An apyrase from Mimosa pudica contains N5,N10‐methenyl tetrahydrofolate and is stimulated by light

Ghosh, Rinku; Biswas, Sumit; Roy, Siddhartha

doi:10.1046/j.1432-1327.1998.2581009.x

Cited by 14 publications

(8 citation statements)

References 7 publications

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“…An apyrase with a molecular mass of 36 kD was previously isolated from Mimosa, and its primary sequence was reported (Ghosh et al, 1998a). In this study, we isolated MP67 as a novel apyrase by monitoring ADP-hydrolyzing activity; therefore, we did Figure 6.…”

Section: Discussionmentioning

confidence: 97%

“…Comparison of hydrolyzing activity toward 14 different phosphate compounds revealed that the enzyme is an NDPase distinct from conventional apyrase (Ishikawa et al, 1984). Isolation and cloning of a 36-kD apyrase from M. pudica have been reported, and this enzyme plays a role in the light response of Mimosa (Ghosh et al, 1998a(Ghosh et al, , 1998b. However, the deduced amino acid sequence of the 36-kD apyrase did not contain an ACR and was quite different from other apyrases (Shibata et al, 2001).…”

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confidence: 99%

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Purification and Biochemical Characterization of a Novel Ecto-Apyrase, MP67, from Mimosa pudica

et al. 2011

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We have previously reported the presence of an apyrase in Mimosa pudica. However, only limited information is available for this enzyme. Thus, in this study, the apyrase was purified to homogeneity. The purified enzyme had a molecular mass of around 67 kD and was able to hydrolyze both nucleotide triphosphate and nucleotide diphosphate as substrates. The ratio of ATP to ADP hydrolysis velocity of the purified protein was 0.01 in the presence of calcium ion, showing extremely high substrate specificity toward ADP. Thus, we designated this novel apyrase as MP67. A cDNA clone of MP67 was obtained using primers designed from the amino acid sequence of trypsin-digested fragments of the protein. In addition, rapid amplification of cDNA ends-polymerase chain reaction was performed to clone a conventional apyrase (MpAPY2). Comparison of the deduced amino acid sequences showed that MP67 is similar to ecto-apyrases; however, it was distinct from conventional apyrase based on phylogenetic classification. MP67 and MpAPY2 were expressed in Escherichia coli, and the recombinant proteins were purified. The recombinant MP67 showed high substrate specificity toward ADP rather than ATP. A polyclonal antibody raised against the recombinant MP67 was used to examine the tissue distribution and localization of native MP67 in the plant. The results showed that MP67 was ubiquitously distributed in various tissues, most abundantly in leaves, and was localized to plasma membranes. Thus, MP67 is a novel ecto-apyrase with extremely high substrate specificity for ADP.

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Section: Discussionmentioning

confidence: 97%

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confidence: 99%

Purification and Biochemical Characterization of a Novel Ecto-Apyrase, MP67, from Mimosa pudica

et al. 2011

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“…However, so far, expression of plant and animal recombinant apyrases in E. coli cells has almost always resulted in formation of inclusion bodies (IB) [20–24], with an only exception of Mimosa pudica where soluble and catalytically active apyrase enzyme synthesis has been demonstrated [17, 25]. The reason for the formation of inclusion bodies is usually a toxic effect of synthesized proteins on bacterial cells or incorrect folding of overexpressed polypeptides resulting from limitations of prokaryotic cells in eukaryotic protein synthesis (errors in the synthesis of a polypeptide chain, reducing environmental bacterial cytosol) and/or very high level expression of heterologous proteins (inefficient intracellular mechanisms of polypeptides folding) [26].…”

Section: Resultsmentioning

confidence: 99%

“…Expression of recombinant NTPDases, both plant and animal, in bacterial cells, almost always resulted in the formation of insoluble and inactive aggregates [20–24]. Only in the case of M. pudica apyrase, a soluble and catalytically active enzyme was achieved, in addition to IB [17, 25]. Soluble and catalytically active NTPDase also obtained in a prokaryotic system was Lp1NTPDase derived from the bacterium Legionella pneumophila .…”

Section: Discussionmentioning

confidence: 99%

Chaperones Are Necessary for the Expression of Catalytically Active Potato Apyrases in Prokaryotic Cells

Porowińska

Czarnecka

Komoszyński

2014

Appl Biochem Biotechnol

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NTPDases (nucleoside triphosphate diphosphohydrolases) (also called in plants apyrases) hydrolyze nucleoside 5′-tri- and/or diphosphate bonds producing nucleosides di or monophosphate and inorganic phosphate. For years, studies have been carried out to use both plant and animal enzymes for medicine. Therefore, there is a need to develop an efficient method for the quick production of large amounts of homogeneous proteins with high catalytic activity. Expression of proteins in prokaryotic cells is the most common way for the protein production. The aim of our study was to develop a method of expression of potato apyrase (StAPY4, 5, and 6) genes in bacterial cells under conditions that allowed the production of catalytically active form of these enzymes. Apyrase 4 and 6 were overexpressed in BL21-CodonPlus (DE3) bacteria strain but they were accumulated in inclusion bodies, regardless of the culture conditions and induction method. Co-expression of potato apyrases with molecular chaperones allowed the expression of catalytically active apyrase 5. However, its high nucleotidase activity could be toxic for bacteria and is therefore synthesized in small amounts in cells. Our studies show that each protein requires other conditions for maturation and even small differences in amino acid sequence can essentially affect protein folding regardless of presence of chaperones.

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“…4), horse gram , potato (Handa and Guidotti 1996), Arabidopsis and (DDBJ/EMBL/ /GenBank AF093604), mimosa (Ghosh et al 1998) and of an ecto-apyrase from rat (Wang and Guidotti 1996) are shown in Fig. 5.…”

Section: Comparison Of Polypeptide Sequences Of Several Plant Apyrasesmentioning

confidence: 99%

Structure of the coding region and mRNA variants of the apyrase gene from pea (Pisum sativum)

2001

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Partial amino acid sequences of a 49 kDa apyrase (ATP diphosphohydrolase, EC 3.6.1.5) from the cytoskeletal fraction of etiolated pea stems were used to derive oligonucleotide DNA primers to generate a cDNA fragment of pea apyrase mRNA by RT-PCR and these primers were used to screen a pea stem cDNA library. Two almost identical cDNAs differing in just 6 nucleotides within the coding regions were found, and these cDNA sequences were used to clone genomic fragments by PCR. Two nearly identical gene fragments containing 8 exons and 7 introns were obtained. One of them (H-type) encoded the mRNA sequence described by Hsieh et al. (1996) (DDBJ/EMBL/GenBank Z32743), while the other (S-type) differed by the same 6 nucleotides as the mRNAs, suggesting that these genes may be alleles. The six nucleotide differences between these two alleles were found solely in the first exon, and these mutation sites had two types of consensus sequences. These mRNAs were found with varying lengths of 3' untranslated regions (3'-UTR). There are some similarities between the 3'-UTR of these mRNAs and those of actin and actin binding proteins in plants. The putative roles of the 3'-UTR and alternative polyadenylation sites are discussed in relation to their possible role in targeting the mRNAs to different subcellular compartments.

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An apyrase from Mimosa pudica contains N5,N10‐methenyl tetrahydrofolate and is stimulated by light

Cited by 14 publications

References 7 publications

Purification and Biochemical Characterization of a Novel Ecto-Apyrase, MP67, from Mimosa pudica

Purification and Biochemical Characterization of a Novel Ecto-Apyrase, MP67, from Mimosa pudica

Chaperones Are Necessary for the Expression of Catalytically Active Potato Apyrases in Prokaryotic Cells

Structure of the coding region and mRNA variants of the apyrase gene from pea (Pisum sativum)

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