Isolation and characterization of the inositol cyclic phosphate products of phosphoinositide cleavage by phospholipase C. Metabolism in cell-free extracts

The phosphoinositides are metabolized by phospholipase C in response to hormone or agonist stimulation in many cell types to produce diglyceride and water-soluble inositol phosphates. We have recently shown that the phospholipase C reaction products include cyclic phosphate esters of inositol. One of these, inositol 1, 2-cyclic 4,5-trisphosphate, is active in promoting Ca2+ mobilization in platelets and in inducing changes in conductance in Limulus photoreceptors similar to those produced by light (Wilson, D. B., Connolly, T. M., Bross, T. E., Majerus, P. W., Sherman, W. R., Tyler, A., Rubin, L. J., and Brown, J. E. (1985) J. Biol. Chem. 260, 13496-13501. In the current study, we have examined the metabolism of the inositol phosphates. We find that both cyclic and non-cyclic inositol trisphosphates are metabolized by inositol 1,4,5-trisphosphate 5-phosphomonoesterase, to inositol 1,2-cyclic bisphosphate and inositol 1,4-bisphosphate, respectively. However, the apparent Km of the enzyme for the cyclic substrate is approximately 10-fold higher than for the non-cyclic substrate. These inositol bisphosphates are more slowly degraded to inositol 1,2-cyclic phosphate and inositol 1-phosphate, respectively. Inositol 1,2-cyclic phosphate is then hydrolyzed to inositol 1-phosphate, which in turn is degraded to inositol and inorganic phosphate by inositol 1-phosphate phosphatase. The human platelet inositol 1,2-cyclic phosphate hydrolase enzyme and a similar rat kidney hydrolase do not utilize the cyclic polyphosphate esters of inositol as substrates. These results suggest that the inositol cyclic phosphates and the non-cyclic inositol phosphates are metabolized separately by phosphatases to cyclic and non-cyclic inositol monophosphates. The cyclic monophosphate is then converted to inositol 1-phosphate by a cyclic hydrolase. We suggest that the enzymes that metabolize the inositol phosphates may serve to regulate cellular responses to these compounds.     

Fructofuranose 2-phosphate is the product of dephosphorylation of fructose 2,6-bisphosphate

Using comparative ion-exchange chromatography on Dowex 1X4, the product of dephosphorylation of fructose 2,6-bisphosphate with purified yeast fructose-2,6-bisphosphate 6-phosphohydrolase, was shown to be identical to the furanose form of fructose 2-phosphate prepared by chemical synthesis according to Pontis and Fischer [Biochem. J. 89, 452-459 (1963)]. As expected for the furanose form of fructose 2-phosphate, the enzymatically formed product consumes 1 mol periodate/mol fructose 2-phosphate, whereas the chemically synthesized pyranose form consumes 2 mol periodate/mol. In addition, it is shown that the enzymatic product behaves identically to the furanose, not the pyranose, form of fructose 2-phosphate in hydrolysis of the ester bond at pH 4 and 37 degrees C, as described previously for the chemically synthesized compounds [Pontis and Fischer (1963) vide supra].     

Synthesis and Some Properties of UDP-(1)fructose

UDP-(1)fructose was synthesized essentially by the method of Michelson or Roseman et al. The product obtained was much more stable to acid than UDP-fructose isolated from Jerusalem artichoke tubers by Umemura et al.⁷⁾ and UDP-glucose. Hydrolysis time curves of UDP-(1)fructose and fructose-1-phosphate in 0.01N HGl and 0.1N HCl both at 100°C are presented. It was concluded from these curves that UDP-(1)fructose was first hydrolyzed into UMP and fructose-1-phosphate, and then fructose-1-phosphate was hydrolyzed more slowly into free fructose and inorganic phosphate.     

Retardation of bean leaf senescence by benzyladenine and its influence on phosphate metabolism

The levels of glucose, sugar phosphates, and adenosine phosphates were determined in primary leaves of intact bean plants during normal senescence and compared to leaves in which senescence was delayed by application of benzyladenine (BA). In both cases there was a rise with time in the levels of glucose 1-phosphate, glucose 6-phosphate, and fructose 6-phosphate, and a decline in 2-phosphoglyceric acid, inorganic phosphate, and the adenosine phosphates (AMP, ADP, ATP). The levels of fructose 1,6-diphosphate remained fairly constant. Although the levels of hexose phosphates, adenosine phosphates, and inorganic phosphate were lower in the BA-treated leaves, the incorporation of ³²P into these compounds by 3- and 6-week-old plants was higher than in the controls. These results suggest that the retardation of leaf senescence by BA in intact bean plants is associated with increased utilization of metabolites, indicating a more rapid turnover of the adenosine phosphates. It is concluded that this effect is brought about by a regulatory coordination of metabolic processes in relation to energy production and utilization.     

Inositol 1,2-cyclic 4,5-trisphosphate is not a product of muscarinic receptor-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis in rat parotid glands.

We have employed a neutral-pH extraction technique to look for inositol 1,2-cyclic phosphate derivatives in [3H]inositol-labelled parotid gland slices stimulated with carbachol. The incubations were terminated by adding cold chloroform/methanol (1:2, v/v), the samples were dried under vacuum and inositol phosphates were extracted from the dried residues by phenol/chloroform/water partitioning. Water-soluble inositol metabolites were separated by h.p.l.c. at pH 3.7. 32P-labelled inositol phosphate standards (inositol 1-phosphate, inositol 1,2-cyclic phosphate, inositol 1,4,5-trisphosphate and inositol 1,2-cyclic 4,5-trisphosphate) were quantitively recovered through both extraction and chromatography steps. Treatment of inositol cyclic phosphate standards with 5% (w/v) HClO4 for 10 min prior to chromatography resulted in formation of the expected non-cyclic compounds. [3H]Inositol 1-phosphate and [3H]inositol 1,4,5-trisphosphate were both present in parotid gland slices and both increased during stimulation with 1 mM-carbachol. There was no evidence for significant quantities of [3H]inositol 1,2-cyclic phosphate or [3H]inositol 1,2-cyclic 4,5-trisphosphate in control or carbachol-stimulated glands. Parotid gland homogenates rapidly converted inositol 1,4,5-trisphosphate to inositol bisphosphate and inositol tetrakisphosphate, but metabolism of the inositol cyclic trisphosphate was much slower. The results suggest that inositol 1,4,5-trisphosphate, but not inositol 1,2-cyclic 4,5-trisphosphate, is the water-soluble product of muscarinic receptor-stimulated phospholipase C in rat parotid glands.     

Cyclic hydrolase-transfected 3T3 cells have low levels of inositol 1,2-cyclic phosphate and reach confluence at low density.

The cDNA that encodes inositol-1,2-cyclic phosphate 2-phosphohydrolase (cyclic hydrolase), an enzyme that converts inositol 1,2-cyclic phosphate (cIns(1,2)P) to inositol 1-phosphate, was expressed in 3T3 cells to investigate the function of inositol cyclic phosphates. Cells with increased cyclic hydrolase activity had lower levels of cIns(1,2)P and grew to a lower density at confluence than control cells. This relationship was strengthened by the demonstration that several cell types with differences in cyclic hydrolase activity had levels of cIns(1,2)P and saturation densities that also correlated inversely with cyclic hydrolase activity. In addition, cyclic hydrolase activity is higher in cells at confluence compared to subconfluence. These results suggest that cellular cIns(1,2)P levels are determined by cyclic hydrolase activity and play a role in the control of cell proliferation.     

Regulation of Acid Phosphatase Synthesis in Baker’s Yeast Protoplast by Phosphate Compounds

The regulation of acid phosphatase synthesis by various phosphate compounds was examined in Baker’s yeast protoplasts. Synthesis was repressed by inorganic phosphate and phosphomonoesters. Phosphomonoesters were hydrolysed by a small amount of non-specific acid phosphatase present in the protoplast membrane. The inorganic phosphate that was liberated and incorporated into protoplasts probably repressed acid phosphatase synthesis. Phosphodiesters, such as 3′, 5′-cyclic AMP, 3′, 5′-cyclic CMP and 3′, 5′-cyclic GMP, promoted acid phosphatase synthesis. The effect of 3′, 5′-cyclic AMP was not to overcome hexose repression, because high hexose did not repress acid phosphatase synthesis. 3′, 5′-cyclic AMP did not overcome repression of the enzyme synthesis by inorganic phosphate. From these observations 3′, 5′-cyclic nucleotides probably had some effect on the yeast acid phosphatase-synthesizing system but the exact role of the nucleotides is obscure.     

Kinetic properties of bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from spinach leaves.

A cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a Spinacia oleracea leaf library and used to express a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells. The insoluble protein expressed in E. coli was purified and used to raise antibodies. Western blot analysis of a protein extract from spinach leaf showed a single band of 90.8 kDa. Soluble protein was purified to homogeneity from S. frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA. The soluble protein had a molecular mass of 320 kDa, estimated by gel filtration chromatography, and a subunit size of 90.8 kDa. The purified protein had activity of both 6-phosphofructo-2-kinase specific activity 10.4-15.9 nmol min(-1) x mg protein (-1) and fructose-2,6-bisphosphatase (specific activity 1.65-1.75 nmol x mol(-1) mg protein(-1). The 6-phosphofructo-2-kinase activity was activated by inorganic phosphate, and inhibited by 3-carbon phosphorylated metabolites and pyrophosphate. In the presence of phosphate, 3-phosphoglycerate was a mixed inhibitor with respect to both fructose 6-phosphate and ATP. Fructose-2,6-bisphosphatase activity was sensitive to product inhibition; inhibition by inorganic phosphate was uncompetitive, whereas inhibition by fructose 6-phosphate was mixed. These kinetic properties support the view that the level of fructose 2,6-bisphosphate in leaves is determined by the relative concentrations of hexose phosphates, three-carbon phosphate esters and inorganic phosphate in the cytosol through reciprocal modulation of 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities of the bifunctional enzyme.     

The partial chemical synthesis of inositol 1,2-cyclic 4,5-trisphosphate

Appreciable amounts of inositol 1,2-cyclic 4,5-trisphosphate (cIP3) are formed on agonist stimulation of secretory cells, e.g., pancreas (1,2) and parotid (3,4). However, the physiological role of this compound is unknown. To obtain sufficient amounts of cIP3, we have developed a synthetic method to produce cIP3 from inositol 1,4,5-trisphosphate (I(1,4,5)P3). The method is an adaptation of the dicyclohexylcarbodiimide (DCCD) method of Khorana et al. (5), which was originally developed to synthesize 2',3'-cyclic ribonucleotides. The method involves treatment of the pyridinium salt of I(1,4,5)P3 with DCCD in pyridine water, which cyclizes part of the 1-phosphate on the inositol ring to the 1,2-cyclic phosphate. The compound identified as cIP3 cochromatographed with authentic cIP3 in two HPLC systems and on ionophoresis. It was converted to I(1,4,5)P3 on mild acid treatment--a characteristic of cyclic inositol phosphates. Inositol 1,2-cyclic 4,5-trisphosphate is then purified by HPLC. Sufficient amounts of cIP3 can be prepared by this method to carry out numerous experiments on its possible cellular role.     

Biosynthesis of bacterial glycogen: characterization of adenosine diphosphate glucose synthetases from Enterobacter hafniae and Aeromonas hydrophila

Enterobacter hafniae and Aeromonas hydrophila ADPglucose synthetases were purified approximately 39-and 61-fold, respectively, over the crude extract. Both enzymes were heat stable at 60°C in the presence of inorganic phosphate. The molecular weights of both enzymes were approximately 200,000 which are similar to other enteric ADPglucose synthetases studied. Based on kinetic results obtained from the partially purified enzymes, the E. hafniae enzyme is activated twofold by phospho-enolpyruvate while the A. hydrophila enzyme is activated twofold by fructose 6-P and 1.5-fold by fructose 1,6 bis-phosphate. The E. hafniae enzyme activity is strongly inhibited by AMP and ADP and the inhibition can be partially reversed by P-enolpyruvate. ADP is the most effective inhibitor of the A. hydrophila enzyme and its inhibiton can be partially overcome by the presence of the activators fructose 6-P and fructose 1,6-P₂. These kinetic results show that the allosteric properties of the E. hafniae enzyme are distinctly different from the ADPglucose synthetases of those previously studied from bacteria of the genus Enterobacter. Although the A. hydrophila enzyme is activated by fructose 1,6-P₂, its allosteric properties are quite different than those observed for ADPglucose synthetase of the Enterobacteriaceae.Abbreviations Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid - glucose 1-P glucose 1-phosphate - Bicine N,N-bis(2 hydroxyethyl)glycine - fructose 6-P fructose 6-phosphate - Mes 2(N-morpholino)-ethane sulfonic acid - fructose 1,6-P₂ fructose 1,6 bis-phosphate - DTE dithioerythritol; pyridoxal-P, pyridoxal-phosphate - fructose 1-P fructose 1-phosphate - P-enolpyruvate phospho-enolpyruvate - 1,6 hexanediol bis-P 1,6 hexanediol bis-phosphate; glucose 6-P, glucose 6-phosphate - dihydroxyacetone-P dihydroxyacetone phosphate - 1-glycerol-3-P 1-glycerol-3-phosphate - erythrose 4-P erythrose 4-phosphate - 2-P-glycerate 2-phosphoglycerate - sedoheptulose 1,7-P₂ sedoheptulose 1,7 bis-phosphate - 3-P-glycerate 3-phosphoglycerate - mannose-6-P mannose-6-phosphate     

Phosphatidylinositol-specific phospholipase C from Bacillus cereus combines intrinsic phosphotransferase and cyclic phosphodiesterase activities: a 31P NMR study

The inositol phosphate products formed during the cleavage of phosphatidylinositol by phosphatidylinositol-specific phospholipase C from Bacillus cereus were analyzed by 31P NMR. 31P NMR spectroscopy can distinguish between the inositol phosphate species and phosphatidylinositol. Chemical shift values (with reference to phosphoric acid) observed are 0.41, 3.62, 4.45, and 16.30 ppm for phosphatidylinositol, myo-inositol 1-monophosphate, myo-inositol 2-monophosphate, and myo-inositol 1,2-cyclic monophosphate, respectively. It is shown that under a variety of experimental conditions this phospholipase C cleaves phosphatidylinositol via an intramolecular phosphotransfer reaction producing diacylglycerol and D-myo-inositol 1,2-cyclic monophosphate. We also report the new and unexpected observation that the phosphatidylinositol-specific phospholipase C from B. cereus is able to hydrolyze the inositol cyclic phosphate to form D-myo-inositol 1-monophosphate. The enzyme, therefore, possesses phosphotransferase and cyclic phosphodiesterase activities. The second reaction requires thousandfold higher enzyme concentrations to be observed by 31P NMR. This reaction was shown to be regiospecific in that only the 1-phosphate was produced and stereospecific in that only D-myo-inositol 1,2-cyclic monophosphate was hydrolyzed. Inhibition with a monoclonal antibody specific for the B. cereus phospholipase C showed that the cyclic phosphodiesterase activity is intrinsic to the bacterial enzyme. We propose a two-step mechanism for the phosphatidyl-inositol-specific phospholipase C from B. cereus involving sequential phosphotransferase and cyclic phosphodiesterase activities. This mechanism bears a resemblance to the well-known two-step mechanism of pancreatic ribonuclease, RNase A.     

Improved rapidity and precision in the determination of brain 2',3'-cyclic nucleotide 3'-phosphohydrolase

A rapid and precise method for the determination of brain 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) activity has been developed. Total brain homogenates were treated with deoxycholate, and CNP activity was measured as inorganic phosphate (phosphomolybdic acid, 410 nm) released from the product, 2′-AMP, by alkaline phosphatase. Measurements were carried out under optimal conditions of temperature (30°C) and pH (6.2) using the whole brain of the rat, chicken, and quaking mouse. The entire assay was applicable to multiple samples and could be completed in less than 1 hr.     

The formation of inositol 1,2-cyclic 4,5-trisphosphate and inositol 1,2-cyclic 4-bisphosphate on stimulation of mouse pancreatic minilobules with carbamylcholine

When [3H]myoinositol-prelabeled pancreatic minilobules were incubated with carbamylcholine (CCh) for 30 min, followed by ionophoresis on paper of the aqueous extracts, there were distinct peaks of radioactivity immediately preceding inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3), which, based on earlier studies with inositol 1,2-cyclic phosphate (IcP), are the expected positions for inositol 1,2-cyclic 4-bisphosphate (IcP2) and inositol 1,2-cylic 4,5-trisphosphate (IcP3). These peaks were essentially absent on ionopherograms of extracts from minilobules not incubated with CCh. Similar results were obtained with high performance liquid chromatography (HPLC), except that the putative inositol cyclic phosphate peaks eluted immediately before the non-cyclic inositol polyphosphates, as to be expected. Taking advantage of the unique acid lability of the inositol cyclic phosphates, we demonstrate that the putative inositol cyclic polyphosphate peaks were specifically eliminated by prior hydrolysis of the aqueous extracts, as shown by either ionophoresis or HPLC. After preparative isolation of putative IcP2 and IcP3 by ionophoresis, acid hydrolysis shifted the positions of putative IcP2 and IcP3 peaks to the positions of standard IP2 and IP3, respectively, as shown by either ionophoresis or HPLC. The amounts of IcP, IcP2, and IcP3 formed on CCh stimulation, as measured by ionophoresis, were 0.7, 6.8, and 29.8% of that of, IP, IP2, and IP3, respectively (average of two experiments which agreed within 10%).     

The plastidic pentose phosphate translocator represents a link between the cytosolic and the plastidic pentose phosphate pathways in plants

Plastids are the site of the reductive and the oxidative pentose phosphate pathways, which both generate pentose phosphates as intermediates. A plastidic transporter from Arabidopsis has been identified that is able to transport, in exchange with inorganic phosphate or triose phosphates, xylulose 5-phosphate (Xul-5-P) and, to a lesser extent, also ribulose 5-phosphate, but does not accept ribose 5-phosphate or hexose phosphates as substrates. Under physiological conditions, Xul-5-P would be the preferred substrate. Therefore, the translocator was named Xul-5-P/phosphate translocator (XPT). The XPT shares only approximately 35% to 40% sequence identity with members of both the triose phosphate translocator and the phosphoenolpyruvate/phosphate translocator classes, but a higher identity of approximately 50% to glucose 6-phosphate/phosphate translocators. Therefore, it represents a fourth group of plastidic phosphate translocators. Database analysis revealed that plant cells contain, in addition to enzymes of the oxidative branch of the oxidative pentose phosphate pathway, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase in both the cytosol and the plastids, whereas the transketolase and transaldolase converting the produced pentose phosphates to triose phosphates and hexose phosphates are probably solely confined to plastids. It is assumed that the XPT function is to provide the plastidic pentose phosphate pathways with cytosolic carbon skeletons in the form of Xul-5-P, especially under conditions of a high demand for intermediates of the cycles.     

Evidence for two catalytic sites on 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Dynamics of substrate exchange and phosphoryl enzyme formation

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase appears to be the only enzyme catalyzing the formation and hydrolysis of Fru-2,6-P2. The enzyme as we isolate it, contains a trace of tightly bound Fru-6-P. In this condition, it exhibited an ATPase activity comparable to its kinase activity. Inorganic phosphate stimulated all of its activities, by increasing the affinity for all substrates and increasing the Vmax of ATP and Fru-2,6-P2 hydrolysis. The enzyme catalyzed ADP/ATP and Fru-6-P/Fru-2,6-P2 exchanges at rates comparable to net reaction rates. It was phosphorylated by both [gamma-32P]ATP and [2-32P] Fru-2,6-P2, and the label from either donor was chased by either unlabeled donor, showing that the bound phosphate is hydrolyzed if not transferred to an acceptor ligand. The rate of labeling of the enzyme by [2-32P]Fru-2,6-P2 was 2 orders of magnitude greater than the maximal velocity of the bisphosphatase and therefore sufficiently fast to be a step in the hydrolysis. Both inorganic phosphate and Fru-6-P increased the rate and steady state of enzyme phosphorylation by ATP. Fru-2,6-P2 inhibited the ATPase and kinase reactions and Fru-6-P inhibited the Fru-2,6 bisphosphatase reaction while ATP and ADP had no effect. Removal of the trace of Fru-6-P by Glu-6-P isomerase and Glu-6-P dehydrogenase reduced enzyme phosphorylation by ATP to very low levels, greatly inhibited the ATPase, and rendered it insensitive to Pi, but did not affect ADP/ATP exchange. (alpha + beta)Methylfructofuranoside-6-P did not increase the rate or steady state labeling by ATP. These results suggest that labeling of the enzyme by ATP involved the production of [2-32P]Fru-2,6-P2 from the trace Fru-6-P. The 6-phosphofructo-2-kinase, fructose 2,6-bisphosphatase, and ATP/ADP exchange were all inhibited by diethylpyrocarbonate, suggesting the involvement of histidine residues in all three reactions. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E-P is labile) and a Fru-2,6 bisphosphatase site which is readily phosphorylated by Fru-2,6-P2.     

Evidence for extracellular enzymic activity of the isolated perfused rat heart

1. The dissimilation of a number of externally added hexose phosphates and 5′-nucleotides by the perfused rat heart is described, and non-specific esterase and 5′-nucleotidase activity associated with the superficial cell membrane or vascular system has been demonstrated. 2. The rate of production of ¹⁴CO₂ from [U-¹⁴C]glucose 6-phosphate suggests that oxidation occurred after hydrolysis to glucose. The incorporation of isotope from [U-¹⁴C]glucose 6-phosphate into glycogen was small, and similar to that obtained with [U-¹⁴C]glucose as substrate. 3. Glucose 6-phosphate was also partially isomerized to fructose 6-phosphate. Similarly, fructose 6-phosphate was converted mainly into glucose 6-phosphate, but also into glucose and inorganic phosphate. When fructose 1,6-diphosphate was added to the perfusate, a mixture of glucose 6-phosphate, fructose 6-phosphate and triose phosphates accumulated in the medium approximately in the equilibrium proportions of the phosphohexose-isomerase and triose phosphate-isomerase reactions, together with inorganic phosphate and some glucose. Glucose 1-phosphate was hydrolysed to glucose, but was not converted into glucose 6-phosphate. Leakage of enzymes out into the perfusion fluid did not occur. 4. This demonstration that phosphohexose isomerase, triose phosphate isomerase and aldolase may react with extracellular substrates at an appreciable rate suggests that these enzymes are attached to the cell membrane.     

Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: phosphate dependence and effects of other oxyanions

The effects of various oxyanions on the activities of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (EC 2.7.1.105/3.1.3.46) were examined. No evidence could be found for an absolute dependence of the kinase activity on inorganic phosphate as was recently reported by M. Laloux, E. Van Schaftingen, and H.-G. Hers ((1985) Eur. J. Biochem. 148, 155-159). Rather, phosphate and arsenate activated the kinase by decreasing the enzyme's Km for fructose 6-phosphate without affecting its Km for ATP or Vmax. The Km of the kinase for fructose 6-phosphate in the presence of inorganic phosphate was found to be significantly lower (6 microM) than previously reported (30 microM) when the hydrolysis of fructose 2,6-bisphosphate by the concomitant bisphosphatase activity at low Fru 6-P concentrations was taken into account. The KA's for phosphate and arsenate activation of the kinase were 0.2 and 0.3 mM, respectively. A number of other oxyanions, including pyrophosphate, sulfate, tungstate, selenate, and molybdate all inhibited the kinase by increasing the Km for fructose 6-phosphate. The apparent Ki's for inhibition of the kinase were in the 0.5-1 mM range. In contrast, all of these oxyanions activated the bisphosphatase, with half-maximal effects requiring millimolar concentrations. Inorganic phosphate was the most potent activator with a KA of 1 mM. In contrast to the other oxyanions, vanadate and meta-periodate inhibited the kinase but had no effect on the bisphosphatase. Vanadate appeared to be a noncompetitive inhibitor since its effects were not overcome by Pi, ATP, or fructose 6-phosphate, and the species responsible was shown to be decavanadate. Like vanadate, meta-periodate had no effect on the bisphosphatase, though it was a potent inhibitor (I0.5 = 30 microM) of the kinase. Its effects were shown to be time-dependent and reversed by dithiothreitol, suggesting that it acted by an oxidative mechanism. These results augment the mounting body of evidence that the enzyme's two reactions are catalyzed at discrete active sites.     

Stereospecific recognition sites for [3H]inositol(1,4,5)-triphosphate in particulate preparations of rat cerebellum

A very high density of stereospecific binding sites for inositol-(1,4,5)P3 have been identified in rat cerebellar membranes using [3H]inositol-(1,4,5)P3 and a rapid centrifugation step to separate free and bound ligand. Binding was shown to be rapid and reversible and of relatively high affinity (KD 23 nM). Incubations were carried out at 4 degrees and under these conditions HPLC analysis demonstrated that there was no significant metabolism of [3H]-(1,4,5)P3 in the presence or absence of ATP over 15 min. The specificity of the site has been carefully evaluated using both natural and novel synthetic inositol phosphates. The stereospecificity is very marked with the D-, DL- and L-isomers of Ins(1,4,5)P3 showing a 1:4:2000 ratio of affinity for the binding site. D-Ins(2,4,5)P3 was the only other phosphate to show relatively high affinity (KD 1500 nM). HPLC-pure Ins(1,3,4)P3 and Ins(1,3,4,5)P4 were substantially weaker and Ins(1,4)P2, Ins-2-P1, Ins-1-P1, Ins(1,2)-cyclic P1 and inositol were totally inactive at concentrations less than 50 microM. These data are discussed in relation to a putative receptor on the endoplasmic reticulum by which Ins(1,4,5)P3 can initiate the release of bound Ca2+.     

31P nuclear magnetic resonance spectroscopy studies of substrate and product binding to fructose-1,6-bisphosphatase.

The enzymatic hydrolysis of fructose 1,6-bisphosphate (Fru-1,6-P2) to fructose 6-phosphate (Fru-6-P) and inorganic phosphate (Pi), which is catalyzed by fructose-1,6-bisphosphatase, has been studied by 31P nuclear magnetic resonance spectroscopy (NMR). At pH 7.5 and 15 degrees C, the equilibrium constant for the central complex K'eq = [E.Fru-6-P.Pi]/[E.Fru-1,6-P2.H2O] is about 2. This observation is in harmony with results obtained with a number of Bi Bi enzyme systems for the determination of K'eq in which a variety of experimental techniques were used (Knowles, J.R. (1980) Annu. Rev. Biochem. 49, 877-919). Significant changes in 31P NMR chemical shifts were observed for both the substrate, Fru-1,6-P2, and the product, Fru-6-P, when bound to the enzyme relative to ligand free in solution. The chemical shifts of the substrate and product were altered further in the presence of Mg2+, the catalytic divalent metal ion. The chemical shifts caused by the addition of metal ion can be reversed in the presence of trans-1,2-diaminocyclohexane- N,N,N',N'-tetraacetic acid (CDTA) or AMP. In the presence of the metal ion chelator or the nucleotide, the substrate had a chemical shift that was about the same as that observed in the absence of metal ion. On the basis of these observations we suggest that AMP and CDTA exhibit similar effects, i.e. they both remove the catalytic metal ion from the enzyme. This finding is supportive of the suggestion (Scheffler, J. E., and Fromm, H.J. (1986) Biochemistry 25, 6659-6665; Liu, F., and Fromm, H.J. (1990) J. Biol. Chem. 265, 7401-7406) that the role of AMP in the regulation of fructose-1,6-bisphosphatase is to prevent binding of the divalent metal activator to the enzyme.     

Phosphate dependency of phosphofructokinase 2

Experiments performed at micromolar concentrations of inorganic phosphate support the conclusion that liver phosphofructokinase 2 would be completely inactive in the absence of inorganic phosphate or arsenate. The concentration of inorganic phosphate that allowed half-maximal activity decreased with increasing pH, being approximately 0.11 mM at pH 6.5 and 0.05 mM at pH 8. The effect of phosphate was to increase V and to decrease Km for fructose 6-phosphate, without affecting Km for ATP. Citrate and P-enolpyruvate inhibited the enzyme non-competitively with fructose 6-phosphate and independently of the concentration of inorganic phosphate. Phosphorylation of the enzyme by the catalytic subunit of cyclic-AMP-dependent protein kinase did not markedly modify the phosphate requirement and its effect of inactivating phosphofructokinase 2 could not be counteracted by excess phosphate. A nearly complete phosphate dependency was also observed with phosphofructokinase 2 purified from Saccharomyces cerevisiae or from spinach leaves. By contrast, the fructose 2,6-bisphosphatase activity of the liver bifunctional enzyme was not dependent on the presence of inorganic phosphate. Phosphate increased this activity about threefold when measured in the absence of added fructose 6-phosphate and a half-maximal effect was reached at approximately 0.5 mM phosphate. Like glycerol phosphate, phosphate counteracted the inhibition of fructose 2,6-bisphosphatase by fructose 6-phosphate, but a much higher concentration of phosphate than of glycerol phosphate was required to reach this effect.