Measurement of the Pyrophosphate Content of Plant Tissues

Smyth, Douglas A.; Black, Clanton C.

doi:10.1104/pp.75.3.862

Cited by 88 publications

(55 citation statements)

References 15 publications

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“…Consequently, there is no thermodynamic obstacle to the conservation of the energy of hydrolysis of PPi in esterification and other reactions. Substrate concentrations of PPi in various tissues of pea and corn (23) suggest that the same may be true of higher plants.…”

Section: Discussionmentioning

confidence: 90%

Proton-Translocating Inorganic Pyrophosphatase in Red Beet (Beta vulgaris L.) Tonoplast Vesicles

Rea

Poole²

1985

Plant Physiol.

160

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ABSTRACrThe substrate and ionic requirements of ATP and inorganic pyrophosphate (PPi) hydrolysis by tonoplast vesicles isolated from storage tissue of red beet (Beta vulgaris L.) were compared with the requirements of ATP-and PPi-dependent proton translocation by the same material. Both ATP hydrolysis and ATP-dependent proton translocation are most stimulated by C-and inhibited by N03-. NaCI and KCI support similar rates of ATP hydrolysis and ATP-dependent proton translocation while K2S04 supports lesser rates for both. PPi hydrolysis and PPi-dependent proton translocation are most stimulated by KV. KCI and K2SO4 support similar rates of PPi hydrolysis and PPi-dependent proton translocation but NaCl has only a small stimulatory effect on both. Since PPi does not inhibit ATP hydrolysis and ATP does not interfere with PPi hydrolysis, it is inferred that the two phosphohydrolase and proton translocation activities are mediated by different tonoplast-associated enzymes. The results indicate the presence of an energy-conserving proton-translocating pyrophosphatase in the tonoplast of red beet.The primary active transport process across the plasma and vacuolar membranes of plant cells is considered to be the ATPdependent electrogenic translocation of protons (19,24). By extension of the chemiosmotic hypothesis (17), the proton electrochemical potential difference generated by proton translocation is thought to provide the requisite motive force for the secondary transport of a wide range of chemical species.While the primacy of ATP for the energization of transport across the membranes of plant cells is not in dispute, the involvement of other phosphoesters, PPi in particular, has yet to be thoroughly evaluated. Walker and Leigh (27) (22). The homogenization medium consisted of 53% (v/v) sterilized coconut milk or 10 mm glycerophosphate, 0.65 M ethanolamine (adjusted to pH 8.0 with concentrated H2SO4), 0.28 M choline chloride, 26 mm K-metabisulfite, 2 mM salicylhydroxamic acid, 0.2% (w/v) BSA (fraction, V, essentially fatty acid-free), 10% (w/v) insoluble PVP, 5 mM DTT, 0.5 mM butylated hydroxytoluene, and 1 mM nupercaine buffered to pH 8.0 with 70 mM Tris-Mes. The suspension medium consisted of 1.1 M glycerol, 1 mm Tris-EDTA, 1 mM nupercaine, 0.5 mM butylated hydroxytoluene, and 5 mM DTT buffered to pH 8.0 with 5 mM Tris-Mes. Choline and ethanolamine were included in the homogenization medium to minimize membrane degradation by phospholipase D while nupercaine and coconut milk, glycerophosphate, or glycerol were added to inhibit phospholipase A and phosphatidic acid phosphatase activity, respectively (22).The KI solution, employed to diminish contamination of the tonoplast vesicles with acid phosphatase (20), and the sucrose density gradient solutions were prepared in suspension medium.Sucrose Density Centrifugation. For the routine preparation of tonoplast vesicles, the 0.25 M KI-treated pellet from 300 to 350 g beet was resuspended in 12 ml suspension medium and 3 ml volumes were layered onto each of four step grad...

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Section: Discussionmentioning

confidence: 90%

Proton-Translocating Inorganic Pyrophosphatase in Red Beet (Beta vulgaris L.) Tonoplast Vesicles

Rea

Poole²

1985

Plant Physiol.

160

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“…But we were surprised also to find pyrophosphatase to be almost equally responsive. At this moment we do not have a hypothesis we can support about the roles of the pyrophosphatase responses but surely expect the roles to relate to the pool of PPi found in plant cells (10,21) and to polymer synthesis. Possibly, the competition of the pyrophosphatases with the UDP-glucopyrophosphorylase or the PPi-PFK is involved in these changes.…”

Section: Enzymes Of Sucrose Metabolism In the Germinating Lima Bean Seedmentioning

confidence: 99%

Sucrose Metabolism in Lima Bean Seeds

Xu¹,

Sung²,

Black³

1989

Plant Physiol.

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Developing and germinating lima bean (Phaseolus lunatus var Cangreen) seeds were used for testing the sucrose synthase pathway, to examine the competition for uridine diphosphate (UDP) and pyrophosphate (PPi), and to identify adaptive and maintenance-type enzymes in glycolysis and gluconeogenesis. In developing seeds, sucrose breakdown was dominated by the sucrose synthase pathway; but in the seedling embryos, both the sucrose synthase pathway and acid invertase were active. UDPase activity was low and seemingly insufficient to compete for UDP during sucrose metabolism in seed development or germination. In contrast, both an acid and alkaline pyrophosphatase were active in seed development and germination. The set of adaptive enzymes identified in developing seeds were sucrose synthase, PPi-dependent phosphofructokinase, plus acid and alkaline pyrophosphatase; and, the adaptive enzymes identified in germinating seeds included the same set of enzymes plus acid invertase. The set of maintenance enzymes identified during development, in the dry seed, and during germination were UDPglucopyrophosphorylase, neutral invertase, ATP and UTP-dependent fructokinase, glucokinase, phosphoglucomutase, ATP and UTP-dependent phosphofructokinase and sucrose-P synthase.Sucrose is a primary nutrient for essentially all higher plant cells. Recently, a new pathway of sucrose breakdown was proposed which is dependent upon UDP and PPi and that involves a cyclic series of reactions to produce the required UDP and PPi (4,13,23,31). When sucrose is broken down by this pathway, which is called the sucrose synthase pathway, both hexoses from sucrose can feed into glycolysis. In addition, we have proposed that in the plant cell cytoplasm, alternative enzymes are present at various steps in glycolysis and gluconeogenesis for the interconversion of sucrose and pyruvate such that they form a network of reactions rather than the classical textbook-type pathways. (3,19,24). In view of these and other recent discoveries such as a substrate level pool of PPi and fructose 2,6-P2 regulation (3, 4, 10, 21, 30), we have reassessed all ofplant glycolysis and gluconeogenesis, beginning with sucrose (24).Because the glycolytic and the gluconeogenic metabolism of sucrose are at a central junction in plant metabolism (7,12,28) MATERIALS AND METHODS All studies were conducted with lima beans, Phaseolus lunatus var Cangreen, purchased locally and grown with good cultural practices either in field plots or in the greenhouse. The studies were conducted over the last 3 years with both field and greenhouse grown plants. Each study was repeated in at least three similar tests and the data presented are illustrative of developmental patterns, recognizing that variations occurred, e.g. seedling emergence in a given study might occur on d 5 or 6, etc. Lima bean has an indeterminate flower production; hence, seed development work based on freshly harvested weights was highly facilitated. And seedling development work also was facilitated because the lima bean...

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“…The pyrophosphorolysis of UDP-glucose was proposed several decades ago (14,18) but never supported experimentally. At that time, and for many years, evidence for the presence of the key substrate, PPi, was missing; however, substrate level amounts of PPi were measured in corn, pineapple (21), and peas (13,21) in 1984 and now PPi has been measured in many plants. Furthermore,' in our work and from the plant literature (10,16,20,27), it was recognized that the ATP-dependent PFK may use other nucleotide triphosphates, e.g.…”

mentioning

confidence: 99%