Role of mevalonate-5-pyrophosphate decarboxylase in the regulation of chick intestinal cholesterogenesis

The response to different dietary conditions of the enzymes responsible for the transformation of mevalonic acid to isopentenyl pyrophosphate has been studied for the first time in the small bowel of the chick to elucidate the role of these enzymes in the regulation of intestinal cholesterogenesis. Feeding a 2% cholesterol diet from hatching resulted in a small but significant inhibition of mevalonate-5-pyrophosphate decarboxylase, while mevalonate kinase and mevalonate-5-phosphate kinase remained unaltered. Similar results were obtained for the three enzymes when 13-day-old chicks fed a standard fat-free diet were switched to a 5% cholesterol diet. Starved chicks exhibited lower intestinal decarboxylase activity than chicks fed a standard diet, while refeeding resulted in levels of activity similar or slightly greater than controls. None of the enzymes effecting the conversion of mevalonate to isopentenyl pyrophosphate in the small intestine presented diurnal variations. Results obtained suggest that mevalonate-5-pyrophosphate decarboxylase may play a significant role in the regulation of cholesterol synthesis in the small intestine.     

Effect of clofibrate on brain mevalonate-5-pyrophosphate decarboxylase

The effect of clofibrate on the activity of the three mevalonate-activating enzymes has been studied for the first time in brain by reactions carried out using [2-¹⁴C] mevalonic acid as substrate and 105,000g supernatants from 14-day-old chick brain. Mevalonate-5-pyrophosphate decarboxylase was clearly inhibited, while mevalonate kinase and mevalonate-5-phosphate kinase were not significantly affected. The effect of clofibrate on decarboxylase activity was progressive with increasing concentrations (1.25–5.00 mM) of the inhibitor. A transient inhibition and a subsequent activation as a function of clofibrate concentration seemed to occur for mevalonate kinase. Direct measurements of decarboxylase activity utilizing [2-¹⁴C] pyrophosphomevalonate as the specific substrate of this enzyme corroborated these results. Kinetic studies showed that clofibrate competes with the substrate ATP.     

Studies on the diurnal rhythm of mevalonate metabolism by sterol and nonsterol pathways and of mevalonate-activating enzymes

Both in vivo and in vitro incorporation of mevalonic acid into nonsaponifiable lipids by 17-day-old chick liver and kidney did not show diurnal rhythm. Using 14CO2 production from MVA as an index of the shunt pathway not leading to sterols, we have demonstrated for the first time that there is no diurnal rhythm in this pathway. No significant differences were found in the specific activities of mevalonate kinase, mevalonate-5-phosphate kinase and mevalonate-5-pyrophosphate decarboxylase from chick liver and kidney throughout a period of 24 hr, using [1-14C]mevalonate as substrate. The absence of diurnal rhythm in the decarboxylase activity was corroborated by further experiments carried out using [2-14C]mevalonate-5-pyrophosphate as specific substrate of this enzyme.     

Studies on pig liver mevalonate-5-diphosphate decarboxylase

A procedure in which three sequential enzymes of cholesterol biosynthesis, mevalonate kinase (ATP: (R)-mevalonate 5-phosphotransferase, EC 2.7.1.36), phosphomevalonate kinase (ATP: (R)-5-phosphomevalonate phosphotransferase, EC 2.7.4.2) and mevalonate-5-diphosphate decarboxylase (ATP: (R)-5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33), from pig liver, could be purified in the one operation is described. Mevalonate kinase and phosphomevalonate kinase were utilized for the enzymic synthesis of mevalonate 5-diphosphate (both 1-14C-labelled and unlabelled), the substrate for mevalonate-5-diphosphate decarboxylase, using excess free ATP4-. A radioactive assay for the enzyme, based on the release of 14CO2 from [1-14C]mevalonate-5-diphosphate, was developed. The assay allowed reassessment of the metal and nucleotide specificity of the decarboxylase. ATP could be partially replaced by GTP and ITP, but no activity was observed with CTP, UTP or TTP. Apparent activation of the enzyme by ATP4- was observed as found for mevalonate kinase (C.S. Lee and W.J. O'Sullivan (1983) Biochim. Biophys. Acta 747, 215-224) and phosphomevalonate kinase (C.S. Lee and W.J. O'Sullivan (1985) Biochim. Biophys. Acta 839, 83-89). The presence of 1 mM excess free ATP4-, above that complexed as the substrate MgATP2-, decreased the Km for MgATP2- from 0.45 mM to 0.15 mM. MgADP- was shown to act as a competitive inhibitor with respect to MgATP2-.     

Quantitative role of different embryonic tissues in mevalonate metabolism by sterol and nonsterol pathways. Relationship with enzyme activities of cholesterogenesis

3-Hydroxy-3-methylglutaryl-CoA reductase, mevalonate kinase, mevalonate-5-phosphate kinase and mevalonate-5-pyrophosphate decarboxylase activities have been determined in brain, liver, intestine and kidneys from 19-day-old chick embryo. Levels of brain reductase and decarboxylase were clearly higher than those found in the other tissues assayed. However, only small differences were observed in the activity of both kinases among the different tissues. Mevalonate metabolism by sterol and nonsterol pathways has been investigated in chick embryo at the same developmental stage. Mevalonate incorporation into total nonsaponifiable lipids was maximal in liver, followed by intestine, brain and kidneys. The shunt pathway of mevalonate not leading to sterols was negligible in both brain and liver, while a clear CO2 production was observed in intestine and kidneys. Sterols running in TLC as lanosterol and cholesterol were the major sterols formed from mevalonate by brain and kidney slices, while squalene and squalene oxide(s) were found to be mainly synthesized by liver slices. Minor differences in the percentage of different sterols were observed in chick embryo intestine. The importance of free and esterified cholesterol accumulation in the different tissues on the inhibition of cholesterogenic activity is discussed.     

Influence of cholestyramine feeding on mevalonate-activating enzymes

Phosphorylation and decarboxylation of mevalonic acid have been measured in different tissues of chicks fed a cholestyramine diet from hatching until 18 days of age. Hepatic and intestinal phosphorylation and decarboxylation of mevalonate were slightly although significantly increased from 15 days of treatment. Direct measurements of 5-pyrophosphomevalonate decarboxylase activity using the specific substrate of this enzyme corroborated these data. Brain enzymatic activities remained unaltered with respect to controls. These results suggest that enzymes responsible for the conversion of mevalonate to isopentenyl pyrophosphate from neonatal chick liver and intestine alter their activities in a coordinate fashion and may play an important role in the regulation of cholesterogenesis in these tissues.     

Effects of clofibrate on the main regulatory enzymes of cholesterogenesis

The in vivo effect of clofibrate on the main regulatory enzymes of cholesterogenesis has been comparatively studied for the first time in chick liver and brain. 3-Hydroxy-3-methylglutaryl-CoA reductase and mevalonate 5-pyrophosphate decarboxylase from chick liver were significantly inhibited by this hypocholesterolenic drug, while mevalonate kinase and mevalonate 5-phosphate kinase were not affected. No enzyme from chick brain was significantly inhibited by the in vivo treatment. However, both liver and brain reductase activity was inhibited in vitro by clofibrate, inhibition that was progressive with increasing concentrations (1.25-5.00 mM) of drug.     

Enzymatic synthesis of mevalonate-5-(2-thiodiphosphate)

Mevalonate-5-(2-thiodiphosphate), a substrate analog for diphosphomevalonate decarboxylase, has been enzymatically prepared from mevalonate-5-phosphate and adenosine-5'-0-(3-thiotriphosphate) using phosphomevalonate kinase as a catalyst, in a 37% yield. The substrate properties of the synthesized compound are compared to those of the normal substrate of the enzyme.     

EFFECT OF PHENYL AND PHENOLIC ACIDS ON MEVALONATE-5-PHOSPHATE KINASE AND MEVALONATE-5-PYROPHOSPHATE DECARBOXYLASE OF THE RAT BRAIN

Abstract— Phenyl and phenolic acids are known to inhibit metabolism of mevalonate in rat brain. The site of inhibition has been found to be mevalonate-5-pyrophosphate decarboxylase. Phenolic acids also inhibited mevalonate-5-phosphate kinase on preincubation. The kinetics showed that p -coumaric acid and isoferulic acid were competing with substrates, mevalonate-5-phosphate or mevalonate-5-pyre phosphate, whereas others showed an uncompetitive type of inhibition. Chlorophenoxyisobutyrate, a hypocholesterolaemic drug, had no effect on these enzymes. An improved method for the synthesis of mevalonate-5-phosphate and mevalonate-5-pyrophosphate, labeled at carbon-1, is described.     

Relationship between changes in free cholesterol and pyrophosphomevalonate decarboxylase activity during myelination

The patterns of cholesterol content in chick brain and liver were studied during embryonic development and compared with the variations in the specific activities of mevalonate-activating enzymes during the same period. Total cholesterol content in both embryonic chick brain and liver increased during incubation. The relative percentage of free cholesterol was always maintained over 85% in brain, while in liver this percentage decreased to less than 10% during the later days of incubation. A straight parallelism was observed between free cholesterol and pyrophosphomevalonate decarboxylase activity in the embryonic brain. On the other hand, the hepatic decarboxylase exhibited a lower specific activity than in brain and did not show significant variations throughout the same period of incubation. Changes in brain pyrophosphomevalonate decarboxylase activity were more pronounced than those observed in both mevalonate kinase and phosphomevalonate kinase activities, in spite of that the specific activity of decarboxylase was the lowest of the three mevalonate-activating enzymes, suggesting that this reaction is one rate-limiting step for cholesterogenesis during myelination.     

STUDIES ON MEVALONATE KINASE, PHOSPHOMEVALONATE KINASE AND PYROPHOSPHOMEVALONATE DECARBOXYLASE IN DEVELOPING RAT BRAIN

Abstract— We have in the present study investigated the properties of mevalonate kinase, phosphomevalonate kinase and pyrophosphomevalonate decarboxylase in the 105,000 g supernatant fractions from rat brain, and determined whether the activities of these enzymes change during brain development. All three enzymes in brain showed a specific requirement for ATP for optimal activity. The presence of Mg²⁺ as divalent cation was also required for optimal activity of mevalonate kinase and phosphomevalonate kinase. Both Mg²⁺ and Mn²⁺ were equally effective divalent metal ions for pyrophosphomevalonate decarboxylase in brain. Mevalonate kinase as well as phosphomevalonate kinase were active in a broad pH range of 6.5–8 while the pH curve for pyrophosphomevalonate decarboxylase showed a peak activity at approx 6. No age-dependent change occurred in the activities of mevalonate kinase and phosphomevalonate kinase in developing brain, whereas pyrophosphomevalonate decarboxylase activity in brain increased during the 1st week after birth, reached a peak value at about the 8th day of age and declined slowly thereafter. The K_m for brain mevalonate kinase in 2, 13 and 52 day old rats were 312, 400 and 434 μM, respectively. The V _max for the kinase in 2, 13 and 52 day old rats were in the range of 45–52 nmol/h/mg protein, respectively. This suggests that, like in liver (R amachandran & S hah , 1976), pyrophosphomevalonate decarboxylase in brain may also be one regulatory step for cholesterol synthesis.     

Hypolipidemic activity of dipyridamole: effects on the main regulatory enzyme of cholesterogenesis.

The in vivo dipyridamole treatment for 16 days produced a significant decrease in chick plasma cholesterol, mainly due to the esterified form. This effect was especially patent in the VLDL + LDL fraction. Similar results were observed in triglyceride content. To our knowledge, this is the first report on this hypolipidemic effects of dipyridamole. Total and esterified cholesterol increased after the same treatment in chick liver, while brain cholesterol content was not affected. Hepatic 3-hydroxy-3- methylglutaryl-CoA reductase activity was drastically reduced, while other secondary regulatory enzymes such as mevalonate kinase, mevalonate 5-phosphate kinase and mevalonate 5-pyrophosphate decarboxylase did not change significantly. No significant differences were found in cholesterol and lipidic phosphorus from liver microsomes, so that the effect of dipyridamole on reductase activity cannot be due to modifications in cholesterol/lipidic phosphorus molar ratio. Neither of these enzyme activities was affected in vitro by dipyridamole.     

Cysteine sulphinate decarboxylase in chick and rat retina during development

The activity, properties and developmental pattern of cysteine sulphinate decarboxylase (CSD) were studied in chick and rat retina. Retinal CSD shows properties similar to those of the enzyme in brain with respect to optimum pH, saturating substrate concentrations and stimulation by pyridoxal phosphate, CSD activity increased 3-fold from the 10th day of embryogenesis to hatching in chicks and in postnatal development in rats. The developmental pattern of CSD activity in both species is coincident with the functional maturation of visual function.     

Effect of phenylalanine derivatives on the main regulatory enzymes of hepatic cholesterogenesis

Phenylalanine, phenylpyruvate and phenylacetate produced a considerable inhibition of chick liver mevalonate 5-pyrophosphate decarboxylase while mevalonate kinase and mevalonate 5-phosphate kinase were not significantly affected. Phenolic derivatives of phenylalanine produced a similar inhibition of decarboxylase activity than that found in the presence of phenyl metabolites. The degree of inhibition was progressive with increasing concentrations of inhibitors (1.25–5.00 mM). Simultaneous supplementation of different metabolites in conditions similar to those in experimental phenylketonuria (0.25 mM each) produced a clear inhibition of liver decarboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase. To our knowledge, this is the first report on the in vitro inhibition of both liver regulatory enzymes of cholesterogenesis in phenylketonuria-like conditions. Our results show a lower inhibition of decarboxylase than that of reductase but suggest an important regulatory role of decarboxylase in cholesterol synthesis.     

3-Hydroxy-3-methylglutaryl-CoA reductase, mevalonate-5-pyrophosphate decarboxylase and acyl-CoA: cholesterol acyl-transferase from mucosa scrapings and isolated enterocytes from chick duodenum, jejunum and ileum

3-Hydroxy-3-methylglutaryl-CoA reductase, mevalonate-5-pyrophosphate decarboxylase and acyl-CoA: cholesterol acyltransferase activities were assayed in mucosal scrapings and isolated enterocytes from chick duodenum, jejunum and ileum. Maximal reductase and decarboxylase specific activities were found in ileum and jejunum, while ileum exhibited the minimal acyltransferase specific activity. The isolated epithelial cells showed levels of reductase and acyltransferase specific activities higher than those found in mucosa scrapings, probably due to the contact of these microsomal proteins with proteolytic enzymes during homogenization of the mucosa. However, no protecting effect of the trypsin inhibitor (2mg/ml) could be observed on reductase activity in mucosa scrapings. The cytosolic location of decarboxylase may account for the similar levels of specific activities found in mucosa scrapings and isolated enterocytes.     

Evidence of essential arginyl residues in chicken liver mevalonate-5-pyrophosphate decarboxylase

Chicken liver mevalonate-5-pyrophosphate decarboxylase (ATP:5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33.) is inactivated by phenylglyoxal in triethanolamine buffer at pH 8.15. The reaction follows pseudo-first-order kinetics with a second-order rate constant of 108 M-1 min-1. Appropriate treatment of the kinetic data for the inactivation reaction indicates that the reaction of a single phenylglyoxal molecule per active unit of the enzyme is enough to completely inactivate the protein. The partially inactivated enzyme shows unaltered Km but decreased V as compared to native mevalonate-5-pyrophosphate decarboxylase. The dissociation constants for the enzyme-substrate complexes were estimated from inactivation reactions at different concentrations of substrates. From the data it is concluded that the modified amino acid is important for the binding of both substrates.     

Modification of the radiochemical assay of rat liver mevalonate-5-diphosphate decarboxylase and induction and stabilization of the activity.

Problems encountered in attempts to purify mevalonate-5-diphosphate decarboxylase from rat liver are addressed. These are the quantitative, facile separation of [14C]isopentenol in the radiochemical assay (2) the instability of the enzyme activity and (3) the very low activity in rat liver. The assay was modified by using Sep Pac C18 filters to bind and release [14C]isopentenol. Authentic isopentenol was quantitated by absorbance at 210 nm wavelength and the extinction coefficient estimated to be epsilon m = 3.26 X 10(3). Recovery of authentic isopentenol from aqueous solution after binding and elution into methanol was quantitative from 10-100 nmols. Recovery of [14C]ispentenol from assay mixtures using 2-[14C]mevalonate-5-diphosphate and alkaline phosphatase to hydrolyze phosphate was quantitative using Sep Pac filter but not using petroleum ether extraction. Enzyme activity was stabilized by phenylmethylsulfonyl fluoride, aprotinin and leupeptin and was stable at -73 degrees C for 3 months. Activity of the decarboxylase was increased by 5-fold after feeding young rats 2.5% cholestyramine for ten days to four weeks.     

Posthatching evolution of in vivo mevalonate metabolism in chick liver

The in vivo mevalonate incorporation into total nonsaponifiable lipids by chick liver was minimal after hatching and drastically increased between 1-5 days. The hepatic synthesis of different cholesterol precursors emerged sequentially after hatching. Between 1-5 days increased strongly the conversion of mevalonate into squalene and also the formation of oxygenated lanosterol derivatives from squalene. The conversion of squalene became completely active at day 8. Cholesterol formation from lanosterol derivatives was completely activated between 8-11 days. Results in this paper demonstrate for the first time the accumulation of a fraction of nonsaponifiable lipids identified as lanosterol derivatives and cholesterol precursors formed from [5-14C]mevalonate in experiments carried out in vivo. Postnatal evolution of these oxysterols may explain the great increase of 3-hydroxy-3-methylglutaryl-CoA reductase activity found in chick liver between 5-11 days, simultaneous or posterior to the diminution of the oxygenated cholesterol precursors.     

Purification and characterization of avian liver mevalonate-5-pyrophosphate decarboxylase

Mevalonate-5-pyrophosphate decarboxylase [ATP:5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33] has been purified 5800 times from chicken liver and obtained in a stable and highly purified form. The protein is a dimer of molecular weight 85400 +/- 1941, and its subunits were not resolved by gel electrophoresis in denaturing conditions. The purified enzyme does not require the presence of SH-containing reagents for either activity or stability. The enzyme shows a high specificity for adenosine 5'-triphosphate (ATP) and requires for activity a divalent metal cation, Mg2+ being most effective. The optimum pH for the enzyme ranges from 4.0 to 6.5. Inhibitory effects for the enzyme activity were detected by citrate, phthalate, and phosphate. The isoelectric point, as determined by column chromatofocusing, is 4.8. The kinetics are hyperbolic for both substrates, showing a sequential mechanism; true Km values of 0.0141 mM and 0.504 mM have been obtained for mevalonate-5-pyrophosphate and ATP, respectively.     

Age-related changes in the mevalonate metabolism in vivo in chick kidneys

The mevalonate incorporation in vivo into total nonsaponifiable lipids by chick kidneys drastically increased after hatching, reaching similar levels to those previously observed in liver. Cholesterol was the major sterol formed from mevalonate from 11 days onward, while a fraction of polar nonsaponifiable lipid(s) was observed as the major compound(s) synthesized at 5-8 days. Relative percentages of squalene, squalene oxide(s) and lanosterol synthesized from mevalonate also increased between 11-18 days after hatching. Results in this paper demonstrate for the first time the accumulation of a fraction of nonsaponifiable lipid(s) identified as lanosterol derivatives and cholesterol precursors formed by kidneys from [5-14C]mevalonate in experiments carried out in vivo, as well as their evolution during postnatal period.