Frank A. Loewus

myo-Inositol metabolism in plants

The multifunctional position supplied by myo-inositol is emerging as a central feature in plant biochemistry and physiology. In this critique, attention is drawn to metabolic aspects and current assessment is made of manifold ways in which myo-inositol and its metabolic products impact growth and development. The fact that a unique enzyme, common to all eukaryotic organisms where such assessment has been undertaken, controls conversion of D-glucose-6-P to 1L-myo-inositol-1-P provides a useful point of departure for this brief metabolic survey. Some aspects such as biosynthesis, phosphate and polyphosphate ester hydrolysis, and O-methylation of myo-inositol have captured the consideration of molecular biologists, yet other aspects including oxidation, conjugation, and transfer to phospholipids remain virtually untouched from this viewpoint. Here, an attempt is made to enlist new interest in all facets of myo-inositol metabolism and its place in plant biology.

Biosynthesis and metabolism of ascorbic acid in plants and of analogs of ascorbic acid in fungi

Abstract Despite the plethora of reviews on l -ascorbic acid, few stray beyond its established roles as an agent in redox-associated reactions or its biomedical importance. Here, an attempt is made to draw attention to l -ascorbic acid biosynthesis and metabolism in plants and to structurally similar compounds in fungi. During the decade since this subject was last addressed, a rational view of ascorbic acid biosynthesis has emerged, fresh evidence of a biosynthetic role for l -ascorbic acid in oxalic acid biosynthesis has been gained, and the biosynthetic and metabolic processes associated with ascorbic acid analogs in fungi have been explored.

L-Ascorbic acid and L-galactose are sources for oxalic acid and calcium oxalate in Pistia stratiotes

Axenic Pistia stratiotes L. plants were pulse-chase labeled with [14C]oxalic acid, L[1-14C]ascorbic acid, L-6-14C]ascorbic acid, D-[1-14C]erythorbic acid, L-[1-14C]galactose, or [1-14C]glycolate. Specific radioactivities of L-ascorbic acid (AsA), free oxalic acid (OxA) and calcium oxalate (CaOx) in labeled plants were compared. Samples of leaf tissue were fixed for microautoradiography and examined by confocal microscopy. Results demonstrate a biosynthetic role for AsA as precursor of OxA and its crystalline deposition product, CaOx, in idioblast cells of P. stratiotes and support the recent discovery of Wheeler, Jones and Smirnoff (Wheeler, G.L., Jones M.A., & Smirnoff, N. (1998). The biosynthetic pathway of vitamin C in higher plants. Nature, 393, 365-369) that L-galactose is a key intermediate in the conversion of D-glucose to AsA in plants. D-[1-14C]erythorbic acid (a diastereomeric analog of AsA) is utilized also by P. stratiotes as a precursor of OxA and its calcium salt deposition product in idioblasts. Labeled OxA is rapidly incorporated into CaOx in idioblasts, but microautoradiography shows there is also significant incorporation of carbon from OxA into other components of growing cells, contrary to the dogma that OxA is a relatively stable end product of metabolism. Glycolate is a poor substrate for synthesis of OxA and CaOx formation, further establishing AsA as th immediate precursor in the synthesis of OxA used for calcium precipitation in crystal idioblasts.

Biosynthesis and metabolism of ascorbic acid in plants

The biosynthesis of L‐ascorbic acid in plants differs from that encountered in ascorbic acid‐synthesizing animals. Enzymic details are sparse, but in vivo studies with tracers clearly establish the stereochemical detail of both processes. Examples of each process are found in separate classes of algae. Plants utilize L‐ascorbic acid as the carbon source for the biosynthesis of two important plant acids, oxalic acid and L‐tartaric acid. Here, cleavage of L‐ascorbic acid between carbons 2 and 3 releases the 2 and 4 carbon intermediates. A second L‐tartaric acid‐synthesizing process peculiar to vitaceous plants, i.e., grape, cleaves ascorbic acid between carbons 4 and 5. The physiological significance of these metabolic interconversions is discussed. Other metabolic processes such as the oxidation/reduction properties of L‐ascorbic acid are also considered.

Isolation and identification of erythroascorbic acid in Saccharomyces cerevisiae and Lypomyces starkeyi

The acid-soluble extract from Lypomyces starkeyi contains an unknown acidic, electrochemically-reactive compound with the retention time relative to ascorbic acid of 0.87 when separated by high performance liquid chromatography (HPLC) on an ion exchange column (Leung and Loewus, Plant Sci., 38 (1985) 65). Incubation of L. starkeyi under anaerobic conditions results in loss of this unknown retention time relative to l-ascorbic acid using HPLC (RAA)0.87 component and accumulation of a new acidic, electrochemically-reactive compound, RAA1.15, identified by gas chromatography/mass spectrometry (GC/MS) as erythroascorbic acid. Erythroascorbic acid, but not the unknown compound RAA0.87, is also found in acid-soluble extracts from Saccharomyces cerevisiae, strain G-25 and a commercial bakers yeast.

myo-Inositol: Biosynthesis and Metabolism

Publisher Summary This chapter discusses the biosynthesis and metabolism in myo -inositol. The awareness about the role of myo -inositol in carbohydrate metabolism emerges not only from an appreciation of the biosynthetic origin and metabolic fate of myo -inositol in higher plants, but also from the realization that hitherto unknown or poorly understood biochemical processes may at present be reconsidered in the light of new observations. Virtually, nothing is known concerning the regulation of myo -inositol biosynthesis, myo -Inositol-1-P synthase appears to be the only enzyme involved in myo-inositol biosynthesis, plant or animal. Conversion of myo -inositol-1-P to inositol must be studied more thoroughly as the free inositol pool is a very important nutritional reserve to the newly germinating seed or the germinating pollen grain. In regard to the metabolism of myo -inositol, there is very little information available regarding the fate of myo -inositol as derived from the reserves of phytic acid in the germinating seed. The information gathered suggests that the core substance of phytic acid is every bit as useful to the new plant as the phosphate it sheds.

Determination of ascorbic acid in algae by high-performance liquid chromatography on strong cation-exchange resin with electrochemical detection☆

Abstract High-performance liquid chromatography on a strong cation-exchange column has been used to determine l -ascorbic acid in algae and in their growth media by electrochemical detection. Work-up procedures, optimized conditions for separation of l -ascorbic acid, and the noninterference of several sugars, lactones, and acids with this assay are described.

Localization of constitutive phytases in lily pollen and properties of the pH 8 form

Abstract Studies on the subcellular localization and selected properties of consitutive phosphohydrolases in pollen from Lilium longiflorum Thunb. (pH 5 phytase, pH 8 phytase, myo -inositol monophosphatase) are described. In anthers, all three enzymes increased in activity during pollen maturation along with levels of phytic acid. These enzymes were localized in mature pollen by the use of lead capture cytochemistry and shown to be associated with the membrane of the organelle previously identified as the storage site of phytic acid in the form of its insoluble salt, phytin. Recovery of the phytase with optimal activity at pH 8 was most complete when detergent or phospholipase C was included in the extracting medium, suggesting that its association with the organelle membrane is more integral than that of the other enzymes. The pH 8 phytase exhibited differences from a pH 5 phytase which has not been previously reported. They include higher substrate specificity for phytate, terminal hydrolysis to myo -inositol trisphosphate (stereochemistry undetermined), and lack of inhibition of fluoride. Our results suggest a subcellular organization of phytic acid metabolism in lily pollen with the pH 8 phytase as an important component.

Partial purification and study of pollen glucuronokinase.

Abstract Glucuronokinase was purified 31-fold from pollen of Lilium longiflorum . The enzyme was inhibited by its product, α- d -glucuronate-1-P, and by UDP- d -glucuronate, and these compounds were competitive inhibitors. Inhibitor constants were 0.18 m m for d -glucuronate-1-P and 0.55 m m for UDP- d -glucuronate. These effects may have regulatory significance; both inhibitors are intermediates in the pathway by which plant cells convert myo -inositol into cell wall uronides and pentoses, and glucuronokinase is a likely step for regulation in this pathway. The enzyme exhibited considerable specificity concerning inhibitors, and an additional 22 compounds were not inhibitory. These included uronic acids other than d -glucuronate and compounds related structurally or metabolically to d -glucuronate.

The C-5 hydrogen isotope-effect in myo-inositol 1-phosphate synthase as evidence for the myo-inositol oxidation-pathway

The hydrogen isotope-effect that occurs in vitro during myo-inositol1-phosphate synthase-catalyzed conversion of D-[5-3H]glucose 6-phosphate into myo[2-3H]inositol 1-phosphate has been used to compare the functional role of the nucleotide sugar oxidation-pathway with that of the myo-inositol oxidation-pathway in germinating lily pollen. Results reveal a significant difference between the 3H/14C ratios of glucosyl and galactosyluronic residues from pectinase-amyloglucosidase hydrolyzates of the 70% ethanol-insoluble fraction of D-[5-3H, 1-14C]glucose-labeled, germinating lily pollen. This isotope effect at C-5 of D-glucose that occurred during its conversion into D-galactosyluronic residues of pectic substance is not explained by loss of 3H when UDP-D-[5-3H, 1-14C]glucose is oxidized by UDP-D-glucose dehydrogenase from germinating lily pollen. The evidence obtained from this study favors a functional role for the myo-inositol oxidation-pathway during in vivo conversion of glucose into galactosyluronic residues of pectin in germinating lily pollen.