Distribution of alpha-Galactosidase in Cucurbita pepo

The distribution of α-galactosidase (α-d-galactoside galactohydrolase [EC 3.2.1.22]) in Cucurbita pepo has been determined in an attempt to assess its involvement in hydrolysis of transport sugars of the raffinose oligosaccharide series ([α-1-6-0-galactopyranosyl]_n sucrose). Extracts prepared from leaves and petioles at different stages of development, roots, flowers, dry and germinating seeds, all contained appreciable levels of α-galactosidase activity. Chromatography of these extracts on DEAE-Sephadex resolved the enzyme into three active isozymic forms. These isozymes were present in all regions of the plant analyzed but their relative proportions varied between tissues and changed within leaf and petiole tissues during development and in seeds during their germination. The level of total α-galactosidase activity in the leaf blade measured on a fresh weight or total protein basis remained constant at all developmental stages analyzed. The occurrence of these isozymes in mature exporting leaves indicates an effective intracellular compartmentation between their location and the sites of galactosyl oligosaccharide biosynthesis, accumulation and movement in the tissue. We have used these results to comment on the transport pathway of galactosyl oligosaccharides between the phloem and surrounding tissues in this plant.     

Characterization of alpha-Galactosidase from Cucumber Leaves

Two forms of alpha-galactosidase (alpha-d-galactoside galactohydrolase, E.C. 3.2.1.22) which differed in molecular weight were resolved from Cucumis sativus L. leaves. The enzymes were partially purified using ammonium sulfate fractionation, Sephadex gel filtration, and diethylaminoethyl-Sephadex chromatography. The molecular weights of the two forms, by gel filtration, were 50,000 and 25,000. The 50,000-dalton form comprised approximately 84% of the total alpha-galactosidase activity in crude extracts from mature leaves and was purified 132-fold. The partially purified 25,000-molecular weight form rapidly lost activity unless stabilized with 0.2% albumin and accounted for 16% of the total alpha-galactosidase activity in the crude extract. The smaller molecular weight form was not found in older leaves.The two forms were similar in several ways including their pH optima which were 5.2 and 5.5 for the 50,000- and 25,000-dalton form, respectively, and activation energies, which were 15.4 and 18.9 kilocalories per mole for the larger and smaller forms. Both enzymes were inhibited by galactose as well as by excess concentrations of p-nitrophenyl-alpha-d-galactoside sub-strate. K(m) values with this substrate and with raffinose and melibiose were different for each substrate, but similar for both forms of the enzyme. With stachyose, K(m) values were 10 and 30 millimolar for the 50,000- and 25,000- molecular weight forms, respectively.     

l-Ornithine:2-Oxoacid Aminotransferase from Squash (Cucurbita pepo, L.) Cotyledons: Purification and Properties

ORNITHINE: 2-oxoacid aminotransferase (EC 2.6.1.13) has been purified over 400-fold with a total recovery of 14% from acetone powders of cotyledons of germinating squash (Cucurbita pepo, L.) seedlings. The pH optimum of the transamination between l-ornithine and alpha-ketoglutarate is 8 and the Michaelis constants are 4.7 mm and 6.3 mm, respectively. The enzyme has a molecular weight of 48,000 as determined by gel filtration. The reaction is essentially specific for alpha-ketoglutarate as the amino group acceptor. The enzyme is inhibited very strongly by hydroxylamine, and less severely by NaCN and isonicotinylhydrazide. No inhibition is observed in the presence of 10 mml-cysteine. The energy of activation is 7.6 kcal/mole. The stability of the enzyme preparation is enhanced by the presence of dithioerythritol and glycerol. The enzyme activity of the most purified fraction is stimulated 30% by the addition of pyridoxal phosphate; however, the evidence for the unequivocal involvement of pyridoxal phosphate was inconclusive.     

Cytokinin-Regulated Mesophyll Cell Division and Expansion during Development of Cucurbita pepo Leaves

The role of cytokinins in the development of mesophyll structure was studied in developing pumpkin Cucurbita pepo L. leaves. Leaves were treated with cytokinins at different stages of growth: when they reached 25 or 50% of their final size (S _max), immediately after leaf growth ceased, and during senescence. At the early stages of leaf development, treatment with exogenous benzyladenine accelerated division of mesophyll cells. At the later stages of development, BA treatment activated expansion of growing cells and those, which have just accomplished their growth. The exogenous cytokinin did not affect the senescent leaf cells. The content of endogenous cytokinins changed during mesophyll development. The juvenile leaves (25% of S _max) were characterized by low level of these phytohormones. In the expanding leaves (50% of S _max), the content of phytohormones increased and decreased when leaf growth ceased. In the senescent leaves, the cytokinin content decreased markedly. It was concluded that the response of mesophyll cells to cytokinin depended on the cell growth phase at the moment of hormone action. Furthermore, in the young leaves, lower cytokinin concentrations were required for division of mesophyll cells in vivo than for cell expansion at the final stage of leaf development.     

Purification and characterization of the trypsin inhibitor from Cucurbita pepo var. patissonina fruits

Three trypsin inhibitor fractions were found in white bush fruits (Cucurbita pepo L. var. patissonina). One of them, CPPTI-fIII, was purified to homogeneity by means of affinity and ion exchange chromatography. It is a cysteine-poor protein with an approximate Mr of 21 000. The inhibitor contains arginine at position P1 of the reactive site and inhibits bovine trypsin, hog pancreatic kallikrein and subtilisin. This inhibitor differs from the inhibitors of white bush dormant seeds, CPPTI-I and CPPTI-II, in its amino-acid composition, molecular mass, amino-acid residue at position P1 of the reactive site and inhibition spectrum.     

Mavicyanin, a blue copper protein from Cucurbita pepo medullosa. Purification and characterization.

1. The copper protein mavicyanin has been isolated and purified from the green squash Cucurbita pepo medullosa. 2. Mavicyanin contains one type-1 copper/18000 Mr, which can be characterized by: intense absorption maximum at 600 nm (epsilon = 5000 M-1 cm-1/Cu, A280/A600 = 8.0 +/- 0.5, A600/A403 = 7.0 +/- 0.25, maximum of fluorescence emission at 335 nM. 3. In the oxidized state the copper of mavicyanin is 100% detectable by electron paramagnetic resonance (EPR). Computer simulation of the rhombic EPR signal gives gz = 2.287, gy = 2.077, gx = 2.025, Az = 3.5 mT, Ay = 2.9 mT and Ax = 5.7 mT. 4. Like other simple type-1 copper proteins, such as stellacyanin, azurin or plastocyanin, mavicyanin is readily reduced by hydroquinone or L-ascorbic acid. Its midpoint potential E'm was determined to be + 285 mV. The reduced protein reacts rather slowly with dioxygen, but is rapidly reoxidized by ferricyanide.     

鸡蛋果叶片细胞质丙酮酸激酶的部分纯化及其调节性质

鸡蛋果叶片细胞质丙酮酸激酶(PK_c)纯化92.6倍.其最适pH为7.2,对热较稳定。PEP的K_m为0.037 mmol/L,ADP的K_m为0.05 mmol/L。ASP、Asn、Cys、α—酮戊二酸和苹果酸均对PK?有轻微的激活作用,但草酸、ATP、CaCl_2则具强烈的抑制作用。     

Partial Purification and Some Properties of Thyroid Peroxidase Solubilized by Alkaline Treatment

Thyroid peroxidase from frozen porcine thyroids has been solubilized by suspension of thyroid “microsomes” in pH 10.5, 0.05 Mcarbonate buffer and centrifugation at 105, 000 × g for 1 hr at 4°C. About 65% of the initial activity is present in the supernatant. Partial purification of the alkaline solubilized TP0 has been achieved bv DEAE cellulose chromatography and ammonium sulfate fractionation. The method results in an 18 to 20 fold purification over the homogenate and recovery of 35 to 40% of TP0 activity. Over 90% of the phospholipids present in the particulate fraction and most of the nucleic acids have been removed. The partially purified preparation catalyzes the oxidation of guaiacol and the lodination of monoiodotyrosine. It is retarded on Sephadex G-200 and has an apparent molecular weight of about 350, 000.     

一种女贞叶抗冻蛋白的分离纯化

根据抗冻蛋白与冰结合的特性,利用碎冰从女贞(Ligustrum lucidum)叶提取液中分离出抗冻蛋白。结果表明,通过碎冰吸附、凝胶过滤和离子交换层析可以获得4个组分的蛋白质,其中的1个经鉴定具有热滞活性。在蛋白质浓度为5mg.mL-1时,它的热滞活性(thermal hysteresis activity,THA)值为0.678°C,对其进行全波长扫描(200-1000nm)发现在975nm处有吸收峰;该蛋白亲水性氨基酸含量较高。     

一种女贞叶抗冻蛋白的分离纯化

根据抗冻蛋白与冰结合的特性, 利用碎冰从女贞(Ligustrum lucidum)叶提取液中分离出抗冻蛋白。结果表明, 通过碎冰吸附、凝胶过滤和离子交换层析可以获得4个组分的蛋白质, 其中的1个经鉴定具有热滞活性。在蛋白质浓度为5 mg. mL-1时, 它的热滞活性(thermal hysteresis activity, THA)值为0.678°C, 对其进行全波长扫描(200-1 000 nm)发现在975nm处有吸收峰; 该蛋白亲水性氨基酸含量较高。     

Purine-containing cucurbitane triterpenoids from Cucurbita pepo cv dayangua

Phytochemical investigation of the fruits of Cucurbita pepo cv dayangua led to the isolation of cucurbitaglycosides A (1) and B (2). This is the first report of cucurbitane triterpenoids with a purine unit. Their structures were elucidated mainly based on interpretation of HRESIMS results, as well as 1D and 2D NMR spectra. Cucurbitaglycosides A and B showed cytotoxic activity against the human epithelial carcinoma cell line HeLa with IC50 of 17.2 and 28.4 microg/mL, respectively.     

Phytochrome and phosphotungstate-chromate-positive vesicles from Cucurbita pepo L.

The phosphotungstic acid-chromic acid (PTA-CrO₃) stain, putatively specific for the plasma membrane of plants, has been used in an attempt to monitor the distribution of this membrane in a 20,000 x g particulate fraction from Cucurbita hypocotyl hooks. On discontinuous sucrose gradients, the relative distributions of the phytochrome and PTA-CrO₃-positive vesicles present in this fraction appear to be correlated. When intact tissue is stained, however, other components, in addition to the plasma membrane, react positively to the stain. These components include prolamellar-body membranes, lipid droplets, and ribosomes. This lack of specificity calls into question the reliability of the technique for the unequivocal identification and accurate quantitation of plasma-membrane fragments in isolated particulate fractions. The present data do not, therefore, provide unambiguous evidence that phytochrome is associated with plasma membrane in tissue homogenates from Cucurbita.Abbreviations PTA-CrO₃ phosphotungstate-chromate - RNP ribonucleoprotein     

Different AT-rich satellite DNAs in Cucurbita pepo and Cucurbita maxima

Summary The AT-rich highly repeated satellite DNA of Cucurbita pepo (zucchini) and Cucurbita maxima (pumpkin) were cloned and their DNA structure was investigated. DNA sequencing revealed that the repeat length of satellite DNA in Cucurbita pepo is 349–352 base pairs. The percentage of AT-base pairs is about 61%. This satellite is highly conserved in restriction enzyme pattern and DNA sequence; sequence heterogeneity is about 10%. In contrast, the satellite DNA of Cucurbita maxima has a repeat length of 168–169 base pairs. This satellite is also rich in AT-base pairs (64%), existing in at least three different variants as revealed by restriction enzyme analysis and DNA sequencing. The sequence heterogeneity between these variants is about 15%. The two satellite DNAs showed no cross-hybridization to each other and sequence homology is only limited. Nevertheless, we found in the C. pepo genome a high amount of sequences resembling the satellite of C. maxima. In contrast, the satellite repeat of C. pepo is found in the C. maxima DNA only in a few copies. These observations were discussed with respect to satellite DNA evolution and compared to the data received from monocotyledonous species.     

Purification and Properties of Alkaline Proteinase from Aspergillus oryzae

Alkaline proteinase was purified from culture extract of a strain of Aspergillus oryzae. The process consists of the Amberlite IRC-50 adsorption, column chromatography on DEAE-cellulose and CM-cellulose and Sephadex G-100 gel filtration. The molecular weight of the enzyme was estimated to be about 23,000 by a gel filtration method. Alkaline proteinase showed neither carboxypeptidase activity nor aminopeptidase activity, but degraded 10101010 poly-l,α-glutamic acid, poly-l-lysine, 10101010 and 10101010. The enzyme was completely inhibited by diisopropylphos-phorofluoridate (10^?2 m) or potato inhibitor (250 μg/ml).     

An alpha-Galactosidase with Hemagglutinin Properties from Soybean Seeds

Soybean (Glycine max L.) seeds contain a galactose-binding protein which displays two activities: (a) an alpha-galactosidase activity and (b) a hemagglutinin activity. The alpha-galactosidase-hemagglutinin was purified to homogeneity by conventional protein purification procedures and also by affinity chromatography. This protein can be easily separated from soybean agglutinin, the N-acetyl-d-galactosamine-specific lectin in soybean. Further, these two agglutinins show no immunological relatedness. The alpha-galactosidase-hemagglutinin can be reversibly converted by pH changes from a tetrameric form which displays both enzymic and hemagglutinin activities to a monomeric form which displays enzymic activity only. Although both the monomeric and tetrameric forms are enzymically active, they display different pH optima and carbohydrate specificities.     

大豆叶片尿囊素酶的纯化和性质

被纯化的 L—ALNase比活是 819 nkat/mg,酶的纯度提高了738倍,得率为27％。最适pH 7.4,K_m为13.7 mmol/L ALN,对还原剂和巯基试剂敏感,但5 mmol/L的抗坏血酸可提高酶活26％。磷酸盐和KCN有些抑制作用,但Mn~(2+)无活化作用。7.5％ PAGE,ANP定位测定,是 R_f 0.62、0.64、0.68、0.70和 0.725个同功酶的混合物。     

Filament formation in vitro of a sieve tube protein from Cucurbita maxima and Cucurbita pepo

Summary Phloem proteins of the sieve tube exudate from Cucurbita maxima Duch. and Cucurbita pepo L. were investigated as to their filament forming ability in vitro. From the two main proteins (116000 dalton, 30000 dalton) only the 116000 dalton protein was found to form reversibly distinct filaments of 6–7 nm diameter upon removal of SH-protecting agents from the buffer, whereas the 30000 dalton protein was precipitated as amorphous material under these conditions. The protein filaments were similar to the filaments ocurring within the sieve tube cells in vivo.Abbreviations SDS sodium dodecyl sulfate - TCA trichloroacetic acid     

Purification and Properties of Two Thermostable Alkaline Xylanases from an Alkaliphilic Bacillus sp.

Two xylanases, designated XylA and XylB, were purified from the culture supernatant of the alkaliphilic Bacillus sp. strain AR-009. The molecular masses of the two enzymes were estimated to be 23 kDa (XylA) and 48 kDa (XylB) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pHs for activity were 9 for XylA and 9 to 10 for XylB. The temperature optima for the activity of XylA were 60°C at pH 9 and 70°C at pH 8. XylB was optimally active at 75°C at pH 9 and 70°C at pH 8. Both enzymes were stable in a broad pH range and showed good stability when incubated at 60 and 65°C in pH 8 and 9 buffers.