Streptavidin and avidin are one of the most successful and widely used as detection reagents in immunology, biochemistry and cell biology due to their high affinity binding to biotin. Biotin found in all organisms is an essential cofactor for a small number of enzymes necessary for CO₂ transfer during carboxylation reactions (Knowles, 1989). In plant cells biotin-dependent enzyme is required for both growth of vegetative tissues and synthesis of storage lipids in developing pea seed (Stumpf, 1980; Harwood, 1988). In yeast (Pichia pastoris) biotin deficiency results in lower growth rates and lower biomass yields in batch cultures and in wash-out in continuous cultures (Jungo, 2007). In animals cells biotin-containing enzymes catalyse ATP-dependent CO₂ fixation and are thus important enzymes in intermediary metabolism (Dakshinamurti and Chauhan, 1989). Furthermore, stored biotin in muscle is of special interest because alterations in mitochondrial biotin-containing enzymes are likely to be involved in most metabolic myopathies (Scholte et al., 1987). In bacteria such as Escherichia coli, Bacillus subtilis, and Bacillus sphaericus, biotin biosynthesis has been elucidated by combined biochemical and genetic studies (Marquet et al., 2001). In higher plants initial genetic information on biotin synthesis came from analysis of the bio1 biotin auxotroph of Arabidopsis that lead to identification of the second biotin auxotroph (Schneider et al., 1989).

It is reported that in pea seeds only one type of seed-specific biotinylated-protein and three biotin-dependent plant carboxylases (Duval et al., 1993; Alban et al., 1993; Harwood, 1988; Wurtele and Nikolau, 1990). And animals cells contain four biotinylated enzyme acetyl-CoA carboxylase (ACC; EC 6.4.1.2), 3-methy-lcrotonyl-CoA carboxylase (MCC; EC 6.4.1.4), propionyl-CoA carboxylase (PCC; EC 6.4.1.3) and pyruvate carboxylase (PC; EC 6.4.1.1) (Zempleni and Mock, 1999). Investigation the level of free biotin in various organs of pea plants found that there is a free biotin in excess of protein-bound biotin (Hoppe and Zacher, 1985; Somers et al., 1993). In addition, changes in the amount of streptavdidn binding protein in dry seeds or in different organs are explained with regard to the possibility that in plants, as in mammals, biotin plays a specialized role in cell growth and differentiation (Hoppe and Zacher, 1985; Somers et al., 1993).

In the present paper streptavidin-alkaline phosphatase conjugate have been used to detect segregation pattern and numbers of isotypes of 78 kD streptavidin biotin-binding proteins (SBP) in the dried pea seeds, germinated seeds, and in different organs including stems, plumules and roots.

1 Results and discussion
Biosynthesis of biotin has been widely investigated in bacteria through combined biochemical and genetic studies (Eisenberg, 1987; Gloeckler et al., 1990). The purpose of this study was to investigate the changes in isotype of 78 kDa streptavidin binding proteins of Pisum sativum L. var Alaska in dormant seeds and during germination.

Extracted protein from dry pea seeds, embryos of 40 hours or different part of 72 hours embryos including plumules, stems, and roots were analyzed by 2D-PAGE, blotted and probed with streptavidinalkaline phosphatase conjugate and results shown in (Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5) respectively.

Figure 1 78 kDa (SBP) isotypes in dry pea seeds

Figure 2 78 kD (SBP) isotypes of embryos of 40 hours germinated pea seeds

Figure 3 78 kD (SBP) isotypes of stems of 72 hours germinated pea seeds

Figure 4 78 kD (SBP) isotypes of plumules of 72 hours germinated pea seeds

Figure 5 78 kD (SBP) isotypes of roots of 72 hours germinated pea seeds

Dry pea seeds have a unique isotypes, showing unsepartated mixture of isotypes with tenth protruding spots with pI value 6.07, 6.08, 6.12, 6.14, 6.20, 6.22, 6.25, 6.27, 6.30, 6.32, respectively. The most acidic and basic isotypes almost separated having a PI values 5.66, 5.82, 5.88, 5.91 and 6.55. Partially separated isotypes in dry pea seeds having a pI value 5.92, 5.94, 5.96 and 5.98 (Figure 1). Embryos of forty hours germinated pea seeds have nine isoytpes with pI 5.76, 5.84, 5.89, 5.94, 6.16, 6.30, 6.42, 6.52 and 6.64. The one with PI value 6.30 is the most prominent and having two spots with slight different molecular weights, the most lower spots almost separated from the upper one (Figure 2). Data presented in figure 3 showed stems parts of 72 hours germinated pea seeds that contain the highest expressed isotypes than other investigated parts. Stem contains eleven isotypes of 87 kD (SBP) with pI values 5.60, 5.88, 5.97, 6.05, 6.13, 6.18, 6.27, 6.30, 6, 39, 6.51 and 6.65. The isotype with PI 6.3 was most abundant and distinguished into three spots with slight different molecular weight and the isotypes with PI 6.39 next most abundant and distinguished into two spots with different molecular weight. Plumules of 72 hours germinated pea seeds have two distinguished unseparated parts of isotypes (Figure 4). The isotypes part with a PI value ranged from 5.78~6.08 having six isotypes with PI values 5.78, 5.87, 5.90, 5.98, 6.02 and 6.08. And the other basic part with PI value ranged from 6.30~6.46 having four isotypes with pI values 6.30, 6.40, 6.42, and 6.46. The isoelectric points of 87 kD (SBP) isotypes in pea roots of 72 hours after germination were located in a range of pI between 5.75 and 6.67 (Figure 5). It contains two parts of isotypes, the acidic one have three isotypes with PI value 5.75, 5.82, and 5.95, and the basic isotypes almost unseparated with PI value varied from 6.27~6.68. The basic part distinguished into three overlapped isotypes and the lower molecular weight spots almost separated from other spot. The data presented here show changes in isotypes pattern of 78 kD (SBP) in dry seed, and during germination and in different parts of pea embryos indicating that post-translational modification was involved or may be caused by progressive phosphorylation during seeds dormancy and during germination.

Since investigating the biochemical properties of 49 kD apyrase isotype obtained from cytoskeleton fraction of germinating pea seeds, found that there were five isotypes with different isoelectric point (5.82, 6.05, 6.30, 6.55, and 6.80), that may be due to post-translational modification (Abe et al., 2002). And another study indicated that formation of similar isotypes caused by progressive phosphorylation with increasing acidity and increasing apparent molecular mass (Duncan and Song, 1999). What might various roles of 87 kD (SBP) isotype be? There is evidence suggesting that biotin and biotin-containing proteins might play specialized roles in regulation of plant development. Thus biotinylated enzymes are required for both growth of vegetative tissues and synthesis of storage lipids in developing seeds (Stumpf, 1980; Harwood, 1988). It is possible that various proteins isotypes function is not merely to hydrolyze nucleoside phosphates, instead they may be involved in some other action, such as mRNA transport along the cytoskeleton (Davies et al., 2001). Changes in tubulin isotypes in rye roots induced by low temperature revealed that the cold stability of microtubules is altered by growth temperature and this cold stability may be related to freezing tolerance (Kerr and Carter, 1990). Additionally, changes in tubulin isotypes in rye roots were noted after only 2 d and 4 d at 4℃, and pronounced changes in the α-tubulins occurred in case β-tubulin isotypes were affected by low temperature (Gregory and John, 1990).

The polymorphism of aldehyde oxidase isoforms observed in both leaves and roots of pea seedlings that changed during plant vegetative development and the activity and protein level of each isoform is regulated not only by environmental conditions but also through plant developmental stages (Zdunek-Zastocka et al., 2004). A proteome study based on 2-D gel electrophoresis followed by mass spectrometric found that 21 spots with more than 1.5-fold altered expression and majority of the differentially expressed proteins belonged to the functional category of signal transduction mechanisms and inorganic ion transport and metabolism (Kumar Swami et al., 2011). This will give an explanation that the isotypes of 78 kDa (SBP) most likely has a multiple roles and most likely inducing a number of specific genes and signal transduction that is leads to protection of cellular structures from damage during dormancy and induce various metabolic process associated with germination. Highly expressed biotin isotypes in pea stem than plumules and roots, probably playing a critical role in cellular differentiation and plant growth. Similarly, it has been reported that the amount of 49 kDa isotypes was higher in the stem than other investigated parts and play a crucial role during germination of pea seed (Moustafa et al., 2003).

2 Conclusion
The present report demonstrates that isotypes of 78 kD streptavidin biotin-binding proteins (SBP) display a specific pI range during seed dormancy and during germination and in different tissues. Further studies on their primary sequences and purification of these isotypes may provide more detailed information on the genetics, biochemical and physiological significance of 78 kD streptavidin binding protein in different plants.

3 Materials and methods
3.1 Sample preparation
Seeds of Alaska peas (Pisum sativum L. var. Alaska) were imbibed for 10 hours and then germinated in vermiculite in plastic boxes in a dark room for 72 hours at 21℃~23℃, as described before (Abe and Davies, 1991; Abe and Davies, 1995). At each time point of 0 hours, 40 hours and 72 hours after germination, three grams of intact embryonic tissue or separate plumules, stems, and roots were dissected from the cotyledons, collected on ice, and ground with a mortar and pestle in 7 volumes of cytoskeleton-stabilizing buffer (CSB) consisting of 5 mmol/L HEPES-KOH (pH 7.5), 10 mmol/L Mg (OAc)₂, 2 mmol/L of ethylenebis (oxyethylenenitrilo) tetraacetic acid, and 1 mmol/L phenylmethylsulphonyl fluoride with the addition of 0.5% polyoxyethylene-10-tridecyl ether and homogenate was filtered through Miracloth (Calbiochem, San Diego, CA, USA).

3.2 Two-dimensional PAGE
An equal amount of protein (40 µg) from each sample was precipitated with 4 volumes of acetone at -20℃ overnight. The precipitate was washed with 80% acetone, dissolved in 8 mol/L urea, 0.5 % NP-40, 2% β-mercaptoethanol, 0.8% Pharmalyte® 3-10 for IEF (Amersham Pharmacia Biotech), and 0.01% BPB, incubated at 39℃ for 1 hour, applied onto a drystrip gel (pI 4-7, Amersham Pharmacia Biotech) equilibrated with 8 mol/L urea, 0.5% Triton-X100, 10 mmol/L DTT, 2 mmol/L acetic acid, and 0.01 mg/mL orange G and subjected to IEF at 22 650 V h using a Multiphor II 2D-Electrophoresis System (Amersham Pharmacia Biotech). The strip was immersed in 6 mol/L urea, 0.05 mol/L Tris-HCl (pH 6.8), 30% glycerol, 1% SDS, and 16 mmol/L DTT for 10 min, followed by 6 mol/L urea, 0.05 mol/L Tris-HCl (pH 6.8), 30% glycerol, 1% SDS, and 0.01% bromophenol blue for 10 min, both with gentle shaking as described by Moustafa et al (2003).

3.3 Immunodetection of 78 kD biotin isotypes
The strip was placed onto a separating slab gel for SDS-PAGE. After electrophoresis, the gels were blotted onto a polyvinylidene fluoride membrane (ImmobilonTM Transfer Membranes, Millipore) and probed with the biotinylated anti-rat Ig species-specific whole antibody from sheep (Amersham Pharmacia Biotech) as the primary antibody. The binding of the primary antibody was detected with streptavidin-alkaline phosphatase conjugate (Amersham Pharmacia Biotech) with 5-Bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium as substrates.

Authors' contributions
Mahmoud F.M. Moustafa conducted all the work.

Acknowledgments
This study was funded by Faculty of Science, South valley University in Qena, and Ministry of Higher Education and the State for Scientific Research in Egypt.

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