scholarly journals Reconstitution of the hexose phosphate translocator from the envelope membranes of wheat endosperm amyloplasts

1996 ◽  
Vol 319 (3) ◽  
pp. 717-723 ◽  
Author(s):  
Ian J TETLOW ◽  
Caroline G BOWSHER ◽  
Michael J EMES
Keyword(s):  
Inorganic Phosphate ◽  
Competitive Inhibitor ◽  
Dihydroxyacetone Phosphate ◽  
Carrier Protein ◽  
Sugar Phosphate ◽  
Wheat Endosperm ◽  
Single Carrier ◽  
Phosphate Translocator ◽  
Envelope Membranes ◽  
Hexose Phosphates

Amyloplasts were isolated and purified from wheat endosperm and the envelope membranes reconstituted into liposomes. Envelope membranes were solubilized in n-octyl β-D-glucopyranoside and mixed with liposomes supplemented with 5.6 mol% cholesterol to produce proteoliposomes of defined size, which showed negligible leakage of internal substrates. Transport experiments with proteoliposomes revealed a counter-exchange of glucose 1-phosphate (Glc1P), glucose 6-phosphate (Glc6P), inorganic phosphate (Pi), 3-phosphoglycerate and dihydroxyacetone phosphate. The Glc1P/Pi counter-exchange reaction exhibited an apparent Km for Glc1P of 0.4 mM. Glc6P was a competitive inhibitor of Glc1P transport (Ki 0.8 mM), and the two hexose phosphates could exchange with each other, indicating the operation of a single carrier protein. Glc1P/Pi antiport in proteoliposomes showed an exchange stoichiometry at pH 8.0 of 1 mol of phosphate transported per mol of sugar phosphate.

scholarly journals Evidence for two types of phosphate translocators in sweet-pepper (Capsicum annum L.) fruit chromoplasts

1996 ◽  
Vol 320 (1) ◽  
pp. 7-10 ◽  
Author(s):  
Paul W. QUICK ◽  
H. Ekkehard NEUHAUS
Keyword(s):  
Physiological Function ◽  
Sweet Pepper ◽  
Dihydroxyacetone Phosphate ◽  
Envelope Membrane ◽  
External Concentration ◽  
Capsicum Annum ◽  
Phosphate Translocator ◽  
Pepper Fruit ◽  
Different Types ◽  
Envelope Membranes

We have investigated whether there is evidence for the presence of different types of phosphate translocators in envelopes purified from pepper-fruit chromoplasts. A method was developed that allowed the purification of envelope membranes from isolated pepper-fruit chromoplasts. Proteoliposomes containing envelope-membrane proteins are able to import inorganic phosphate (Pi) or glucose 6-phosphate (Glc6P). In both cases, the rate of import is strongly dependent upon preloading of proteoliposomes with either Pi, dihydroxyacetone phosphate (DHAP) or Glc6P. This demonstrates the presence of a phosphate translocator activity catalysing a counter exchange of phosphorylated intermediates. Interestingly, a high external concentration of Glc6P does not strongly inhibit Pi uptake into proteoliposomes preloaded with DHAP, whereas external Glc6P strongly inhibits Pi uptake into proteoliposomes preloaded with Glc6P. This observation strongly indicates that two types of phosphate translocator are present in chromoplast envelopes from red-pepper fruits. These data are discussed with respect to the possible physiological function of two types of phosphate translocator in one type of plastid.

scholarly journals Analysis of carbohydrate transport across the envelope of isolated cauliflower-bud amyloplasts

1995 ◽  
Vol 307 (2) ◽  
pp. 521-526 ◽  
Author(s):  
T Möhlmann ◽  
O Batz ◽  
U Maass ◽  
H E Neuhaus
Keyword(s):  
Envelope Protein ◽  
Starch Synthesis ◽  
Specific Inhibitor ◽  
Competitive Inhibitor ◽  
Transport Proteins ◽  
Dihydroxyacetone Phosphate ◽  
Envelope Membrane ◽  
Phosphate Translocator ◽  
Low Concentrations ◽  
Carbohydrate Transport

Using isolated amyloplasts from cauliflower buds, we have characterized the interaction and transport of various carbohydrates across the envelope membrane of a heterotrophic plastid. According to our results, glucose 6-phosphate (Glc6P) and glucose 1-phosphate (Glc1P) do not share the same transport protein for uptake into cauliflower-bud amyloplasts. Glc6P-dependent starch synthesis is strongly inhibited in the presence of dihydroxyacetone phosphate (DHAP) or 4,4′-di-isothiocyano-2,2′- stilbenedisulphonic acid (DIDS), whereas Glc1P-dependent starch synthesis is hardly affected by these compounds. Analysis of the Glc6P uptake into proteoliposomes reconstituted from the envelope proteins of cauliflower-bud amyloplasts indicate that Glc6P is taken up in a counter-exchange mode with Pi, DHAP or Glc6P, whereas Glc1P does not act as a counter-exchange substrate. Pi is a strong competitive inhibitor of Glc6P uptake (Ki 0.8 mM) into proteoliposomes, whereas Glc1P does not significantly inhibit Glc6P transport. Beside a hexose-phosphate translocator, these amyloplasts possess an envelope protein mediating the transport of glucose across the membrane. This translocator exhibits an apparent Km for glucose of 2.2 mM and is inhibited by low concentrations of phloretin, known to be a specific inhibitor of glucose-transport proteins. Maltose inhibits the uptake of glucose (Ki 2.3 mM), indicating that both carbohydrates share the same translocator.

scholarly journals Fructose 1,6-bisphosphate aldolase from rabbit muscle. The isomerization of the enzyme-dihydroxyacetone phosphate complex

1977 ◽  
Vol 167 (2) ◽  
pp. 361-366 ◽  
Author(s):  
E Grazi ◽  
M Blanzieri
Keyword(s):  
Competitive Inhibitor ◽  
Dihydroxyacetone Phosphate ◽  
Rabbit Muscle ◽  
Keto Form ◽  
First Order ◽  
Phosphate Complex ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Dead End ◽  
Fructose 1,6 Bisphosphate

The formation and dissociation of the aldolase-dihydroxyacetone phosphate complex were studied by following changes in A240 [Topper, Mehler & Bloom (1957), Science 126, 1287-1289]. It was shown that the enzyme-substrate complex (ES) slowly isomerizes according to the following reaction: (formula: see text) the two first-order rate constants for the isomerization step being k+2 = 1.3s-1 and k-2 = 0.7s-1 at 20 degrees C and pH 7.5. The dissociation of the ES complex was provoked by the addition of the competitive inhibitor hexitol 1,6-bisphosphate. At 20 degrees C and pH 7.5, k+1 was 4.7 X 10(6)M-1-S-1 and k-1 was 30s-1. Both the ES and the ES* complexes react rapidly with 1.7 mM-glyceraldehyde 3-phosphate, the reaction being practically complete in 40 ms. This shows that the ES* complex is not a dead-end complex. Evidence was also provided that aldolase binds and utilizes only the keto form of dihydroxyacetone phosphate.

scholarly journals Glycogen synthesis in amphibian oocytes: evidence for an indirect pathway

1996 ◽  
Vol 315 (2) ◽  
pp. 455-460 ◽  
Author(s):  
Eduardo KESSI ◽  
Victoria GUIXÉ ◽  
Ana PRELLER ◽  
URETA URETA
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Glycogen Synthesis ◽  
Lactate Production ◽  
Dihydroxyacetone Phosphate ◽  
Indirect Pathway ◽  
Ic50 Value ◽  
Glucose Incorporation ◽  
Relationship Of ◽  
Hexose Phosphates

Glycogen is the main end product of glucose metabolism in amphibian oocytes. However, in the first few minutes after [U-14C]glucose microinjection most of the label is found in lactate. The burst of lactate production and the shape of the time curves for the labelling of glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate and glycogen suggest a precursor–product relationship of lactate with respect to glycogen and its intermediates. Expansion (by microinjection) of the pool of glycolytic intermediates, such as dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 3-phosphoglycerate or phosphoenolpyruvate, results in a marked decrease in [U-14C]glucose incorporation into glycogen. After co-injection of doubly labelled glucoses, extensive detritiation (93%) of the glucosyl units of glycogen was observed with [2-3H,U-14C]glucose and partial detritiation with [3-3H,U-14C]glucose (34%) or [5-3H,U-14C]glucose (46%). After injection of [6-3H,U-14C]glucose, a small but significant and reproducible detritiation (13%) in glycogen was observed. Co-injection of [U-14C]glucose and 3-mercaptopicolinate resulted in marked inhibition of glycogen labelling. Half-maximal inhibition was observed at 0.58 mM 3-mercaptopicolinate, which agrees with the IC50 value (0.47 mM) for the inhibition in vitro of phosphoenolpyruvate carboxykinase activity. We conclude that in frog oocytes most of the glucosyl units are incorporated into glycogen by an indirect pathway involving breakdown of glucose to lactate, which is then converted into glycogen via gluconeogenesis. Both processes, glycolytic degradation of glucose to lactate and subsequent reconversion of the latter into hexose phosphates and glycogen, occur in the same cell.

scholarly journals Regulatory Properties of Apple Leaf Cytosolic Fructose-1,6-bisphosphatase

HortScience ◽  
2004 ◽  
Vol 39 (4) ◽  
pp. 761C-761
Author(s):  
Rui Zhou* ◽  
Lailiang Cheng
Keyword(s):  
Enzyme Activity ◽  
Adenosine Monophosphate ◽  
Competitive Inhibitor ◽  
Inhibitory Effect ◽  
Carbon Partitioning ◽  
Dihydroxyacetone Phosphate ◽  
Saturation Curve ◽  
Fruit Species ◽  
Apparent Homogeneity ◽  
Fructose 1,6 Bisphosphatase

Cytosolic fructose-1,6-bisphosphatase (cytoFBPase) (EC 3.1.3.11) occupies a strategic site in sucrose synthesis and has been demonstrated to play a key role in carbon partitioning between sucrose and starch in non-sorbitol forming plants. In addition to sucrose and starch, Sorbitol is the primary photosynthetic end product in the leaves of many tree fruit species in the Rosaceae family. To understand the biochemical regulation of photosynthetic carbon partitioning between sorbitol, sucrose and starch in sorbitol synthesizing species, we purified cytoFB-Pase to apparent homogeneity from apple leaves. The enzyme was a homotetramer with a subunit mass of 37 kDa. It was highly specific for fructose-1,6-bisphosphate with a Km of 3.1 μm and a Vmax of 48 units/mg protein. Either Mg2+ or Mn2+ was required for its activity with a Km of 0.59 mm and 62 μM, respectively. Li+, Ca2+, Zn2+, Cu2+ and Hg2+ inhibited whereas Mn2+ enhanced the Mg2+-activated enzyme activity. Fructose-6-phosphate was found to be a mixed type inhibitor with a Ki of 0.47 mm. Fructose 2,6-bisphosphate (F2,6BP) competitively inhibited the enzyme activity and changed the substrate saturation curve from hyperbolic to sigmoidal. Adenosine monophosphate (AMP) was a non-competitive inhibitor for the enzyme. F2,6BP interacted with AMP to inhibit the enzyme in a synergistic way. Dihydroxyacetone phosphate did not have inhibitory effect on apple leaf cytosolic FBPase activity. Sorbitol increased the susceptibility of the enzyme to the inhibition by F1,6BP. The presence of sorbitol in the reaction mixture led to a reduction in the enzyme activity.

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