The effect of retinoids on the activity of the membrane form of galactosyltransferase, studied in an enzyme/liposome model system.

In the present study the effect of retinoids on the membrane form of galactosyltransferase was tested. A model system consisting of pure bovine milk galactosyltransferase and phosphatidylserine vesicles was used for this investigation. Retinol, retinal and retinylphosphate were able to overcome the modulating effect of phosphatidylserine, that is, activated the enzyme. Retinoic acid and retinylpalmitate were ineffective in this system.

Conversion of the amphiphilic galactosyltransferase from human mammary carcinoma cells to an active hydrophilic enzyme form by limited proteolysis.

As analyzed by a phase-separation technique, the Triton X-114 extract of human mammary carcinoma cells (MCF-7 cells) contain an amphiphilic form of galactosyltransferase (UDPgalactose: D-glucose 4-beta-D-galactosyltransferase, EC 2.4.1.22), while the galactosyltransferase activity released by these cells represents a hydrophilic form of the enzyme. When the amphiphilic galactosyltransferase was subjected to limited proteolysis with thermolysin, this treatment generated a hydrophilic form of the enzyme. With respect to Km for UDPgalactose the kinetic data were very similar for the amphiphilic, for the released and the hydrophilic galactosyltransferases produced by proteinase treatment. Differences were detected in electrophoretic and gel chromatographic properties. The hydrophilic enzymes showed a greater electrophoretic mobility on non-denaturing polyacrylamide gels than did the amphiphilic form. On Sepharose 6B column chromatography, the amphiphilic galactosyltransferase appeared to be of higher molecular weight than the hydrophilic enzyme.

Semicarbazide-sensitive amine oxidase catalyzes endothelial cell-mediated low density lipoprotein oxidation.

OBJECTIVE: Deamination products of semicarbazide-sensitive amine oxidases (SSAO), i.e. aldehydes, superoxide and ammonia have been shown to initiate vascular damage. SSAOs are copper-enzymes, present in endothelial (EC), smooth muscle cells (SMC) and in blood. Transition metals ions (Cu, Fe) mediate the oxidative (atherogenic) modification of LDL by SMC and EC. The physiological source of the active metal ions is still under debate. We hypothesize that SSAOs may catalyze LDL oxidation by endothelial cells via enzyme-complexed Cu++. METHODS: EC isolated from human umbilical veins and cultured in 35 mm wells in RPMI-1640 medium were used as LDL oxidation system. RESULTS: Diamine oxidase (DAO), a SSAO which activity is elevated in tissues and sera of diabetic patients, catalyzes the oxidation of LDL by EC. In the presence of purified DAO (0.07 to 70 U/l) LDL oxidation was increased up to 10-fold as measured by thiobarbituric acid reactive substance (TBARS) formation as well as apoprotein modification of LDL. Chemical blockage of the SSAO substrate binding site did not inhibit the catalytic effect of DAO on LDL oxidation. Denaturation of the enzyme did not destroy the ability of the preparation to facilitate LDL oxidation by EC. The potential of the enzyme to catalyze LDL oxidation was not suppressed in the presence of serum. However, selective removing of enzyme-copper completely abolished the ability of the enzyme to trigger cell-mediated LDL oxidation. CONCLUSION: DAO, beside generating angiopathic deamination products, has the potential to act as a pathophysiological catalyst of LDL atherogenic modification by vascular cells.

Tyrosine phosphorylation blocks tyrosine free radical formation and, hence, the hormonogenic iodination reaction.

It is known that a tyrosine free radical is produced during the hormonogenic iodination reaction of tyrosine residues on thyroglobulin. In the hormonogenic region of thyroglobulin, phosphorylated tyrosine residues have been detected. Using an vitro tyrosine iodinating system we report that the hormonogenic reaction cannot go off if tyrosine becomes phosphorylated. Enzymatic dephosphorylation of the modified amino acid restored the ability of the molecule to become iodinated. Considering the mechanism of the tyrosine free radical formation, these observations are due to the inability of the phosphorylated amino acid to form a free radical. Our data may suggest a putative regulatory mechanism in thyroid hormone synthesis by phosphorylation of hormonogenic tyrosine residues on thyroglobulin.

The salicylate metabolite gentisic acid, but not the parent drug, inhibits glucose autoxidation-mediated atherogenic modification of low density lipoprotein.

Oxidation of low density lipoprotein (LDL) by glucose-derived radicals may play a role in the aetiology of atherosclerosis in diabetes. Salicylate was shown to scavenge certain radicals. In the present study, aspirin, salicylate and its metabolites 2,5- and 2, 3-dihydroxybenzoic acid (DHBA) were tested for their ability to impair LDL oxidation by glucose. Only the DHBA derivatives, when present during LDL modification, inhibited LDL oxidation and the increase in endothelial tissue factor synthesis induced by glucose oxidised LDL. The LDL glycation reaction was not affected by DHBA. The antioxidative action of DHBA may be attributed to free radical scavenging and/or chelation of transition metal ions catalysing glucose autoxidation.

Genistein prevents the glucose autoxidation mediated atherogenic modification of low density lipoprotein.

Hyperglycemia has been assumed to be responsible for oxidative stress in diabetes. In this respect, glucose autoxidation and advanced glycation end products (AGE) may play a causal role in the etiology of diabetic complications as e.g. atherosclerosis. There is now growing evidence that the oxidative modification of LDL plays a potential role in atherogenesis. Glucose derived oxidants have been shown to peroxidise LDL. In the present study, genistein, a compound derived from soy with a flavonoid chemical structure (4', 5, 7-trihydroxyisoflavone) has been evaluated for its ability to act as an antioxidant against the atherogenic modification of LDL by glucose autoxidation radical products. Daidzein, (4',7-dihydroxyisoflavone) an other phytoestrogen of soy, was tested in parallel. Genistein--in contrast to daidzein--effectively prevented the glucose mediated LDL oxidation as measured by thiobarbituric acid-reactive substance formation (TBARS), alteration in electrophoretic mobility, lipid hydroperoxides and fluorescence quenching of tryptophan residues of the lipoprotein. In addition the potential of glucose-oxidized LDL to increase tissue factor (TF) synthesis human endothelial cells (HUVEC) was completely inhibited when genistein was present during LDL oxidative modification by glucose. Both phytoestrogens did not influence the nonenzymatic protein glycation reaction as measured by the in vitro formation of glycated LDL. As the protective effect of genistein on LDL atherogenic modification was found at glucose/genistein molar ratios which may occur in vivo, our findings support the suggested beneficial action of a soy diet in preventing chronic vascular diseases and early atherogenic events.

Modulation of biological tyrosine reactions by tyrosine phosphorylation.

This review focuses on the influence of tyrosine phosphorylation on the biological reactions of tyrosine. Reactions that are modulated by this amino acid modification include dityrosine formation, thyroid hormone synthesis, and DOPA formation. In addition, we show that the reactivity of tyrosine in the common Lowry method of determination of protein concentrations is lost upon phosphorylation of the amino acid.

The uremic toxin indoxyl sulfate acts as a pro- or antioxidant on LDL oxidation.

Uremic toxins have been shown to play a role in chronic kidney disease (CKD) associated oxidative stress. Oxidative stress and inflammation have been associated with increased risk of cardiovascular disease in uraemia. The oxidative modification of LDL may play a role in early atherogenesis. Enhanced LDL oxidation has been found in uremic patients which may account for accelerated atherosclerosis observed in CKD. The uremic toxin indoxyl sulfate (IS) has been reported to exert oxidative and antioxidative activity. Thus, in the present study we have investigated the influence of IS on the atherogenic modifications of LDL exposed in vitro to different oxidising systems. The transition metal ion (Cu(2+)) and hemin/H2O2 induced lipid oxidation reactions monitored by conjugated diene formation, were inhibited by the presence of IS, which points to possible antioxidant effects by this uremic toxin. A protective effect of IS on LDL apoprotein modification by the exposure to the product of the myeloperoxidase/H2O2/Cl(-) system HOCl, was also observed as estimated by protein carbonyl formation. In contrast, a marked increase in conjugated dienes and lipid hydroperoxides was observed when lipid oxidation was initiated by the free radical generator AAPH in presence of IS. The GC-MS analysis revealed the formation of indole-2,3-dione and 6,12-dihydro-6,12-dioxo-indolo[2,1-b]quinazoline (tryptanthrin) in IS/AAPH reaction. A scheme for the generation of tryptanthrin from IS via indoxyl radicals is proposed, which may facilitate LDL lipid oxidation. Our observations add further insight in the Janus-faced properties of this important uremic toxin.

Phorbol ester, retinoic acid and diacylglycerol decrease ecto- but increase total basal-protein kinase activity of human fibroblasts.

Studies have been carried out on intact human lung fibroblasts (HLF) in situ to investigate the effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the anti-tumor promoter retinoic acid (RA) on ecto-protein kinases. The ecto-kinase reaction of the HLF-cells was cAMP-independent, showed an apparent Km for ATP of 6.99 +/- 0.35 (microM) and was substantially inhibited by TPA and RA. With the notable exception of a approximately 57 kD phosphoprotein both compounds decreased the overall phosphorylation of intact cells. In contrast to RA-treatment, however, TPA caused the release of a high molecular weight (approximately 210 kD) phosphoprotein from the HLF. RA was the most potent retinoid in reducing ecto-kinase activity. The physiological modulators of protein kinase C: 1,2- and 1,3-diolein as well the synthetic 1,2-dioctanoylglycerol decreased the ecto-kinase activity to an extent similar to that of TPA. The drop in ecto-kinase activity of HLF-cells in situ caused by TPA, RA and the diacylglycerols was accompanied by an increase in total basal-(Mg++-dependent) protein kinase activity present in extracts of treated cells. The results suggest an important role of ecto-kinase in the response of intact cells to TPA and RA.

Co2+ is able to substitute for Mn2+ in some exogenous and endogenous galactosyltransferase reactions.

The ability of Co2+ to substitute for Mn2+ in exogenous and endogenous galactosyltransferase reactions was tested. Exogenous transfer was measured towards different high and low molecular weight galactose acceptors using galactosyltransferase from the following sources: crude serum, the serum enzyme partially purified by affinity chromatography and a pure enzyme preparation from milk. Endogenous transfer was estimated in preparations from human urinary bladder tumor cells and from rat liver microsomal fractions. The results show that Co2+ is able to substitute for Mn2+ in some exogenous and endogenous galactosyltransferase reactions. This ability seems to depend on the molecular structure of the galactose acceptor as well as on the nature of the enzyme.

Galactosyltransferase activities in cultured urinary bladder tumor cells.

After demonstrating that three bladder cancer cell lines (human bladder transitional cell carcinoma, MGH-U1; rat bladder transitional cell carcinoma, RBTCC; Nara rat bladder epidermal carcinoma, NBT-II) had galactosyltransferase (GT) activity in their cell surfaces, we investigated the effect of increasing cell density on the activity of this enzyme. All three cell lines responded to increased cell density by increased activity of cell-surface GT towards endogenous acceptor. By the use of exogenous acceptor, we showed that in the two transitional cell carcinoma lines (human and rat), the increased activity was probably caused by increased levels of endogenous acceptor rather than enzyme. In the rat bladder epidermal carcinoma line, on the other hand, increased GT activity seemed to be the result of increased levels of the enzyme. These conclusions were supported by the increased shedding of GT into the medium with increasing cell density in case of the epidermal carcinoma cells, but not the two transitional cell carcinoma lines. Total cell-associated GT activity would indicate that, in contrast to the two transitional cell carcinoma lines, the bladder epidermal carcinoma cells may have an increased rate of synthesis of GT as confluence is approached.

Influence of ultraviolet light, various temperatures, and zinc ions on anthralin (dithranol). Biochemical and chemical investigations.

The changes of anthralin under various physical conditions (temperature, ultraviolet irradiation) were investigated by biochemical assay (inhibition of G-6-PDH activity), by oxygen monitor (increased oxygen consumption in the presence of zinc ions), and by recording the absorption spectra. Higher temperatures and exposure to ultraviolet light provoke the formation of a biochemically highly active compound within short periods of time. In clinical therapy, this compound may easily be formed when anthralin is used together with ultraviolet irradiation (Ingram method). Changes in the biochemical activity of anthralin are accompanied by changes in the absorption spectra. Oxidation (e.g. in the presence of zins ions) or inhibition of oxidation (e.g. in the presence of salicylic acid) may easily be detected by spectroscopic assay.

Phosphatidylserine modulates calf intestine alkaline phosphatase activity towards low and high molecular weight substrate.

Pretreatment of calf intestine alkaline phosphatase with phosphatidylserine resulted in an inhibition of the phosphatase activity towards low - (p-nitrophenylphosphate) and high (phosphohistone) molecular weight substrate. Phosphatidylcholine, irrespectively of the substrate used did not cause enzyme modulation. 12-O-tetradecanoylphorbol-13-acetate, 1,2-diolein as well certain retinoids, known to effect phosphatidylserine-sensitive enzyme systems (Castagna, M. et al. 1982, J. Biol. Chem. 257, 7847-7851; Gmeiner, B. 1986, Biochim. Biophys. Acta 856, 392-394) had no influence on the modulated phosphatase. The lipid interacting drug trifluoperazine inhibited the enzyme activity towards phosphohistone, but not towards p-nitrophenylphosphate as a substrate. The results indicate that acidic phospholipid may play a role in activity modulation of calf intestine membranous alkaline phosphatase activity.

Measurement of phosphotyrosine phosphatase activity using the Folin-Ciocalteu phenol reaction.

An assay for the estimation of phosphotyrosine phosphatase using the Folin-Ciocalteu phenol reaction to monitor enzyme activity is presented. The method is based on the property of the substrate phosphotyrosine not to react as a phenol until it is dephosphorylated. The method is sensitive, there is no interference from the use of detergents and it does not rely on special laboratory equipment to distinguish tyrosine from phosphotyrosine.

Enzyme inhibition in human skin homogenates by hydrocortisone, hydrocortisone acetate and hydrotisone butyrate.

In fresh human skin homogenates, the activities of four enzymes, lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G-6-PDH), "acid" phosphatase (AcP), and "leucine aminopeptidase" (LAP) were assayed following an incubation with hydrocortisone, hydrocotisone acetate, or hydrocortisone-17-butyrate, respectively. Concentration of the three compounds measured 2.75 mMol/l. Hydrocortison butyrate inhibited LDH-G-6-PDH-, and AcP-activities. Hydrocortisone and hydrocortisone acetate exerted a significant inhibitory action only in the case of G-6-PDH-activity.--On pure G-6-PDH from yeast, the inhibition exerted by hydrocortisone butyrate was significantly stronger than the inhibition exerted by the two other steroids. Time/action diagrams revealed the fact that hydrocortisone butyrate is superior to the other two compounds from the beginning of the incubation period.--The date sustain the assumption that hydrocortisone butyrate exerts biochemical-pharmacological actions of its own and that it may not be considered just as an esterified transport form of hydrocortisone.

Influences of retinoic acid and retinoid on skin metabolism. Investigations of oxygen consumption and enzymatic activities of human skin.

All-trans retinoic acid and its derivative retinoid, two new compounds with expanding therapeutic spectrum in dermatology, were investigated in biochemical assays. Both substances provoke an increase in oxygen consumption of rat skin whereas in human skin only retinoid was found active in this respect. In resting yeast cells, both substances failed to exert any significant influence on oxygen consumption.--Pure G-6-PDH was inhibited by retinoic acid and retinoid in concentrations as low as 5 mug/ml. In human skin homogenates, LDH-, GAPDH-, and G-6-PDH-activities were inhibited by retinoic acid whereas GOT-, LAP-, and ALD-activites remained practically unchanged following an incubation with retinoic acid in concentrations between 1 and 100 mug/ml for 60 min.--The data collected in this study were briefly discussed with regard to the use of retinoic acid and its derivatives in psoriasis.

Inhibition of glucose-6-phosphate dehydrogenase activity by betamethasone and three of its esters with dermatological importance.

Betamethasone, betamethasone-17-valerate, betamethasone-17-benzoate, and betamethasone-17,21-diproprionate were investigated for their inhbitory action on glucose-beta-phosphate dehydrogenase (G-6-PDH) activity (pure enzyme from yeast, enzyme from human skin homogenate). Between these four compounds, marked differences were encountered which could not be attributed to the presence of an esterified or unesterified steroid. According to these data it does not seem to be justified to consider betamethasone esters simply as the transport forms of the topically inactive betamethasone but one must consider the betamethasone esters having biochemical actions of their own.

The influence of D-penicillamine on enzymatic activities: glucose-6-phosphate dehydrogenase. Correlation with serum levels measured in humans.

The influence of D-penicillamine on glucose-6-phosphate dehydrogenase of yeast (pure enzyme), human hemolysate, and human skin homogenate were determined. In high concentrations, D-penicillamine inhibits glucose-6-phosphate dehydrogenase activity (concentrations above 6.7 mmol/l, i. e. l g/l). In low concentrations, D-penicilliamine exerts an indirect influence by removing some inhibiting metal ions, such as zinc. In human skin homogenates, an activating action of D-penicillamine on glucose-6-phosphate dehydrogenase activity occurs due to the chelation of metal ions.

Paracetamol catalyzes myeloperoxidase-initiated lipid oxidation in LDL.

The oxidative modification of LDL may play a significant role in atherogenesis. Myeloperoxidase (MPO) expressed in human atherosclerotic plaques has been suggested to be operative in vivo, making LDL atherogenic. Tyrosyl radicals generated by MPO have been shown to act as physiological pro-oxidants of lipid peroxidation in LDL. Assuming that a variety of phenolic compounds are able to form phenoxyl radicals when exposed to peroxidases, we tested the ability of paracetamol, a known analgesic drug with a tyrosine-like monophenolic structure, to act as a pro-oxidant of lipid peroxidation in LDL. Spectroscopic analyses indicated that paracetamol, similar to tyrosine, could undergo peroxidase-induced phenoxyl radical formation, which was inhibited by the radical scavenger ascorbic acid as well as by heme poisons and catalase. Measurement of conjugated dienes and lipid hydroperoxides in LDL preparations exposed to MPO/H2O2 in the absence or presence of paracetamol revealed that the drug could act as a catalyst of lipid oxidation in LDL. Similar results were found when LDL oxidation was performed with activated human neutrophils, which use MPO to promote lipid peroxidation. In conclusion, the results suggest that paracetamol could act, via a phenoxyl radical, as a catalyst of LDL oxidative modification by MPO.