Phytases are the primary enzymes responsible for the hydrolysis of phytic acid, myo-inositol-1,2,3,4,5,6-hexakisphosphate (I-1,2,3,4,5,6-P,). A number of phytases with varying specificities, properties, and localizations hydrolyze phytic acid present in cells. l h e specificity of hydrolysis of phytic acid by alkaline phytase from lily (Lilium longiflorum 1.) pollen is described. Structures of the intermediate inositol phosphates and the final product were established by a variety of nuclear magnetic resonance techniques ('H-, 31P-, and 3'P-'H-detected multiple quantum coherence spectroscopy, and total correlation spectroscopy). O n the basis of the structures identified we have proposed a scheme of hydrolysis of phytic acid. Initial hydrolysis of the phosphate ester occurs at the D-5 position of phytic acid to yield the symmetrical I-1,2,3,4,6-P5. The two subsequent dephosphorylations occur adjacent to the D-5 hydroxyl group to yield I-1,2,3-P3 as the final product. Alkaline phytase differs from other phytases in the specificity of hydrolysis of phosphate esters on the inositol ring, its high substrate specificity for phytic acid, and biochemical properties such as susceptibility to activation by calcium and inhibition by fluoride. l h e physiological significance of alkaline phytase and the biological role of I-1,2,3-P3 remain to be identified.Phytic acid, myo-inositol hexakisphosphate, is a major constituent of seeds and pollen grains (1-5% of dry weight) (Loewus, 1990;Raboy, 1990). In mature lily (Lilium longiflorum L.) pollen and seeds, phytic acid is localized in membrane-bound phytic-rich granules. The presence of phytic acid in plant cells has been known for some time (Loewus, 1990;Raboy, 1990). However, it was only recently recognized that phytic acid is present in virtually all mammalian cells in concentrations higher than most other inositol phosphates (Menniti et al., 1993) and may function as a neurotransmitter (Vallejo et al., 1988). The discovery that I-1,4,5-P3 plays a crucial role in calcium cellular signaling (Bemdge et al., 1989) has greatly increased interest in the structure, metabolism, and biological role of inositol phosphates, including phytic acid.The primary enzymes responsible for the degradation of phytic acid are phytases. Phytases are a special class of phosphatases that catalyze the sequential hydrolysis of phytic acid to inositol phosphates and, in some cases, to inositol. Phytases occur in a variety of organisms including plants, fungi, and animals (reviewed by Cosgrove, 1980a). A variety of phytases differing in pH optima, substrate specificity, and * Corresponding author; fax 1-906-487-2061. specificity of hydrolysis have been identified in plants and fungi (Cosgrove, 1980a(Cosgrove, , 1980b. Acid phytases from wheat bran and Aspergilli have been extensively studied and the stereospecificity of hydrolysis has been well established (Cosgrove, 1980b). Based on the specificity of initial hydrolysis, two classes of acid phytases are recognized by the Intemational Union of Pure a...

Alkaline phytase activity, with a pH optimum of 8, was recovered from detergent extracts of dormant seeds of nine vaneties of Phaseolus vulgaris L., Pisum sativum L. var. Early Alaska, and Medicago sativa L. This alkaline phytase of legume seeds was activated by calcium and differed from most seed phytases in its relative insensitivity to inhibition by fluoride. activity differed from the pH 5 phytase in its relative insensitivity to inhibition by fluoride and the more pronounced enhancement of its activity by calcium. MATERIALS AND METHODS SeedsMany plant seeds contain significant amounts of phytic acid (myo-inositolhexakisphosphoric acid) which is degraded during germination by one or more phytases (myo-inositolhexakisphosphoric acid phosphohydrolase) (15). Phytic acid thus serves as an important store of phosphate and inositol, which is mobilized during seed germination. There is evidence that phytic acid also serves as an important antioxidant, contributing to the longevity of dormant seeds by preventing lipid peroxidation (8).Seeds contain both constitutive phytase activity and phytases that are synthesized de novo during germination (14). Most seed phytases which have been studied to date belong to a special class ofnonspecific acid phosphatases with optimal activity between pH 4.0 and 5.6. In addition to phytic acid hydrolysis, these enzymes are able to hydrolyze a variety of natural and synthetic phosphate esters. In terms of the rate of hydrolysis, phytic acid has occasionally been shown to be one of the poorer substrates for seed phytase (1 1, 13).Phytases which are relatively specific for phytic acid have been isolated from Typha lattifolia pollen (9) Preparation of Seed ExtractsFor preparation of extracts for phytase analysis, 10 g of seeds were ground to a fine powder in a prechilled mortar and pestle. To this powder, 100 mL of chilled extraction buffer [0.02 M Tris-HCl (pH 7.6) containing 0.1 % Triton X-100] was added along with 5 g of acid-washed sand. The mixture was homogenized until a uniform slurry was obtained and then centrifuged at 12,000g for 30 min at 4°C. The supernatant was dialyzed overnight at 4°C against the TrisHCl buffer (pH 7.6) without Triton X-100. The dialyzed extracts were used for phytase assays without further purification. Phytase AssaysThe reaction mixture for phytase assay contained 0.

The inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ compartment of endocrine cells was studied with alpha-toxin- and digitonin-permeabilized rat insulinoma (RINA2) and rat pheochromocytoma (PC12) cells. The Ca2+ uptake was ATP-dependent, and submicromolar concentrations of IP3 specifically released the stored Ca2+. Half-maximal Ca2+ release was observed with 0.25-0.5 mumol of IP3/l, and the amount of Ca2+ released due to IP3 could be enhanced by additional loading of the Ca2+ compartment. Consecutive additions of the same concentration of IP3 for 1-2 h always released the same amount of Ca2+ without desensitization, providing an ideal basis to further characterize the IP3-induced Ca2+ release. Here we describe for the first time a reversible inhibitory effect of decavanadate on the IP3-induced Ca2+ release. Among the vanadium species tested (decavanadate, oligovanadate and monovanadate), only decavanadate was inhibitory, with a half-maximal effect at 5 mumol/l in both cell types. The effect of decavanadate could be overcome by increasing the amount of sequestered Ca2+ or added IP3. Decavanadate did not affect the ATP-driven Ca2+ uptake but oligovanadate was inhibitory on Ca2+ uptake. p-Hydroxymercuribenzoate (pHMB) at concentrations between 10 and 30 mumol/l also inhibited the Ca2+ release due to IP3. Thiol compounds such as dithiothreitol (DTT; 1 mmol/l) added before pHMB removed all its inhibitory effect on the IP3-induced Ca2+ release, whereas the inhibition caused by decavanadate was unaffected by DTT. Thus, the decavanadate-dependent inhibition functions by a distinctly different mechanism than pHMB and could serve as a specific tool to analyse various aspects of the IP3-induced Ca2+ release within endocrine cells.

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