Purification and Characterization of Molecular and Immunological Properties of Rice Ferredoxin-Nitrite Reductase

Ferredoxin-nitrite reductase [EC 1.7.7.1] has been purified to apparent homogeneity from rice (Oryza satira cv. Kinmaze) leaves by a procedure used for the spinach enzyme [S. Ida and B. Mikami, Biochim. Biophys. Acta, 681, 167 (1986)]. The rice enzyme consists of a single polypeptide of % molecular weight of 60,000 with 536 amino acid residues. The enzyme showed nearly identical absorption, circular dichroism, and magnetic circular dichroism spectra to those of the spinach enzyme, indicating the presence of the same prosthetic groups and protein conformation in both enzymes. The apparent Km values for nitrite and methyl viologen were 360 μm and 63 μm, respectively. The pH optimum was 7.6. These kinetic parameters are indistinguishable from those reported for spinach nitrite reductase. Monospecific antiserum against purified rice enzyme cross-reacted with nitrite reductases from a variety of higher plants and some phylogenetically divergent plants. Immunological comparisons indicated the rice enzyme is much more closely related to the other monocot enzymes in antigenic structure than to the dicot enzyme proteins. The results lend further support to our previous study [S. Ida, Plant Sci., 49, 111 (1987)] that spinach ferredoxin-nitrite reductase is serologically more related to the dicot enzymes than to the monocot nitrite reductases. Conspicuous differences between the rice and spinach enzymes were found in their molecular sizes and antigenicity. Relatedness of amino acid compositions of the enzyme proteins is discussed in relation to antigenic properties of ferredoxin-nitrite reductase.     

Biosynthesis of Ferredoxin-Nitrite Reductase in Rice Seedlings

Changes in ferredoxin-nitrite reductase [EC 1.7.7.1 [EC] ] in etiolatedrice seedlings were followed during induction by nitrate andlight. Etiolated seedlings showed maximal induction of the enzymeactivity during greening with nitrate, while the enzyme activityin etiolated seedlings receiving nitrate in darkness increasedhalf as much as that in nitrate-treated greening plants. Theincrease in nitrite reductase activity during induction coincidedwith an increase in the content of proteins immunoprecipitatedby antibodies raised against spinach nitrite reductase. Lighthad no effect on the induction of the extractable nitrite reductasein the absence of nitrate. Poly(A)⁺-RNA extracted from nitrate-treatedgreening shoots directed the synthesis in a rabbit reticulocyte-lysateof polypeptides immunoprecipitated by spinach nitrite reductaseantibodies. One major polypeptide larger than the native enzymewas found among the translation products, suggesting that nitritereductases in greening rice shoots are synthesized as an precursorform. Analysis of two-dimensional electrophoretograms indicatedthe existence of isoforms of nitrite reductase in rice seedlingswhich had been immunoprecipitated with spinach nitrite reductaseantibodies. ¹To whom all correspondence should be sent. (Received May 15, 1987; Accepted September 7, 1987)     

海枣曲霉木聚糖酶的纯化及末端序列研究

海枣曲霉麸曲经水浸提、硫酸铵盐析、凝胶过滤、离子交换层析及ＨＰＬＣ分子排阻层析制备了ＰＡＧＥ,ＳＤＳ－ＰＡＧＥ、ＰＡＧＥ酶谱及ＨＰＬＣ纯的木聚糖酶。     

Partial cDNA Cloning and Nucleotide Sequence of Rice Dwarf Virus Genome

Rice dwarf virus (RDV) was isolated and purified from infected rice leaves with chloro form extraction, PEG precipitation and sucrose gradient centrifugation. Total RDV RNA ge nome was separated in the agarose gel and segments of RDV RNA genome were purified. The cDNAs of several segments were synthesized with oligo dT as primer. Through cDNA mapping, subcloning and sequencing, we have obtained partial DNA sequence of those segments. Here we report the cloning and partial DNA sequence of segment 8 from RDV RNA genome.     

陆地棉脂肪酶基因克隆及氨基酸序列分析

根据陆地棉(Gossypium hirsutum L.)EST序列设计一对引物,采用RT-PCR方法从萌发的棉籽中获得了脂肪酶(triacylglycerol acylhydrolases,EC 3.1.1.3)基因,该基因编码483个氨基酸;比对结果显示,棉籽脂肪酶与拟南芥、水稻、蓖麻等脂肪酶相似性较低,具有由Ser-Asp-His组成的三联体催化活性中心,在亲核Ser残基周围有GXSXG保守序列.软件预测显示该脂肪酶为可溶性蛋白,分子量约为55.4 kD,等电点为9.07,不含N端信号肽,亚细胞定位可能是过氧化物酶体或胞质溶胶.     

极端嗜盐硫解酶基因的克隆和氨基酸组成分析

根据嗜盐菌(Halobacterium salinarum)NRC\|34001中硫解酶的基因序列信息,采用PCR技术从菌株Halobacterium sp.ZP\|6中克隆了极端嗜盐硫解酶的基因,并对此酶的氨基组成进行了分析。同非嗜盐硫解酶相比,极端嗜盐硫解酶不但含有较多的负电荷氨基酸,较少的正电荷氨基酸和强疏水氨基酸,而且同类氨基酸中的小氨基酸含量明显增高。这表明极端嗜盐硫解酶的嗜盐特性不单来自形成的分子静电屏蔽网和疏水作用的调节,且与分子表面张力减小密切相关。     

Amino Acid Sequence of Triculamin

The amino acid sequence of triculamin is established from the amino acid composition of the peptides, which have been obtained by partial acid hydrolysis. It is a branched peptides, and a possible structure of the branched parts is proposed.     

Molecular Cloning and Sequence Analysis of a Gene Encoding Rice Proteinase Inhibitor

With the primers designed basing on the terminal amino acid sequences of rice proteinase inhibitors and the preferred codons of rice genes, a new gene coding for a rice proteinase inhibitor has been amplified and cloned from Oryza sativa var. japonica (cv. Zhonghua 8) using PCR technique. The gene contains 408 basepairs and encodes 133 amino acid residues. The deduced amino acid sequence with duplicated Bowman-Birk type structure and active sites specific to trypsin has relatively high homology with that of proteinase inhibitors from wheats, beans etc. As for rice, the new gene shares 74.8% homology with a rice bran trypsin inhibitor reported previously. The evolutionary characteristics of the proteinase inhibitor family has also been discussed.     

Rice Embryo Globulins: Amino-Terminal Amino Acid Sequences, cDNA Cloning and Expression

A globulin fraction prepared from rice embryos contained polypeptidesor polypeptide groups of 49 kDa (designated REG1), 46 kDa (designatedREG2), about 35 kDa, 32 kDa and 25 kDa. The amino-terminal sequencesof REG1 and the major polypeptide in the 35-kDa group were identical,suggesting that the REG1 polypeptide undergoes partial proteolyticprocessing that removes a carboxy-terminal region. A cDNA clone,designated pcREG2, encoding REG2 was isolated, and its nucleotidesequence was determined. The deduced amino acid sequence ofREG2 was found to be 68% identical to that of the maize GLB2globulin. Reg2 mRNA was present at high levels during embryodevelopment for up to 14 days after flowering (DAF). Lower levelswere found 20 DAF when the maturation of embryos was almostcompleted, and at the dry mature stage. Reg2 mRNA almost disappearedupon imbibition of isolated dry mature embryos but it was re-inducedat a low level by further treatment with ABA. The expressionof Reg2 was not induced by ABA in suspension-cultured cells,unlike that of Osem, one of the late embryogenesis abundantprotein (LEA) genes. (Received November 6, 1995; Accepted April 22, 1996)     

五步蛇蛇毒类凝血酶N端的部分氨基酸序列

从五步蛇蛇毒中纯化得到的类凝血酶,在SDS-PAGE及IEF均为一条带,且分子质量约38 ku,等电点约为4.0。测定该酶N端15个氨基酸的序列是VIGGVECDINEHRFL,与其他的蛇毒类凝血酶有高度同源性。     

Production, Characterization, and Partial Amino Acid Sequence of Xylanase A from Schizophyllum commune

[This corrects the article on p. 804 in vol. 36.].     

Molecular Cloning and Sequence Analysis of a Gene Encoding Rice 10 kD Prolamin

Using PCR technique, two prolamin genes from Oryza sativa var. indica (cv. Guanglu′ ai) and O. sativa var. japonica (cv. Zhonghua 8) were amplified and cloned. The prolamin gene contained 525 base pairs and encoded 134 amino acid residues. The two genes cloned from two different rice cultivars exhibited 100% homology and were highly homologous with the 10 kD prolamin gene in other rice species amountin an homology ranging from 96.6% to 100%. The deduced amino acid sequence shared 34.2% homology with that of maize 10 kD prolamin. As for dicots, only two types of storage protein shared some homology with rice 10 kD prolamin. One was from Brazil nut and the other from castor bean. Analysis on the signal peptide of rice 10 kD prolamin showed that it shared higher homology with that of storage proteins in some monocots such as maize, sorghum and oat. No similar sequence was found in dicots. The gene sequences of "Guangluai” and "Zhonghua 8” 10 kD prolamin would appear in EMBL data-base under the accession number L36604 and L36605 respectively.     

cDNA Cloning of Monodehydroascorbate Radical Reductase from Cucumber: a High Degree of Homology in Terms of Amino Acid Sequence between This Enzyme and Bacterial Flavoenzymes

The FAD-enzyme monodehydroascorbate (MDA) reductase catalyzesthe regeneration of ascorbate from the MDA radical using NAD(P)Has the electron donor [Hossain and Asada (1985) J. Biol. Chem.260: 12920]. We cloned a cDNA of MDA reductase from cucumberseedlings and deduced its entire sequence of amino acid residues.The cDNA library from cucumber seedlings in the expression vectorwas screened with an antiserum against cucumber MDA reductase.Inserts from three immunoscreened clones hybridized with twooligonucleotide probes designed on the basis of the sequencesof two peptide fragments from the cucumber enzyme. The nucleotidesequences of these three clones were determined and the longestone contained an open reading frame of 1,302 bp in length. Themolecular mass of the translation product predicted from theopen reading frame was 47 kDa, the same as that determined forthe purified enzyme. The amino acid sequences determined fromfragments of lysyl endopeptidase-digested MDA reductase couldbe aligned with that deduced from the open reading frame, althoughsubstitution of several residues was apparent. Thus, the openreading frame encoded an isozyme of MDA reductase of cucumberdifferent from the purified enzyme. MDA reductase has the FAD-and NAD(P)H-binding domains of flavoproteins but shares onlylimited homology in terms of amino acid sequence with flavoenzymesfrom eukaryotes. ¹Research Institute of Innovative Technology for the Earth (RITE),Kizu, Kyoto, 619-02 Japan     

核酸、氨基酸分类和蛋白质二级结构关系的分析与分子设计

核酸序列中包含一定的蛋白质结构信息。根据通常情况下遗传密码表中密码子中间位的碱基配对时产生的氢键数目,尝试将20种氨基酸划分为两类,并用自编的计算机软件对蛋白质二级结构数据库中两类氨基酸的类聚现象进行了统计分析。结果表明,使用这种方法对氨基酸进行划分后,氨基酸残基具有较大概率与划入同一类的氨基酸残基相邻出现,并且这种聚集体对二级结构具有一定的偏好性。最后按照该方法设计了一段氨基酸序列并给出了预测服务器预测得到的结构。     

核酸、氨基酸分类和蛋白质二级结构关系的分析与分子设计

核酸序列中包含一定的蛋白质结构信息。根据通常情况下遗传密码表中密码子中间位的碱基配对时产生的氢键数目,尝试将20种氨基酸划分为两类,并用自编的计算机软件对蛋白质二级结构数据库中两类氨基酸的类聚现象进行了统计分析。结果表明,使用这种方法对氨基酸进行划分后,氨基酸残基具有较大概率与划入同一类的氨基酸残基相邻出现,并且这种聚集体对二级结构具有一定的偏好性。最后按照该方法设计了一段氨基酸序列并给出了预测服务器预测得到的结构。     

米曲霉木糖还原酶基因的克隆及序列分析

木糖还原酶（XR, EC 1.1.1.21）是真菌微生物代谢木糖的关键酶之一。本文以米曲霉基因组DNA为模板,克隆木糖还原酶基因（xr,GenBank登录号：FJ957890.1）,并对XR的序列、系统进化树、理化性质及蛋白结构等进行生物信息学分析。结果表明： xr基因序列长1449 bp,其中开放阅读框长960 bp,编码319个氨基酸,蛋白质分子质量35.9 kDa,等电点为5.78;米曲霉XR与其他菌种XR有较高的同一性,含有醛酮还原酶家族的两个指纹结构和一个参与辅酶结合活性位点指纹结构,以及醛酮还原酶家族典型的（β/α）8 TIM桶结构,说明米曲霉XR属于醛酮还原酶家族。     

小麦CEN/TFL1-like cDNA克隆及其编码序列分析

以水稻、金鱼草、拟南芥CEN/TFL1蛋白质序列为参考,以小麦中CEN/TFL1基因同源EST片段为依据设计特异引物,利用RT-PCR从普通小麦‘中国春’幼穗总RNA中扩增出549 bp的特异片段。测序结果表明,该片段包含了完整的ORF,编码产物为典型的CEN/TFL1-like蛋白家族成员。序列比对表明,该基因编码产物与黑麦草LpTFL1、水稻FDR2和FDR1以及玉米ZmTFL1亲缘关系最近,分别具有96.5%、96.0%、93.6%和95.4%的相似性;与哺乳动物Rattus norveqicus磷脂酰乙醇胺结合蛋白(PEBP)具有38.9%相似性。空间结构预测表明,该蛋白具有PEBP家族成员的典型三维结构。     

Purification and Partial Amino Acid Sequence of the Major 70,000-Dalton Heat Shock Protein in Neurospora crassa

Fracella, F., Mohsenzadeh, S., and Rensing, L. 1993. Purification and partial amino acid sequence of the major 70,000-Dalton heat shock protein in Neurospora crassa. Experimental Mycology , 17, 362-367. The major heat shock protein of 70 kDa (hsp70) from heat-shocked mycelial extracts of Neurospora crassa was purified to near homogeneity employing DEAE anion-exchange chromatography followed by affinity chromatography on ATP-agarose. The isolated hsp70 migrates as a single band on one-dimensional sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE), with a molecular mass of ∼69 kDa. On two-dimensional gels it is resolved into two polypeptides with isoelectric points in the acidic range of ∼pH 5.2. The first 53 amino terminal amino acids of the major protein were sequenced and compared with hsp70 of other species. The amino acids aspartic acid, arginine, and phenylalanine occur at positions 27, 28, and 44 (from the methionine terminus) in contrast to the main consensus sequence. These three differing amino acids are shared by yeast, and, in addition, the first two by Arabidopsis, petunia, and maize.     

蛋白质内含子在特定氨基酸序列中的剪接

基因工程技术已经被广泛应用于抗体的生产。但是由于抗体的分子量较大,导致合成抗体较为困难。蛋白质内含子是前体蛋白质中的一段氨基酸序列,能够将自身剪切出来,并将两端的外显子连接形成成熟的蛋白质。将抗体的Fab(antigen binding fragment)和Fc(crystalline fragment)分别与蛋白质内含子(intein) 的N端(IN)和C端(IC)融合表达,利用蛋白质内含子的剪接功能,可形成完整的抗体分子。KSCDKTH是存在于抗体铰链区(hinge region)的一段氨基酸序列,如果在KSCDKTH序列中筛选到高效剪接的蛋白质内含子,即可通过蛋白质剪接,将抗体分子的Fab和Fc剪接形成完整抗体。本文筛选发现,Ssp DnaX的3种断裂蛋白质内含子(S0, S1, S11)具有在KSCDKTH序列中高效剪接的能力,这一研究结果为抗体的剪接合成提供了可行性。     

Purification and Partial Amino Acid Sequence of Brevicin 27, a Bacteriocin Produced by Lactobacillus brevis SB27

Brevicin 27, a bacteriocin produced by Lactobacillus brevis SB27, is inhibitory mainly against closely related Lactobacillus brevis and Lactobacillus büchneri strains. It was purified from the culture supernatant by a four-step purification procedure including ammonium sulfate precipitation, cation exchange, hydrophobic interaction, and reverse-phase, high performance liquid chromatographies. The purified bacteriocin was subjected to mass spectrometry, amino acid composition analysis, and sequencing by Edman degradation. It was shown to be an about 5200-Da basic protein containing a high proportion of lysine and of hydrophobic amino acids. The partial N-terminal amino acid sequence (25 residues) was unique when compared with the Protein Data Bank (PDB), Swiss Prot, and Protein Information Resource (PIR) data banks and to the translated Gen Bank. Received: 24 July 1996 / Accepted: 10 September 1996