Phosphoglucose isomerase from bananas: partial characterization and relation to main changes in carbohydrate composition during ripening.

Some characteristics of phosphoglucose isomerase (PGI, EC 5.3.1.9) from banana were measured during fruit ripening of three banana cultivars. In banana, PGI was present as two dimeric isoenzymes, named PGI1 and PGI2, which had similar native molecular masses but differed in relation to heat stability and isoelectric point. Total PGI activity showed a distinct two-step change during fruit ripening. Before the climacteric period, PGI activity gradually decreased with the starch content, then its activity began to increase with sucrose accumulation. The ratio of PGI1, and PGI2 was constant, indicating that both enzymes would be involved in starch degradation and sucrose synthesis. PGI activity and changes in carbohydrate composition suggests the existence of some control to fit the requirements of the intense carbon flow from starch to sucrose.     

Non-Complexed Four Cascade Enzyme Mixture: Simple Purification and Synergetic Co-stabilization

Cell-free biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it was essential to produce (purified) enzymes at low costs and stabilize them for a long time so to decrease biocatalyst costs. We studied the stability of the four recombinant enzyme mixtures, all of which originated from thermophilic microorganisms: triosephosphate isomerase (TIM) from Thermus thermophiles, fructose bisphosphate aldolase (ALD) from Thermotoga maritima, fructose bisphosphatase (FBP) from T. maritima, and phosphoglucose isomerase (PGI) from Clostridium thermocellum. It was found that TIM and ALD were very stable at evaluated temperature so that they were purified by heat precipitation followed by gradient ammonia sulfate precipitation. In contrast, PGI was not stable enough for heat treatment. In addition, the stability of a low concentration PGI was enhanced by more than 25 times in the presence of 20 mg/L bovine serum albumin or the other three enzymes. At a practical enzyme loading of 1000 U/L for each enzyme, the half-life time of free PGI was prolong to 433 h in the presence of the other three enzymes, resulting in a great increase in the total turn-over number of PGI to 6.2×10⁹ mole of product per mole of enzyme. This study clearly suggested that the presence of other proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme PGI due to in vitro macromolecular crowding effect. Also, this result could be used to explain why not all enzymes isolated from thermophilic microorganisms are stable in vitro because of a lack of the macromolecular crowding environment.     

Effects of PGI2 on the inactivation of thrombin, factor Xa, and plasmin by antithrombin-III and heparin.

The influence of PGI2 on the activity and on the inactivation of enzymes participating in blood coagulation (thrombin and Factor Xa) and fibrinolysis (plasmin) were investigated. According to the results PGI2 has no effect on the activity of Factor Xa and plasmin nor on the inactivation of these enzymes by antithrombin-III in the absence and presence of heparin at a concentration of PGI2 up to 400 micrograms/ml. An acceleration of the inactivation of thrombin by antithormbin-III was found in the presence of PGI2 within a concentration of 100-400 micrograms/ml without any effect on the heparin-accelerated inactivation of thrombin by antithrombin. We got similar results using clotting tests for the assay and the application of synthetic substrate for thrombin. This inactivation-accelerating effect of PGI2 on thrombin was only demonstratable at a concentration five magnitudes higher than that of the anti-aggregation effect on platelets.     

Prostacyclin synthase and phospholipases in the vascular wall of experimental hypertensive rats

To reveal the role of enzymes involved in PGI2 synthesis for vascular PGI2 generation in experimental hypertensive models, we defined PGI2 synthase and phospholipases activities in the aortic wall of two different experimental hypertensive rats, e.g. spontaneously hypertensive rats (SHR) and desoxycorticosterone acetate (DOCA)-salt hypertensive rats. In the stage of established hypertension both of the hypertensive models had a significantly large capacity of the vascular wall to produce PGI2, as compared to respective control rats. PGI2 synthase activities in the vascular wall were significantly increased by 27% for SHR and by 80% for DOCA-salt hypertensive rats. Moreover, the enzymatic activities were closely related to the blood pressure values for both of the models. On the other hand, phospholipase C or phospholipase A2 activities were increased or unchanged in SHR, respectively, whereas both of the phospholipases were significantly decreased in DOCA-salt hypertensive rats. Thus, it is indicated that PGI2 synthase is partly responsible for the increased PGI2 generation in the vascular wall of SHR and DOCA-salt hypertensive rats, and that vascular phospholipase C is playing a more important role in providing arachidonate for PGI2 synthesis in SHR.     

Recovery of prostacyclin capacity of irradiated endothelial cells and the protective effect of vitamin C

Ionizing irradiation has been reported to affect prostacyclin (PGI2) production by intact blood vessels and cultured endothelial cells (EC) due to damage of enzymes of the arachidonate cascade. In the present study, we investigated whether EC can recover from radiation injury and regain their capacity to produce PGI2. Bovine aortic EC were exposed to radiation doses of 3 and 6 Gy and their capacity to produce PGI2 in response to stimulation with arachidonic acid was tested, at various times after irradiation. The results of these experiments showed clearly that EC exposed to single or fractionated irradiation could recover their capacity to produce PGI2 depending on the radiation dose and the time period following radiation. Radiation damage is associated with oxidant stress and the production of free radicals. We therefore tested the ability of an oxygen radical scavenger, vitamin C, to protect the capacity of irradiated EC to produce PGI2. Pretreatment of EC with low concentrations of vitamin C inhibited the radiation induced release of PGI2 to the culture medium. Vitamin C also enhanced the capacity of irradiated EC to produce PGI2 following short stimulation with arachidonic acid. Treatment with this scavenger however, did not protect the cells against the cytopathic effects of radiation.     

R,R,R-alpha-tocopherol potentiates prostacyclin release in human endothelial cells. Evidence for structural specificity of the tocopherol molecule

Human umbilical vein endothelial cells (HUVEC) in culture synthesize prostacyclin (PGI2) as the predominant metabolite of arachidonic acid which is derived from the deacylation of phospholipids. Under basal-unstimulated condition, PGI2 release from HUVEC is extremely low; however, when endothelial monolayers were preincubated with the natural vitamin E (R,R,R-alpha-tocopherol), we found a dose-dependent potentiation of basal PGI2 release. When HUVEC were stimulated with arachidonate or ionophore A23187, there was a dose-dependent increase of PGI2 release in response to tocopherol enrichment. When HUVEC were labelled with [Me-3H]choline followed by A23187 stimulation, a significantly higher lysophosphatidylcholine was found in the tocopherol-enriched cells, suggesting a change in enzymes involved in phosphatidylcholine metabolism. Analysis of these enzymes revealed that phospholipase A2 activity was enhanced by tocopherol enrichment, whereas lysophospholipase and acyl-CoA acyltransferase were unaffected. To determine the specificity of the tocopherol molecule, different analogues were tested for their PGI2 potentiating activity. Results showed that the free hydroxyl group on the chromanol ring as well as the phytyl side-chain are absolutely required to stimulate PGI2 release, whereas, different methyl locations and substituents on the chromanol ring had no effect. These studies demonstrated that tocopherol potentiates basal PGI2 release in HUVEC and in contrast to its reported inhibitory role in rat platelets, myocardium and neutrophils, tocopherol stimulates phospholipase activity in HUVEC.     

IL-1 and related cytokines enhance thrombin-stimulated PGI2 production in cultured endothelial cells without affecting thrombin-stimulated von Willebrand factor secretion or platelet-activating factor biosynthesis

We examined the effects of various cytokines on alpha-thrombin-stimulated prostaglandin (PG) I2 production, von Willebrand factor (vWF) secretion, and platelet-activating factor (PAF) synthesis in cultured human umbilical vein endothelial cells (HUVEC). A 24-h pretreatment with IL-1 beta doubled the low level of constitutive PGI2 production. In contrast, alpha-thrombin increased PGI2 production fivefold in untreated HUVEC. The most striking increase in PGI2 production was observed in IL-1 beta-treated HUVEC that were subsequently stimulated with thrombin. PGI2 production was two to three times greater than in untreated, thrombin-stimulated HUVEC and nearly eightfold greater than in IL-1 beta-treated but unstimulated HUVEC. Enhanced thrombin-stimulated PGI2 production was also observed in HUVEC pretreated with the related cytokines IL-1 alpha, TNF, or lymphotoxin. This cytokine effect was selective for PGI2 production because none of these cytokines altered either constitutive or thrombin-stimulated vWF secretion or PAF biosynthesis. IL-1 beta enhancement of thrombin-stimulated PGI2 production was concentration and time dependent and required protein synthesis. IL-1 beta pretreatment also enhanced PGI2 production in response to another agonist, histamine, and to exogenously added substrates, arachidonic acid or PGH2. Our results indicate that activation by IL-1 and related cytokines selectively primes endothelial cells for enhanced PGI2 production, but not vWF secretion or PAF synthesis, in response to thrombin and histamine. The evidence suggests that this effect is mediated through specific induction of biosynthetic enzymes for PGI2.     

Phosphoglucose Isomerase from Bananas: Partial Characterization and Relation to Main Changes in Carbohydrate Composition during Ripening

Some characteristics of phosphoglucose isomerase (PGI, EC 5.3.1.9) from banana were measured during fruit ripening of three banana cultivars. In banana, PGI was present as two dimeric isoenzymes, named PGI₁ and PGI₂, which had similar native molecular masses but differed in relation to heat stability and isoelectric point. Total PGI activity showed a distinct two-step change during fruit ripening. Before the climacteric period, PGI activity gradually decreased with the starch content, then its activity began to increase with sucrose accumulation. The ratio of PGI₁, and PGI₂ was constant, indicating that both enzymes would be involved in starch degradation and sucrose synthesis. PGI activity and changes in carbohydrate composition suggests the existence of some control to fit the requirements of the intense carbon flow from starch to sucrose.     

Engineering of a protein with cyclooxygenase and prostacyclin synthase activities that converts arachidonic acid to prostacyclin

Prostacyclin (PGI2), a vascular protector with vasodilation and antithrombotic properties, is synthesized by coupling reactions of cyclooxygenase (COX, the first enzyme) with PGI2 synthase (PGIS, the second enzyme) using arachidonic acid (AA) as an initial substrate. The first COX product, prostaglandin H2 (PGH2) is also a command substrate for other prostanoid enzymes that produce distinct eicosanoids, such as thromboxane A2 (TXA2). The actions of TXA2 to cause vasoconstriction and platelet aggregation oppose the vasodilatory and anti-aggregatory effects of PGI2. Specifically upregulating PGI2 biosynthesis is an ideal model for the prevention and treatment of the TXA2-mediated thrombosis involved in strokes and myocardial infarctions. Here, we report that a single protein was constructed by linking COX-2 and PGIS together to form a new fusion enzyme through a transmembrane domain with 10 or 22 residues. The engineered protein expressed in HEK293 and COS-7 cells was able to continually convert AA to prostaglandin (PG) G2 (catalytic step 1), PGH2 (catalytic step 2), and PGI2 (catalytic step 3). The studies first demonstrate that a single protein with three catalytic functions could directly synthesize PGI2 from AA with a Km of approximately 3.2 microM. Specific upregulation of PGI2 biosynthesis through expression of the engineered single protein in the cells has shown strong activity in inhibiting platelet aggregation induced by AA in vitro, which creates a great potential for the fusion enzyme to be used as one of the new therapeutic interventions for strokes and heart attacks. The studies have also provided a model linking COX with its downstream enzymes to specifically regulate biosynthesis of eicosanoids which have potent biological functions.     

The effects of nitric oxide on prostanoid production and release by human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC) express and synthesize both constitutive and inducible nitric oxide synthase (NOS) and cyclo-oxygenase (COX) enzymes, and have been extensively used as an in vitro model to investigate the role of these enzymes in the patho-physiology of placenta-fetal circulation. In this study we investigated the role of NO in regulating prostanoid production and release from HUVEC. Both untreated and IL-1beta-treated HUVEC were exposed to various NOS inhibitors and NO donors in short-term (1 or 3 hours) experiments, and the effects on prostanoid production were evaluated through the measurement of prostaglandins (PG) I2, E2 and F2alpha released in the incubation medium. We found that the inhibition of inducible NOS but not endothelial NOS antagonizes the IL-1beta-induced increase in PGI2 release. However, NOS inhibitors do not modify baseline PGI2 production. Pharmacological levels of NO, obtained with various NO donors, inhibit basal and IL-1beta-stimulated PG release.     

Effect of ozone exposure on prostacyclin synthesis in lung

The effect of ozone exposure on prostacyclin (PGI2) synthesis in the rat lung was studied. Male Wistar rats were exposed to 0.2, 0.4, 0.8, 1.2 and 1.8 ppm ozone for 24h. The higher concentration (1.8 ppm) significantly depressed-PGI2 synthesizing activity of lung homogenates. Time-courses (1, 3, 5, 7, 14 and 28 days) of the effect of ozone (0.4 and 0.8 ppm) exposure on the PGI2-synthesizing activity of lung homogenates were studied. The PGI2-synthesizing activity of the lung decreased, reaching a maximum at 5 days and then gradually returning to normal by day 14, and remaining normal at day 28, even though the ozone exposure continued. The formation of lipid peroxides due to ozone exposure may cause the depression of PGI2-synthesizing activity of lung. Induction of anti-oxidative enzymes may relate to the recovery of the PGI2-synthesizing activity.     

Human follicular dendritic cells interact with T cells via expression and regulation of cyclooxygenases and prostaglandin E and I synthases

PGE(2) inhibits mature T cell proliferation and protects T cells from activation-induced cell death (AICD). We have previously demonstrated that human follicular dendritic cells (FDC) strongly express PGI synthase. In this study, the hypothesis that FDC have regulatory roles on germinal center T cells by controlling production of PGE(2) and PGI(2) was tested. Confocal microscopic analyses of human tonsil tissues revealed that FDC indeed expressed PGE synthase in addition to PGIS. To confirm these results, we studied the regulation mechanism of PG production in FDC, using an established human FDC-like cell line, HK. Specifically in response to TNF-alpha, TGF-beta, and LPS, protein expression of cyclooxygenase (COX)-2 and downstream PGE synthase was up-regulated with coordinate kinetics, whereas COX-1 and PGIS were constitutively expressed. The increase of these enzymes was reflected in actual production of PGE(2) and PGI(2). Interestingly, IL-4 almost completely abrogated the stimulatory activity of TNF-alpha, TGF-beta, and LPS in PG production. Furthermore, the up-regulation of PGE(2) and PGI(2) production was markedly down-regulated by indomethacin and a selective COX-2 inhibitor. PGI(2) analog and PGE(2) inhibited proliferation and AICD of T cells in dose- and time-dependent manners. Finally, coculture experiments revealed that HK cells indeed inhibit proliferation and AICD of T cells. Put together, these results show an unrecognized pathway of FDC and T cell interactions and differential mechanisms for PGE(2) and PGI(2) production, suggesting an important implication for development and use of anti-inflammatory drugs.     

A tale of two isomerases: compact versus extended active sites in ketosteroid isomerase and phosphoglucose isomerase

Understanding the catalytic efficiency and specificity of enzymes is a fundamental question of major practical and conceptual importance in biochemistry. Although progress in biochemical and structural studies has enriched our knowledge of enzymes, the role in enzyme catalysis of residues that are not nearest neighbors of the reacting substrate molecule is largely unexplored experimentally. Here computational active site predictors, THEMATICS and POOL, were employed to identify functionally important residues that are not in direct contact with the reacting substrate molecule. These predictions then guided experiments to explore the active sites of two isomerases, Pseudomonas putida ketosteroid isomerase (KSI) and human phosphoglucose isomerase (PGI), as prototypes for very different types of predicted active sites. Both KSI and PGI are members of EC 5.3 and catalyze similar reactions, but they represent significantly different degrees of remote residue participation, as predicted by THEMATICS and POOL. For KSI, a compact active site of mostly first-shell residues is predicted, but for PGI, an extended active site in which residues in the first, second, and third layers around the reacting substrate are predicted. Predicted residues that have not been previously tested experimentally were investigated by site-directed mutagenesis and kinetic analysis. In human PGI, single-point mutations of the predicted second- and third-shell residues K362, H100, E495, D511, H396, and Q388 show significant decreases in catalytic activity relative to that of the wild type. The results of these experiments demonstrate that, as predicted, remote residues are very important in PGI catalysis but make only small contributions to catalysis in KSI.     

COMPARISON OF THE BEHAVIOUR OF A SOLUBLE AND A MEMBRANE-BOUND ENZYME IN TRANSECTED PERIPHERAL NERVES

Phosphoglucoisomerase (PGI), a soluble enzyme, and AChE, a membrane-bound enzyme were studied in transected peroneal nerves of dog and in isolated segments of these nerves. Although activities of both enzymes increased at the ends of transected nerves, marked differences in their behaviour were observed. The increment in AChE activity was much sharper than that of PGI and continued to grow with time whereas the increase in PGI developed fully within the initial hours after transection and did not change thereafter. In an isolated nerve segment AChE accumulated at both ends with a concomitant decrease in the middle part, whereas changes in PGI activity appeared only in the terminal parts, the rest of the nerve remaining at the normal level. The terminal increase of PGI did not, contrary to that of AChE, depend on the length of the isolated segment. The changes in PGI activity may be features of a local peritraumatic reaction whereas those of AChE indicate involvement of the whole segment along which the enzyme containing organelles are transported.     

Heparin and acidic fibroblast growth factor interact to decrease prostacyclin synthesis in human endothelial cells by affecting both prostaglandin H synthase and prostacyclin synthase

Prostaglandin production by cultured human endothelial cells varies with growth conditions. We observed a marked diminution in both spontaneous and inducible production of prostacyclin (PGI2) by human umbilical vein and saphenous vein endothelial cells when they were cultured in the presence of the heparin-binding growth factor, acidic fibroblast growth factor (aFGF) and heparin, compared with PGI2 production during culture in medium lacking these factors. Decreased PGI2 production was related to duration of exposure of the cells to aFGF and heparin and depended on the concentration of both substances. Heparin (1-100 micrograms/ml) strongly potentiated the effects of aFGF but had a limited and variable effect alone. The decrease in PGI2 production correlated with a reduction in the cellular content of immunoreactive prostaglandin H synthase and prostacyclin synthase. Arachidonate deacylation was not decreased. In addition, the eicosanoid profile of endothelial cells was changed by exposure to aFGF and heparin. These studies indicate that heparin acts as a modulator of prostaglandin synthesis in endothelial cells through its interaction with aFGF, mediated by alterations in two key enzymes in the arachidonate metabolic pathway.     

Predicting the adaptive evolution of Thermoanaerobacterium saccharolyticum

A fully evolved metabolic network can be described as a weighted sum of elementary modes where the usage probabilities of modes are distributed according to the Boltzmann distribution law (Srienc and Unrean, 2010). An organism presumably achieves the fully evolved state through adaptive changes in the kinetics of rate-controlling enzymes. Metabolic control analysis identifies reactions catalyzed by such enzymes. Comparison of the experimentally determined metabolic flux distributions of Thermoanaerobacterium saccharolyticum AS411 with the predicted flux distribution of a fully evolved metabolic network identified phosphoglucose isomerase (PGI) as the enzyme with the greatest flux control, the rate-controlling enzyme. The analysis predicts that an increased activity of PGI would enable the metabolic network to approach the fully evolved state and result in a faster specific growth rate. The prediction was confirmed by experimental results that showed an increased specific activity of PGI in a culture of strain AS411 that adaptively evolved over 280 generations. Sequencing of the gene confirmed the occurrence of a group of mutations clustered in the subunit binding domain of the dimeric enzyme. The results indicate that the evolutionary path is predictable as the strain AS411 adapted toward the fully evolved state by increasing the PGI activity. This experimental finding confirms that enzymes with predicted highest metabolic flux control are the targets of adaptive metabolic pathway evolution.     

Phosphoglucose isomerase gene duplication in the bony fishes: An evolutionary history

Electrophoretic patterns of phosphoglucose isomerase (PGI) in bony fishes provide strong evidence for a model of genetic control by two independent structural gene loci, most likely resulting from a gene duplication. This model is confirmed by a comparison of certain kinetic and molecular properties of the PGI homodimers (PGI-1 and PGI-2) isolated from extracts of the teleost Astyanax mexicanus. In addition, in most higher teleosts examined, the PGI enzymes show a regular pattern of tissue distribution, with PGI-2 predominant in muscle, the heterodimer often strongest in the heart, and PGI-1 predominant in liver and other organs. An examination of 53 species of bony fishes belonging to 38 families indicates a widespread occurrence of duplicate PGI loci and an early origin of the gene duplication, perhaps in the Leptolepiformes. The apparent presence of three PGI loci in trout and goldfish exemplifies how new loci can be incorporated into the genome through polyploidization.This research was supported in part by a NSF graduate traineeship to J.C.A., by the Clayton Foundation for Research in Biochemistry (G.B.K.), by NSF Grant GB-15644 and NIH Grant GM-15769 to Robert K. Selander, and by contract AT(38-1)-310 between the University of Georgia and the U.S. Atomic Energy Commission.     

A novel phosphoglucose isomerase (PGI)/phosphomannose isomerase from the crenarchaeon Pyrobaculum aerophilum is a member of the PGI superfamily: structural evidence at 1.16-A resolution

The crystal structure of a dual specificity phosphoglucose isomerase (PGI)/phosphomannose isomerase from Pyrobaculum aerophilum (PaPGI/PMI) has been determined in native form at 1.16-A resolution and in complex with the enzyme inhibitor 5-phosphoarabinonate at 1.45-A resolution. The similarity of its fold, with the inner core structure of PGIs from eubacterial and eukaryotic sources, confirms this enzyme as a member of the PGI superfamily. The almost total conservation of amino acids in the active site, including the glutamate base catalyst, shows that PaPGI/PMI uses the same catalytic mechanisms for both ring opening and isomerization for the interconversion of glucose 6-phosphate (Glc-6-P) to fructose 6-phosphate (Fru-6-P). The lack of structural differences between native and inhibitor-bound enzymes suggests this activity occurs without any of the conformational changes that are the hallmark of the well characterized PGI family. The lack of a suitable second base in the active site of PaPGI/PMI argues against a PMI mechanism involving a trans-enediol intermediate. Instead, PMI activity may be the result of additional space in the active site imparted by a threonine, in place of a glutamine in other PGI enzymes, which could permit rotation of the C-2-C-3 bond of mannose 6-phosphate.