Metallo-proteinase from the seedlings of kale (Brassica oleracea L. var. sabellica):

Metallo-proteinase from 8-d-old seedlings of kale was isolated. The enzyme was extracted with 1% NaCl, concentrated by ammonium sulfate and finally purified by high-performance liquid chromatography. The isolated enzyme had a molecular weight of 22.4 kDa and showed a maximum activity at pH 9.0 using casein as a substrate. Proteolytic activity of proteinase was inhibited by chelators. The inhibition by ethylenediaminetetraacetate (EDTA) was abolished by some divalent metals ions, especially by Zn²⁺. The enzyme showed activity against the synthetic peptides Suc-Ala-Ala-Pro-Leu-pNA and Suc-Ala-Ala-Pro-Phe-pNA, and hydrolized the following peptide bonds in the oxidized insulin B-chain: Leu6-Cya7, Leu15-Tyr16, Leu17—Val18 and Phe25-Tyr26.Abbreviations EDTA ethylenediaminotetraacetic acid - HPLC high-performance liquid chromatography - NEM N-ethylmaleimide - PCMB p-mecuribenzoic acid - PMSF phenylmethylsulfonyl fluoride This work was supported by the University Science Programme, Ministry of National Education, and Polish Academy of Science, Warsaw, Poland.     

Isolation and characterization of a new trypsin inhibitor from Crotalaria paulina seeds

A new trypsin inhibitor (CPTI) has been isolated from Crotalaria paulina seeds. Purification of the inhibitor was carried out by gel filtration, ion-exchange chromatography, and subsequent reversed-phase HPLC. The presence of a single polypeptide chain, with a molecular mass of 20 kDa and isoelectric point 4.0, was detected. The trypsin inhibitor had a Ki value of 4.5 x 10(-8) M and was capable of acting on human, bovine, and porcine trypsin and weakly on bovine chymotrypsin. Amino acid analysis showed that CPTI has a high content of aspartate, glutamate, leucine, serine, and glycine, having 177 amino acid residues in its composition. These data suggest that the protein belongs to the Kunitz-type trypsin inhibitors.     

Isolation and characterization of a trypsin inhibitor fromVigna sinensis seeds

The major trypsin inhibitor ofVigna sinensis cv. seridó was isolated and shown to be devoid of chymotrypsin inhibiting activity. It has a molecular weight of 9800 as determined by gel electrophoresis and a pI of 5.0. The activity of the inhibitor was decreased by treatment with trinitrobenzenesulfonic acid, suggesting that it isa LYS-X type trypsin inhibitor. Selfassociation of the molecule was demonstrated both in 1% sodium dodecylsulfate and inacidic (pH 2.4) conditions.     

Isolation and partial amino acid sequence of the trypsin inhibitor from the seeds of Cucurbita maxima

Trypsin inhibitor from squash (Cucurbita maxima) seed was extracted with 0.1 M-acetate buffer, pH 4.5, purified on immobilized trypsin, and separated by SE-Sephadex C-50 chromatography into three active fractions. All of them inhibited trypsin to the same extent, showed no antichymotrypsin or antikallikrein activity, had a similar molecular weight (about 3300), and contained no tryptophan, phenylalanine or threonine. The partial amino acid sequence of tryptic and peptic peptides of fraction III was determined by Edman degradation procedure.     

Isolation and partial characterization of a low molecular weight trypsin inhibitor from human urine

A low molecular weight glycoprotein which completely inhibited trypsin at a 1 : 1 molar ratio was isolated from human urine. It was generated from a precursor molecule which in turn derived from plasma inter-alpha-trypsin inhibitor. It had one polypeptide chain with a molecular weight of about 20 000 and a high content of half-cystine residues. Its amino-terminal amino-acid sequence was Val-Thr-Glu-Val-Thr-X-Leu-Glu-Asp-.     

Isolation and partial characterization of the pancreatic secretory trypsin inhibitor in the rat

Isolation in a 55% yield of the low molecular weight pancreatic secretory trypsin inhibitor was achieved by gel filtration of an acid extract of whole inactive rat pancreas juice on Sephadex G-50 at pH 2.5 followed by desalting and ion-exchange chromatography on SP Sephadex C-50 at pH 4.5. Two distinct chromatographic fractions were obtained, labeled fraction 1 and 2. Fractions 1 and 2 showed three, respectively two, distinct closely migrating cationic bands on gel electrophoresis in barbital buffer, pH 8.6. Each fraction demonstrated one band on polyacrylamide disc electrophoresis at pH 4.6. The inhibitor is homogenous on gel filtration and on the basis of its stoichiometry with active site titrated rat anionic trypsin. Its molecular weight is approx. 6024. The amino acid composition is included. Rat pancreatic secretory trypsin inhibitor is trypsin-specific and interacts on a 1:1 molar basis with rat trypsin. It is good inhibitor of bovine trypsin but a poor inhibitor of human cationic trypsin and its binding to trypsin is reversible by acidification. Like other inhibitors of this sort, it is present in about 0.1–0.2% of the total protein content of the juice, and normally exists in its free form. A simple procedure for the production of antiserum to the inhibitor which is a poor antigen is also described.     

A trypsin and chymotrypsin inhibitor from Caesalpinia bonduc seeds: isolation, partial characterization and insecticidal properties.

Evolution of proteinase inhibitor diversity in leguminous plants of tropical rainforests is under immense pressure from the regular upregulation of proteolytic machinery of their pests. The present study illustrates the isolation and bioinsecticidal potency of a serine proteinase inhibitor from the seeds of Caesalpinia bonduc (CbTI), inhabiting Great Nicobar Island, India. Following initial fractionation by ammonium sulfate precipitation, CbTI was purified to homogeneity by ion exchange, gel filtration and trypsin affinity chromatography. SDS-PAGE of gel filtrated CbTI showed a couple of proteins CbTI-1 ( approximately 16kDa) and CbTI-2 (20kDa) under non-reducing conditions, which subsequent to trypsin affinity chromatography yielded only CbTI-2. Both Native PAGE as well as iso-electric focusing showed 2 iso-inhibitors of CbTI-2 (pI values of 5.35 and 4.6). CbTI exhibited tolerance to extremes of temperatures (0-60 degrees C) and pH (1-12). A 1:1 stoichiometric ratio was noted during CbTI-2-trypsin complex formation, which was absent on binding with chymotrypsin. Further, SDS-PAGE analysis also showed that CbTI-1 has affinity only towards chymotrypsin, whereas both trypsin and chymotrypsin formed complexes with CbTI-2. Dixon plot analysis of CbTI-2 yielded inhibition constants (K(i)) of 2.75 x 10(-10)M and 0.95 x 10(-10)M against trypsin and chymotrypsin activity respectively. Preliminary investigations on the toxicological nature of CbTI revealed it to be a promising bioinsecticidal candidate.     

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.     

Purification and characterization of a trypsin inhibitor from Putranjiva roxburghii seeds

A highly stable and potent trypsin inhibitor was purified to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family by acid precipitation, cation-exchange and anion-exchange chromatography. SDS-PAGE analysis, under reducing condition, showed that protein consists of a single polypeptide chain with molecular mass of approximately 34 kDa. The purified inhibitor inhibited bovine trypsin in 1:1 molar ratio. Kinetic studies showed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 1.4x10(-11) M. The inhibitor retained the inhibitory activity over a broad range of pH (pH 2-12), temperature (20-80 degrees C) and in DTT (up to100 mM). The complete loss of inhibitory activity was observed above 90 degrees C. CD studies, at increasing temperatures, demonstrated the structural stability of inhibitor at high temperatures. The polypeptide backbone folding was retained up to 80 degrees C. The CD spectra of inhibitor at room temperature exhibited an alpha, beta pattern. N-terminal amino acid sequence of 10 residues did not show any similarities to known serine proteinase inhibitors, however, two peptides obtained by internal partial sequencing showed significant resemblance to Kunitz-type inhibitors.     

Isolation and characterization of a trypsin inhibitor from finger millet

A trypsin inhibitor was isolated from finger millet (Eleusine coracana) by ammonium sulphate fractionation, chromatography on CM-Sephadex and Sepha     

Agrobacterium rhizogenes-mediated transformation of Brassica oleracea var. sabauda and B. oleracea var. capitata

Agrobacterium rhizogenes A4M70GUS-mediated transformation of Savoy cabbage (Brassica oleracea L. var. sabauda) and two local lines of cabbage (B. oleracea L. var. capitata) was obtained using hypocotyl and cotyledon explants. The percentage of explants which formed roots was very high in all genotypes: 92.3 % in Savoy Gg-1, 64.4 % in cabbage P₂₂I₅, and 87.2 % in P₃₄I₅. Spontaneous shoot regeneration of excised root cultures grown on the hormone-free medium occurred in all three genotypes. In cabbage lines P₂₂I₅ and P₃₄I₅ shoot regeneration was higher (9.3 and 2.6 % respectively) than in Savoy cabbage Gg-1 (1.3 %). Transgenic nature of hairy root-derived plants was evaluated by GUS histological test and PCR analysis. All the tested cabbage shoots were GUS positive whilst in a Savoy cabbage GUS expression was registered only in 55 % of tested clones. PCR analysis demonstrated the presence of the GUS gene in regenerated shoot clones and in T₁ progeny.     

Isolation and partial characterization of a lectin from Euphorbia heterophylla seeds.

An N-acetylgalactosamine-specific lectin was isolated from Euphorbia heterophylla seeds by affinity chromatography on cross-linked arabinogalactan. It is a dimeric protein of two identical subunits of Mr 32 000, and differs structurally from all previously known Euphorbiaceae lectins. Its distribution over the seed is typical in that it is merely confined to the primary axes.     

Purification and characterization of peroxidase from cauliflower (Brassica oleracea L. var. botrytis) buds

Peroxidases (EC 1.11.1.7; donor: hydrogen peroxide oxidoreductase) are part of a large group of enzymes. In this study, peroxidase, a primer antioxidant enzyme, was purified with 19.3 fold and 0.2% efficiency from cauliflower (Brassica oleracea L.) by ammonium sulphate precipitation, dialysis, CM-Sephadex ion-exchange chromatography and Sephadex G-25 purification steps. The substrate specificity of peroxidase was investigated using 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulphonic acid) (ABTS), 2-methoxyphenol (guaiacol), 1,2-dihydroxybenzene (catechol), 1,2,3-trihyidroxybenzene (pyrogallol) and 4-methylcatechol. Also, optimum pH, optimum temperature, optimum ionic strength, stable pH, stable temperature, thermal inactivation conditions were determined for guaiacol/H(2)O(2), pyrogallol/H(2)O(2), ABTS/H(2)O(2), catechol/H(2)O(2) and 4-methyl catechol/H(2)O(2) substrate patterns. The molecular weight (M(w)) of this enzyme was found to be 44 kDa by gel filtration chromatography method. Native polyacrylamide gel electrophoresis (PAGE) was performed for isoenzyme determination and a single band was observed. K(m) and V(max) values were calculated from Lineweaver-Burk graph for each substrate patterns.     

结球甘蓝和青花菜小孢子胚植株再生

结球甘蓝（Brassica oleracea var.capitata）和青花菜（Brassica oleracea var.italica）小孢子胚再生植株频率低是目前影响游离小孢子培养技术有效应用的关键问题之一,研究其小孢子胚植株再生频率的影响因素,提高胚再生植株频率,对促进游离小孢子培养技术在甘蓝类蔬菜育种中更好地应用具有重要意义。该文以结球甘蓝中甘11和青花菜TI-111等基因型为试材,对影响游离小孢子胚再生成植株的固体培养基类型、琼脂浓度、胚的类型及胚在液体培养基中的滞留时间等因素进行了研究。结果表明：游离小孢子培养25天的子叶胚在琼脂浓度为1%–1.25%的B5培养基上植株再生频率最高。进一步通过8个不同基因型对上述实验结果进行了验证,结果显示,游离小孢子培养25天的子叶胚在1%琼脂浓度的B5培养基上植株再生频率达77.8%–97.2%。     

Purification and characterization of a trypsin inhibitor from seeds of Murraya koenigii

A protein with trypsin inhibitory activity was purified to homogeneity from the seeds of Murraya koenigii (curry leaf tree) by ion exchange chromatography and gel filtration chromatography on HPLC. The molecular mass of the protein was determined to be 27 kDa by SDS-PAGE analysis under reducing conditions. The solubility studies at different pH conditions showed that it is completely soluble at and above pH 7.5 and slowly precipitates below this pH at a protein concentration of 1 mg/ml. The purified protein inhibited bovine pancreatic trypsin completely in a molar ratio of 1:1.1. Maximum inhibition was observed at pH 8.0. Kinetic studies showed that Murraya koenigii trypsin inhibitor is a competitive inhibitor with an equilibrium dissociation constant of 7 × 10^{? 9} M. The N-terminal sequence of the first 15 amino acids showed no similarity with any of the known trypsin inhibitors, however, a short sequence search showed significant homology to a Kunitz-type chymotrypsin inhibitor from Erythrina variegata.     

Purification and characterization of a trypsin inhibitor from seeds of Murraya koenigii

A protein with trypsin inhibitory activity was purified to homogeneity from the seeds of Murraya koenigii (curry leaf tree) by ion exchange chromatography and gel filtration chromatography on HPLC. The molecular mass of the protein was determined to be 27 kDa by SDS-PAGE analysis under reducing conditions. The solubility studies at different pH conditions showed that it is completely soluble at and above pH 7.5 and slowly precipitates below this pH at a protein concentration of 1 mg/ml. The purified protein inhibited bovine pancreatic trypsin completely in a molar ratio of 1:1.1. Maximum inhibition was observed at pH 8.0. Kinetic studies showed that Murraya koenigii trypsin inhibitor is a competitive inhibitor with an equilibrium dissociation constant of 7 x 10(-9) M. The N-terminal sequence of the first 15 amino acids showed no similarity with any of the known trypsin inhibitors, however, a short sequence search showed significant homology to a Kunitz-type chymotrypsin inhibitor from Erythrina variegata.     

结球甘蓝和青花菜小孢子胚植株再生

结球甘蓝(Brassica oleracea var. capitata)和青花菜(Brassica oleracea var. italica)小孢子胚再生植株频率低是目前影响游离小孢子培养技术有效应用的关键问题之一, 研究其小孢子胚植株再生频率的影响因素, 提高胚再生植株频率, 对促进游离小孢子培养技术在甘蓝类蔬菜育种中更好地应用具有重要意义。该文以结球甘蓝中甘11和青花菜TI-111等基因型为试材, 对影响游离小孢子胚再生成植株的固体培养基类型、琼脂浓度、胚的类型及胚在液体培养基中的滞留时间等因素进行了研究。结果表明: 游离小孢子培养25天的子叶胚在琼脂浓度为1%–1.25%的B5培养基上植株再生频率最高。进一步通过8个不同基因型对上述实验结果进行了验证, 结果显示, 游离小孢子培养25天的子叶胚在1%琼脂浓度的B5培养基上植株再生频率达77.8%–97.2%。     

Purification and characterization of a new trypsin inhibitor from Dimorphandra mollis seeds

A second trypsin inhibitor (DMTI-II) was purified from the seed of Dimorphandra mollis (Leguminosae-Mimosoideae) by ammonium sulfate precipitation (30–60%), gel filtration, and ion-exchange and affinity chromatography. A molecular weight of 23 kDa was estimated by gel filtration on a Superdex 75 column SDS-PAGE under reduced conditions showed that DMTI-II consisted of a single polypeptide chain, although isoelectric focusing revealed the presence of three isoforms. The dissociation constant of 1.7 × 10^–9 M with bovine trypsin indicated a high affinity between the inhibitor and this enzyme. The inhibitory activity was stable over a wide pH range and in the presence of DTT. The N-terminal sequence of DMTI-II showed a high degree of homology with other Kunitz-type inhibitors.