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PubMed1992

Amino-acid sequence and predicted three-dimensional structure of pea seed (Pisum sativum) ferritin.

The iron storage protein, ferritin, is widely distributed in the living kingdom. Here the complete cDNA and derived amino-acid sequence of pea seed ferritin are described, together with its predicted secondary structure, namely a four-helix-bundle fold similar to those of mammalian ferritins, with a fifth short helix at the C-terminus. An N-terminal extension of 71 residues contains a transit peptide (first 47 residues) responsible for plastid targetting as in other plant ferritins, and this is cleaved before assembly. The second part of the extension (24 residues) belongs to the mature subunit; it is cleaved during germination. The amino-acid sequence of pea seed ferritin is aligned with those of other ferritins (49% amino-acid identity with H-chains and 40% with L-chains of human liver ferritin in the aligned region). A three-dimensional model has been constructed by fitting the aligned sequence to the coordinates of human H-chains, with appropriate modifications. A folded conformation with an 11-residue helix is predicted for the N-terminal extension. As in mammalian ferritins, 24 subunits assemble into a hollow shell. In pea seed ferritin, its N-terminal extension is exposed on the outside surface of the shell. Within each pea subunit is a ferroxidase centre resembling those of human ferritin H-chains except for a replacement of Glu-62 by His. The channel at the 4-fold-symmetry axes defined by E-helices, is predicted to be hydrophilic in plant ferritins, whereas it is hydrophobic in mammalian ferritins.

Amino Acid Sequence↗

PubMed1994

Localization and characterization of two structurally different forms of acetyl-CoA carboxylase in young pea leaves, of which one is sensitive to aryloxyphenoxypropionate herbicides.

Young pea leaves contain two structurally different forms of acetyl-CoA carboxylase (EC 6.4.1.2; ACCase). A minor form, which accounted for about 20% of the total ACCase activity in the whole leaf, was detected in the epidermal tissue. This enzyme was soluble and was purified to homogeneity from young pea leaf extracts. It consisted of a dimer of two identical biotinyl subunits of molecular mass 220 kDa. In this respect, this multifunctional enzyme was comparable with that described in other plants and in other eukaryotes. A predominant form was present in both the epidermal and mesophyll tissues. In mesophyll protoplasts, ACCase was detected exclusively in the soluble phase of chloroplasts. This enzyme was partially purified from pea chloroplasts and consisted of a freely dissociating complex, the activity of which may be restored by combination of its separated constituents. The partially purified enzyme was composed of several subunits of molecular masses ranging from 32 to 79 kDa, for a native molecular mass > 600 kDa. One of these subunits, of molecular mass 38 kDa, was biotinylated. This complex subunit structure was comparable with that of microorganisms and was referred to as a 'prokaryotic' form of ACCase. Biochemical parameters were determined for both ACCase forms. Finally, both pea leaf ACCases exhibited different sensitivities towards the grass ACCase herbicide, diclofop. This compound had no effect on the 'prokaryotic' form of ACCase, while the 'eukaryotic' form was strongly inhibited.

Acetyl-CoA Carboxylase↗

PubMed1995

Purification and characterization of recombinant pea-seed ferritins expressed in Escherichia coli: influence of N-terminus deletions on protein solubility and core formation in vitro.

Plant ferritin subunits are synthesized as precursor molecules; the transit peptide (TP) in their NH2 extremity, responsible for plastid targeting, is cleaved during translocation to this compartment. In addition, the N-terminus of the mature subunit contains a plant-specific sequence named extension peptide (EP) [Ragland, Briat, Gagnon, Laulhère, Massenet, and Theil, E.C. (1990) J. Biol. Chem. 265, 18339-18344], the function of which is unknown. A novel pea-seed ferritin cDNA, with a consensus ferroxidase centre conserved within H-type animal ferritins has been characterized. This pea-seed ferritin cDNA has been engineered using oligonucleotide-directed mutagenesis to produce DNA fragments (1) corresponding to the wild-type (WT) ferritin precursor, (2) with the TP deleted, (3) with both the TP and the plant specific EP sequences deleted and (4) containing the TP but with the EP deleted. These four DNA fragments have been cloned in an Escherichia coli expression vector to produce the corresponding recombinant pea-seed ferritins. Expression at 37 degrees C led to the accumulation of recombinant pea-seed ferritins in inclusion bodies, whatever the construct introduced in E. coli. Expression at 25 degrees C in the presence of sorbitol and betaine allowed soluble proteins to accumulate when constructs with the TP deleted were used; under this condition, E. coli cells transformed with constructs containing the TP were unable to accumulate recombinant protein. Recombinant ferritins purified from inclusion bodies were found to be assembled only when the TP was deleted; however assembled ferritin under this condition had a ferroxidase activity undetectable at acid pH. On the other hand, soluble recombinant ferritins with the TP deleted and expressed at 25 degrees C were purified as 24-mers containing an average of 40-50 iron atoms per molecule. Despite the conservation in the plant ferritin subunit of a consensus ferroxidase centre, the iron uptake activity in vitro at pH 6.8 was found to be lower than that of the recombinant human H-ferritin, though it was much more active than the recombinant human L-ferritin. The recombinant ferritin with both the TP and the EP deleted (r delta TP/EP) assembled correctly as a 24-mer; it has slightly higher ferroxidase activity and decreased solubility compared with the wild-type protein with the TP deleted (r delta TP). In addition, on denaturation by urea followed by renaturation by dialysis the r delta TP/EP protein showed a 25% increase in core-formation in vitro compared with the r delta TP protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1995

Sequence and expression of the mRNA encoding HSP22, the mitochondrial small heat-shock protein in pea leaves.

A 3 h treatment at 40 degrees C of pea (Pisum sativum var. Douce Provence) plants induces production and accumulation of a small heat-shock protein of 22 kDa apparent molecular mass, designated HSP22, in the matrix compartment of mitochondria [Lenne and Douce (1994) Plant Physiol. 105, 1255-1261]. We show here that the HSP22 precursor (i.e. the mature protein plus the transit peptide) has an apparent molecular mass of 26 kDa after in vitro translation of mRNA extracted from heat-stressed pea plants and immunodetection. We have isolated, cloned and sequenced the full-length cDNA encoding the precursor of the mitochondrial HSP22. An analysis of the amino acid sequence of the mitochondrial HSP22 reveals that this protein is a representative member of the low-molecular-mass heat shock protein (HSP) superfamily, exhibiting the specific consensus regions that are typical of the small HSPs. Most importantly, comparison of the mitochondrial HSP22 sequence with that of chloroplast small HSPs indicates that HSP22 does not contain the typical chloroplast consensus region III. We have also analysed the kinetics of HSP22 induction, and report results on the temporal expression of HSP22 at the transcriptional level. HSP22 mRNA was detected as soon as 10 min after the temperature was raised to a high temperature of 40 degrees C. Then the amount of HSP22 mRNA declined considerably even though pea plants were still submitted to the heat treatment. These results are discussed in light of the translation data previously published [Lenne and Douce (1994) Plant Physiol. 105, 1255-1261], particularly concerning the physiological behaviour of mitochondria when plants are heat-stressed. Furthermore, we have studied the dependence of HSP22 accumulation with temperature and demonstrate that the pea mitochondrial heat-shock response is only developed under extreme environmental growth conditions.

Amino Acid Sequence↗

PubMed1996

The pea chloroplast membrane-associated protein, IEP96, is a subunit of acetyl-CoA carboxylase.

Two forms of acetyl-CoA carboxylase (ACCase) have been characterized in pea (Pisum sativum L.) leaves; a heteromeric chloroplast enzyme and a homomeric, presumably cytosolic enzyme. The biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and beta-carboxyltransferase (CT) subunits of the plastidial-ACCase have recently been characterized and cloned. To further characterize the carboxyltransferase, an improved assay for CT was developed and used to follow its partial purification. CT activity co-purifies with ACCase activity during gel permeation chromatography. However, upon anion-exchange chromatography or native PAGE, CT separates from the BC and BCCP subunits of plastidial-ACCase and ACCase activity is lost. In addition, it is demonstrated that a previously sequenced pea chloroplast cDNA of unknown function (IEP96) with a predicted molecular weight of 91 kDa encodes the alpha-CT subunit of the MS-ACCase. Antibodies raised against the first 404 amino acids of IEP96 protein detected a polypeptide with molecular weight of 91 kDa that co-eluted during gel permeation chromatography with plastidial CT and ACCase activities. These antibodies also immunoprecipitated the activities of both ACCase and CT with the concomitant precipitation of the beta-CT subunit. Furthermore, antibodies against beta-CT immunoprecipitated the IEP96 protein. Two-dimensional PAGE and DEAE purification of ACCase protein demonstrated that the beta-CT forms a tight association with the IEP96 protein. Pea leaf was fractionated into soluble and membrane fractions and the alpha-CT subunit was primarily associated with the membrane fraction. Together, these data demonstrate that IEP96 is the alpha-CT subunit of pea chloroplast ACCase.

Acetyl-CoA Carboxylase↗

PubMed1997

The LS locus of pea encodes the gibberellin biosynthesis enzyme ent-kaurene synthase A.

Gibberellins (GAs) are hormones required for several aspects of plant development, including internode elongation and seed development in pea (Pisum sativum L.). The first committed step in the GA biosynthesis pathway is the conversion of geranylgeranyl diphosphate (GGDP) to ent-kaurene via copalyl diphosphate (CDP). These two reactions are catalyzed by the cyclases ent-kaurene synthase A (KSA) and ent-kaurene synthase B (KSB), respectively. Previous genetic and biochemical analysis of the GA-responsive ls-1 mutant of pea suggested that GA levels are reduced in a developmental- and organ-specific manner due to reduced GA biosynthesis. Analysis of cellfree enzyme preparations from WT and ls-1 embryos at contact point reveals that ls-1 reduces the activity of KSA but not KSB. To characterize the ls-1 mutation in more detail, a cDNA coding for a pea KSA was cloned and shown to be encoded by the LS locus. The ls-1 mutation results from an intronic G to A substitution that causes impaired RNA splicing. To determine the activity of the KSAs encoded by the LS and ls-1 alleles, a new in vitro assay for combined KSA and KSB activity has been developed using the KSB gene of pumpkin. Using recombinant WT KSA and KSB fusion proteins, GGDP is converted to ent-kaurene in vitro. Based on the sequence of RT-PCR products, three different truncated KSA proteins are predicted to exist in ls-1 plants. The most abundant mutant KSA protein does not possess detectable activity in vitro. Nevertheless, the ls-1 allele is not null and is able to encode at least a partially functional KSA since a more severe is allele has been identified. The ls-1 mutation has played a key role in identifying a role for GAs in pea seed development in the first few days after fertilization, but not in older seeds. KSA expression in seeds is developmentally regulated and parallels overall GA biosynthesis, suggesting that KSA expression may play an important role in the regulation of GA biosynthesis and seed development.

Alkyl and Aryl Transferases↗

PubMed1997

Myb26: a MYB-like protein of pea flowers with affinity for promoters of phenylpropanoid genes.

Plant Myb proteins represent a group of transcription factors which have a DNA-binding domain similar to that found in the products of the animal myb proto-oncogenes. Members of the Myb family regulate the biosynthesis of phenylpropanoids, including anthocyanin and phlobaphene pigments, in several species. In this study, PCR with degenerate primers was used to analyse the presence of myb-like genes in pea (Pisum sativum L.). A fragment representing a flower bud-expressed gene, designated myb26, was recovered, and a full length cDNA was isolated from a pea flower bud cDNA library. The predicted protein is 217 amino acids long and its Myb-domain and carboxy terminal region show extensive homology to the snap-dragon proteins Myb305 and Myb340, both of which regulate phenylpropanoid biosynthesis. Expression of myb26 is flower-specific and parallels the expression of flavonoid biosynthetic genes, increasing as the flower bud matures and intensifies in colour. However, myb26 represents neither of the two known regulatory genes for anthocyanin production in pea, nor does it complement mutants of the myb-like anthocyanin regulatory gene an2 in petunia. Myb26 was expressed in E. coli as a fusion protein. It was shown that in vitro Myb26 recognizes the c-Myb and P-box-like binding sites representing cis-elements in the promoter regions of several phenylpropanoid biosynthetic genes. The results suggest that myb26 is a previously undefined gene involved in regulation of some aspect of phenylpropanoid production in pea.

Amino Acid Sequence↗

PubMed1999

Cloning and characterisation of PGA1 and PGA2: two G protein alpha-subunits from pea that promote growth in the yeast Saccharomyces cerevisiae.

We report here on the cloning and characterization of two G protein alpha-subunits from pea: PGA1 and PGA2. Based on DNA gel blot analysis, PGA1 and PGA2 are the only Galpha homologous sequences in pea. RT-PCR analysis reveals that PGA1 and PGA2 transcripts are present in a variety of adult pea tissues. However, PGA2 mRNA is consistently detected at a lower level than PGA1 and demonstrates some degree of tissue specificity relative to PGA1. In the apical bud of pea seedlings, PGA1 and PGA2 transcripts decrease in response to 24 h of white light following growth for 6 days in darkness. The G protein mediated, yeast mating pathway was used to analyse the function of PGA1 and PGA2 in vivo. PGA1 downregulates the mating pathway, but through a mechanism that is independent of Gbetagamma sequestration. Unexpectedly, both PGA1 and PGA2 promote growth through a mating pathway independent mechanism.

Amino Acid Sequence↗

PubMed1999

A heat shock transcription factor in pea is differentially controlled by heat and virus replication.

Since some heat-inducible genes [heat shock (hs) genes] can be induced by virus infection in pea [e.g. Hsp70; Aranda et al. 1996, Proc. Natl Acad. Sci. USA 93, 15289-15293], we have investigated the effect that heat and virus replication may have on the expression of a heat-shock transcription factor gene (Hsf). We have characterized what appears to be the only member of the Hsf family in pea, PsHsfA. Similar to Hsp70, PsHsfA is heat-inducible in vegetative and embryonic tissues, which is concordant with the presence of heat shock elements (HSEs) and stress responsive elements (STREs) on its promoter sequence. The expression of PsHsfA during virus replication was studied in pea cotyledons and leaves, and compared to that of Hsp70. In situ hybridization experiments showed that whereas Hsp70 is induced, there is no detectable increased accumulation of PsHsfA RNA associated with the replication of pea seed-borne mosaic potyvirus (PSbMV). These experiments indicate that there is a selective control of virus-induced hs gene expression, and suggest that different regulatory pathways control hs gene expression during heat shock and virus replication.

Amino Acid Sequence↗

PubMed2001

A pea homologue of human DNA helicase I is localized within the dense fibrillar component of the nucleolus and stimulated by phosphorylation with CK2 and cdc2 protein kinases.

DNA helicases catalyse the transient opening of duplex DNA during nucleic acid transactions. Here we report the isolation of a second nuclear DNA helicase (65 kDa) from Pisum sativum (pea) designated pea DNA helicase 65 (PDH65). The enzyme was immunoaffinity purified using an antihuman DNA helicase I (HDH I) antibody column. The purified PDH65 showed ATP- and Mg(2+)-dependent DNA and RNA unwinding activities, as well as ssDNA-dependent ATPase activity. The direction of DNA unwinding was 3' to 5' along the bound strand. Antibodies against HDH I recognized the purified PDH65, and immunodepletion with these antibodies removed the DNA and RNA unwinding and ATPase activities from purified preparations of PDH65. The DNA and RNA unwinding activities were upregulated after phosphorylation of PDH65 with CK2 and cdc2 protein kinases. By incorporation of BrUTP into pea root tissue, followed by double immunofluorescence labelling and confocal microscopy, PDH65 was shown to be localized within the dense fibrillar component of pea root nucleoli in the regions around the rDNA transcription sites. These observations suggest that PDH65 may be involved both in rDNA transcription and in the early stages of pre-rRNA processing.

Antibodies↗

PubMedDate not supplied

Effect of pig bodyweight on ileal amino acid endogenous losses after ingestion of a protein-free diet enriched in pea inner fibre isolates.

The present study was conducted to evaluate whether bodyweight and the micronisation of dietary fibre affect the endogenous nitrogen and amino acid losses (ENL and EAAL) in pigs. The effect of the micronising process was tested by providing pigs with 90 g DM x kg(-1) BW0.75 of a N-free diet supplemented with isolated pea inner fibres, presented in native or micronised form and with a water-holding capacity of 12 and 4 g water g(-1) DM, respectively. ENL and EAAL were measured on pigs weighing 24, 62 and 105 kg. In all cases, daily ENL increased linearly (P < 0.05) with BW, for the majority of the AA and total N. As BW increased, daily ENL, total EAAL and the majority of EAAL increased linearly independently of micronisation (P < 0.05). When expressed per kg DMI, total EAAL and the majority of each EAA decreased curvilinearly and reached nadir at around 100 kg BW. For ENL expressed per kg DMI, micronisation resulted in a curvilinear decrease with increasing BW, as compared to a linear decrease for pigs fed the native pea fibre diet (non-micronised). Micronisation of pea inner fibres did not decrease ENL or EAAL daily, except for proline. When the losses were expressed as g x k(-1)g DMI, micronisation did not decrease ENL but decreased (P < 0.05) endogenous losses for a majority of AA as well as for total AA. The results suggest that small pigs excrete more endogenous N per kg DMI than large pigs and that pea fibre micronisation reduces EAAL but not ENL when expressed per kg DMI.

Amino Acids↗

PubMed2000

Dissection of nodulation signaling using pea mutants defective for calcium spiking induced by nod factors and chitin oligomers.

Changes in intracellular calcium in pea root hairs responding to Rhizobium leguminosarum bv. viciae nodulation (Nod) factors were analyzed by using a microinjected calcium-sensitive fluorescent dye (dextran-linked Oregon Green). Within 1-2 min after Nod-factor addition, there was usually an increase in fluorescence, followed about 10 min later by spikes in fluorescence occurring at a rate of about one spike per minute. These spikes, corresponding to an increase in calcium of approximately 200 nM, were localized around the nuclear region, and they were similar in terms of lag and period to those induced by Nod factors in alfalfa. Calcium responses were analyzed in nonnodulating pea mutants, representing seven loci that affect early stages of the symbiosis. Mutations affecting three loci (sym8, sym10, and sym19) abolished Nod-factor-induced calcium spiking, whereas a normal response was seen in peas carrying alleles of sym2(A), sym7, sym9, and sym30. Chitin oligomers of four or five N-acetylglucosamine residues could also induce calcium spiking, although the response was qualitatively different from that induced by Nod factors; a rapid increase in intracellular calcium was not observed, the period between spikes was lower, and the response was not as sustained. The chitin-oligomer-induced calcium spiking did not occur in nodulation mutants (sym8, sym10, and sym19) that were defective for Nod-factor-induced spiking, suggesting that this response is related to nodulation signaling. From our data and previous observations on the lack of mycorrhizal infection in some of the sym mutants, we propose a model for the potential order of pea nodulation genes in nodulation and mycorrhizal signaling.

Calcium↗

PubMed1980

Vanadate inhibition of auxin-enhanced H secretion and elongation in pea epicotyls and oat coleoptiles.

In experiments carried out to investigate the acid secretion theory of auxin action, we utilized sodium orthovanadate, an agent found to be a selective inhibitor of a plasma membrane-associated H(+)-pumping ATPase in Neurospora [Bowman, B. J. & Slayman, C. W. (1979) J. Biol. Chem. 245, 2928-2934]. At 1 mM, vanadate inhibited auxin-enhanced medium acidification by pea epicotyl segments within 5 min, whether added 0.5 or 2.5 hr after auxin. Inhibition of acidification was total after 10-15 min but could be reversed within 10 min after vanadate removal. When given as a 40-min pretreatment, vanadate completely prevented any auxin-enhanced acidification. Vanadate inhibition of medium acidification by oat coleoptile segments was also total and reversible, but both inhibition and reversal occurred after longer lag times than in pea. Inhibitory effects of vanadate on elongation in pea and oat tissue closely paralleled its effects on acidification, and the inhibitory effect of vanadate on elongation could be reversed by an acidic buffer. Vanadate did not inhibit respiration or protein synthesis in pea epicotyl segments, although it strongly inhibited L-[(14)C]leucine uptake. These results indicate the importance of cell wall acidification for short- and long-term auxin-enhanced growth and suggest the participation in wall acidification of a plasma membrane-associated ATPase acting as an H(+) pump.

Journal Article↗

PubMed1981

Rearrangements in the chloroplast genomes of mung bean and pea.

We have mapped all the cleavage sites for the restriction endonucleases BstEII, Kpn I, Pst I, Pvu II, Sac I, Sal I, Sma I, and Xho I on the circular chloroplast chromosomes from mung bean and pea. The mung bean chloroplast genome measures 150 kilobase pairs (kb) in length; it includes two identical sequences of 23 kb that contain the ribosomal genes and are arranged as an inverted repeat separated by single-copy regions of 21 and 83 kb. The pea chloroplast genome is only 120 kb in size, has only one set of ribosomal genes, and does not possess any detectable repeated sequences. The mung bean inverted repeat structure is common to all other nonleguminous higher plant chloroplast genomes studied, whereas the pea structure has been found only in the closely related legume Vicia faba. We conclude from these data that loss of one copy of the inverted repeat sequence has occurred only rarely during the evolution of the Angiosperms, and in the case of the legumes after the divergence of the mung bean line from the pea-Vicia line. We present hybridization data indicating that rearrangements that change the linear order of homologous sequences within the chloroplast genome have been quite frequent during the course of legume evolution.

Journal Article↗

PubMed1986

Developmental regulation of two genes encoding ribulose-bisphosphate carboxylase small subunit in pea and transgenic petunia plants: Phytochrome response and blue-light induction.

We describe the effects of light quality on the light-induced expression of two genes (rbcS-3A and rbcS-3C) encoding the ribulose-1,5-bisphosphate carboxylase small subunit of pea plants. These two genes exhibit distinctive photoresponses depending on the developmental stages of the leaves. In etiolated primary leaves, changes in the rbcS-3A and rbcS-3C transcript levels are regulated by phytochrome. In mature green leaves, on the other hand, the transcript levels are modulated by a putative blue-light photoreceptor working in concert with phytochrome. We transferred the two pea rbcS genes into the petunia genome and found that their phytochrome (rbcS-3A and -3C) and blue-light responses (rbcS-3C) are recapitulated in the appropriate developmental stages of the transgenic plants. Moreover, the transgenic plants express the pea rbcS genes in leaves at levels close to those in pea leaves. Our results show that 0.4 kilobases of the rbcS-3A and 2 kilobases of the rbcS-3C upstream regions are sufficient for these photoresponses.

Journal Article↗

PubMed1990

Cell division in higher plants: a cdc2 gene, its 34-kDa product, and histone H1 kinase activity in pea.

The mitotic cell cycle of yeast and animal cells is regulated by the cdc2 gene and its product, the p34 protein kinase, and by other components of the MPF or histone H1 kinase complex. We present evidence that cdc2, p34, and a histone H1 kinase also exist in higher plants. Protein extracts from 10 plant species surveyed display a 34-kDa component recognized by a monoclonal antibody directed against an evolutionarily conserved epitope of fission yeast p34. Nondenatured protein extracts of mitotic Pisum sativum (garden pea) tissues were fractionated by gel filtration, electrophoretically separated under denaturing conditions, and immunoblotted. p34 crossreactive material was apparent in both low and high molecular mass fractions, indicating that pea p34 occurs as both a monomer and as part of a high molecular mass complex. Histone H1 kinase activity was found predominantly in the higher molecular mass fractions, those to which the least phosphorylated form of pea p34 was confined. We also report the cloning of the pea homologue of cdc2 by polymerase chain reaction. DNA sequence analysis reveals perfect conservation of the hallmark "PSTAIR" sequence motif found in all cdc2 gene products analyzed to date.

Amino Acid Sequence↗

PubMed1993

Molecular cloning and characterization of rho, a ras-related small GTP-binding protein from the garden pea.

The rho proteins, members of the ras superfamily of small GTP-binding proteins, play a central role in the modulation of cellular functions involving the actin cytoskeleton such as in the establishment of cell polarity and morphology. As a first step in elucidating signal transduction pathways leading to processes mediated by the actin cytoskeleton in plants, we initiated cloning and characterization of rho proteins from pea. One rho-related, partial cDNA clone of 167 bp was isolated utilizing a polymerase chain reaction-based cloning strategy, using degenerate primers that correspond to conserved domains within the rho proteins. A full-length cDNA was isolated by screening a pea cDNA library using the 167-bp cDNA as a probe. The Rho1Ps cDNA contains an open reading frame encoding a polypeptide (Rho1Ps) of 197 amino acids that shows 45-64% sequence identity to members of the rho family and about 30% identity to other members of the ras superfamily. In addition to the nucleotide-binding and GTPase domains, Rho1Ps shares conserved residues and motifs unique to the rho proteins. Purified Rho1Ps protein expressed in Escherichia coli retains specific GTP-binding activity. These data indicate that Rho1Ps encodes a small GTP-binding protein of the rho family. The Rho1Ps transcript is expressed in all organs of pea seedlings, being more abundant in root tips and apical buds. DNA gel blot analyses show that the rho proteins in pea are encoded by a multigene family.

Amino Acid Sequence↗

PubMed1995

Cloning and molecular analysis of the pea seedling copper amine oxidase.

A pea seedling amine oxidase cDNA has been isolated and sequenced. A single long open reading frame has amino acid sequences corresponding to those determined from active site peptide (Janes, S.M., Palcic, M.M., Scaman, C.H., Smith, A.J., Brown, D.E., Dooley, D.M., Mure, M., and Klinman, J.P. (1992) Biochemistry 31, 12147-12154) and N-terminal sequencing experiments. The latter reveals the protein to have a 25-amino acid leader sequence with characteristics of a secretion signal peptide, as expected for this extracellular enzyme. Comparisons of the amino acid sequence of the mature pea enzyme (649 amino acids) with that of the mature lentil enzyme (569 amino acids; Rossi, A., Petruzzelli, R., and Finazzi-Agrò, A. (1992) FEBS Lett. 301, 253-257) reveal important and unexpected differences particularly with regard to protein length. Sequencing of part of the lentil gene identified several frameshift differences within the coding region resulting in a mature lentil protein of exactly the same length, 649 amino acids, as the pea enzyme. Multiple alignments of 10 copper amine oxidase sequences reveal 33 completely conserved residues of which 10 are found within 41 aligned residues at the C-terminal tails, the region missing from the original lentil sequence. One of only four conserved histidines is found in this region and may represent the third ligand to the copper. The pea enzyme contains around 3-4% carbohydrate as judged by deglycosylation experiments. We have also demonstrated by hybridization analysis that copper amine oxidase genes are present in a range of mono- and dicotyledonous plants.

Amine Oxidase (Copper-Containing)↗