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11.
DNA Methylation Occurred around Lowly Expressed Genes of Plastid DNA during Tomato Fruit Development 总被引:5,自引:1,他引:4
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We have analyzed DNA methylation of plastid DNA from fully ripened red fruits, green mature fruits, and green leaves of tomato (Lycopersicon esculentum var. Firstmore). Essentially identical restriction profiles were obtained between chromoplast and chloroplast DNAs by EcoRI digestion. BstNI/EcoRII and HpaII/MspI are pairs of isoschizomers that can discriminate between methylated and unmethylated DNAs. These endonucleases produced different restriction patterns of plastid DNAs from tomato fruits compared to tomato leaves. Moreover, we have found from Southern blots that methylation was not detected in DNA fragments containing certain genes that are actively expressed in chromoplasts, whereas DNA fragments bearing genes that are barely transcribed in chromoplasts are methylated. 相似文献
12.
Characterization of an ATPase Associated with the Inner Envelope Membrane of Amyloplasts from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.) 总被引:2,自引:2,他引:0
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Amyloplast envelope membranes isolated from cultured, white-wild cells of sycamore (Acer pseudoplatanus L.) have been found to contain a Mg2+-ATPase, ranging in specific activity from 5 to 30 nanomoles per minute per milligram protein. This ATPase hydrolyzes a broad range of nucleoside triphosphates, whereas it hydrolyzes nucleoside mono- and diphosphates poorly, if at all. The ATPase activity was stimulated by several divalent cations, including Mg2+, Mn2+ and Ca2+, whereas it was not affected by Sr2+, K+, or Na+. The Km for total ATP was 0.6 millimolar, and the activity showed a broad pH optimum between 7.5 and 8.0. The ATPase was insensitive to N,N′-dicyclohexylcarbodiimide and oligomycin, but it was inhibited by vanadate. All these characteristics are basically similar to those reported previously for the Mg2+-ATPase of the chloroplast inner-envelope membrane. Likewise, the amyloplast envelope enzyme was shown to be located specifically on the inner envelope membrane. The amyloplast envelope membranes were chemically modified with a series of unique affinity labeling reagents, the adenosine polyphosphopyridoxals (M Tagaya, T Fukui 1986 Biochemistry 25: 2958-2964). About 90% of the ATPase activity was lost when the envelope membranes were preincubated with 0.1 millimolar adenosine triphosphopyridoxal. Notably, the enzyme was protected completely from inactivation in the presence of its substrate, ATP. In contrast, both adenosine diphosphopyridoxal and pyridoxal phosphate caused much less of an inhibitory effect. This greater relative reactivity of the triphosphopyridoxal analog is similar to that reported previously with Escherichia coli F1 ATPase (T Noumi et al. 1987 J Biol Chem 262: 7686-7692). 相似文献
13.
14.
ADP-Glucose Transport by the Chloroplast Adenylate Translocator Is Linked to Starch Biosynthesis 总被引:1,自引:0,他引:1
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In organello starch biosynthesis was studied using intact chloroplasts isolated from spinach leaves (Spinacia oleracea). Immunoblot analysis using a specific antiserum against the mitochondrial adenylate (ADP/ATP) translocator of Neurospora crassa shows the presence of an adenylate translocator protein in the chloroplast envelope membranes, similar to that existing in mitochondria and amyloplasts from cultured cells of sycamore (Acer pseudoplatanus). The double silicone oil layer-filtering centrifugation technique was employed to study the kinetic properties of adenylate transport in the purified chloroplasts; ATP, ADP, AMP, and most importantly ADP-Glc were shown to be recognized by the adenylate translocator. Similar to the situation with sycamore amyloplasts, only ATP and ADP-Glc uptake was inhibited by carboxyatractyloside, an inhibitor of the mitochondrial adenylate translocator. Evidence is presented to show that the ADP-Glc transported into the chloroplast stroma is utilized for starch synthesis catalyzed by starch synthase (ADP-Glc:1,4-α-d-glucan 4-α-d-glucosyltransferase). The high activity of sucrose synthase producing ADP-Glc observed in the extrachloroplastic fractions suggests that starch biosynthesis in chloroplasts may be coupled with the direct import of ADP-Glc from the cytosol. 相似文献
15.
16.
Takashi Saito Kensuke Futatsugi Daisuke Miki Hiroshi Suzuki Kiyoshi Yasukawa 《Biotechnology Techniques》1992,6(4):365-370
Summary Murine interleukin-6 (mIL-6) was expressed inEscherichia coli as human growth hormone (hGH) fusion protein. The products were cleaved by thrombin to liberate mIL-6. Monoclonal and polyclonal
antibodies specific to mIL-6 were prepared by immunizing rats with mIL-6 thus obtained. ELISA for the quantitation of mIL-6
was also established, which could detect mIL-6 in a quantity as low as 2 ng/ml. 相似文献
17.
Preferential secretion of R-type alpha-amylase molecules in rice seed scutellum at high temperatures
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Exposure of fresh scutella excised from 4-day-old rice seedlings to higher temperatures, (40-42°C), drastically reduced the biosynthesis of α-amylase as determined by the incorporation of [35S]methionine into the immunoprecipitable product. However, the intracellular transport and extracellular secretion of the enzyme molecules were enhanced at high temperatures, indicating that the biosynthesis and secretion of α-amylase are distinguishable in their temperature dependency. At the higher temperature regime (40°C), the complex-type α-amylase isoform, resistant to hydrolytic digestion by endo-β-N-acetylglucosaminidase H (Endo-β-H) was predominantly secreted, whereas at lower temperatures (15°C), the isoform susceptible to Endo-β-H attack was the major molecular form secreted. 相似文献
18.
Shoei Furukawa Isao Kamo Yoshiko Furukawa Saeko Akazawa Eijiro Satoyoshi Koji Itoh Kyozo Hayashi 《Journal of neurochemistry》1983,40(3):734-744
Abstract: A sensitive two-site enzyme immunoassay system for mouse β nerve growth factor (NGF) was developed, based on the sandwiching of the antigen between anti-mouse β NGF antibody IgG coated to a polystyrene tube and anti-mouse β NGF antibody Fab'-linked β- d -galactosidase (β- d -galactoside hydrolase, EC 3.2.1.23). This method has the following advantages: (a) the procedures are simple and rapid compared to bioassay or two-site radioimmunoassay; (b) antibody Fab'-β- d -galactosidase complex is more stable than 125 I-labeled antibody; (c) purified β NGF is detectable at a concentration as low as 10 pg/ml. Our enzyme immunoassay was used to examine the levels of NGF in some tissues of mice. The submaxillary gland contained a high concentration of NGF. However, other tissues, such as the heart, brain, and skeletal muscle, and serum did not contain detectable NGF. These results support recent findings by other investigators that NGF was not found in the organs/tissues other than the submaxillary gland of mice. 相似文献
19.
Further proof for the catalytic role of the larger subunit in the spinach leaf ribulose-1,5-diphosphate carboxylase 总被引:4,自引:0,他引:4
The catalytically active oligomeric form of the larger subunit, Am, obtained from spinach leaf ribulose-1,5-diphosphate carboxylase by pretreatment with p-mercuribenzoate at pH 7.5 followed by incubation at pH 9.0, was free of the smaller subunit based on C-terminal amino acid analyses. Valine was the predominant C-terminus of the Am preparations, the release of tyrosine being negligibly small [cf. Sugiyama and Akazawa, 9 (1970) 4499]. The pH optimum of the ribulose-1,5-diphosphate carboxylase reaction by Am was about 8.5, in comparison to the native enzyme which showed an alkaline pH optimum only in the absence of Mg2+. The substrate saturation curve of the catalytic subunit with respect to bicarbonate followed the Michaelis-Menten equation, as contrasted to the anomalous reaction kinetics of the native ribulose-1,5-diphosphate carboxylase molecule reported previously. These overall results indicate that the allosteric properties of spinach ribulose-1,5-diphosphate carboxylase are possibly conveyed by a unique structural conformation that requires the presence of the smaller subunit in association with the larger catalytic subunit component of the enzyme molecule. 相似文献
20.
Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds II. Scutellum as the Site of Sucrose Synthesis 总被引:8,自引:6,他引:2
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In a close parallel to the developmental pattern of α-amylase activity, a rapid increase of maltase activity occurred in the endosperm tissue of germinating rice seeds after about 4 days of the seed imbibition. The overall pattern of the 2 hydrolytic enzyme activities strongly suggest that amylolytic breakdown is the major metabolic route of starch utilization in the germinating rice seeds. Results of the chemical analyses of sugar constituents as well as the measurements of sucrose synthetase activity show that the scutellum is the site of sucrose synthesis in the germinating rice seeds. It is thus supported that glucose derived from the reserve starch in endosperm is transported to scutellum, where it is converted to sucrose. Sucrose is further mobilized to the growing tissues, shoots and roots. 相似文献