Soybean Root Nodule Acid Phosphatase

Abstract

Acid phosphatases are ubiquitous enzymes that exhibit activity against a variety of substrates in vitro, although little is known about their intracellular function. In this study we report the isolation, characterization, and partia1 sequence of the major acid phosphatase from soybean (Clycine max 1.) root nodules. l h e phosphatase was purified predominantly as a heterodimer with subunits of 28 and 31 kD; homodimers of both subunits were also observed and exhibited phosphatase activity. In addition to the general phosphatase substrate, pnitrophenyl phosphate, the heterodimeric form of the enzyme readily hydrolyzed 5'-nucleotides, flavin mononucleotide, and O-phospho-L-Tyr. Low or negligible activity was observed with ATP or polyphosphate. Purified nodule acid phosphatase was stimulated by magnesium, inhibited by calcium and EDTA, and competitively inhibited by cGMP and cAMP with apparent Ki values of 7 and 1 2 PM, respectively. Partia1 N-terminal and interna1 sequencing of the nodule acid phosphatase revealed homology to the soybean vegetative storage proteins. There was a 17-fold increase in enzyme activity and a noticeable increase in protein levels detected by immunoblotting methods during nodule development. Both of these parameters were low in young nodules and reached a peak in mature, functional nodules, suggesting that this enzyme is important for efficient nodule metabolism.General phosphohydrolases are classified as alkaline phosphatases or acids based solely upon whether their optimal activity is above or below pH 7.0 (Vincent et al., 1992). Whereas plant alkaline phosphatases have been characterized as having specific metabolic roles, most plant ACPs have been shown to hydrolyze a wide variety of phosphorylated substrates in vitro, making their cellular role(s) difficult to define (for review, see Duff et al. [1994]). One method of determining the substrate specificity of an ACP is to determine the kinetic parameters of specificity such as V,,,lK, with numerous substrates. This approach was taken by Duff et al. (1989aDuff et al. ( , 1989bDuff et al. ( , 1991 to characterize an ACP from Brassica nigra suspension-cultured cells as a PEP phosphatase.The difficulty in classifying plant ACPs is further compounded by a lack of available sequence information: Only

Legume nodules from nutrient-poor soils exhibit high plasticity of cellular phosphorus recycling and conservation during variable phosphorus supply

“…Furthermore, the increased intracellular and extracellular phosphohydrolase (APase and phytase) activity, would serve as an additional route of Pacquisition from organically-bound sources of P, during conditions of limiting soil P. The importance of increased APase activity for P metabolism has been extensively reported (Gilbert et al 1998, Miller et al 2001. Intracellular APases break down P nucleotides, sugar-P and P-monoesters and recycle P through the supply of P for amino acid biosynthesis and nodule metabolism during P-deprivation (Penheiter et al 1997). Previous studies have also shown that an increase in intracellular nodule APase activity may constitute an adaptive mechanism for N 2 fixing legumes to tolerate P deficiency (Kouas et al 2008, Bargaz et al 2012).…”

Chitinases from the Plant Disease Biocontrol Agent, Stenotrophomonas maltophilia C3

“…The major stained bands were excised, and the N-terminal amino acid sequence of the proteins were determined at the University of Nebraska-Lincoln Protein Core Facility (Biosystems Procise 494; Perkin-Elmer Applied Biosystems, Foster City, CA). Proteins that were blocked at the N terminus were subjected to enzymatic digestion and peptide separation according to the method of Penheiter et al (23). Ten bands of a protein (≈30 µg) were cut from 12% SDS-PAGE separation gels and digested with 0.3 µg of trypsin (Sigma Chemical) at 37°C for 20 h. The resultant peptides were fractionated on a 2.1 × 250 mm C18 column (Model 218 TP52, VYDAC, Hesperia, CA) (1 column volume = 0.866 ml) with a linear gradient from 2% CH 3 CN in 0.1% trifluoroacetic acid to 60% buffer B (90% CH 3 CN in 0.09% trifluoroacetic acid) over 15 column-volumes at 0.2 ml/min on a high performance liquid chromatography (Microbore; ISCO, Lincoln, NE).…”

Nonsymbiotic hemoglobins in rice are synthesized during germination and in differentiating cell types

“…Polyclonal antibodies were raised against this r-ns-Hb1 in rabbits by the Antibody Core Facility of the University of Nebraska-Lincoln. Serum titers were analyzed by Western blotting techniques (Penheiter et al 1997).…”