Cloning and characterization of purple acid phosphatase phytases from wheat, barley, maize, and rice

Barley (Hordeum vulgare) and wheat (Triticum aestivum) possess significant phytase activity in the mature grains. Maize (Zea mays) and rice (Oryza sativa) possess little or virtually no preformed phytase activity in the mature grain and depend fully on de novo synthesis during germination. Here, it is demonstrated that wheat, barley, maize, and rice all possess purple acid phosphatase (PAP) genes that, expressed in Pichia pastoris, give fully functional phytases (PAPhys) with very similar enzyme kinetics. Preformed wheat PAPhy was localized to the protein crystalloid of the aleurone vacuole. Phylogenetic analyses indicated that PAPhys possess four conserved domains unique to the PAPhys. In barley and wheat, the PAPhy genes can be grouped as PAPhy_a or PAPhy_b isogenes (barley, HvPAPhy_a, HvPAPhy_b1, and HvPAPhy_b2; wheat, TaPAPhy_a1, TaPAPhy_a2, TaPAPhy_b1, and TaPAPhy_b2). In rice and maize, only the b type (OsPAPhy_b and ZmPAPhy_b, respectively) were identified. HvPAPhy_a and HvPAPhy_b1/b2 share 86% and TaPAPhya1/a2 and TaPAPhyb1/b2 share up to 90% (TaPAPhy_a2 and TaPAPhy_b2) identical amino acid sequences. despite of this, PAPhy_a and PAPhy_b isogenes are differentially expressed during grain development and germination. In wheat, it was demonstrated that a and b isogene expression is driven by different promoters (approximately 31% identity). TaPAPhy_a/b promoter reporter gene expression in transgenic grains and peptide mapping of TaPAPhy purified from wheat bran and germinating grains confirmed that the PAPhy_a isogene set present in wheat/barley but not in rice/maize is the origin of high phytase activity in mature grains.     

紫萍P143品系植物rbcS基因的cDNA克隆与表达

紫萍P14 3品系离体半叶状体在黑暗下培养 4d以后 ,rbcS基因的表达比完整植株显著降低 ;半叶状体在黑暗下培养 10d ,后再转移到长日照条件下培养 ,rbcS基因的表达能恢复到较高水平     

Isolation and Characterization of Chloroplast DNA from the Duckweed Spirodela oligorrhiza

Chloroplast DNA of the duckweed Spirodela oligorrhiza, isolated by CsCl gradient centrifugation, was characterized by its buoyant density, guanine + cytosine content, melting behavior, circularity, and contour length. In all these characteristics, chloroplast DNA of S. oligorrhiza is similar to the chloroplast genomes of other higher plants, except that it has a significantly larger size.     

Purification and characterization of a purple acid phosphatase from rat spleen

An acid phosphatase species which is activated by Fe2+ was purified 3,700-fold from rat spleen by chromatography on columns containing Blue-Sepharose, concanavalin A-Sepharose, Sephadex G-100, and CM-Sephadex. The enzyme hydrolyzed aryl phosphates, nucleoside di- and triphosphates, phosphoproteins, and thiamine pyrophosphate with Km values of 10(-4) to 10(-3) M at an optimal pH of 5.0-5.8. Co-purification of the acid phosphatase and acid phosphoprotein phosphatase indicated that they were identical. The purified enzyme was glycoprotein in nature, showing four heterogeneous forms on acid polyacrylamide gel electrophoresis (pI values, 7.8, 8.0, 8.3, and 8.5), but it gave a molecular weight of 33,000 on sodium dodecyl sulfate-gel electrophoresis and gel permeation chromatography. The enzyme had a purple color (lambda max 545 nm) and contained 2 iron atoms per enzyme molecule. Among reductants, ascorbic acid and Fe2+ were the best activators, although their combined effect was not additive. Fe2+ and ascorbic acid both changed the purple enzyme into the same active form (lambda max 515 nm), giving almost the same kinetic constants for substrates and for inhibitors such as molybdate, phosphate and fluoride. However, low concentrations of Fe2+, from 0.01 mM to 1.0 mM, immediately and reversibly activated the enzyme, whereas high concentrations of ascorbic acid over 1 mM were required for maximal activation, which was slow and irreversible.     

Purification and characterization of purple acid phosphatase from developing rat bone

Tartrate-resistant acid phosphatase active on nucleoside di- and triphosphate substrates was isolated from developing rat bone and purified 2500-fold. The enzyme concentration had a purple coloration and activity that was sensitive to reducing agents. Mild reducing agents such as ferrous ion and ascorbic acid caused loss of purple color and increased activity toward substrates severalfold; however, a strong reductant such as dithionite caused loss of both color and activity which were partially restored by addition of ferrous ion and ascorbic acid. Enzyme activity was homogeneous with protein during the final gel permeation steps of chromatography and gave an apparent molecular size of about 40,000 Da. Determination of iron in the most pure preparation revealed the presence of 1.3 atoms of iron per molecule of the tartrate-resistant enzyme E2. Other properties of the purified enzyme include a pI of approximately 9.5 and sensitivity to inhibition by ions of copper, zinc, fluoride, and molybdate. Antibody prepared to the pre-concanavalin A (Con A)-Sepharose purified enzyme reacted with all protein from the Con A step, but it did not react with tartrate-sensitive acid phosphatase from rat bone or with potato acid phosphatase. Purple acid phosphatase from rat bone has many properties that parallel the iron-containing purple acid phosphatases from rat spleen, bovine spleen, and pig uterine secretions.     

Protein, nucleic acid and starch metabolism in the duckweed Spirodela oligorrhiza, treated with cytokinins

Bacteria-free cultures of Spirodela oligorrhiza continue to increase in frond number for 2 to 3 days after transfer to darkness. There is then no further increase in frond number for 3 to 4 weeks, although DNA, RNA and protein synthesis continue at decreased rates and starch accumulates in the plants. We refer to such ;non-growing' plants in darkness as dormant. Adding kinetin to dormant Spirodela initiated increased DNA, RNA and protein synthesis within 1h, although new fronds were not detected until 24h after the addition of kinetin. The frond number then continued to increase. Starch accumulated in dormant plants. Accumulation of starch appeared to be a consequence of inhibition of growth rather than the converse. No evidence was obtained for a block in [(14)C]glucose metabolism that might explain the lack of growth in darkness in the absence of kinetin. In darkness, more ribosomes were membrane-bound in dormant Spirodela than in Spirodela growing with kinetin. Similarities between the response of Spirodela to darkness, stringent control in bacteria and pleiotypic controls in animal cells are discussed. It is suggested that all three processes are ultimately controlled by specific protein kinases that are individually sensitive to different effectors.     

Expression and distribution of tartrate-resistant purple acid phosphatase in the rat nervous system.

Tartrate-resistant purple acid phosphatase (TRAP) of osteoclasts and certain cells of the monocyte-macrophage lineage belongs to the family of purple acid phosphatases (PAPs). We provide here evidence for TRAP/PAP expression in the central and peripheral nervous systems in the rat. TRAP/PAP protein was partially purified and characterized from the trigeminal ganglion, brain, and spinal cord. The TRAP activity (U/mg tissue) in these tissues was about 10-20 times lower than in bone. Reducing agents, e.g. ascorbate and ferric iron, increased the TRAP activity from the neural tissues (nTRAP) and addition of oxidizing agents completely inactivated both bone and nTRAP. The IC(50) for three known oxyanion inhibitors of TRAP/PAP was similar for bone and nTRAP with the same rank order of potency (molybdate > tungstate > phosphate). This indicates that the redox-sensitive binuclear iron center characteristic of mammalian PAPs is present also in nTRAP. Western blots of partially purified nTRAP revealed a band with the expected size of 35 kD. The expression of TRAP in the trigeminal ganglion, brain, and spinal cord was confirmed at the mRNA level by RT-PCR. In situ hybridization histochemistry demonstrated TRAP mRNA expression in small ganglion cells of the trigeminal ganglion, in alpha-motor neurons of the ventral spinal cord, and in Purkinje cells of the cerebellum. TRAP-like immunoreactivity was encountered in the cytoplasm of neuronal cell bodies in specific areas of both the central and the peripheral nervous system. Together, the data demonstrate that active TRAP/PAP is expressed in certain parts of the rat nervous system.     

Induction of high affinity phosphate transporter in the duckweed Spirodela oligorrhiza

Duckweed plants (Spirodela oligorrhiza) grown under phosphate (Pi)-deficient conditions (- P plants) exhibited more than 50-fold higher Pi uptake activity than plants grown under Pi-sufficient conditions (+ P plants). The Pi uptake activity of - P plants measured using (32)Pi was significantly inhibited by carbonylcyanide m-chlorophenylhydrazone, indicating that Pi uptake is energized by the electrochemical proton gradient across the plasma membrane (PM). When Pi uptake was examined at various concentrations of Pi, more active uptake of Pi was observed in - P plants than in + P plants, irrespective of the Pi concentrations. An immunoblot analysis of the PM proteins using antiserum against the conserved sequence of the high-affinity Pi transporter recognized the occurrence and large accumulation of a novel protein band at 48 kDa in - P plants. The protein was almost completely extracted with chloroform-methanol (2:1, v/v), but only a trace amount of the protein was detected in + P plants. Immunohistochemical studies of plant roots using the same antiserum demonstrated a large accumulation of high-affinity Pi transporters at the outermost cortical cells of - P plants, but not of + P plants. When an immunoblot analysis of PM proteins was performed using antiserum against the PM H(+)-ATPase, a positive band of about 96 kDa was detected in both plants with a similar signal intensity. Furthermore, ATP-hydrolytic and ATP-dependent H(+)-transporting activities of PM H(+)-ATPase in - P plants were not higher than those in + P plants. However, kinetic analyses showed that the PM H(+)-ATPase in - P plants had a lower K(m) value and a higher coupling efficiency between ATP hydrolysis and H(+) pumping than the corresponding values in + P plants. These results suggest that the significant stimulation of Pi uptake in - P plants may be due mainly to the induction and accumulation of the high-affinity Pi transporter in the PM, and that the electrochemical proton gradient across the PM may be generated by the high-ATP-affinity and energy-efficient H(+) pump in - P plants. This would facilitate the acquisition of Pi in S. oligorrhiza under Pi-depleted conditions.     

Isolation and characterization of purple acid phosphatase gene during seedling development in mungbean

Purple acid phosphatases (PAPs), which are normally found in plant tissues, can hydrolyze a broad spectrum of phosphate esters. In this study, a mungbean [Vigna radiata (L.) Wilczek cv. KPS1] acid phosphatase gene (VrPAP1) was isolated from seedling cotyledons. The full-length of VrPAP1 cDNA contained an open reading frame of 1 644 bp encoding 547 amino acid residues with a predicted molecular mass of 62.07 kDa. Sequence analysis showed that VrPAP1 is purple acid phosphatase. RNA blot analyses indicated that the VrPAP1 accumulated during the first hour in cotyledons of germinating seeds and reached a maximum expression after 24 h and then decreased. The VrPAP1 mRNA was observed in cotyledons, hypocotyls and leaves but not in radicles or dry seeds. DNA blot analysis indicated that VrPAP1 is a single copy gene in the mungbean genome.     

Stimulation of Nitrate Reductase by Light and Ammonium in Spirodela oligorrhiza

Light-enhanced nitrate reductase (NR) activity was 8 times greaterthan the dark control. Exogenous application of sucrose, glucoseand fructose increased the induction of NR in the light as wellas in the dark, whereas glycolate had no effect. DCMU [3-(3,4-dichlorophenyl)-1, 1-dimethyl urea] completely inhibited thedevelopment of NR in light. Sucrose, when added with DCMU, reversedthis inhibitory effect NR in vivo was more stable in light thanin darkness, the half-lives being 9.6 h and 6.4 h, respectively.The addition of sucrose did not change the half-life of NR ineither light or darkness. Ammonium, the end product of the inorganicnitrogen assimilatory pathway, stimulated the NR activity whereasamino acids decreased it. Key words: Spirodela oligorrhiza, nitrate reductase, ammonium, light     

Cloning and characterization of a Rhizobium meliloti nonspecific acid phosphatase

Nodulated legumes require high levels of phosphorus for optimal symbiotic performance. However, the basis for this elevated phosphorus requirement is poorly understood, and very little information regarding bacteroid phosphorus metabolism is available. To develop an understanding of the relative importance of organic and inorganic phosphorus sources for bacteroids, we investigated phosphatase activity in Rhizobium meliloti. An R. meliloti plasmid library clone that complemented an Escherichia coli phosphatase mutant was isolated, and the clone was sequenced. The complementing fragment contained a 337-amino-acid open reading frame that has a potential leader sequence and processing sites characteristic of periplasmic proteins. The phosphatase activity was located in the periplasm of R. meliloti and of E. coli containing the cloned gene. The subunit molecular mass of the cloned phosphatase was 33 kDa, and gel filtration indicated the active enzyme was a 66-kDa homodimer. Lack of substrate specificity suggests the cloned gene, napD, encodes a nonspecific acid phosphatase with a pH optimum of approximately 6.5. An R. meliloti napD transposon-insertion mutant was constructed, and its symbiotic phenotype was determined to be Fix⁺ regardless of the level of phosphorus provided to the host plant. Received: 26 August 1997 / Accepted: 4 February 1998     

Heterologous expression and characterization of recombinant purple acid phosphatase from red kidney bean

Purple acid phosphatases (PAPs) are dinuclear metallohydrolases of widespread occurrence. In a first step to understand structure-function relationship of PAP from red kidney bean (kbPAP), we cloned its cDNA and functionally expressed the enzyme in insect cells. kbPAP cDNA encodes a protein of 459 amino acids with 99% identity to the published primary structure (T. Klabunde et al., Eur. J. Biochem. 226 (1994) 369-375). N-terminally the cDNA encodes 27 amino acids with characteristics for a signal directing the nascent protein to the endoplasmic reticulum. A baculovirus vector was constructed containing cDNAs of kbPAP and green fluorescent protein, the latter to serve as transfection and infection marker. Heterologous expression in High Five insect cells afforded a dimeric, disulfide-linked phosphatase of 110 kDa, identical to the mass of native kbPAP. Purification in three steps yielded 1.5 mg recombinant protein per liter of culture medium with a specific activity of 266 units/mg, slightly exceeding that of native kbPAP. The recombinant protein was functionally indistinguishable from native kbPAP, despite differences in glycosylation and sensitivity to redox reagents.     

Porcine purple acid phosphatase: heterologous expression, characterization, and proteolytic analysis

Uteroferrin is an iron-binding glycoprotein, which is abundantly synthesized in porcine uterine glandular endometrium and believed to be involved in maternal/fetal iron transport. In the present study, uteroferrin has been cloned and functionally expressed using baculovirus-infected insect host cells Spodoptera frugiperda. The work also addresses the possible role of proteolytic cleavage to facilitate the release of uteroferrin-bound iron. The enzyme secreted in culture medium exhibits a molecular mass and catalytic properties similar to native porcine uteroferrin. The specific activity was estimated at 233 U/mg using p-nitrophenyl phosphate as substrate. Partial cleavage of the enzyme with trypsin resulted in a 1.7-fold enhancement in specific activity and a two-subunit polypeptide as observed in preparations of most mammalian purple acid phosphatases. Digestion with the aspartic protease pepsin resulted in a 2.5-fold enzyme inactivation correlated with the appearance of low molecular weight polypeptide fragments and the release of enzyme-bound iron.     

Rhythmicity of L-phenylalanine ammonia-lyase activity in Spirodela oligorrhiza. Effects of darkening, abscisic acid and 1-amino-2-phenylethylphosphonic acid

L-Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in Spirodela oligorrhiza (Kurz) Hegelm. cultivated in continuous light showed ca 48-h rhythmicity in the activity. Abscisic acid shifted the oscillation by ca 24 h. 1-Amino-2-phenylethylphosphonic acid, a competitive inhibitor of the enzyme in vitro, markedly increased PAL activity in vivo and nullified the oscillation. Rhythmicity in PAL activity did not occur in darkness. Illumination was a prerequisite for maintaining high PAL activity as well as for the occurrence of the oscillations in the enzyme activity. 'Average'enzyme activity decreased proportionally to time of cultivation of plants in the stationary culture. Transfer of the plants to fresh medium was connected with the increase of PAL activity to a maximum during the following 1-2 days. Diurnal oscillation of PAL activity with a maximum at 1800–1900 h, dependent on the presence of glucose or sucrose in the nutrient medium, was also recorded.     

Isolation and characterization of four cell wall purple acid phosphatase genes from tobacco cells

Four full-length cDNAs were isolated from a cDNA library prepared from tobacco cultured cells and designated NtPAP4, NtPAP12, NtPAP19 and NtPAP21, which could correspond to purple acid phosphatase (PAP). Levels of both NtPAP12 and NtPAP21 mRNA in the protoplasts immediately increased after the protoplasts were transferred to a medium for cell wall regeneration, and the accumulation of the mRNA was correlated with cell wall regeneration for 3 h. It is likely that the NtPAP12 and NtPAP21 gene products are wall-bound PAPs at the early stage of regenerating walls in tobacco protoplasts.     

Purification and characterization of purple acid phosphatase PAP1 from dry powder of sweet potato

Purple acid phosphatase (PAP) was purified from sweet potato dry powder, which is used as a food additive. Spectrometric and enzymatic analyses, and analysis of the amino-terminal sequence indicated that the purified purple acid phosphatase was PAP1. High activity in neutral and acidic conditions, broad substrate specificity, and good thermal stability of PAP1 suggest the possibility of practical applications of PAP1.     

Cloning and characterization of the human colipase cDNA

Pancreatic lipase hydrolyzes dietary triglycerides to monoglycerides and fatty acids. In the presence of bile salts, the activity of pancreatic lipase is markedly decreased. The activity can be restored by the addition of colipase, a low molecular weight protein secreted by the pancreas. The action of pancreatic lipase in the gut lumen is dependent upon its interaction with colipase. As a first step in elucidating the molecular events governing the interaction of lipase and colipase with each other and with fatty acids, a cDNA encoding human colipase was isolated from a lambda gt11 cDNA library with a rabbit polyclonal anti-human colipase antibody. The full-length 525 bp cDNA contained an open reading frame encoding 112 amino acids, including a 17 amino acid signal peptide. The predicted protein sequence contains 100% of the published protein sequence for human colipase determined by chemical methods, but predicts the presence of five additional NH2-terminal amino acids and four additional COOH-terminal amino acids. Comparison of the predicted protein sequence with the known sequences of colipase from other species reveals regions of extensive identity. In vitro translation of mRNA transcribed from the cDNA gave a protein of the expected molecular size that was processed by pancreatic microsomal membranes. Sequence analysis of the in vitro translation product after processing demonstrated signal peptide cleavage and the presence of a human procolipase, as exists in the pig and horse colipases. DNA blot analysis was consistent with the presence of a single gene for colipase. RNA blot analysis demonstrated tissue-specific expression of colipase mRNA in the pancreas. Thus, we report, for the first time, a cDNA for colipase.(ABSTRACT TRUNCATED AT 250 WORDS)     

A model of the acid sphingomyelinase phosphoesterase domain based on its remote structural homolog purple acid phosphatase

Sequence profile and fold recognition methods identified mammalian purple acid phosphatase (PAP), a member of a dimetal-containing phosphoesterase (DMP) family, as a remote homolog of human acid sphingomyelinase (ASM). A model of the phosphoesterase domain of ASM was built based on its predicted secondary structure and the metal-coordinating residues of PAP. Due to the low sequence identity between ASM and PAP (approximately 15%), the highest degree of confidence in the model resides in the metal-binding motifs. The ASM model predicts residues Asp 206, Asp 278, Asn 318, His 425, and His 457 to be dimetal coordinating. A putative orientation for the phosphorylcholine head group of the ASM substrate, sphingomyelin (SM), was made based on the predicted catalysis of the phosphorus-oxygen bond in the active site of ASM and on a structural comparison of the PAP-phosphate complex to the C-reactive protein-phosphorylcholine complex. These complexes revealed similar spatial interactions between the metal-coordinating residues, the metals, and the phosphate groups, suggesting a putative orientation for the head group in ASM consistent with the mechanism considerations. A conserved sequence motif in ASM, NX3CX3N, was identified (Asn 381 to Asn 389) and is predicted to interact with the choline amine moiety in SM. The resulting ASM model suggests that the enzyme uses an SN2-type catalytic mechanism to hydrolyze SM, similar to other DMPs. His 319 in ASM is predicted to protonate the ceramide-leaving group in the catalysis of SM. The putative functional roles of several ASM Niemann-Pick missense mutations, located in the predicted phosphoesterase domain, are discussed in context to the model.     

Cloning and sequencing of bovine kidney alkaline phosphatase cDNA

The molecular cloning and nucleotide sequencing of bovine kidney alkaline phosphatase is reported. The homology with the human enzyme is about 90% at both the nucleotide and amino acid levels. The only significant sequence differences occur at the respective C termini. The high degree of homology also extends into the 5' and 3' untranslated regions of the two cDNAs.