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.     

Interactions of inorganic phosphate and sulfate anions with collagen

We use direct infrared measurements to determine the number of binding sites, their dissociation constants, and preferential interaction parameters for inorganic phosphate and sulfate anions in collagen fibrils from rat tail tendons. In contrast to previous reports of up to 150 bound phosphates per collagen molecule, we find only 1-2 binding sites for sulfate and divalent phosphate under physiological conditions and approximately 10 binding sites at low ionic strength. The corresponding dissociation constants depend on NaCl concentration and pH and vary from approximately 50 microM to approximately 1-5 mM in the physiological range of pH. In fibrils, bound anions appear to form salt bridges between positively charged amino acid residues within regions of high excess positive charge. In solution, we found no evidence of appreciable sulfate or phosphate binding to isolated collagen molecules. Although sulfate and divalent phosphate bind to fibrillar collagen at physiological concentrations, our X-ray diffraction and in vitro fibrillogenesis experiments suggest that this binding plays little role in the formation, stability and structure of fibrils. In particular, we demonstrate that the previously reported increase in the critical fibrillogenesis concentration of collagen is caused by preferential exclusion of "free" (not bound to specific sites) sulfate and divalent phosphate from interstitial water in fibrils rather than by anion binding. Contrary to divalent phosphate, monovalent phosphate does not bind to collagen. It is preferentially excluded from interstitial water in fibrils, but it has no apparent effect on critical fibrillogenesis concentration at physiological NaCl and pH.     

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 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.     

Effect of phosphate and other inorganic anions on the activity of chicken liver cytosolic aspartate aminotransferase

Chicken liver aspartate aminotransferase was inhibited by several inorganic anions. The inhibitory effect of the anions was related to their chaotropic character. Apparent Km (2-oxoglutarate) and Km (L-aspartate) values depended on the molarity of the buffer. The profile of the curves obtained did not depend on the nature of the enzyme sample assayed. Phosphate slightly inhibited the holoaspartate aminotransferase and was a strong inhibitor of apoaspartate aminotransferase with respect to pyridoxal phosphate.     

Activation of cyanobacterial RuBP-carboxylase/oxygenase is facilitated by inorganic phosphate via two independent mechanisms.

Orthophosphate (Pi) modulates the activity and activation of ribulose 1,5-bis-phosphate carboxylase/oxygenase (RuBisCO) via a mechanism that is still controversial. Whereas its effects on the higher plant enzyme have been described, little is known about Pi regulation of the structurally similar, yet kinetically different cyanobacterial enzyme. We found that RuBisCO of Synechocystis PCC6803 was affected by Pi in a paradoxical fashion. On the one hand, Pi inhibited catalysis by competing with the substrate RuBP, and on the other hand it stimulated enzyme activation in a dual manner manifested by multiphasic kinetics, which differed from the effect on activation of the higher plant enzyme. Pi concentrations > 5 mM promoted the carbamylation of the cyanobacterial enzyme and the binding of Mg2+ to the carbanion at suboptimal concentrations of CO2 and Mg2+. Surprisingly, Pi also increased the activation level of the carbamylated enzyme via another putative site of interaction. In contrast with the higher plant RuBisCO, RuBP did not inhibit the stimulatory effect of phosphate on activation of the cyanobacterial enzyme, suggesting a Pi effect through a site other than the sugar binding site. The dual effect on activation could be distinguished by the phosphate analogue vanadate, which inhibited only the stimulation achieved at high phosphate concentrations. The elevation of RuBisCO activation at suboptimal levels of CO2 and high concentrations of RuBP suggests that in cyanobacteria Pi may have a role analogous to that of RuBisCO activase in higher plants.     

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.     

Activation of nitrate reductase by oxidation

The activation of the inactive form of the nitrate reductase (NADH: nitrate oxidoreductase, EC 1.6.6.1) present in cell-free extracts of Chlorella vulgaris Beijerinck requires an oxidizing agent. Ferricyanide causes conversion of the proenzyme to active enzyme within a few minutes, even at 0°C. Molecular oxygen causes a slow activation which requires many hours at room temperature, and never reaches the high activity level attained with ferricyanide. In unfractionated extracts, CO inhibits the activation by molecular O₂. The sensitivity of this activation to CO may account for the in vivo sensitivity of nitrate reduction to CO in these algae.     

Activation of angiotensin converting enzyme by monovalent anions

The angiotensin converting enzyme catalyzed hydrolysis of furanacryloyl-Phe-Gly-Gly is activated by monovalent anions in the order C1- greater than Br- greater than F- greater than NO3- greater than CH3COO-. In the alkaline pH region, increasing anion concentrations decrease the KM but do not change the kcat. This behavior is characteristic of an ordered bireactant mechanism in which the anion binds to the enzyme prior to the substrate. At acidic pH values, however, the anion activation is a result of both a decrease in KM and an increase in kcat, implying a bireactant mechanism in which anion and substrate bind randomly. For both the ordered and the bireactant mechanisms the anion serves as an essential activator. The effect of chloride on enzyme activity was studied over the pH range 5-10 under kcat/KM conditions and demonstrates that the apparent chloride binding constant increases from 3.3 mM at pH 6.0 to 190 mM at pH 9.0. The kcat vs. pH profile exhibits two pK values of 5.6 and 9.6, while the variation of KM with pH is characterized by a pK of 8.9 and a 2-fold increase between pH 6.5 and 7.5. The chloride activation of the hydrolysis of furanacryloyl-Phe-Gly-Gly is compared with that of the physiological substrates angiotensin I and bradykinin.     

Regulation of nebramycin biosynthesis by inorganic phosphate

The effect of inorganic phosphate on the biosynthesis of nebramycin factors2, 4 and5′ was studied inStreptomyces tenebrarius strain A (forming2, 4 and5′ in natural ratios) and its mutants B (forming predominantly2), C (forming2 as the only major product) and D (forming predominantly5′). In phosphate-supplemented complex media, the production of2 in A, B and C was reduced by 20–70%, while the yields of5′ remained unchanged in A and decreased by 30–60% in B. The production of4 increased by 50–90% in A and was fully suppressed in B. In D the biosynthesis of the three factors was inhibited completely.     

Activation of nitrate reductase by calcium and calmodulin

Nitrate reductase prepared from the leaves of Amranthus is activated by calcium and a small M, protein factor prepared from spinach by the procedures used for calmodulin preparation. The activation is considerably enhanced if both Ca²⁺ and the protein factor are present. This activation is inhibited by EGTA, a Ca²⁺ specific chelator and by anticalmodulin compounds like chlorpromazine. The effect of EGTA is reversed by C²⁺. The protein factor was identified as calmodulin. The enzyme is also activated by commercially available calmodulin. Calmodulin activation seems to be manifested in the FMNH₂-NR moiety of the enzyme molecule.     

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.     

Activation of HMG-CoA reductase by microsomal phosphatase

HMG-CoA reductase activity can be modulated by a reversible phosphorylation-dephosphorylation with the phosphorylated form of the enzyme being inactive and the dephosphorylated form, active. Phosphatases from diverse sources, including cytosol, have been shown to dephosphorylate and activate HMG-CoA reductase. The present study demonstrates phosphatase activity capable of activating HMG-CoA reductase that is associated with purified microsomes. The incubation of microsomes at 37 degrees C for 40 min results in a twofold stimulation of HMG-CoA reductase activity, and this stimulation is blocked by sodium fluoride or phosphate. The ability of microsomes to increase HMG-CoA reductase activity occurs regardless of whether microsomes are prepared by ultracentrifugation or calcium precipitation. Additionally, phosphatases capable of activating HMG-CoA reductase are present in both the smooth and rough endoplasmic reticulum. Freeze-thawing does not prevent microsomes from activating HMG-CoA reductase but preincubation results in a significant decrease in the ability of microsomes to increase HMG-CoA reductase activity. Thus, the present study demonstrates that purified liver microsomes contain phosphatase activity capable of activating HMG-CoA reductase.     

Divalent metal ion, inorganic phosphate, and inorganic phosphate analogue binding to yeast inorganic pyrophosphatase

Four different techniques, equilibrium dialysis, protection of enzymatic activity against chemical inactivation, 31P relaxation rats, and water proton relaxation rates, are used to study divalent metal ion, inorganic phosphate, and inorganic phosphate analogue binding to yeast inorganic pyrophosphatase, EC 3.6.1.1. A major new finding is that the binding of a third divalent metal ion per subunit, which has elsewhere been implicated as being necessary for enzymatic activity [Springs, B., Welsh, K. M., & Cooperman, B. S. (1981) Biochemistry (in press)], only becomes evident in the presence of added inorganic phosphate and that, reciprocally, inorganic phosphate binding to both its high- and low-affinity sites on the enzyme is markedly enhanced in the presence of divalent metal ions, with Mn2+ causing an especially large increase in affinity. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide evidence against divalent metal ion inner sphere binding to phosphate for enzyme subunits having one or two divalent metal ions bound per subunit and evidence for a conformational change restricting active-site accessibility to solvent on the binding of a third divalent metal ion per subunit.