A rapid procedure has been developed for the isolation of the photosystem two reaction centre complex (PS II RC) from a double mutant of Chlamydomonas reinhardtii, F54-14, which lacks the Photosystem one complex and the chloroplast ATPase. Thylakoid membranes are solubilised with 1.5% (w/v) Triton X-100 and the PS II RC purified by anion-exchange chromatography using TSK DEAE-650(S) (Merck). The complex has a pigment stoichiometry of approximately six chlorophyll a: two pheophytin a: one cytochrome b-559: one to two -carotene. It photoaccumulates reduced pheophytin and oxidised P680 in the presence of sodium dithionite and silicomolybdate, respectively. Immunoblotting experiments have confirmed the presence of the D1 and D2 polypeptides in this complex. The -subunit of cytochrome b-559 was identified by N-terminal sequencing. Comparison of the complex with the PS II RC from pea using SDS-polyacrylamide gel electrophoresis showed that their polypeptide compositions were similar. However, the -subunit of cytochrome b-559 from C. reinhardtii has a lower apparent molecular weight than the pea counterpart whereas the -subunit is larger.Abbreviations DM
n-dodecyl -d-maltoside
- RC
reaction centre
- SiMo
silicomolybdate, SiMo12O404–
- TAP
Tris-acetate-phosphate 相似文献
The Ole e 6 allergen from olive tree pollen has been isolated by combining gel permeation and reverse-phase chromatographies. It is a single and highly acidic (pI 4.2) polypeptide chain protein. Its NH2-terminal amino acid sequence has been determined by Edman degradation. Total RNA from the olive tree pollen was isolated, and a specific cDNA was amplified by the polymerase chain reaction using a degenerate oligonucleotide primer designed according to the NH2-terminal sequence of the protein. The nucleotide sequencing of the cDNA rendered an open reading frame encoding a 50 amino acid polypeptide chain, in which two sets of the sequential motif Cys-X3-Cys-X3-Cys are present. No sequence similarity has been found between this protein and other previously described polypeptides. 相似文献
Shikimate dehydrogenase (SKDH, EC 1.1.1.25) was extracted from seedlings of pepper ( Capsicum annuum L.) and purified 347-fold. The purification procedure included precipitation with ammonium sulphate and chromatography in columns of Reactive Red-agarose, Q-Sepharose and Sephadex G-100. Pepper SKDH isozymes are separable only using PAGE. The purified enzyme has a relative molecular mass of 67 000 as estimated by gel filtration. The optimum pH of enzyme activity is 10.5 and the optimum temperature is 50°C, but the enzyme is quickly inactivated at temperatures higher than 40°C. The purified enzyme exhibited typical Michaelis-Menten kinetics and Km values are 0.087 m M for shikimic acid and 0.017 m M for NADP. The mechanism of reaction is sequential considering NADP as a cosubstrate. Ions such as Ca2+, Mg2+ and Mn2+ activate the enzyme, but Zn2+ and Cu2+ are strong inhibitors. Some phenolic compounds such as guaiacol, protocatechuic acid and 2,4-D are competitive inhibitors of pepper SKDH, showing Ki values of 0.38 m M , 0.27 m M and 0.16 m M , respectively. 相似文献
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
Light signals perceived by photoreceptors are transduced into the nucleus to regulate gene expression and development. The COP9 protein complex localized in the nucleus has a central role in modulating light-regulated development, and may have a role in regulating development in general in all organisms. This review considers recent advances in the study of the COP9 complex and its sub-units in plant and animal systems, and discusses the possible functional implications. 相似文献
Two isoenzymes of glutamine synthetase (EC 6.3.1.2), GS1 and GS2, have been purified from cells of Emiliania huxleyi using Cibacron blue dye ligand chromatography and gel filtration, separated by ion-exchange chromatography on Mono-Q and partly characterized. Each enzyme is a homohexamer with a molecular mass of 402 kDa for GS1 and 501 kDa for GS2. The molecular mass of the subunits of GS1 and GS2 was estimated to be 61 and 78 kDa, respectively. As in higher plants, GS1 is slightly more thermostable than GS2 and much less stimulated by thiols than GS2. For these reasons, GS1 was designated as the cytosolic enzyme and GS2 as the chloroplastic one. Although the Kms for NH2OH are about the same, GS2 possesses a much higher affinity for glutamine than GS1. As in bacteria, ATP appears to play an important role in the allosteric regulation of GS2. l-Ala and CTP are potent inhibitors of GS1 activity. CTP, carbamoyl-phosphate and l-Ala exert a cumulative inhibitory effect on GS1 activity. GS2 is also inhibited to some extent by l-Ala and l-His. NH2-terminal sequence analysis of GS2 did not show any homology with bacteria, cyanobacteria or higher plants. 相似文献
Degradation and extraction of high molecular weight DNA from formaldehyde fixed tissues suitable for gene analysis are presented. We previously reported that DNase might play an important role in the degradation of DNA extracted from formaldehyde fixed tissues (Tokuda et al. 1990). In the present study, DNase activity of the supernatant from rat tissues fixed in buffered formaldehyde at room temperature was negligible within 3 hr. Analysis of DNA extracted from reconstituted chromatin revealed that the degradation increased in the absence of DNase depending on the duration of the formaldehyde fixation. Furthermore, high molecular weight DNA could be extracted from tissues devoid of DNase activity fixed in buffered formaldehyde containing EDTA. These results demonstrated that DNA degradation was due mainly to a mechanism other than DNAse which was inhibited by EDTA. For clinical application, v-H-ras gene was successfully detected by Southern blotting from rat spleen tissues fixed in buffered formaldehyde especially at 4 C. Fixation at low temperature is useful for gene analysis. 相似文献
Cathepsin D was purified from ovaries of Xenopus laevis by both QAE-cellulose and pepstatin-Sepharose chromatography and then characterized and compared with Xenopus liver cathepsin D. Ovary cathepsin D appeared predominantly as a 43-kilodalton (kDa) molecular mass, as revealed by SDS-polyacrylamide gel electrophoresis, whereas the liver enzyme was obtained exclusively as a 36-kDa protein. The purified 43-kDa ovary enzyme cleaved vitellogenin limitedly to produce yolk proteins at pH 5.6. The specific activity of ovary cathepsin D was five to six times lower than that of the liver enzyme, as measured by hemoglobin-hydrolysis at pH 3, but the ovary enzyme was shown to be superior to the liver enzyme in terms of vitellogenin-cleaving activity, as examined at pH 5.6. Ovarian enzyme preparations contained variable amounts of 36-kDa species; this form was considered to be an autolytic product of the 43-kDa form arising during purification, because it was not detected in oocyte extracts but was generated by incubation of the purified 43-kDa enzyme alone in an acid solution. The conversion of the 43-kDa form by hepatic factors was accompanied by a marked increase in hemoglobin-hydrolytic activity. 相似文献
Cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices was purified 22-fold by QAE- and SP-Sephadex chromatography. The pH optimum of the enzyme was 8.0 in either Tris-HCl or barbital buffer. The κm values of oxaloacetate and NADH were 0.400 ± 0.018 and 0.410 ± 0.038 mM, respectively. The enzyme lost about 90% of its activity when heated for 2 min at 65°C. A 61.4% inhibition of the enzyme was noted at 4 mM concentration of diethyl pyrocarbonate. A 3 mM concentration of fructose 1,6-diphosphate inhibited the enzyme by 76.5%. The inhibition was non-competitive with respect to NADH with a κi value of 0.85 mM. A 75% inhibition of the enzyme was noted at 1 mM concentration of mebendazole that inhibited the enzyme upon competing with NADH with a κi value of 0.176 mM. A 2-mM concentration of citrate almost doubled the enzyme activity. The enzyme was inhibited at high concentrations of either substrate. The enzyme was not inhibited by p-hydroxymercuribenzoate or fumarate. The enzyme was absolutely specific for NADH as a cofactor. The properties of this enzyme are compared with those of the enzyme from the host liver, the cyst fluid and some other animal sources. The results are discussed in terms of the differences among the properties of the host liver, the cyst fluid and the protoscolices enzymes. The biochemical basis for the use of mebendazole in the treatment of echinococcosis is also elucidated. 相似文献
This article describes some of the dose assessment issues that should be considered when planning and executing a soil remedial activity. What is the proper dose scenario/ model? What are the appropriate cleanup criteria? How is the data gathered and analyzed (both before and after) cleanup?
By describing the features and aspects of how these issues and others were considered or not considered in planning for the remedial action underway at Maralinga, Australia (former site of the Nuclear Weapons Testing Program of the United Kingdom) when compared with historical international experience of this type, the author attempts to illustrate that it is almost nonsensical to preselect a single soil value for Pu in soil (for national or international use), particulariy when expressed as a soil concentration (pCi/g or bq/g). This is especially so when the problem is Pu on the surface or near the surface of the soil. This is the situation common at nuclear test sites where “one point safe tests” were conducted. At these locations aspects of resuspension (i.e., area size, particle size, wind speed, etc.) become the dominant drivers for the development of cleanup criteria, sampling regimes, data gathering, and analysis regimes, etc. These particular elements and others are discussed and illustrated. 相似文献