Activation of a plant invertase by inorganic phosphate

Activation by orthophosphate of a plant invertase from root nodules of Lupinus luteus L. has been demonstrated. The activation affects an increase in maximum velocity (V(max)) of the reaction. Activation was also achieved with a number of similar anions and it has been possible to infer a broad classification of anions capable of serving as activators. The possibility of orthophosphate activation in vivo has been considered, and there is some evidence to suggest that this could regulate invertase activity under physiological conditions.     

Activation of biliverdin-IXalpha reductase by inorganic phosphate and related anions

The N-terminal region of a 32 kDa cell-surface-binding protein, encoded by the D8L gene of vaccinia virus, shows sequence homology to CAs (carbonic anhydrases; EC 4.2.1.1). The active CAs catalyse the reversible hydration of CO2 to bicarbonate participating in many physiological processes. The CA-like domain of vaccinia protein [vaccCA (vaccinia virus CA-like protein)] contains one of the three conserved histidine residues required for co-ordination to the catalytic zinc ion and for enzyme activity. In the present study, we report the engineering of catalytically active vaccCA mutants by introduction of the missing histidine residues into the wild-type protein. The wild-type vaccCA was inactive as a catalyst and does not bind sulfonamide CA inhibitors. Its position on a phylogram with other hCAs (human CAs) shows a relationship with the acatalytic isoforms CA X and XI, suggesting that the corresponding viral gene was acquired from the human genome by horizontal gene transfer. The single mutants (vaccCA N92H/Y69H) showed low enzyme activity and low affinity for acetazolamide, a classical sulfonamide CA inhibitor. The activity of the double mutant, vaccCA N92H/Y69H, was much higher, of the same order of magnitude as that of some human isoforms, namely CA VA and CA XII. Moreover, its affinity for acetazolamide was high, comparable with that of the most efficient human isoenzyme, CA II (in the low nanomolar range). Multiplication of vaccinia virus in HeLa cells transfected with the vaccCA N92H/Y69H double mutant was approx. 2-fold more efficient than in wild-type vaccCA transfectants, suggesting that the reconstitution of the enzyme activity improved the virus life cycle.     

Ribulose diphosphate carboxylase/oxygenase. IV. Regulation by phosphate esters.

The stimulation or inhibition of ribulose diphosphate oxygenase by a variety of compounds is compared with the reported effects on these compounds on the ribulose diphosphate carboxylase activity. A possible transition state analog of ribulose diphosphate, 2-carboxyribitol 1, 5-diphosphate, at a molar ratio of inhibitor to enzyme of 10 to 1, irreversibly inactivates the oxygenase and carboxylase activities. This is consistent with the hypothesis that there may be a single active site for both the carboxylase and oxygenase activities. Several compounds of the reductive pentose photosynthetic carbon cycle act as effectors of the ribulose diphosphate oxygenase in a manner complementary to their reported effect upon the carboxylase. Ribose 5-phosphate inhibits the oxygenase with an apparent Ki of 1.8 mM, but it is reported to activate the carboxylase; fructose 6-phosphate and glucose 6-phosphate act similarly but are less effective than ribose 5-phosphate. Fructose 1. 6-diphosphate stimulates the oxygenase at low magnesium ion concentrations. The stimulatory effect of 6-phosphogluconate on the oxygenase is associated with a 3-fold reduction of the Km (Mg2+). ATP inhibits the oxygenase but has been reported to stimulate the carboxylase; pyrophosphate acts in an opposite manner. From these results it appears that the ratio of carboxylase to oxygenase activity may be a variable factor with predictable subsequent alteration in the ratio between photosynthetic CO2 fixation and photorespiration.     

Energy transduction in photosynthetic bacteria. VIII. Activation of the energy-transducing ATPase by inorganic phosphate

ATPase activity and ATP-induced energization of photosynthetic membranes from Rhodopseudomonas capsulata are stimulated by phosphate; the maximum stimulatory effect occurs at a concentration between 1 and 2 mM.The sensitivity of the ATPase to oligomycin increases in the presence of phosphate since all the P_i-stimulated activity is inhibited by this antibiotic. Aurovertin, which has no effect on ATPase in the absence of phosphate, inhibits completely the activity elicited by this anion.The addition of P_i induces a substantial increase in the V of ATPase activity without changing the affinity of the enzyme for ATP or ADP.Arsenate, at the same concentrations, produces effects very similar to those of phosphate. The stimulation by arsenate of the transfer of energy from ATP to the membrane suggests a non-hydrolytic role of this anion as a modifier of the ATPase activity.     

Energy transduction in photosynthetic bacteria. VIII. Activation of the energy-transducing ATPase by inorganic phosphate.

ATPase activity and ATP-induced energization of photosynthetic membranes from Rhodopseudomonas capsulata are stimulated by phosphate; the maximum stimulatory effect occurs at a concentration between 1 and 2 mM. The sensitivity of the ATPase to oligomycin increases in the presence of phosphate since all the Pi-stimulated activity is inhibited by this antibiotic. Aurovertin, which has no effect on ATPase in the absence of phosphate, inhibits completely the activity elicited by this anion. The addition of Pi induces a substantial increase in the V of ATPase activity without changing the affinity of the enzyme for ATP or ADP. Arsenate, at the same concentrations, produces effects very similar to those of phosphate. The stimulation by arsenate of the transfer of energy from ATP to the membrane suggests a non-hydrolytic role of this anion as a modifier of the ATPase activity.     

Chloride transport by lobster hepatopancreas is facilitated by several anion antiport mechanisms

Three anion antiporters have previously been demonstrated in lobster hepatopancreatic basolateral membrane vesicles (BLMV) to perform vital physiological functions in the crustacean. Cl(-) was shown to be transported by all three of the documented antiporters. The stilbene, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, also known as SITS, strongly inhibited Cl(-)/SO(4)(2-), Cl(-)/oxalate(2-) and Cl(-)/HCO(3)(-) exchange. It was concluded that Cl(-) could be transported by different modes of the documented existing anion antiporters in the lobster hepatopancreatic BLMV.     

Nitrite-dependent vasodilation is facilitated by hypoxia and is independent of known NO-generating nitrite reductase activities

The reduction of circulating nitrite to nitric oxide (NO) has emerged as an important physiological reaction aimed to increase vasodilation during tissue hypoxia. Although hemoglobin, xanthine oxidase, endothelial NO synthase, and the bc(1) complex of the mitochondria are known to reduce nitrite anaerobically in vitro, their relative contribution to the hypoxic vasodilatory response has remained unsolved. Using a wire myograph, we have investigated how the nitrite-dependent vasodilation in rat aortic rings is controlled by oxygen tension, norepinephrine concentration, soluble guanylate cyclase (the target for vasoactive NO), and known nitrite reductase activities under hypoxia. Vasodilation followed overall first-order dependency on nitrite concentration and, at low oxygenation and norepinephrine levels, was induced by low-nitrite concentrations, comparable to those found in vivo. The vasoactive effect of nitrite during hypoxia was abolished on inhibition of soluble guanylate cyclase and was unaffected by removal of the endothelium or by inhibition of xanthine oxidase and of the mitochondrial bc(1) complex. In the presence of hemoglobin and inositol hexaphosphate (which increases the fraction of deoxygenated heme), the effect of nitrite was not different from that observed with inositol hexaphosphate alone, indicating that under the conditions investigated here deoxygenated hemoglobin did not enhance nitrite vasoactivity. Together, our results indicate that the mechanism for nitrite vasorelaxation is largely intrinsic to the vessel and that under hypoxia physiological nitrite concentrations are sufficient to induce NO-mediated vasodilation independently of the nitrite reductase activities investigated here. Possible reaction mechanisms for nitrite vasoactivity, including formation of S-nitrosothiols within the arterial smooth muscle, are discussed.     

Activation of tumor tissue glycolysis by inorganic phosphate at low pH values]

It was shown that the activating effect of inorganic phosphorus on glucose utilization by the tumour tissue in vitro was retained at low pH of the incubation medium. The 0.15M Na2HPO4 in tris-buffer solutions infusion at the moment of cessation of the tumour selfacidity under glucose infusion led to further decrease of the tumour tissue pH. The tumour tissue pH values of about 4.5--4.6 were obtained. It is recommended to use the solutions with inorganic phosphorus for acidifying the tumour tissue in optimisation of the complex therapy schemes.     

Activation by phosphate of yeast phosphofructokinase.

The activity of yeast phosphofructokinase assayed in vitro at physiological concentrations of known substrates and effectors is 100-fold lower than the glycolytic flux observed in vivo. Phosphate synergistically with AMP activates the enzyme to a level within the range of the physiological needs. The activation by phosphate is pH-dependent: the activation is 100-fold at pH 6.4 while no effect is observed at pH 7.5. The activation by AMP, phosphate, or both together is primarily due to changes in the affinity of the enzyme for fructose-6-P. Under conditions similar to those prevailing in glycolysing yeast (pH 6.4, 1 mM ATP, 10 mM NH4+) the apparent affinity constant for fructose-6-P (S0.5) decreases from 3 to 1.4 mM upon addition of 1 mM AMP or 10 mM phosphate; if both activators are present together, S0.5 is further decreased to 0.2 mM. In all cases the cooperativity toward fructose-6-P remains unchanged. These results are consistent with a model for phosphofructokinase where two conformations, with different affinities for fructose-6-P and ATP, will present the same affinity for AMP and phosphate. AMP would diminish the affinity for ATP at the regulatory site and phosphate would increase the affinity for fructose-6-P. The results obtained indicate that the activity of phosphofructokinase in the shift glycolysis-gluconeogenesis is mainly regulated by changes in the concentration of fructose-6-P.     

Butylated hydroxytoluene and inorganic phosphate plus Ca2+ increase mitochondrial permeability via mutually exclusive mechanisms

Mitochondria undergo a permeability transition (PT), i.e., become nonselectively permeable to small solutes, in response to a wide range of conditions/compounds. In general, opening of the permeability transition pore (PTP) is Ca²⁺- and P_i-dependent and is blocked by cyclosporin A (CsA), trifluoperazine (TFP), ADP, and butylated hydroxytoluene (BHT). Gudz and coworkers have reported [7th European Bioenergetics Conference, EBEC Short Reports (1992)7, 125], however, that, under some conditions, BHT increases mitochondrial permeability via a process that may not share all of these characteristics. Specifically, they determined that the BHT-induced permeability transition was independent of Ca²⁺ and was insensitive to CsA. We have used mitochondrial swelling to compare in greater detail the changes in permeability induced by BHT and by Ca²⁺ plus P_i with the following results. (1) The dependence of permeability on BHT concentration is triphasic: there is a threshold BHT concentration (ca. 60 nmol BHT/ mg mitochondrial protein) below which no increase occurs; BHT enhances permeability in an intermediate concentration range; and at high BHT concentrations (> 120 nmol/mg) permeability is again reduced. (2) The effects of BHT depend on the ratio of BHT to mitochondrial protein. (3) Concentrations of BHT too low to induce swelling block the PT induced by Ca²⁺ and P_i. (4) The dependence of the Ca²⁺-triggered PT on P_i concentration is biphasic. Below a threshold of 50–100 M, no swelling occurs. Above this threshold swelling increases rapidly. (5) P_i levels too low to support the Ca²⁺-induced PT inhibit BHT-induced swelling. (6) Swelling induced by BHT can bestimulated by agents and treatments that block the PT induced by Ca²⁺ plus P_i. These data suggest that BHT and Ca²⁺ plus P_i, increase mitochondrial permeability via two mutually exclusive mechanisms.     

Catalysis by Escherichia coli ribonuclease HI is facilitated by a phosphate group of the substrate

To investigate the role of the phosphate group 3' to the scissile phosphodiester bond of the substrate in the catalytic mechanism of Escherichia coli ribonuclease HI (RNase HI), we have used modified RNA-DNA hybrid substrates carrying a phosphorothioate substitution at this position or lacking this phosphate group for the cleavage reaction. Kinetic parameters of the H124A mutant enzyme, in which His(124) was substituted with Ala, as well as those of the wild-type RNase HI, were determined. Substitution of the pro-R(p)-oxygen of the phosphate group 3' to the scissile phosphodiester bond of the substrate with sulfur reduced the k(cat) value of the wild-type RNase HI by 6.9-fold and that of the H124A mutant enzyme by only 1. 9-fold. In contrast, substitution of the pro-S(p)-oxygen of the phosphate group at this position with sulfur had little effect on the k(cat) value of the wild-type and H124A mutant enzymes. The results obtained for the substrate lacking this phosphate group were consistent with those obtained for the substrates with the phosphorothioate substitutions. In addition, a severalfold increase in the K(m) value was observed by the substitution of the pro-R(p)-oxygen of the substrate with sulfur or by the substitution of His(124) of the enzyme with Ala, suggesting that a hydrogen bond is formed between the pro-R(p)-oxygen and His(124). These results allow us to propose that the pro-R(p)-oxygen contributes to orient His(124) to the best position for the catalytic function through the formation of a hydrogen bond.     

Activation of Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase by inorganic phosphate

Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17], one of the metal ion-requiring aldolases, is markedly activated by Pi. The activation is reversible and can be observed in every step of enzyme purification. The extent of activation is almost independent of the metal ion used, but varies with each substrate. The cleavage of l-4-carboxy-4-hydroxy-2-oxoadipate, a physiological substrate of the enzyme, is most strongly activated: Pi gives a hyperbolic activation curve with an activation constant of 0.36 mM and a maximum activation of about 65-fold. Arsenate, phosphorous acid, bicarbonate, acetyl phosphate, thiamine diphosphate, ADP, PPi, and ATP are also effective to various extents. These anions appear to be effective in the free form but not in the metal ion-complex. Many organic and inorganic anions are ineffective. Pi causes parallel increases in Vmax and in Km for substrate or metal ion with a concomitant shift of the optimum pH toward the alkaline side, and the enhancement of activity is closely correlated with the shift of optimum pH. Pi induces no gross change of molecular form of the enzyme protein as evaluated from gel filtration, PAGE, UV, fluorescence, and CD spectral data. Based on these findings, the mechanism and the physiological meaning of the observed activation are discussed.     

Activation of sea urchin eggs by inositol phosphates is independent of external calcium.

We investigated the contribution of external calcium ions to inositol phosphate-induced exocytosis in sea urchin eggs. We show that: (a) inositol phosphates activate eggs of the sea urchin species Lytechinus pictus and Lytechinus variegatus independently of external calcium ions; (b) the magnitude and duration of the inositol phosphate induced calcium changes are independent of external calcium; (c) in calcium-free seawater, increasing the volume of inositol trisphosphate solution injected decreased the extent of egg activation; (d) eggs in calcium-free sea water are more easily damaged by microinjection; microinjection of larger volumes increased leakage from eggs pre-loaded with fluorescent dye. We conclude that inositol phosphates do not require external calcium ions to activate sea urchin eggs. This is entirely consistent with their role as internal messengers at fertilization. The increased damage caused to eggs in calcium-free seawater injected with large volumes may allow the EGTA present in the seawater to enter the egg and chelate any calcium released by the inositol phosphates. This may explain the discrepancy between this and earlier reports.     

Active-site histidines in recombinant cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase examined by site-directed mutagenesis

The functions of His²⁹¹, His²⁹⁵ and His³²⁴ at the active-site of recombinant A. nidulans ribulose-1,5-bisphosphate carboxylase/ oxygenase have been explored by site-directed mutagenesis. Replacement of His²⁹¹ by K or R resulted in unassembled proteins, while its replacement by E, Q or N resulted in assembled but inactive proteins. These results are in accord with a metal ion-binding role of this residue in the activated ternary complex by analogy to x-ray crystallographic analyses of tobacco and spinach enzymes.His³²⁴ (H327 in spinach), which is located within bonding distance of the 5-phosphate of bound bi-substrate analog 2-carboxyarabinitol 1,5-bisphosphate in the crystal structures, has been substituted by A, K, R, Q and N. Again with the exception of the H324K and R variants, these changes resulted in detectable assembled protein. The mutant H324A protein exhibited no detectable carboxylase activity, whereas the H324Q and H324N changes resulted in purifiable holoenzyme with 2.0 and 0.1% of the recombinant wild-type specific carboxylase activity, respectively. These results are consistent with a phosphate binding role for this residue.The replacement of His²⁹⁵, which has been suggested to aid in phosphate binding, with Ala in the A. nidulans enzyme leads to a mutant with 5.8% of the recombinant wild-type carboxylase activity. All other mutations at this position resulted in unassembled proteins. Purified H295A and H324Q enzymes had elevated K_m(RuBP) values and unchanged CO₂/O₂ specificity factors compared to recombinant wild-type.Abbreviations CABP D-2-carboxyarabinitol 1,5 bisphosphate - IPTG isopropyl-b-d-thiogalactopyranoside - L large subunit of rubisco - PAGE polyacrylamide gel electrophoresis - rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-P₂, ribulose 1,5 bisphosphate - S small subunit of rubisco - SDS sodium dodecyl sulfate - X-gal 5-bromo-4-chloro-3-indolyl-b-d-galactoside     

Activation of alkyldihydroxyacetone phosphate synthase by detergents.

The protein pairs S3-S4, S4-S5, and S4-S20 isolated from the 30 S subunit of the Escherichia coli ribosome were investigated by the method of sedimentation equilibrium for possible intermolecular interactions in the reconstitution buffer. The pairs S3-S4 and S4-S5 were found to interact in TMK buffer (0.03 m Tris, 0.02 m MgCl₂, 0.35 m KCl, pH 7.4) at 4 °C with free energies of interaction of ?5.1 and ?4.8 kcal/mol, respectively. The pair S4-S20 demonstrated no tendency to complex under the same conditions.