sulfhydryl blocking agents. The Km was determined to be 5 × 10?5m. 相似文献
12.
Danny M. Gee Gary M. Hathaway Richard H. Palmieri Ernst A. Noltmann 《Journal of molecular biology》1980,142(1):29-42
As a corollary to X-ray crystallographic work performed by H. Muirhead, detailed studies on crystalline pig muscle phosphoglucose isomerase have been conducted to establish its basic physical and chemical properties. The enzyme species being investigated by X-ray diffraction has been determined to be isoenzyme III. Its molecular weight in the native state was found to be 132,000, its s020,w value to be 7·25 S. The enzyme is composed of two subunits of equal molecular weight (66,000). Its amino acid composition is largely similar to that of rabbit muscle phosphoglucose isomerase, with the significant exception that the pig muscle isomerase contains only three sulfhydryl groups per polypeptide chain (two of them accessible to titration with p-mercuribenzoate) as compared with twice that number for the rabbit muscle enzyme. This low number of sulfhydryl groups is interpreted as being responsible for the ease with which heavy-atom, isomorphous derivatives could be prepared for the pig muscle enzyme by Shaw & Muirhead (1977). 相似文献
13.
A purification procedure employing normal chromatographic techniques is outlined for isolating phytochrome from etiolated oat (Avena sativa L.) seedlings. Yields in excess of 20% (25 milligrams or more) of phytochrome in crude extract were obtained from 10- to 15-kilograms lots. The purified oat phytochrome had an absorbance ratio (A280 nm/A665 nm) of 0.78 to 0.85, comparable to reported values, and gave a single major band with an estimated molecular weight of 62,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. A modification of the oat isolation procedure was used to isolate phytochrome from etiolated rye Secale cereale cv. Balbo) seedlings. During isolation rye phytochrome exhibited chromatographic profiles differing from oat phytochrome on diethylaminoethyl cellulose and on molecular sieve gels. It eluted at a higher salt concentration on diethylaminoethyl cellulose and nearer the void volume on molecular sieve gels. Yields of 5 to 10% (7.5-10 milligrams) of phytochrome in crude extract were obtained from 10- to 12-kilogram seedling lots. The purified rye phytochrome had an absorbance ratio of 1.25 to 1.37, significantly lower than values in the literature and gave a single major band with an estimated molecular weight of 120,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. It is suggested that the absorbance ratio and electrophoretic behavior of rye phytochrome are indices of purified native phytochrome, and that oat phytochrome as it has been described is an artifact which arises as a result of endogenous proteolysis during isolation. A rationale is provided for further modifications of the purification procedure to alleviate presumed protease contaminants. 相似文献
14.
p-Nitrophenoxycarbonyl methyl disulfide has been synthesized for use as a quantitating agent for methanethiolation of protein sulfhydryl groups. This reagent reacts specifically and quantitatively with cysteine residues of proteins to yield an unsymmetrical disulfide containing a CH3S group and concomitantly releases the chromophore, p-nitrophenol. Titration of the sulfhydryl groups of glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) with this reagent has been studied. Incorporation of CH3S as measured by the release of p-nitrophenol paralleled the loss of sulfhydryl group dependent activity of the enzyme. The enzyme was found inactive on modification of four of the eight sulfhydryl groups present in the enzyme. Stability of p-nitrophenoxycarbonyl methyl disulfide has also been studied in different buffer systems. The rate of decomposition of the p-nitrophenyl ester due to hydrolysis was found negligible below a pH of 8.0 compared to its rate of reaction with free sulfhydryl groups. 相似文献
15.
Subunit structure of -acetohydroxy acid isomeroreductase from Salmonella typhimurium 总被引:4,自引:0,他引:4
α-Acetohydroxy acid isomeroreductase, purified from Salmonella typhimurium, has a molecular weight of 220,000. The native enzyme consists of a tetramer of four identical subunits on the basis of the following criteria: (1) SDS gel electrophoresis revealed a single component of molecular weight 55,000 (2) carboxypeptidase digestion of the enzyme revealed 4 moles of glycine released per mole of enzyme; (3) amino acid analysis of the native enzyme indicated 204 moles of lysine and arginine; (4) after tryptic digestion, a total of 51 peptides were detected by high voltage electrophoresis and descending chromatography. In the native enzyme, it was possible to tititrate 8 sulfhydryl groups per mole of enzyme. Neither the rate nor extent of sulfhydryl titration was affected by substrates or products. After denaturation with SDS or urea, 8 additional sulfhydryls per mole of enzyme were titrated. 相似文献
16.
A phytochrome radioimmunoassay with a detection limit of about 2 nanograms has been developed. The radioimmunoassay does not suffer from the potential drawbacks of the commonly used spectral assay and requires less than 1 microliter of crude extract from dark-grown plants for quantitation of phytochrome. Measurement of phytochrome in crude extracts by radioimmunoassay gives values about 25% greater than those obtained by spectral assay. The amount of phytochrome detected in crude extracts of light-grown oats by radioimmunoassay is approximately 1% of that detected in comparable extracts from dark-grown oats. General interference by crude plant extracts with radioimmunoassays was also observed and corrected for. 相似文献
17.
Purification and initial characterization of ubiquitin from the higher plant, Avena sativa 总被引:10,自引:0,他引:10
Ubiquitin is a highly conserved, 76-amino acid polypeptide recently demonstrated to be involved in ATP-dependent protein degradation in mammalian cells. From immunoblot analyses with anti-human-ubiquitin antibodies we have detected the presence of free ubiquitin in green leaves, etiolated shoots, and dry seeds of the higher plant, oats (Avena sativa L.). We also find that crude oat extracts contain protease(s) that rapidly degrade both oat and human ubiquitin (t1/2 approximately 10 min at 27 degrees C). This proteolysis apparently cleaves ubiquitin at the carboxyl-terminal glycine dipeptide and results in inactivation of the molecule with respect to ligation but does not affect its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using homogenization conditions that preclude this proteolysis (low pH and the addition of the protease inhibitor p-chloromercuribenzoate) and immunoblotting as an assay for the protein, a procedure for the purification of ubiquitin from etiolated oat shoots was developed. Characterization of purified oat ubiquitin by absorption spectra, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, radioimmunoassay with anti-human-ubiquitin antibodies, and kinetic analyses using the ubiquitin activating enzyme isolated from rabbit liver indicates that this protein is remarkably similar to the mammalian form. Small differences between the oat and human proteins have been observed by amino acid compositional analyses indicating that the two forms are not totally homologous. Immunoblotting of crude oat extracts has revealed the presence of high molecular weight proteins recognized by anti-ubiquitin antibodies that represent ubiquitin-protein conjugates formed in vivo. Taken together, these data provide evidence that higher plants contain a ubiquitin-dependent proteolytic pathway that is mechanistically identical to that present in animals. 相似文献
18.
A.M. El-Badry 《BBA》1974,333(2):366-377
Hexosediphosphatase (d-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) has been isolated, purified, and crystallized, from previously isolated spinach chloroplasts. The effects of various anions, cations, and sulfhydryl compounds were tested, and activation by Mg2+, glycine, HCO3?, and sulfhydryl compounds is described. The purified enzyme is very specific for fructose 1,6-diphosphate and does not attack sedoheptulose-1,7-bisphosphate. The s20 value of the enzyme was 7.7, from which the molecular weight of the enzyme was estimated as 140 000. 相似文献
19.
While two monoclonal antibodies directed to phytochrome from etiolated oat (Avena sativa L.) shoots can precipitate up to about 30% of the photoreversible phytochrome isolated from green oat shoots, most precipitate little or none at all. These results are consistent with a report by J.G. Tokuhisa and P.H. Quail (1983, Plant Physiol. 72, Suppl., 85), according to which polyclonal rabbit antibodies directed to phytochrome from etiolated oat shoots bind only a small fraction of the phytochrome obtained from green oat shoots. The immunoprecipitation data reported here indicate that essentially all phytochrome isolated from green oat shoots is distinct from that obtained from etiolated oat shoots. The data indicate further that phytochrome from green oat shoots might itself be composed of two or more immunochemically distinct populations, each of which is distinct from phytochrome from etiolated shoots. Phytochrome isolated from light-grown, but norflurazon-bleached oat shoots is like that isolated from green oat shoots. When light-grown, green oat seedlings are kept in darkness for 48 h, however, much, if not all, of the phytochrome that reaccumulates is like that from etiolated oat shoots. Neither modification during purification from green oat shoots of phytochrome like that from etiolated oat shoots, nor non-specific interference by substances in extracts of green oat shoots, can explain the inability of antibodies to recognize phytochrome isolated from green oat shoots. Immunopurified polyclonal rabbit antibodies to phytochrome from etiolated pea (Pisum sativum L.). shoots precipitate more than 95% of the photoreversible phytochrome obtained from etiolated pea shoots, while no more than 75% of the pigment is precipitated when phytochrome is isolated from green pea shoots. These data indicate in preliminary fashion that an immunochemically unique pool of phytochrome might also be present in extracts of green pea shoots.Abbreviation ELISA
enzyme-linked immunosorbent assay
- mU
milliunit
- Pfr
far-red-absorbing form of phytochrome
- Pr
red-absorbing form of phytochrome 相似文献
20.
A long chain acyl-CoA reductase of Clostridium butyricum has been partially purified from the 100,000g supernatant fraction of cell extracts. The enzyme reduces acyl-CoA derivatives to aldehydes in the presence of NADH. It is stable in dithiothreitol-containing buffers at 4 °C, heat-labile, and sensitive to sulfhydryl reagents. It is active with palmitoyl-CoA, stearoyl-CoA, oleoyl-CoA, and myristoyl-CoA. Its apparent molecular weight on Sephadex G-100 column chromatography is 50,000. In crude extracts and at low purification, an NADPH-dependent reduction of palmitaldehyde to cetyl alcohol was also observed. An acyl-CoA hydrolase was also observed in crude extracts. 相似文献