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1.
Heterologous hybridization of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) restores the enzyme activities 总被引:3,自引:0,他引:3
A Incharoensakdi T Takabe T Takabe T Akazawa 《Biochemical and biophysical research communications》1985,126(2):698-704
The catalytic core (A8) and small subunit (B) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were isolated from two species of cyanobacteria (Aphanothece halophytica and Synechococcus ACMM 323) as well as from the photosynthetic purple sulfur bacterium, Chromatium vinosum. The subunit B is essential for the activity of all three enzymes. The heterologous hybridization of RuBisCO molecules from the three organisms was attempted and the reconstitution of the catalytically active hybrid was achieved between A8 derived from either Aphanothece or Synechococcus and subunit B from Aphanothece, Synechococcus or Chromatium. However, reconstitution of the enzymically active hybrid between A8 from Chromatium and B subunits from the cyanobacteria could not be achieved. Experiments by using high performance liquid column chromatography also showed the formation of a heterologous hybrid possessing RuBP carboxylase activity. 相似文献
2.
Centrifugal filtration through a double layer of silicone oil was applied to determine the intermembrane space of organelles enclosed by double envelope membranes, i.e. proplastids, chloroplasts, mitochondria and amyloplasts. The organelles, capable of transporting adenylates by an adenylate translocator located in the inner envelope membrane, were incubated with increasing concentrations of adenylates while maintaining their specific radioactivities constant. Intermembrane spaces were estimated by extrapolation of radioactivities recovered after filtration of the organelles. The values estimated were compared to those obtained employing the classical method measuring the intake of [14C]-sucrose and [14C]-sorbitol which are impermeable to the inner membranes of organelles. The intermembrane space determined by the present method was shown to be uniformly smaller than the sucrose-permeable space which was always smaller than the sorbitol-permeable space. 相似文献
3.
The catalase molecule in germinating pumpkin cotyledons is synthesizedas a precursor (59-kDa) form, whose relative molecular massis larger than the mature enzyme (55-kDa). Although both typesof molecules are localized in the microbodies, the 59-kDa specieshas been shown to be present predominantly in the leaf peroxisomesisolated from green cotyledons, while the 55-kDa species ispredominantly in the glyoxysomes from etiolated cotyledons [Yamaguchiet al. (1984) Proc. Natl. Acad. Sci. USA, 81: 4809]. We examinedthe distribution of the 59- and 55-kDa catalase molecules indark- and light-grown tissues of pumpkin seedlings as well asin other plant species, using the immunoblotting technique.The ratios of the 59- and 55-kDa catalase species differed inthe pumpkin tissues examined. Light interferes with the conversionof the 59-kDa precursor to the 55-kDa form, especially in thecotyledons. The effect of light was less pronounced in the rootsand hypocotyls, indicating that the light regulation of theconversion is tissue-specific. Dark- and light-grown cotyledonsfrom cucumber and watermelon seedlings showed a similar lightregulation, suggesting that cucurbitaceous plants possess similarlight-regulatory mechanism. From the analysis of catalase proteinfrom various plant tissues, a limited correlation between molecularforms of catalase and different microbody populations was observed. (Received September 6, 1986; Accepted December 4, 1986) 相似文献
4.
Takashi Yutsudo Hisao Kurazono Chihiro Sasakawa Masanosuke Yoshikawa Makoto Iwaya Tae Takeda Yoshifumi Takeda 《FEMS microbiology letters》1987,48(1-2):273-276
Abstract A Vero toxin (VT2 or Shiga-like toxin II)-converting phage was isolated from Escherichia coli 0157: H7 strain J-2. Nontoxigenic E. coli C600 produced VT2 when lysogenized with the toxin-converting phage. Eco RI fragments of the phage DNA were ligated with Eco RI-digested pBR322 or pUC118 and were transformed into E. coli MC1061 or MV1184. Transformants exhibiting VT2 production commonly contained a 4.6 kb Eco RI fragment. It was found that a 2.3 kb Kpn I- Sph I fragment coded VT2 production and that this fragment hybridized weakly with the 2.1 kb fragment encoding VT1. 相似文献
5.
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
6.
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
7.
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
8.
Dr. Tomonobu Kusano Kazuyuki Sugawara Chihiro Inoue Nobuhiro Suzuki 《Current microbiology》1991,22(1):35-41
The genes coding ford-ribulose-1,5-bisphosphate carboxylase (RuBPCase) from an iron-oxidizing bacterium,Thiobacillus ferrooxidans, were cloned into anEscherichia coli plasmid, pUC18. The recombinant plasmid, termed pTR11, contained a 4.0-kb PstI fragment including the entire coding regions for both large and small subunits of RuBPCase.Escherichia coli carrying pTR11 did not show any CO2-fixing activity. However, a derivative plasmid with an appropriate deletion, which was placed under the control of atac promoter, conferred ribulose bisphosphate-dependent CO2-fixing activity on the host cell. Analysis of gel-filtration chromatography of the RuBPCase synthesized inE. coli revealed that it had a hexadecameric form like the native enzyme ofT. ferrooxidans. 相似文献
9.
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
10.
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