Marica Bakovic

Transcriptional activation of the murine CTP:phosphocholine cytidylyltransferase gene (Ctpct): combined action of upstream stimulatory and inhibitory cis-acting elements.

CTP:phosphocholine cytidylyltransferase plays a key role in regulating the rate of phosphatidylcholine biosynthesis. However, the proximal regulatory elements for the gene (Ctpct) that encode this enzyme and the cognate transcription factors involved have not been characterized. Ctpct promoter activities were deduced from promoter deletion constructs linked to a luciferase reporter and transiently transfected into C3H10T1/2 and McArdle RH7777 cells. Positive regulatory elements were located between -130 and -52 bp from the transcription start site. Basal expression resided downstream between -52 and +38 bp. DNase I protection and electromobility-shift assays indicated that Sp1-related nuclear factors bind to a stimulatory, a possible inhibitory and minimal promoter element. Gel-shift assays confirmed that all three regulatory regions bound Sp1. Sp1 was further implicated when Sp1-deficient Drosophila cells were co-transfected with promoter-reporter constructs and an Sp1 construct. DNase I assays also indicated that the Ap1 binding elements could be occupied in the proximal activator and minimal promoter regions. Gel-shift assays demonstrated that the distal activator region could bind Ap1 and an unknown transcription factor. We conclude that Sp1, Ap1 and an unknown transcription factor have important roles in regulating expression of the Ctpct gene.

Identification of transcriptional enhancer factor-4 as a transcriptional modulator of CTP:phosphocholine cytidylyltransferase alpha.

CTP:phosphocholine cytidylyltransferase (CCT) is the rate-limiting and regulated enzyme of mammalian phosphatidylcholine biosynthesis. There are three isoforms, CCTα, CCTβ1, and CCTβ2. The mouse CCTα gene promoter is regulated by an enhancer element (Eb) located between −103 and −82 base pairs (5′-GTTTTCAGGAATGCGGAGGTGG-3′) upstream from the transcriptional start site (Bakovic, M., Waite, K., Tang, W., Tabas, I., and Vance, D. E. (1999) Biochim. Biophys. Acta 1436, 147–165). To identify the Eb-binding protein(s), we screened a mouse embryo cDNA library by the yeast one-hybrid system and obtained 19 positive clones. Ten cDNA clones were identified as transcriptional enhancer factor-4 (TEF-4). The TEF-binding consensus sequence, 5′-(A/T)(A/G)(A/G)(A/T)ATG(C/T)(G/A)-3′, was identified within the Eb binding region. Gel-shift analysis using radiolabeled Eb fragment as a probe showed that cell extracts from yeast expressing hemagglutinin-tagged TEF-4 caused a marked band retardation that could be prevented with an anti-hemagglutinin antibody. When COS-7 cells were transfected with TEF-4, CCTα promoter-luciferase reporter activity and CCTα mRNA levels increased. A TEF-4 deletion mutant containing a DNA-binding domain, mTEA(+), stimulated the CCTα promoter activity, whereas protein lacking the DNA binding domain, mTEA(−), did not. Unexpectedly, when the ATG core of the TEF-4 binding consensus within the Eb region was mutated, promoter activity was enhanced rather than decreased. Thus, TEF-4 might act as a dual transcriptional modulator as follows: as a suppressor via its direct binding to the Eb element and as an activator via its interactions with the basal transcriptional machinery. These results provide the first evidence that TEF-4 is an important regulator of CCTα gene expression.

Oxidation kinetics of caffeic acid by prostaglandin H synthase: Potential role in regulation of prostaglandin biosynthesis

The naturally occurring catechol derivative caffeic acid is a moderate stimulator of prostaglandin H synthase cyclooxygenase activity and a good reducing substrate for prostaglandin H synthase-compounds I and II. The discrepancy between the two properties is explained by a specific peroxidative mechanism that includes the formation of an inhibitory complex of caffeic acid with native enzyme followed by a three-step irreversible ping-pong peroxidation. The concentration of caffeic acid necessary to produce 50% stimulation of 0.2 mM arachidonic acid oxidation is 0.8 +/- 0.1 mM. The rate constant for the reaction of prostaglandin H synthase with hydrogen peroxide, determined from steady-state results, is (5.68 +/- 0.1) x 10(5) M-1 s-1. The rate constant for the reaction of prostaglandin H synthase-compound II with caffeic acid is (1.25 +/- 0.1) x 10(6) M-1 s-1. The dissociation constant of caffeic acid from the inhibitory complex is 35 +/- 10 microM. In diluted enzyme solutions, caffeic acid binding is diminished and the enzyme exhibits higher peroxidase activity. Our results suggest that caffeic acid is not a O-demethylation product of ferulic acid degradation catalyzed by prostaglandin H synthase, nor a chelating agent for the heme iron. The oxidation of caffeic acid could be important in the regulation of both prostaglandin H synthase and lipoxygenase activities and hence prostaglandin and leukotriene biosynthesis.

Oxygenation reactions, of prostaglandins endoperoxide synthase and soybean lipoxygenase. Surprising stoichiometry in the formation of hydroperoxy and hydroxy derivatives of cis,cis-eicosa-11,14-dienoic acid

The stoichiometry of the oxygenation reaction of cis,cis-eicosa-11,14-dienoic acid catalyzed by prostaglandin endoperoxide synthase and soybean lipoxygenase has been investigated by using steady-state initial rate measurements. The rate of product formation (conjugated diene hydroperoxy and hydroxy derivatives) was followed spectrophotometrically at 235 nm, and the rate of oxygen consumption was measured polarographically. The ratio of the two rates, d[conjugated diene/-d[O2], is 2/1 for the prostaglandin endoperoxide synthase catalyzed reaction and 1/1 for the lipoxygenase reaction. The 2/1 ratio can be explained by two interrelated routes, each of which results in formation of the conjugated diene hydroxy derivative of the acid. One route, initiated by hydrogen atom abstraction from the acid by Compound I, results in formation of the conjugated diene hydroperoxy derivative. The latter is converted to the hydroxy derivative by regenerating Compound I from the native enzyme. The other route involves direct oxygen atom insertion into the acid by the tyrosyl radical form of Compound I. The decrease in absorbance at 235 nm obtained in the presteady-state phase suggests that during the initial contact of hydroperoxide and enzyme an epoxy-hydroxy fatty acid-enzyme complex may be formed.

Pre-steady-state kinetics and modelling of the oxygenase and cyclooxygenase reactions of prostaglandin endoperoxide synthase

The pre-steady-state kinetics of the prostaglandin endoperoxide synthase oxygenase reaction with eicosadienoic acids and the cyclooxygenase reaction with arachidonic acid were investigated by stopped-flow spectrophotometry at 426 nm, an isosbestic point between native enzyme and compound I. A similar reaction mechanism for both types of catalysis is defined from combined kinetic experiments and numerical simulations. In the first step a fatty acid hydroperoxide reacts with the native enzyme to form compound I and the fatty acid hydroxide. In the second step the fatty acid reduces compound I to compound II and a fatty acid carbon radical is formed. This is followed by two fast steps: (1) the addition of either one molecule of oxygen (the oxygenase reaction) or two molecules of oxygen (the cyclooxygenase reaction) to the fatty acid carbon radical to form the corresponding hydroperoxyl radical, and (2) the reaction of the hydroperoxyl radical with compound II to form the fatty acid hydroperoxide and a compound I-protein radical. A unimolecular reaction of the compound I-protein radical to reform the native enzyme is assumed for the last step in the cycle. This is a slow reaction not significantly affecting steps 1 and 2 under pre-steady-state conditions. A linear dependence of the observed pseudo-first-order rate constant, k(obs), on fatty acid concentration is quantitatively reproduced by the model for both the oxygenase and cyclooxygenase reactions. The simulated second order rate constants for the conversion of native enzyme to compound I with arachidonic or eicosadienoic acids hydroperoxides as a substrate are 8 x 10(7) and 4 x 10(7) M(-1) s(-1), respectively. The simulated and experimentally obtained second-order rate constants for the conversion of compound I to compound II with arachidonic and eicosadienoic acids as a substrate are 1.2 x 10(5) and 3.0 x 10(5) M(-1) s(-1), respectively.

Effect of Trolox C on the oxygenation reaction of prostaglandin endoperoxide synthase with cis,cis-eicosa-11, 14-dienoic acid

Trolox C, a water-soluble derivative of alpha-tocopherol, stimulates the oxygenation of cis,cis-eicosa-11, 14-dienoic acid (AH) by prostaglandin endoperoxide synthase at lower concentrations and suppresses the stimulated reaction at higher concentrations. Surprisingly, Trolox C does not affect the stoichiometric ratio between the rate of formation of the oxygenation product 11-hydroxy-12-trans, 14-cis-eicosadienoic acid (AOH) and the rate of disappearance of molecular oxygen. The ratio of the two rates, d[AOH]/-d[O2], remains constant at 2/1 for a series of Trolox C concentrations and in the absence of Trolox C. Results indicate that AH reacts preferentially with Compound I of the enzyme and that Trolox C does not compete for Compound I. Enzyme inactivation begins with formation of an unproductive Compound I-tyrosyl radical (Compound I-X.) which has the same number of oxidizing equivalents as the conventional peroxidase Compound I. The stimulating effect of low concentrations of Trolox C can be explained by reduction of the oxyferryl heme so that Compound I-X. is reduced to a Compound II-X.species, the Compound II analog of Compound I-X.. Thus heme bleaching is prevented. A further one-electron reduction by Trolox C of Compound II-X. reforms the native enzyme, which permits enzyme recycling. Large concentrations of Trolox C inhibit reformation of native enzyme, reducing the extent of stimulation.