银杏种仁中抗菌蛋白的纯化及性质

银杏果仁提取上清液经硫酸铵沉淀、CM—52离子交换柱层析和Sephadex G-50凝胶过滤层析后分离纯化出一种抗菌蛋白。SDS—PAGE分析结果表明,此蛋白分子量为13000,对热稳定;氨基酸组分分析表明,该蛋白含18种不同氨基酸,尤富含丙氨酸(Ala)和精氨酸(His)等,缺乏半胱氨酸(Cys);纯化的蛋白对黄瓜镰刀孢菌(Fusarium oxysporum)、瓜类炭疽菌(Colletotrichum orbiculare)、小麦全蚀病菌(Gaeumannomyces graminis)等真菌有很强的抑制作用,而且对金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia Coli)等细菌也有一定的抑制作用。     

Molecular cloning and characterization of a glutathione S-transferase gene from Ginkgo biloba.

Glutathione S-transferases (GSTs) play an important role in the response of plants to changing environmental conditions. Here, we report the cloning of the GST gene for GST from Ginkgo biloba, a native medicinal plant species in China, by rapid amplification of cDNA ends (RACE). The full-length cDNA (designated as GbGST) was 1008 bp and contained a 684 bp open reading frame (ORF) encoding a polypeptide of 228 amino acids. The genomic sequence of GbGST was also obtained. Semi-quantitative RT-PCR analysis revealed that GbGST expressed in all tested tissues of G. biloba, including leaf, root and stem and the expression of GbGST could be induced by UV, MJ and drought treatments, suggesting that GbGST was potentially involved in plant's stress tolerance. To our knowledge, this is the first GST cDNA cloned from Ginkgoaceae. Based on comparative analyses of amino acid sequence, phylogeny, predicted three-dimensional structure together with the gene structure, the GbGST should be classified into the tau class.     

Molecular cloning, characterization and expression of a novel jasmonate-dependent defensin gene from Ginkgo biloba

A novel defensin gene was isolated from Ginkgo biloba. The full-length cDNA of G. biloba defensin (designated as Gbd) was 534bp. The cDNA contained a 240-bp open reading frame encoding an 80-amino acid protein of 5.68 kDa with a potential 30 aa signal peptide. The putative GbD mature protein showed striking similarity to other plant defensins, representing low molecular size antimicrobial polypeptides. Eight cysteine sites conserved in plant defensins were also found in GbD at similar positions. Three-dimensional structure modeling showed that GbD strongly resembled defensin from tobacco (NaD1) and consisted of an alpha-helix and a triple-strand antiparallel beta-sheet that were stabilized by four intramolecular disulfide bonds, implying GbD may have functions similar to NaD1. The genomic DNA gel blot indicated that Gbd belonged to a multigene family. Expression analysis revealed that Gbd was up-regulated by wounding and methyl jasmonate treatments, suggesting that Gbd is potentially involved in plant resistance or tolerance to pathogens during wounding.     

Purification and characterization of a co(II)-sensitive alpha-mannosidase from Ginkgo biloba seeds

An alpha-mannosidase was purified from developing Ginkgo biloba seeds to apparently homogeneity. The molecular weight of the purified alpha-mannosidase was estimated to be 120 kDa by SDS-PAGE in the presence of 2-mercaptoethanol, and 340 kDa by gel filtration, indicating that Ginkgo alpha-mannosidase may function in oligomeric structures in the plant cell. The N-terminal amino acid sequence of the purified enzyme was Ala-Phe-Met-Lys-Tyr-X-Thr-Thr-Gly-Gly-Pro-Val-Ala-Gly-Lys-Ile-Asn-Val-His-Leu-. The alpha-mannosidase activity for Man(5)GlcNAc(1) was enhanced by the addition of Co(2+), but the addition of Zn(2+), Ca(2+), or EDTA did not show any significant effect. In the presence of cobalt ions, the hydrolysis rate for pyridylaminated Man(6)GlcNAc(1) was significantly faster than that for pyridylaminated Man(6)GlcNAc(2), suggesting the possibility that this enzyme is involved in the degradation of free N-glycans occurring in developing plant cells (Kimura, Y., and Matsuo, S., J. Biochem., 127, 1013-1019 (2000)). To our knowledge, this is the first report showing that plant cells contain an alpha-mannosidase, which is activated by Co(2+) and prefers the oligomannose type free N-glycans bearing only one GlcNAc residue as substrate.     

Purification, characterization, and molecular cloning of the gene of a seed-specific antimicrobial protein from pokeweed

A small cysteine-rich protein with antimicrobial activity was isolated from pokeweed (Phytolacca americana) seeds and purified to homogeneity. The protein inhibits the growth of several filamentous fungi and gram-positive bacteria. The protein was highly basic, with a pI higher than 10. The entire amino acid sequence of the protein was determined to be homologous to antimicrobial protein (AMP) from Mirabilis jalapa. The cDNA encoding the P. americana AMP (Pa-AMP-1) and chromosomal DNA containing the gene were cloned and sequenced. The deduced amino acid sequence shows the presence of a signal peptide at the amino terminus, suggesting that the protein is synthesized as a preprotein and secreted outside the cells. The chromosomal gene shows the presence of an intron located within the region encoding the signal peptide. Southern hybridization showed that there was small gene family encoding Pa-AMP. Immunoblotting showed that Pa-AMP-1 was only present in seeds, and was absent in roots, leaves, and stems. The Pa-AMP-1 protein was secreted into the environment of the seeds during germination, and may create an inhibitory zone against soil-borne microorganisms. The disulfide bridges of Pa-AMP-1 were identified. The three-dimensional modeling of Pa-AMP-1 indicates that the protein has a small cystine-knot folding, a positive patch, and a hydrophobic patch.     

Purification, characterization, and molecular cloning of a chitinase from the seeds of Benincasa hispida

A chitinase was purified from the seeds of Benincasa hispida, a medicinal plant also called white gourd, and a member of the Cucurbitaceae family. Purification was done by using a procedure consisting of only two fractionation steps: an acid denaturation step followed by ion-exchange chromatography. The sequence of the N-terminal forty amino acid residues was analyzed and the sequence indicated that the enzyme is a class III chitinase. The enzyme, which is a basic chitinase, is one of at least five chitinases detected in the seed extract of B. hispida. Like other class III chitinases, this enzyme also has lysozyme activity. A genomic clone of the gene encoding the enzyme was isolated and sequenced. The gene has the potential to encode a protein of 301 amino acid residues. The deduced amino acid sequence of the protein, as expected from the N-terminal amino acid sequence, shares high degrees of similarity with other class III chitinases.     

Molecular cloning, characterization and expression of atpA and atpB genes from Ginkgo biloba

The chloroplast ATP synthase (ATPase) utilizes the energy of a transmembrane electrochemical proton gradient to drive the synthesis of ATP from ADP and phosphate. The chloroplast ATPase α and β subunits are the essential components of multisubunit protein complex. In this paper, the full-length cDNA and genomic DNA of ATPase α (designated as GbatpA) and β (designated as GbatpB) subunit genes were isolated from Ginkgo biloba. The GbatpA and GbatpB genes were both intronless. The coding regions of GbatpA and GbatpB were 1530 bp and 1497 bp long, respectively, and their deduced amino acid sequences showed high degrees of identity to those of other plant ATPase α and β proteins, respectively. The expression analysis by RT-PCR revealed that GbatpA and GbatpB both expressed in tissue-specific manners in G. biloba and might involve in leaf development. The recombinant GbATPB protein was successfully expressed in E. coli strain using pET28a vector with ATPase activity as three times high as the control, and the results showed that the molecular weight of the recombinant protein was about 54 kDa, a size that was in agreement with that predicted by bioinformatics analysis. This study provides useful information for further studying on overall structure, function and regulation of the chloroplast ATPase in G. biloba, the so-called “living fossil” plant as one of the oldest gymnosperm species. These authors contributed equally to this work     

Characteristics and antifungal activity of a chitin binding protein from Ginkgo biloba

An antifungal peptide from leaves of Ginkgo biloba, designated GAFP, has been isolated. Its molecular mass of 4244.0 Da was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation. GAFP exhibited antifungal activity towards Pellicularia sasakii Ito, Alternaria alternata (Fries) Keissler, Fusarium graminearum Schw. and Fusarium moniliforme. Its activities differed among various fungi. GAFP could also cause increased hyphal membrane permeabilization and a rapid alkalization of the medium when applied at 100 microgram/ml to Pellicularia sasakii Ito hyphae. The amino acid sequence of GAFP shows characteristics of the cysteine/glycine-rich chitin binding domain of many chitin binding proteins. The cysteine residues are well conserved.     

Purification,characterization, and molecular cloning of an outer layer protein of carp fertilization envelope

An outer layer protein of carp fertilization envelope (FE), FEO‐1, was purified from carp oocytes. The cDNAs encoding FEO‐1 were cloned. The mature protein of FEO‐1 is 21 kDa in molecular weight and contains 177 amino acid residues whose sequence has 58% identity to the outer layer protein of chick vitelline membrane. In situ hybridization and immunocytochemistry show that FEO‐1 is expressed in oocytes and liver. In oocytes, FEO‐1 is stored in the cortical granules. During cortical reaction, it is exocytosed to the perivitelline space and then gradually added to the outer layer of FE (FE_o). FEO‐1 first appears as discrete deposits along FE_o, then merges to form a continuous layer. The thickness of FE_o increases as cortical reaction proceeds. In addition to FEO‐1, FE_o contains cystatin, fibroin‐like substance (FLS), and cathepsin‐like substance (CLS) as well. They are stored in the cortical granules and are exocytosed to FE_o simultaneously with FEO‐1 during cortical reaction. In FE_o, FEO‐1 is present in monomer form and can be completely extracted by sodium dodecyl sulfate (SDS)‐mercaptoethanol (MSH). On the other hand, the cystatin, FLS, and CLS present in FE_oare cross‐linked together. They are partially extracted by SDS‐MSH but can be completely extracted by guanidium thiocyanate‐lauroylsarcosine. Mol. Reprod. Dev. 54:186–193, 1999. © 1999 Wiley‐Liss, Inc.     

Purification, characterization and antifungal properties of a lipid-transfer protein from sunflower (Helianthus annuus) seeds

An antifungal protein from Helianthus annuus L. seeds (Ha-AP10) has been purified to homogeneity and characterized. Ha-AP10 purification was performed by gel filtration, cation exchange chromatography and reverse phase HPLC. Its molecular mass was estimated to be 10 kDa and western blot analyses suggest that it has an extracellular location. The N-terminal sequence of Ha-AP10 showed strong homology to some plant lipid-transfer proteins (LTPs). Antifungal tests have demonstrated that Ha-AP10 exerts a fungistatic effect. It completely inhibits the germination of spores of the fungal pathogen Fusarium solani f. sp. eumartii at a concentration of 40 μg ml⁻¹ and produces a 50% growth inhibition at 6.5 μg ml⁻¹ (0.65 μ M ). These data place Ha-AP10 among the most potent antifungal LTPs described so far.     

Purification, molecular cloning and functional characterization of swine phosphatidylethanolamine-binding protein 4 from seminal plasma

Phosphatidylethanolamine-binding proteins (PEBPs) are found in various species and have multiple functions. In this study, we purified the swine homolog of human PEBP4 (sPEBP4) from swine seminal plasma, cloned the sPEBP4 cDNA and functionally characterized this protein. The molecular mass of the purified protein was calculated to be 25 kDa by SDS-polyacrylamide gel electrophoresis under reducing conditions. The full-length cDNA of sPEBP4 contains 815 bp with an open reading frame of 669 bp that encodes a protein 222 residues in length. sPEBP4 contains a putative phosphatidylethanolamine-binding domain between residues 79 and 195; however, this domain did not show lipid binding activity. The overall amino acid sequence identity of PEBP4s from swine, human, mouse, bovine and canine ranges between 56.1% and 82.4%. Immunohistochemical staining and western blotting analysis showed that sPEBP4 is secreted from epithelial cells in the epididymis to the seminal plasma. To explore the role of sPEBP4 in the seminal plasma, we tested the effect of sPEBP4 on swine sperm motility. Sperms suspended in phosphate-buffered saline began to swim after the addition of purified sPEBP4, but not when swine serum albumin was added, indicating that sPEBP4 promotes sperm motility.     

Purification, characterization and molecular cloning of prophenoloxidases from Sarcophaga bullata

Prophenoloxidase (PPO) is a key enzyme associated with both melanin biosynthesis and sclerotization in insects. This enzyme is involved in three physiologically important processes viz., cuticular hardening, defense reactions and wound healing in insects. It was isolated from the larval hemolymph of Sarcophaga bullata and purified by employing ammonium sulfate precipitation, Phenyl Sepharose chromatography, DEAE-Sepharose chromatography, and Sephacryl S-200 column chromatography. The purified enzyme exhibited two closely moving bands on 7.5% SDS-PAGE under denaturing conditions. From the estimates of molecular weight on Sephacryl S-100, TSK-3000 HPLC column and SDS-PAGE, which ranged from 90,000 to 100,000, it was inferred that the enzyme is made up of a single polypeptide chain. Activation of PPO (K(a)=40 microM) was achieved by the cationic detergent, cetyl pyridinium chloride below its critical micellar concentration (0.8 mM) indicating that the detergent molecules are binding specifically to the PPO and causing the activation. Neither anionic, nor nonionic (or zwitterionic) detergents activated the PPO. The active enzyme exhibited wide substrate specificity and marked thermal unstability. Using primers designed to conserved amino acid sequences from known PPOs, we PCR amplified and cloned two PPO genes from the sarcophagid larvae. The clones encoded polypeptides of 685 and 691 amino acids. They contained two distinct copper binding regions and lacked the signal peptide sequence. They showed a high degree of homology to dipteran PPOs. Both contained putative thiol ester site, two proteolytic activation sites and a conserved C-terminal region common to all known PPOs.     

Purification, characterization, and molecular cloning of tyrosinase from Pholiota nameko

Tyrosinase (monophenol, 3,4-dihydroxy L-phenylalanine (L-DOPA):oxygen oxidoreductase, EC 1.14.18.1) was isolated from fruit bodies of Pholiota nameko and purified to homogeneity. The purified enzyme was a monomer with a molecular weight of 42,000 and contained 1.9 copper atoms per molecule. The N-terminal of the purified enzyme could not be detected by Edman degradation, probably due to blocking, while the C-terminal sequence of the enzyme was determined to be -Ala-Ser-Val-Phe-OH. The amino acid sequence deduced by cDNA cloning was made up of 625 amino acid residues and contained two putative copper-binding sites highly conserved in tyrosinases from various organisms. The C-terminal sequence of the purified enzyme did not correspond to that of the deduced sequence, but agreed with Ala384-Ser385-Val386-Phe387 in sequence. When the encoded protein was truncated at Phe387, the molecular weight of the residual protein was calculated to be approximately 42,000. These results suggest that P. nameko tyrosinase is expressed as a proenzyme followed by specific cleavage to produce a mature enzyme.     

银杏种仁中一种抗氧化活性蛋白的纯化及性质

银杏种仁经破碎,提取缓冲液4℃浸取后离心得上清液。上清液经硫酸铵沉淀,DEAE-52离子交换层析,MonoQ离子交换层析,UltroGelACA-54凝胶过滤层析后,分离得到一种具有抗氧化活性的蛋白。该蛋白经UltroGelACA-54凝胶过滤层析测定分子量为60 kD,经PAGE和SDS-PAGE鉴定均为单一蛋白质条带。SDS-PAGE测定其亚基分子量为10 kD。该蛋白具有一定的还原能力和清除超氧阴离子自由基及DPPH自由基能力,并在30～60℃温度下具有良好的稳定性。