ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 4, с. 529-535
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
APYRASE, STREPTAVIDIN-BINDING PROTEIN, AND ANTIMICROBIAL
ACTIVITY IN Pisum sativum1 © 2014 M. F. M. Moustafa***
*Botany Department, Faculty of Science, South Valley University, Egypt **Department of Biological Science, Faculty of Sciences, King Khalid University, Abha, Saudi Arabia
Received October 11, 2013
The 49 kD apyrase (EC 3.6.1.5), streptavidin-binding protein, and antimicrobial activity in the subcellular fractions from different seed parts of Pisum sativum L. var. Alaska were examined. Except cotyledons, all subcellular fractions contained 49 kD apyrase, and a considerable relationship was found between 49 kD apyrase and NTPase activities that increased with increasing time of germination. The bulk of 49 kD apyrase and NTPase activities was found in the nucleus pellets and cytoskeleton-enriched fraction, indicating their physiological importance. At 72 h of germination, all subcellular fractions of primary stems have a greater amount of 49 kD apyrase and NTPase than primary leaves and much more than primary roots and cotyledonary stalks. All seed parts showed antimicrobial activities, and the bulk of inhibition activities was found in the cytoskeleton-en-riched and nucleus pellets, which was greater in the primary stems and leaves than in other parts. Current findings reveal that apyrases have important roles in metabolic activities in all parts of the pea plants except cotyledons. Cotyledons contained much streptavidin-binding proteins, which might have different physiological roles than apyrases.
Keywords: Pisum sativum — 49 kD apyrase — streptavidin-binding proteins — antimicrobial activity
DOI: 10.7868/S0015330314040137
INTRODUCTION
Apyrases are enzymes with the unifying properties having the ability to hydrolyze both the y- and P-phos-phate ofATP and ADP [1]. Ecto-apyrases have diverse physiological functions, including the salvagion of extracellular nucleotides [2], the modulation of ATP-mediated immunoresponses [3], blood platelet aggregation [4], protein glycosylation [5], the quenching of the ATP neurotransmitter signal [6], and the enhancement of soybean nodulation [7]. The pea (Pisum sativum L.) apyrase used in this study was identified as ATP diphosphohydrolyase (EC 3.6.1.5) based on its ability to hydrolyze both ADP and ATP to AMP and by its cDNA sequence [8, 9]. Recently, the 49 kD apyrase from pea called PsAPYl (DDBJ/EMBL/GenBank BAB40230) and its homologs have been found in Ara-bidopsis thaliana, Medicago truncatula, and in several other plants species [10]. Our previous study showed that APY1 activity in the pea homogenate increased
1 This text was submitted by the author in English.
Abbreviations: BCIP — 5-bromo-4-chloro-3-indolyl phosphate; BPB — bromophenol blue; CSB — cytoskeleton-stabilizing buffer; DMSO - dimethysulfate; NBT - nitro blue tetrazolium; PMSF -phenylmethylsulfonyl fluoride; PTE — polyoxyethylene-10-tride-cyl ether; SBP — streptavidin-binding protein. Corresponding author: Mahmoud F. M. Moustafa. Department of Biological Science, Faculty of Sciences, King Khalid University, Abha, Saudi Arabia; fax: 209-6521-1279; e-mail: hamdony@yahoo.com
with germination time in terms of its transcript accumulation and an enzymatic activity [11]. Shibata et al. [12] showed that PsAPYl in P. sativum was localized in the cell wall, nucleus, and around the filamentous structures. In the present study, sequential centrifuga-tion, antigen to the anti-APYl, and enzyme activity were used to evaluate the subcellular localization of 49 kD apyrase in all pea seed parts during the early seedling stage. This study also aimed to find if there is any correlation between 49 kD apyrase and antimicrobial activity in various pea seed parts.
MATERIALS AND METHODS
Plant materials and sample preparation. Pea (Pisum sativum L., var. Alaska) seeds were soaked in tap water for 10 h and sown in vermiculite to allow germination in the dark at 21 °C for various times. Three grams of cotyledons, embryos, epicotyls, cotyledonary stalks, primary stems, primary leaves, and primary roots (hy-pocotyls + root) were dissected at 24, 48, and 72 h after sowing and then ground with a mortar and pestle at 4°C in 7 volumes of cytoskeleton-stabilizing buffer (CSB) consisting of 5 mM Hepes-KOH (pH 7.5), 10 mM Mg(OAc)2, 2 mM EGTA, and 1 mM PMSF with the addition of 0.5% polyoxyethylene-10-tridecyl ether (PTE). The homogenate from each sample was filtered through two layers of Miracloth ("Calbio-
chem"), and then centrifuged at 250 g for 5 min to obtain crude nuclei, and the resulting supernatant was centrifuged at 27 000 g for 15 min to obtain the cytosk-eleton-enriched pellet and the post-cytoskeleton supernatant [13]. The 250 g and 27000 g pellets were re-suspended using CSB in a volume equivalent to the su-pernatants.
SDS-PAGE and western blotting. Fifteen microliters from each fraction were analyzed by SDS-PAGE. After electrophoresis, each gel was transferred onto a PVDF membrane (ImmobilonTM Transfer Membranes, "Millipore") and treated with the anti-apyrase antibody from rats as the primary antibody [8] and bi-otinylated anti-rat Ig species-specific whole antibody from sheep ("Amersham Pharmacia Biotech", United Kingdom) as the secondary antibody. Detection of secondary antibody binding was done with streptavi-din—alkaline phosphatase conjugate ("Amersham Pharmacia Biotech") with 5-bromo-4-chloro-3-in-dolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) as substrates. In order to identify the streptavi-din-binding proteins (SBP), reactions were done without anti-apyrase antibody [11].
Apyrase activity in subcellular fractions. Enzyme activity from each seed part was measured as released phosphate using nucleoside triphosphate, nucleoside diphosphate, and nucleoside monophosphate as substrates [8]. Aliquots (1 ^L) from each sample diluted to the appropriate concentration with CSB were added to 83.3 ^L of the assay mixture (100 mM Tricine— NaOH (pH 7.5), 10 mM substrate (ATP, CTP, GTP, TTP, UTP, ADP, and AMP), and 10 mM CaCl2, and incubated at 25°C for 15 min. 50% (v/v) TCA (16.7 ^L) was added and chilled on ice. 500 ^L of the ferrous sulfate-ammonium molybdate reagent was added to each sample, and the activity was measured with a Beckman DU640 spectrophotometer. Each experiment was performed three times and expressed as means ± standard errors.
Antimicrobial activity of the various subcellular fractions. 10.6 g ofpea cotyledons, embryos, epicotyls, cotyledonary stalks, primary stems, primary leaves, and primary roots were dissected and ground with a mortar and pestle. Each sample was centrifuged at 250 g for 5 min, and the resulting supernatant was centrifuged at 27000 g for 15 min. Samples of the superna-tants and pellets were resuspended in the 20 mL of methanol and placed on the shaker for 48 h. Methanol was evaporated by incubating the extracts in oven at 45°C for 48 h; the samples were weighed and dissolved in 3 mL of sterile dimethyl sulfoxide (DMSO) and analyzed for antimicrobial activity by agar well diffusion methods [14]. Petri plates were prepared by pouring 10 mL of sterilized nutrient agar and allowed to solidify, and wells were made with the aid of a sterile cork borer in the solidified agar. One milliliter of the logphase cultures of the pathogenic Pseudomonas aeruginosa and Candida sp. were seeded and uniformly
spread on the surface of the nutrient agar medium, and 0.53 g/mL from each of the extract samples was separately introduced into separate holes. Cefoxitin (30 ^g) was used as a positive control for pathogenic bacteria, and fluconazole (30 ^g) for pathogenic Candida sp., while DMSO was used as a negative control. All inoculated plates were incubated aerobically at 29 °C for 24 h for bacterial strains and for 48 h for Candida sp., and inhibition zones were measured.
Statistical analysis. Each antimicrobial assay was performed in triplicate and means ± standard deviations were calculated. Differences at p < 0.05 were considered statistically significant using one-way ANOVA, v. 10.
RESULTS AND DISCUSSION
To verify the distribution pattern of pea apyrase in the crude nuclei, the cytoskeleton-enriched fraction, and the post-cytoskeleton supernatant in developing pea parts, extracted proteins from each fraction were electrophoresed and probed with anti-apyrase antibody. The amount of 49 kD apyrase in each fraction was quantified densitometrically and expressed as OD x band area (fig. 1, table 1). The immunological reaction of anti-apyrase serum against pea cotyledon proteins (fig. 1, panels a1, a2, and a3) showed that there was no 49 kD apyrase signal in the nuclei (lane 1), the cy-toskeleton-enriched fraction (lane 3), and the post-cytoskeleton supernatant (lane 4) in the course of germination. This is in accordance with the results obtained by Julano and Varner [15], who found that only two enzymes were present in the pea cotyledons during germination, e.g., phosphorylase and a-amylase. At 24, 48, and 72 h, the antigen to the anti-APY1 serum was detected in all fractions in the tissues of the dissected embryos after 24 h of germination (fig. 1A), in the dissected epicotyls (fig. 1B), cotyledonary stalks (fig. 1C), and primary roots after 48 h of germination (fig. 1D), and in the primary stems (fig. 1E), primary leaves (fig. 1F), cotyledonary stalks (fig. 1G), and in the primary roots after 72 h of germination (fig. 1H). In all tissues, the relative amount of 49 kD apyrase was abundantly present in the crude nuclei (lane 1) and in the cytoskeleton-enriched pellets (lane 3), and the amount increased progressively during germination time. At 24 h, the total amount of 49 kD apyrase was less in advanced embryos, while at 48 h, it was higher in the epicotyls than those in the cotyledonary stalks and primary roots. At 72 h, the amount of 49 kD apy-rase in the primary stems was higher than those in
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