Kinetic mechanisms of linoleic acid oxidation by 5-lipoxygenase from Solanum tuberosum L

Linoleic acid oxidation by 5-lipoxygenase from Solanum tuberosum has been studied as affected by sodium dodecylsulfate (Ds-Na). The reaction system consisted of 5-lipoxygenase and mixed micelles of linoleic acid and Lubrol PX. It contained varying amounts of the enzyme effector--Ds-Na. The enzyme showed a pronounced cooperativity, and the reaction was governed by the Hill equation with h = 3.7. On the other side, increasing amounts of Ds-Na added to the system caused a tremendous increase of enzyme activity and simultaneous decline of h, with was proportional to Ds-Na concentration. Ds-Na had dual effect on 5-lipoxygenase--there was an optimal concentration of the compound (0.34 mM Lubrol PX; 0.2 mM LA; 0.13 mM Ds-Na; pH = 6.3) causing the 4-fold highest activation and h = 1.6. The further increase of Ds-Na led to the enzyme inhibition. If Ds-Na was 0.5 mM, h became 1. At this point, each molecule of 5-lipoxygenase bound 3 molecules of Ds-Na and 1 molecule of linoleic acid, thus the total number of occupied binding sites was 4. A kinetic scheme of 5-lipoxygenase reaction has been proposed. It was found that the enzyme's kinetic behaviour could be explaine if assumed an existence of a special noncatalytic binding centre capable of binding several (up to 3) molecules of either substrate, or effector. Such a centre can serve as an anchoring site facilitating the enzyme binding to the surface of lipid aggregates containing insolubilized substrate molecules. Replacing linoleic acid in the binding site, Ds-Na activates the enzyme, possibly due to the much more effective translocation of 5-lipoxygenase to the surface of lipid aggregates. This mechanism can be an universal alternative to the FLAP-type regulation of 5-lipoxygenase activities.     

Purification of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. An iron-sulfur protein

1. The (R)-2-hydroxyglutaryl-CoA dehydratase system from Acidaminococcus fermentans was separated by chromatography of cell-free extracts on Q-Sepharose into two components, an activator and the actual dehydratase. The latter enzyme was further purified to homogeneity by chromatography on blue-Sepharose. It is an iron-sulfur protein (Mr 210,000) consisting of two different polypeptides (alpha, Mr 55,000, and beta, Mr 42,000) in an alpha 2 beta 2 structure with probably two [4Fe-4S] centers. After activation this purified enzyme catalysed the dehydration of (R)-2-hydroxyglutarate only in the presence of acetyl-CoA and glutaconate CoA-transferase, demonstrating that the thiol ester and not the free acid is the substrate of the dehydration. The result led to a modification of the hydroxyglutarate pathway of glutamate fermentation. 2. The activation of the dehydratase by the flow-through from Q-Sepharose concentrated by ultrafiltration required NADH, MgCl2, ATP and strict anaerobic conditions. This fraction was designated as Ao. Later when the concentration was performed by chromatography on phenyl-Sepharose, an NADH-independent form of the activator, designated as A*, was obtained. This enzyme, which required only ATP for activation of the dehydratase, was purified further by affinity chromatography on ATP-agarose. It contains neither iron nor inorganic sulfur. A*, as well as the activated dehydratase, were irreversibly inactivated by exposure to air within less than 15 min. The activated dehydratase but not A* was also inactivated by 1 mM hydroxylamine or by 0.1 mM 2,4-dinitrophenol. 3. The (R)-2-hydroxyglutaryl-CoA dehydratase system is closely related the that of (R)-lactoyl-CoA dehydratase from Clostridium propionicum as described by R. D. Kuchta and R. H. Abeles [(1985) J. Biol. Chem. 260, 13,181-13,189].     

Regulatory effects of fatty acyl-coenzyme A derivatives on phosphate-activated pig brain and kidney glutaminase in vitro.

1. Fatty n-acyl-CoA derivatives in the concentration range 5muM-0.1mM and with 5-18 fatty acyl carbons have dual effects on phosphate-activated glutaminase from pig brain and kidney. Generally, fatty acyl-CoA derivatives in low concentrations activate the enzyme, but inhibit at higher concentrations; phosphate and citrate potentiate the activation, displaying positive co-operatively, and protect against inactivation. The fatty acyl-CoA derivatives affect glutaminase similarly to Bromothymol Blue, but differently from acetyl-CoA, which activates the enzyme only at very low phosphate or citrate concentrations. 2. Saturated fatty acyl-CoA derivatives, with 5-10 fatty acyl carbons, only activate the enzyme in the concentration range 0-0.1 mM. When the fatty acyl chain is elongated, the fatty acyl-CoA derivatives gradually become more powerful inhibitors of glutaminase at the expense of their activating capacity. In particular, palmitoyl-CoA and stearoyl-CoA are strong inhibitors at concentrations (10 muM) at which the corresponding free fatty acids and fatty acyl-carnitine derivatives have no effect. 3. The unsaturated fatty acyl-CoA derivatives, oleoyl-CoA and linoleoyl-CoA, behave as potent activators in the lower part of the concentration range tested (0-0.05mM), and as inhibitors in the upper part of this range (0.02-0.10mM). Oleic acid and linoleic acid have similar properties, but their activating capacity is less pronounced. 4. Phosphate both prevented and reversed the inhibition, but no restoration of activity was possible once the enzyme became inactivated. 5. By changing the pH from 7.0 to 8.0 the activating capacity of the fatty acyl-CoA derivatives is increased, as is their concentration range for activation. 6. The fatty acyl-CoA derivatives are somewhat more potent activator for brain glutaminase, but otherwise they affect the two enzymes similarly.     

Effect of cis-unsaturated fatty acids on aortic protein kinase C activity.

Long-chain cis-unsaturated fatty acids could substitute for phosphatidylserine and activate bovine aortic protein kinase C in assays with histone as substrate. The optimal concentration was 24-40 microM for oleic, linoleic and arachidonic acids. With arachidonic acid, the Ka for Ca2+ was 130 microM and kinase activity was maximal at 0.5 mM-Ca2+. Diolein only slightly activated the oleic acid-stimulated enzyme at low physiological Ca2+ concentrations (0.1 and 10 microM). Oleic acid also stimulated kinase C activity, determined with a Triton X-100 mixed-micellar assay. Under these conditions, the fatty acid activation was absolutely dependent on the presence of diolein, but a Ca2+ concentration of 0.5 mM was still required for maximum kinase C activity. The effect of fatty acids on protein kinase C activity was also investigated with the platelet protein P47 as a substrate, since the properties of kinase C can be influenced by the choice of substrate. In contrast with the results with histone, fatty acids did not stimulate the phosphorylation of P47 by the aortic protein kinase C. Activation of protein kinase C by fatty acids may allow the selective phosphorylation of substrates, but the physiological significance of fatty acid activation is questionable because of the requirement for high concentrations of Ca2+.     

Proline hydroxylation by soybean lipoxygenase.

The lipoxygenase-catalyzed hydroxylation of proline was studied in vitro in the presence of linoleic acid. The rate of reaction exhibited dependence on the concentration of proline, linoleic acid and the enzyme. The magnitude of hydroxyproline formed per mg of protein was time-dependent. Nordihydroguaiaretic acid at 0.1 mM concentration completely inhibited this reaction. No proline hydroxylation was detected during peroxidation of linoleic acid by vanadyl(IV). It is suggested that free-radical products of linoleic acid peroxidation may co-oxygenate proline in the presence of lipoxygenase.     

Analysis of the kinetics of phospholipid activation of cobra venom phospholipase A2

A kinetic analysis of the "dual phospholipid model" for cobra venom phospholipase A2 (Hendrickson, H. S., and Dennis, E. A. (1984) J. Biol. Chem. 259, 5734-5739) was applied to the activation of phospholipase A2-catalyzed hydrolysis of a thiol ester analog of phosphatidylethanolamine (thio - PE) in Triton X - 100/phospholipid mixed micelles by various phosphorylcholine-containing activators. Activation of thio-PE hydrolysis by didecanoylphosphatidylcholine (PC) was found to be a function of the surface concentration of activator rather than bulk concentration. Its presence did not affect the initial binding of enzyme to phospholipid in the micelle surface as determined kinetically. After initial binding of enzyme to the surface, the activation appears to be due to enzyme-lipid binding in the surface. Activation does not appear to affect the affinity of the enzyme for phospholipid substrate, but rather affects the catalytic efficiency of the enzyme as characterized by the value of Vmax. The monomeric phospholipid dibutyryl-PC, when used as an activator at 57 mM (bulk concentration), also showed effects of surface dilution with Triton X-100, which would not be expected unless the lipid is incorporated into the micelles to some extent at these high concentrations. A thiol ester analog of phosphatidylcholine, thio-PC, was less effective than didecanoyl-PC as an activator, but appeared to be more effective than decylphosphorylcholine. A conformational change of the enzyme upon binding of the activator, after enzyme is bound to substrate at the interface, is discussed as a possible mechanism for this activation.     

Stimulation of guanylate cyclase activity by several fatty acids.

Guanylate cyclase of plasma membrane of isolated rat fat cells was activated 7 to 11 fold by oleic acid, linoleic acid, linolenic acid or arachidonic acid. The activation of the enzyme by linoleic acid or oleic acid was influenced by the concentration of enzyme protein and that of the fatty acid. At 158 μg/ml of enzyme protein, 0.6 mM linoleic acid produced maximal activation of 12 fold which was partially reversed by washing. Particulate guanylate cyclase of cerebral cortex and liver was also activated by linoleic acid.     

Characterization of a phospholipase A2 in hamster lung and in vitro and in vivo effects of bleomycin on this enzyme

Phospholipase A2 (PLA2) activity from adult hamster lung was characterized using L-alpha-1-palmitoyl-2-arachidonyl-[arachidonyl-1-14C]-phosphatidylcholine as the substrate. The released [14C]-arachidonic acid was separated by TLC. The enzyme activity increased with increasing incubation time (0-120 minutes), calcium ion concentration (0-25.0 mM) and protein (0-2.0 mg). The optimum pH was 8.0. Deoxycholate had a concentration dependent (0.1 to 0.5 mM) inhibitory effect on the activity. PLA2 specific activity was the highest in mitochondrial fraction. PLA2 activity following incubation with bleomycin was increased in a dose related fashion. In vivo study showed that both PLA2 activity and collagen content in hamster lung were significantly elevated at 14 days followed intratracheal instillation of bleomycin. The activation of PLA2 may play an important role in bleomycin-induced pulmonary toxicity.     

Activities of branched-chain 2-oxo acid dehydrogenase and its components in skin fibroblasts from normal and classical-maple-syrup-urine-disease subjects.

1. Comparisons of the activity and kinetics of the branched-chain 2-oxo acid dehydrogenase in cultured skin fibroblasts from normal and classical maple-syrup-urine-disease (MSUD) subjects provide a kinetic explanation for the enzyme defect. 2. In the intact cell assays, normal fibroblasts demonstrated hyperbolic kinetics with 3-methyl-2-oxo[1-14C]butyrate as a substrate. Intact fibroblasts from four classical MSUD patients showed no decarboxylation over a substrate concentration range of 0.25 to 5.0 mM, and thiamin (4 mM) was without effect. 3. The overall reaction of the multienzyme complex was efficiently reconstituted by using a disrupted-cell system. Normals again showed typical hyperbolic kinetics at the 2-oxo acid concentrations of 0.1 to 5 mM. The Vmax. and apparent Km values were 0.10 +/- 0.02 m-unit/mg of protein and 0.05-0.1 mM respectively, with 3-methyl-2-oxobutyrate. In contrast, classical MSUD patients exhibited sigmoidal kinetics (Hill coefficient, 2.5) with activity approaching 40-60% of the normal value at 5 mM substrate. The K0.5 values from the Hill plots for MSUD patients were 4-7 mM. 4. The E1 (branched-chain 2-oxo acid decarboxylase) component of the multienzyme complex was measured in disrupted-particulate preparations. Normals again showed hyperbolic kinetics with the 2-oxo acid, whereas MSUD preparations exhibited sigmoidal kinetics with the activity of E1 strictly dependent on substrate concentration. Apparent Km or K0.5 were 0.1 and 1.0 mM for normal and MSUD subjects respectively. 5. Measurements of E2 (dihydrolipoyl transacylase) and E3 (dihydrolipoyl dehydrogenase) in MSUD preparations showed them to be in the normal range. 6. The above data suggest a defect in the E1 step of branched-chain 2-oxo acid dehydrogenase in classical MSUD patients.     

Enzymatic dephosphorylation of retinyl monophosphate by rat liver

Rat liver has been shown to contain an enzyme that catalyzes the dephosphorylation of retinyl monophosphate. This activity was extracted with 0.1 M Tris buffer (pH 7.5). Maximum reaction rate was observed at a pH range of 7.0-7.5. It did not require metal ions for activity and was sensitive to fluoride ion. The retinyl monophosphate phosphatase activity was proportional to time and protein and substrate concentration. Triton X-100 (range of 0.05-0.10%) increased the activity 100%, whereas other detergents (Tween 80, cholate, and deoxycholate) did not activate the enzyme. A number of phosphorylated compounds tested as inhibitors of retinyl monophosphatase activity, such as glucose 6-phosphate (20 mM), glycerophosphate (20 mM), phosphatidic acid (8 mM), and dolichyl phosphate (3 mM), did not compete with retinyl monophosphate as substrate. However, at 20 mM concentration, ATP, ADP, 5'-AMP, and pyrophosphate were inhibitors of the enzyme. It is not possible at present to give further details about the specificity of the phosphatase activity. The enzyme described could play a regulatory role in retinol-mediated glycosylations, by altering the endogenous level of retinyl monophosphate.     

Discovery of a novel activator of 5-lipoxygenase from an anacardic acid derived compound collection

Lipoxygenases (LOXs) and cyclooxygenases (COXs) metabolize poly-unsaturated fatty acids into inflammatory signaling molecules. Modulation of the activity of these enzymes may provide new approaches for therapy of inflammatory diseases. In this study, we screened novel anacardic acid derivatives as modulators of human 5-LOX and COX-2 activity. Interestingly, a novel salicylate derivative 23a was identified as a surprisingly potent activator of human 5-LOX. This compound showed both non-competitive activation towards the human 5-LOX activator adenosine triphosphate (ATP) and non-essential mixed type activation against the substrate linoleic acid, while having no effect on the conversion of the substrate arachidonic acid. The kinetic analysis demonstrated a non-essential activation of the linoleic acid conversion with a K_A of 8.65 μM, αK_A of 0.38 μM and a β value of 1.76. It is also of interest that a comparable derivative 23d showed a mixed type inhibition for linoleic acid conversion. These observations indicate the presence of an allosteric binding site in human 5-LOX distinct from the ATP binding site. The activatory and inhibitory behavior of 23a and 23d on the conversion of linoleic compared to arachidonic acid are rationalized by docking studies, which suggest that the activator 23a stabilizes linoleic acid binding, whereas the larger inhibitor 23d blocks the enzyme active site.     

Pseudomonas aeruginosa acid phosphatase. Activation by divalent cations and inhibition by aluminium ion.

In Pseudomonas aeruginosa, the effect of different cations on the acid phosphatase activity was studied in order to acquire more information related to a previously proposed mechanism, involving the coordinated action of this enzyme with phospholipase C. Although the natural substrate of this enzyme is phosphorylcholine, in order to avoid the possible interaction of its positive charge and those of the different cations with the enzyme molecule, the artificial substrate p-nitrophenylphosphate was utilized. Kinetic studies of the activation of acid phosphatase (phosphorylcholine phosphatase) mediated by divalent cations Mg2+, Zn2+ and Cu2+ revealed that all these ions bind to the enzyme in a compulsory order (ordered bireactant system). The Km values obtained for p-NPP in the presence of Mg2+, Zn2+ and Cu2+ were 1.4 mM, 1.0 mM and 3.5 mM, respectively. The KA values for the same ions were 1.25 mM, 0.05 mM and 0.03 mM, respectively. The Vmax obtained in the presence of Cu2+ was about twofold higher than that obtained in the presence of Mg2+ or Zn2+. The inhibition observed with Al3+ seems to be a multi-site inhibition. The K'app and n values, from the Hill plot, were about 0.25 mM and 4.0 mM, respectively, which were independent of the metal ion utilized as activator. It is proposed that the acid phosphatase may exert its action under physiological conditions, depending on the availability of either one of these metal ions.     

Control of phosphofructokinase from rat skeletal muscle. Effects of fructose diphosphate, AMP, ATP, and citrate.

Under conditions used previously for demonstrating glycolytic oscillations in muscle extracts (pH 6.65, 0.1 to 0.5 mM ATP), phosphofructokinase from rat skeletal muscle is strongly activated by micromolar concentrations of fructose diphosphate. The activation is dependent on the presence of AMP. Activation by fructose diphosphate and AMP, and inhibition by ATP, is primarily due to large changes in the apparent affinity of the enzyme for the substrate fructose 6-phosphate. These control properties can account for the generation of glycolytic oscillations. The enzyme was also studied under conditions approximating the metabolite contents of skeletal muscle in vivo (pH 7.0, 10mM ATP, 0.1 mM fructose 6-phosphate). Under these more inhibitory conditions, phosphofructokinase is strongly activated by low concentrations of fructose diphosphate, with half-maximal activation at about 10 muM. Citrate is a potent inhibitor at physiological concentrations, whereas AMP is a strong activator. Both AMP and citrate affect the maximum velocity and have little effect on affinity of the enzyme for fructose diphosphate.     

Optimization of Large Scale Preparation of 13-(S)-Hydroperoxy-9Z, 11E-Octadecadienoic Acid Using Soybean Lipoxygenase. Application to the Chemoenzymatic Synthesis of (+)-Coriolic Acid

The optimization of 13-S-hydroperoxy-9Z, 11E-octadecadienoic acid synthesis is described using lipoxygenase-1 from soybeans at high substrate concentration. The optimal values of the tested parameters are as follows: oxygen pressure 2.5 bar, temperature 5°C, pH 11, enzyme concentration 4 mg/ml and substrate concentration 0.1 M. All these values were used in a single reaction, allowing chemoenzymatic synthesis of gram amounts of (+)-coriolic acid (99%, e.e. 97%) with a 54% yield starting from linoleic acid.     

Interfacial catalysis by lipases

We designed a convenient, specific, sensitive and continuous lipase activity assay using natural long-chain triacylglycerols (TAGs). Oil was extracted from Parinari glaberrimum seed kernels and the purified TAGs used as a substrate for detecting low levels of lipase activities. The purified TAGs are naturally fluorescent. The presence of detergents above their critical micellar concentration dramatically increases the fluorescence of the parinaric acid released by various lipases. This increase is linear with time and proportional to the amount of lipase added. Quantities as low as 0.1 ng of pure pancreatic lipase could be detected under standard conditions (pH 8).

The interfacial activation of human pancreatic lipase (HPL) probably involves the motion of a lid covering the active site of the enzyme. We observed that the presence of either bile salts or a small proportion of water-miscible organic solvents (called activator compounds) considerably enhances the enzymatic activity of HPL on a monomeric solution of tripropionin. This finding suggests that the activator compounds may favor the opening of the lid. This hypothesis was checked by comparing the immunoreactivity of HPL and HPL with a mini-lid (HPL(-lid)) towards anti-HPL monoclonal antibodies (mAbs), in the presence and absence of the activator compounds.     

A continuous spectrophotometric assay for phospholipase A(2) activity

This paper describes a simple continuous spectrophotometric method for assaying phospholipase A(2) (PLA(2)) activity. The procedure is based on a coupled enzymatic assay, using dilinoleoyl phosphatidylcholine as phospholipase substrate and lipoxygenase as coupling enzyme. The linoleic acid released by phospholipase was oxidized by lipoxygenase and then phospholipase activity was followed spectrophotometrically by measuring the increase in absorbance at 234 nm due to the formation of the corresponding hydroperoxide from the linoleic acid. The optimal assay concentrations of hog pancreatic phospholipase A(2) and lipoxygenase were established. PLA(2) activity varied with pH, reaching its optimal value at pH 8.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (V(m)=1.8 microM/min, K(m)=4.5 microM, V(m)/K(m)=0.4 min(-1)). This assay also allows PLA(2) inhibitors, such as p-bromophenacyl bromide or dehydroabietylamine acetate, to be studied. This method was proved to be specific since there was no activity in the absence of phospholipase A(2). It also has the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase A(2).     

Ascorbate activates soluble guanylate cyclase via H2O2-metabolism by catalase

Conditions necessary for the activation by ascorbic acid of soluble guanylate cyclase purified from bovine lung have been examined. Ascorbic acid (0.1-10 mM) did not directly activate the enzyme, nonetheless, pronounced activation by ascorbate (3-10 mM) was observed in incubation mixtures containing 1 microM bovine liver catalase. Superoxide dismutase (SOD) and mannitol did not affect the catalase-dependent activation of guanylate cyclase elicited by ascorbate, suggesting that superoxide anion and hydroxyl radical were not mediating the activation of the enzyme. However, SOD enhanced the relatively low level activation of the enzyme elicited by catalase in the absence of added ascorbate. Pronounced inhibition (both with and without added ascorbate) was observed of catalase-dependent activation of guanylate cyclase by either ethanol (100 mM) or a fungal catalase preparation. Neither ethanol nor fungal catalase inhibited activation of guanylate cyclase by S-nitrosyl-N-acetyl-penicillamine (SNAP), a source of the nitric oxide free radical. These observations indicate that autoxidation of ascorbic acid or thiols present with the guanylate cyclase preparation leads to generation of H2O2, and its metabolism by bovine liver catalase mediates the concomitant activation of guanylate cyclase. The mechanism of activation appears to be associated with the presence of Compound I of catalase and to be inhibited by superoxide anion.     

Stimulation of human fat cell adenylate cyclase by GDP and guanosine 5'-O-(2-thiodiphosphate)

GDP regulation of basal and receptor-mediated catecholamine-sensitive human fat cell adenylate cyclase was studied using purified plasma membrane preparations and assay conditions selected to minimize conversion of GDP to GTP. Under ordinary assay conditions (low NaCl concentration) and with App(NH)p as substrate to prevent GDP conversion to GTP, basal enzyme activity was stimulated up to 2-fold by GDP (0.1 mM) while addition of epinephrine (0.1 mM) eliminated stimulation by GDP and reduced basal adenylate cyclase activity. With ATP as substrate, the enzyme was not responsive to hormone in the absence of guanyl nucleotides and GDP augmentation of basal activity was small (0-1.5-fold) while stimulatory effects of epinephrine and isoproterenol were minimally but definitely exhibited (1.5-fold over basal). Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), a GDP analog resistant to phosphorylation and hydrolysis and an antagonist of GTP, stimulated enzyme activity more than did GDP but did not promote epinephrine action. Rather, inhibition of GDP beta S-stimulated adenylate cyclase activity was seen with both epinephrine and isoproterenol and also with GTP. In the presence of NaCl (200 mM), which alone produced 2-3-fold increase in basal enzyme activity, GDP (0.1 mM) and GDP beta S (50 microM) produced 8- and 15-fold increases of activity, respectively. Addition of UDP, to prevent possible conversion of GDP to GTP, had no effect on NaCl-enhanced activation by GDP. The results indicate that the human fat cell adenylate cyclase system is unique in responding to GDP and its analog GDP beta S by stimulation in the absence of hormone but suggest that as in other systems catecholamine-mediated stimulation is normally dependent on GTP. Salts (Na+) appear to stimulate the enzyme by facilitating the interaction of the guanyl nucleotide regulatory protein (N8) with the catalytic unit.     

Potassium ion-activated hydrolysis of p-nitrophenyl phosphate in pancreatic islet-cell membranes.

Hydrolysis of p-nitrophenyl phosphate was measured in a fraction enriched in plasma membranes from pancreatic islets of non-inbred ob/ob mice. Hydrolysis was stimulated by K+ (10mM) in the pH range 5--10; a small peak of K+-induced activation was observed between pH7.5 and 8. Both the K+-induced activation and the hydrolysis in the absence of K+ were Mg2+-dependent; maximum activation was obtained with 10mM-K+ plus 5 mM-Mg2+. Rb+ was as effective an activator as K+. Ouabain was inhibitory, the effect being inversely related to the K+ concentration; 0.1--0.2mM-ouabain caused about 50% inhibition in the presence of 1 mM-K+, but had no demonstrable effect in the presence of 4--5mM-K+. The K+-stimulated activity was markedly inhibited by 0.1mM-ATP, 35--140 MM-Na+, or 0.01 mM-p-chloromercuribenzenesulphonic acid. Similarities to Rb+ accumulation suggest that catalysis of univalent cation flow in pancreatic beta-cells may be coupled to a phosphoryl-transfer reaction with ATP as natural substrate or regulator.     

Phosphoenolpyruvate carboxylase of Escherichia coli. Specificity of some compounds as activators at the site for fructose 1,6-bisphosphate, one of the allosteric effectors

An investigation was performed to elucidate some unusual phenomena which had been observed with phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of Escherichia coli. (i) Fructose 1,6-bisphosphate (Fru-1,6-P2) and GTP--the allosteric activators--were competitive with each other in the activation. (ii) Some analogs of PEP such as DL-2-phospholactate and 2-phosphoglycolate, which behaved as inhibitors in the presence of the activator (acetyl-CoA or dioxane), activated the enzyme to some extent in the absence of the activator. (iii) Ammonium sulfate deprived the enzyme of sensitivity to Fru-1,6-P2 or GTP but had no effect on the sensitivity to other effectors. It was found that the activation by the analogs was lost upon desensitization of the enzyme to Fru-1,6-P2 by reaction with 2,4,6-trinitrobenzene sulfonate. The activation by the analogs was not observed in the presence of 200 mM ammonium sulfate. In the presence of lower concentrations (0.1 mM) of PEP, ammonium sulfate activated the enzyme at concentrations less than 700 mM but had an inhibitory effect on the desensitized enzyme. These findings suggest that the unusual phenomena described above are a result of binding of the phosphate esters and sulfate ions with the Fru-1,6-P2 site of the enzyme or the active site depending on the reaction conditions.