Isolation and characterization of six pathogenesis-related (PR) proteins of Samsun NN tobacco

The purification to homogeneity of pathogenesis-related (PR) proteins R and S from Nicotiana tabacum cv. Samsun NN leaves has been achieved by using a combination of conventional and high-performance chromatographic supports. The same procedure allowed the purification and the characterization of four other proteins which displayed some properties characteristic of tobacco PR proteins and were shown to accumulate in tobacco leaves in response to virus infection. They can be, therefore, considered as new tobacco PR proteins which we designate as PR-s₁,-s₂,-r₁ and-r₂. The relative electrophoretic mobilities (Rf) under non-denaturing conditions were estimated to 0.30 for PR-r₁ and-r₂, 0.25 for Pr-R, 0.20 for PR-s₁ and-s₂ and 0.15 for PR-S. On SDS gels PR proteins R and S possessed the same apparent molecular weight (M _r 24000) as did PR-proteins s₁ and r₁ (M _r 14500) and PR-s₂ and-r₂ (M _r 13000). However, proteins s₁, s₂, r₁ and r₂ had identical electrophoretic mobilities on SDS gels when the loading sample buffer contained no reducing agent. Polyclonal antisera were raised against PR proteins R and S and used in immunoblotting experiments. Proteins R and S were shown to be serologically closely related. No cross-reaction was detected with any of the four new tobacco PR proteins r₁, r₂, s₁ and s₂ or with the previously described PR proteins, i.e. PR-1a,-1b,-1c,-2,-N,-O,-P and-Q.     

Isolation and characterization of cDNA clones encoding pathogenesis-related proteins from tobacco mosaic virus infected tobacco plants.

Infection of the tobacco cultivar Samsun NN by tobacco mosaic virus (TMV) results in a hypersensitive response. During this defense reaction several host encoded proteins, known as pathogenesis-related proteins (PR-proteins), are induced. Poly(A)+ RNA from TMV infected tobacco plants was used to construct a cDNA library. Thirty two cDNA clones were isolated and after digestion with different restriction endonucleases, twenty clones were found to code for PR-1a, six clones for PR-1b, and four clones for PR-1c. Two independent cDNA clones of each class were further characterized by DNA sequence analysis. All clones analyzed contained the 138 amino acid coding regions of their respective mature proteins, but only partial sequences of the signal peptides. Minor differences between the nucleotide sequences for clones belonging to the same class were detected. Comparison of the amino acid sequence for PR-1a deduced from its nucleotide sequence with published data obtained by Edman degradation of the protein showed four differences. Analysis of the 3' ends of the cDNA clones indicates that various alternate poly(dA) addition sites are used. Southern blot analysis using these cDNAs as probes suggests the presence of multiple PR-protein genes in the genomes of tobacco and tomato plants.     

Virus-induced synthesis of messenger RNAs for precursors of pathogenesis-related proteins in tobacco

Infection of Samsun NN tobacco with tobacco mosaic virus (TMV) induces a number of host-encoded, so-called pathogenesis-related (PR-) proteins, which are found in the intercellular space of the leaf and are associated with induced resistance. By immunoprecipitation of their in vitro translation products we were able to detect the mRNAs corresponding to a number of PR-proteins in TMV-infected tobacco, but not in healthy plants. Analysis by the Northern blot technique using cloned cDNA of PR1-mRNAs as probe showed that the mRNAs for the closely related proteins PR1a, 1b and 1c occur at a low level in healthy tobacco; upon TMV infection this level is increased > 100-fold. The PR1-specific probe did no hybridize to mRNAs corresponding to other PR-proteins. Sequencing of the 5'-terminal region of PR1-mRNAs showed that PR1-proteins are derived from precursors by removal of an N-terminal signal peptide of 30 amino acids.     

Purification and some properties of an extracellular ribonuclease with antiviral activity against tobacco mosaic virus from <Emphasis Type="Italic">Bacillus cereus</Emphasis>

A new ribonuclease (RNase) with tobacco mosaic virus inhibition was isolated and purified from Bacillus cereus ZH14 through ammonium sulfate precipitation, ultrafiltration, ion-exchange chromatography of DEAE-Sephadex A-50 column, and gel chromatography of Sephacryl S-200HR column. The enzyme was purified approximately 134-fold with a recovery of 9.2%. The RNase had an MW of 75.6 kDa in SDS-PAGE, which differed from RNases reported previously. The inhibitory activity of the RNase in the purification process against tobacco mosaic virus was tested, and the percentage inhibition of the purified RNase (48 U/ml) reached 90%. The protein could tolerate 90°C and pH 4.0.     

Analysis ofcis-regulatory elements involved in induction of a tobacco PR-5 gene by virus infection

cis-Regulatory elements involved in tobacco mosaic virus (TMV)-inducible expression were indentified in a tobacco PR-5 gene, encoding an acidic thaumatin-like protein. By fusing upstream sequences of the PR-5 gene to the GUS reporter gene and analysing transgenic plants containing these fusions for local and systemic induction of GUS activity by TMV, it was found that sequences between-1364 and-718 are involved in TMV induction of PR-5 gene expression.     

北京棒状杆菌AS1.299谷氨酰胺合成酶的提纯和性质

用热变性、硫酸铵分段沉淀和DEAE纤维素柱层析部分纯化了北京棒状杆菌AS1.299(Corynebacterium pekinense)谷氨酰胺合成酶,并用垂直平板电泳对酶进一步精制。酶对谷氨酰胺的Km值为16.4mM,对羟胺的Km值为43.5mM;酶的沉降系数为21S,SDS凝胶电泳测得酶的亚基分子量为56,000。化学修饰表明,该酶活力与硫氢基、色氨酸、精氨酸残基无关;酪氨酸修饰剂的作用引起酶活力下降,初步证明,组氨酸残基是该酶活力的必需基因。     

Large quantities of recombinant PR-5 proteins from the extracellular matrix of tobacco: Rapid production of microbial-recalcitrant proteins

A procedure for the extraction of large quantities of PR-5 proteins that have been recalcitrant to microbial-based expression systems is described. Targeting of the recombinant proteins to the extracellular matrix allowed efficient protein extraction by a vacuum infiltration/centrifugation system. Approximately 1 kg of fresh leaves from transgenic tobacco plants overexpressing either truncated osmotin (Liu et al., 1996) or A9 fromAtriplex nummularia L. (Casas et al., 1991) yielded between 3 and 5 mg of purified proteins that fully retained their antifungal activity. The entire system of overexpression, extraction, and purification could be easily scaled up for the production of several grams of protein.     

Purification and characterization of cholesterol sulfotransferase from rat skin.

Previous work has demonstrated that the activity of the enzyme cholesterol sulfotransferase is rapidly and dramatically increased upon squamous differentiation of a variety of epithelial cells in culture, including epidermal keratinocytes. As a step toward understanding the molecular mechanisms underlying this differentiation-related change, we now report the partial purification and characterization of this enzyme activity from rat skin. Supernatant solutions from rat skin homogenates were subjected to a series of column chromatography steps including anion exchange, gel filtration, chromatofocusing and hydrophobic interaction chromatography. The purification procedure resulted in cholesterol sulfotransferase activity purified 2,700-fold with a 11% recovery. The most purified preparation yielded a major Coomassie blue-stained band on denaturing polyacrylamide gel electrophoresis of an apparent molecular weight (MW) of 40,000 Da. Photoaffinity labeling with the donor substrate, 3'-phosphoadenosine-5'-phospho-[35S]-sulfate resulted in a single radiolabeled protein band on denaturing polyacrylamide gel electrophoresis, again of apparent MW 40,000 Da, strongly suggesting that the major Coomassie blue-stained band in the most purified preparation is the cholesterol sulfotransferase protein. Among 3beta-hydroxysteroids with a A5 double bond that were tested, each served as a substrate, while androgens, estrogens, corticosteroids, p-nitrophenol and DOPA did not serve as substrates. Apparent Michaelis constants for the 3beta-hydroxysteroid substrates ranged from 0.6 to 8 microM.     

Pathogenesis-related PR-1 proteins are antifungal. Isolation and characterization of three 14-kilodalton proteins of tomato and of a basic PR-1 of tobacco with inhibitory activity against Phytophthora infestans.

Three distinct basic 14-kD proteins, P14a, P14b, and P14c, were isolated from tomato (Lycopersicon esculentum Mill. cv Baby) leaves infected with Phytophthora infestans. They exhibited antifungal activity against P. infestans both in vitro (inhibition of zoospore germination) and in vivo with a tomato leaf disc assay (decrease in infected leaf surface). Serological cross-reactions and amino acid sequence comparisons showed that the three proteins are members of the PR-1 group of pathogenesis-related (PR) proteins. P14a and P14b showed high similarity to a previously characterized P14, whereas P14c was found to be very similar to a putative basic-type PR-1 from tobacco predicted from isolated DNA clones. This protein, named PR-1 g, was purified from virus-infected tobacco (Nicotiana tabacum Samsun NN) leaves and characterized by amino acid microsequencing, along with the well-known acidic tobacco PR-1a, PR-1b, and PR-1c. The various tomato and tobacco PR-1 proteins were compared for their biological activity and found to display differential fungicidal activity against P. infestans in both the in vitro and in vivo assays, the most efficient being the newly characterized tomato P14c and tobacco PR-1g.     

Purification and serological characterization of three basic 15-kilodalton pathogenesis-related proteins from tomato

In tomato (Lycopersicon esculentum) several acidic and basic apoplastic pathogenesis-related (PR) proteins are induced upon inoculation with virulent or avirulent races of Cladosporium fulvum (Cooke) (syn. Fulvia fulva [Cooke] Cif). One of the most predominant and best characterized tomato PR proteins is P14, a basic protein that shows homology to the tobacco (Nicotiana tabacum) PR-1 protein family. To investigate whether, by analogy with these tobacco PR-1 proteins, P14 also belongs to a family of differently charged isomers, the abundantly occurring PR proteins with molecular masses around 15 kilodaltons (kD) were purified from apoplastic fluids isolated from C. fulvum-infected tomato. Three basic proteins migrating similarly to P14 on sodium dodecyl sulfate polyacrylamide gels were purified to homogeneity by gel filtration followed by high resolution liquid chromatography. Two proteins (15.5 kD, isoelectric point [pl] 10.9 and 10.7 appeared to be serologically related to each other and to the tobacco PR-1 proteins. A third protein (15 kD, pl 10.4) was not serologically related to any other tomato PR protein but was found to be related to PR-R from tobacco.     

Induction of pathogenesis-related proteins by spermidine exogenously supplied to detached tobacco leaves

Continuous treatment with spermidine or 1-aminocyclopropane-1-carboxylic acid stimulated ethylene production and ethylene-forming enzyme activity and accelerated chlorophyll breakdown in detached tobacco leaves. The treatments also induced the production of eleven major acidic pathogenesis-related proteins, which were also produced during the hypersensitive reaction to tobacco necrosis virus. A delay between the onset of the stimulated ethylene increase and the detection of PR-proteins was found; ethylene production was stimulated after a few hours of treatment, whereas one, three and all the eleven PR-proteins were detected by polyacrylamide gel electrophoresis of fluid extracts after 2, 4 and 6 days of treatments, respectively. The possible causal relationship between stimulation of ethylene production and PR-protein accumulation is discussed.     

Enzymatic synthesis of lignin: purification to homogeneity of the three O-methyltransferases of tobacco and production of specific antibodies

Three o-diphenol-O-methyltransferases (OMTs; EC 2.1.1.6) involved in the biosynthesis of lignin have been purified to homogeneity from tobacco leaves. Seven different fractionation steps which included (NH4)2 SO4 precipitation, conventional low-pressure chromatography on Ultrogel AcA34 and DEAE-cellulose columns, high-performance liquid chromatography (HPLC) on three different supports (Mono Q, Mono P, and TSK G-3000 SW columns), and finally preparative electrophoresis were necessary. At each step of purification, the protein content of the enzymatic fractions was analyzed by electrophoresis on polyacrylamide gels under denaturing conditions. Purified OMT I appeared on sodium dodecyl sulfate-polyacrylamide gel as a doublet with electrophoretic mobilities corresponding to molecular weights of 38,500 +/- 2000 and 39,500 +/- 2000. The other two enzymes migrated as single but rather broad bands with molecular weights of 42,000 (OMT II) and 43,000 (OMT III). Polyclonal antibodies were raised in rabbits. The titers of antibodies were measured by an indirect enzyme-linked immunosorbent assay method, and their specificity was demonstrated by immunoblotting enzyme preparations at different stages of purification. Immunodetection of the three enzymes with a specific antiserum suggested serological relationships between the three OMTs of tobacco.     

Characterization of a 29-kDa beta-1,3-glucanase from Trichoderma harzianum

A beta-1,3-glucanase, from culture filtrates of Trichoderma harzianum, was purified in sequential steps by gel filtration, hydrophobic interaction and ion exchange chromatography. A typical procedure provided 69-fold purification with 0.32% yield. The molecular mass of the protein was found to be approximately 29 kDa, as estimated by SDS-PAGE on a 10% slab gel. The K(M) and V(max) values for beta-1,3-glucanase, using laminarin as substrate, were 1. 72 mg ml(-1) and 3.10 U ml(-1), respectively. The pH optimum for the enzyme was pH 4.4 and maximum activity was obtained at 50 degrees C. The enzyme was strongly inhibited by HgCl(2) and SDS. These results suggest that each beta-1,3-glucanase produced by T. harzianum is different and is probably encoded by different genes.     

Purification of rabbit and human serum paraoxonase.

Rabbit serum paraoxonase/arylesterase has been purified to homogeneity by Cibacron Blue-agarose chromatography, gel filtration, DEAE-Trisacryl M chromatography, and preparative SDS gel electrophoresis. Renaturation (Copeland et al., 1982) and activity staining of the enzyme resolved by SDS gel electrophoresis allowed for identification and purification of paraoxonase. Two bands of active enzyme were purified by this procedure (35,000 and 38,000). Enzyme electroeluted from the preparative gels was reanalyzed by analytical SDS gel electrophoresis, and two higher molecular weight bands (43,000 and 48,000) were observed in addition to the original bands. This suggested that repeat electrophoresis resulted in an unfolding or other modification and slower migration of some of the purified protein. The lower mobility bands stained weakly for paraoxonase activity in preparative gels. Bands of each molecular weight species were electroblotted onto PVDF membranes and sequenced. The gas-phase sequence analysis showed that both the active bands and apparent molecular weight bands had identical amino-terminal sequences. Amino acid analysis of the four electrophoretic components from PVDF membranes also indicated compositional similarity. The amino-terminal sequences are typical of the leader sequences of secreted proteins. Human serum paraoxonase was purified by a similar procedure, and ten residues of the amino terminus were sequenced by gas-phase procedures. One amino acid difference between the first ten residues of human and rabbit was observed.     

Pathogenesis-related proteins and their genes in cereals

Pathogenesis-related proteins (PR-proteins) are induced in plants in response to attack by microbial or insect pests. They have been classified into several groups (PR-1 through PR-14 at present) based on their amino acid sequences and biochemical functions. Many of these proteins that have been purified from infected plants or seed extracts possess antifungal or insecticidal activity. Genes and cDNA clones for all classes of PR-proteins have been isolated from a variety of cereals. Some of these genes/cDNAs have been used to transform cereals. This review presents a summary of the PR-proteins and their genes characterized from rice, wheat, barley, sorghum and maize. Efforts to improve disease or insect resistance of these cereal plants by genetic engineering using genes for PR-proteins also are discussed. In many cases, the expression of the PR-proteins either singly or in combination appears to improve resistance to fungi or insects. In addition, chromosomal location of the PR-protein genes indicates that members of the same family of PR-protein genes or sometimes even several families of PR-protein genes often are clustered in the cereal genome, suggesting coordinate regulation. Some of these PR-protein genes map closely to quantitative traits loci. Some concerns regarding the use of genes encoding PR-proteins for genetic modification of cereals also are addressed.     

Purification and detection of multiple forms of histidine decarboxylase in rat gastric mucosa (author's transl)]

A specific histidine decarboxylase from rat gastric mucosa has been obtained at high purity and good yield (purification about 600-fold). The purification procedure included double (NH4)2SO4 fractionation, ion-exchange chromatography, preparative isoelectric focusing in a granulated gel and gel filtration. Only the specific histidine enzyme was obtained by that procedure; DOPA decarboxylase, a non-specific enzyme, was absent in our final preparation. Each step of the purification was visualized by polyacrylamide gel electrophoresis and analytical isoelectric focusing. The purified enzyme was apparently homogenous by criteria of electrophoresis and gel filtration and has a molecular weight of 94 000. Several protein bands appeared after isoelectric focusing and the enzyme activity was localized in 3 distinct peaks. The gastric enzyme consists of 3 active forms which could be distinguished by their isoelectric points: 5.4, 5.75 and 6. Moleculare weights estimated by SDS polyacrylamide gel electrophoresis were 97 000, 93 000 and 90 000, and no subunits were observed. Pyridoxal phosphate was required as a coenzyme and resolution of the holoenzyme agreed with a portion of the coenzyme tightly bound to the apoenzyme. The purified enzyme was stable at low ionic strength, near neutral pH; concentrated reducing agents inhibit the enzyme.     

Structure of tobacco genes encoding pathogenesis-related proteins from the PR-1 group.

Infection of Samsun NN tobacco with tobacco mosaic virus (TMV) was found to induce the synthesis of mRNA encoding a basic protein with a 67% amino acid sequence homology to the known acidic pathogenesis-related (PR) proteins 1a, 1b and 1c. By Southern blot hybridization it was shown that the tobacco genome contains at least eight genes for acidic PR-1 proteins and a similar number of genes encoding the basic homologues. Clones corresponding to three of the genes for acidic PR-1 proteins were isolated from a genomic library of Samsun NN tobacco. The nucleotide sequence of these genes and their flanking sequences were determined. One clone was found to correspond to the PR-1a gene; the two other clones do not correspond to known TMV-induced PR-1 mRNA's and may represent silent genes. Compared to the PR-1a gene, these genes contain an insertion or deletion in the putative promoter region and mutations affecting the PR-1 reading frame.     

Alkaline phosphatase isozymes in the midgut of silkworm: purification of high pH-stable microvillus and labile cytosolic enzymes

Summary Genetically defined alkaline phosphatase (ALP) isozymes from the larval midgut tissues ofBombyx mori were purified and characterized. The membrane-bound form (m-ALP) was solubilized with 1% Triton X-100, then purified by DEAE-Sephacel, Con A-Sepharose 4B and Ultrogel AcA 34 column chromatography. The soluble form (s-ALP) was purified by DEAE-Sephacel, epoxy Toyopearl coupled with phosphonic acid and Ultrogel AcA 34 column chromatography. About 840- and 650-fold purification were achieved for m-ALP and s-ALP, respectively, and both ALPs were homogeneous as judged by poly-acrylamide gel electrophoresis. Both forms were found to be similar (MW=68 000 in gel permeation chromatography, and a single subunit as a monomer in denaturing SDS-polyacrylamide gels with MW=58 000 for m-ALP and MW=61 000 for s-ALP). The pH optima of ALPs were shown to lie at 10.9 (m-ALP) and 9.8 (s-ALP), the former being extremely stable even in pH 10–12 which accords with the physiological milieu inBombyx midgut lumen. The K_m values of the m-ALP and s-ALP forp-nitrophenyl phosphate were 1.99 and 1.49 mM, respectively. Both ALPs had a similar substrate specificity.l-Cysteine strongly inhibited both ALPs, but inhibitory effects ofl-phenylalanine,l-homoarginine andl-leucine were undetectable for s-ALP and very weak for m-ALP. A comparison of enzymatic properties on two ALPs suggested that each isozyme plays different roles.Abbreviations m-ALP membrane-bound alkaline phosphatase - s-ALP soluble alkaline phosphatase