Inositol-1 (or 4)-monophosphatase from Glycine max embryo axes is a phosphatase with broad substrate specificity that includes phytate dephosphorylation

• Published in: Biochimica et biophysica acta Vol. 1770; no. 4; pp. 543 - 550
• Main Authors: Islas-Flores, Ignacio, Villanueva, Marco A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2007
• Subjects: Adenosine Triphosphate - chemistry; Adenosine Triphosphate - metabolism; Amino Acid Sequence; Cations, Divalent - chemistry; Diphosphates - chemistry; Diphosphates - metabolism; Enzyme Induction; Enzyme Stability; Germination; Glycine max; Glycine max - embryology; Glycine max - enzymology; Hot Temperature; Hydrolysis; IMPase; Inositol Phosphates - chemistry; Inositol Phosphates - metabolism; Kinetics; Lithium Chloride - chemistry; Molecular Sequence Data; Molecular Weight; Phosphatase; Phosphoric Monoester Hydrolases - antagonists & inhibitors; Phosphoric Monoester Hydrolases - biosynthesis; Phosphoric Monoester Hydrolases - chemistry; Phosphoric Monoester Hydrolases - isolation & purification; Phosphorylation; Phytate; Phytic Acid - chemistry; Phytic Acid - metabolism; Protein Denaturation; Seeds; Seeds - enzymology; Sodium Fluoride - chemistry; Substrate Specificity; Time Factors; Seeds; DTT; Phosphatase; ADP; glucose 6-P; NaPPi; p-NPP; Pi; MSB; SDS; AMP; Germination; PAGE; IEF; EDTA; PIPES; IMP; Glycine max; EGTA; IMPase; Phytate; DEAE; ATP; β-ME; PPase
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2006.12.001

A phosphate-hydrolyzing activity from Glycine max embryo axes was purified by a series of chromatographic steps and electroelution from activity gels, and demonstrated to be an inositol-1 (or 4)-monophosphatase by partial internal amino acid sequence. This enzyme hydrolyzed ATP, sodium pyrophosphate (NaPPi), inositol hexakisphosphate, and inositol 1-monophosphate, but not p-nitrophenyl phosphate, ADP, AMP or glucose 6-P. Using NaPPi as substrate, the highly purified protein hydrolyzed up to 0.4 mmol phosphate min − 1  mg − 1 protein and had a Km avg of 235 μM for NaPPi. Since NaPPi is relatively inexpensive and readily available, we used this as substrate for the subsequent characterization. We observed the following: (a) specific inhibition by Li and NaF but not by butanedione monoxime, or orthovanadate; (b) activation by Cu 2+ and Mg 2+; (c) optimum activity at pH 7.4; and (d) temperature stability after 1-h incubations at 37–80 °C, with maximum activity at 37 °C. The partially purified protein was detected by in-gel activity assays and the band was electroeluted to yield a highly purified protein. Analysis by SDS-PAGE and native IEF-PAGE yielded a single major polypeptide of 29 kDa and pI ∼ 5.9, respectively. In addition, in-gel activity from embryo axes and whole hypocotyls at early germination times revealed one high and one intermediate molecular weight isoform, but only the intermediate one corresponded to IMPase. Throughout the post-imbibition period, the activity of the high molecular weight isoform disappeared and IMPase increased, indicating an increasing expression of the enzyme as germination and growth proceeded. These data indicate that the inositol-1 (or 4)-monophosphatase present in the embryo axis of G. max has a wide phosphate substrate specificity, and may play an important role in phosphate metabolism during the germination process.