Kinetic characterization of recombinant mouse retinal dehydrogenase types 3 and 4 for retinal substrates

Background

Retinal dehydrogenases (RALDHs) catalyze the dehydrogenation of retinal into retinoic acids (RAs), which are required for embryogenesis and tissue differentiation. This study sought to determine the detailed kinetic properties of 2 mouse RALDHs, namely RALDH3 and 4, for retinal isomer substrates, to better define their specificities in RA isomer synthesis.

Methods

RALDH3 and 4 were expressed in Escherichia coli as His-tagged proteins and affinity-purified. Enzyme kinetics were performed with retinal isomer substrates. The enzymatic products were analyzed by high pressure liquid chromatography.

Results

RALDH3 oxidized all-trans retinal with high catalytic efficiency (V_max/K_m = 77.9) but did not show activity for either 9-cis or 13-cis retinal substrates. On the other hand, RALDH4 was inactive for all-trans retinal substrate, exhibited high activity for 9-cis retinal oxidation (V_max/K_m = 27.4), and oxidized 13-cis retinal with lower catalytic efficiency (V_max/K_m = 8.24). β-ionone, a potent inhibitor of RALDH4 activity, suppressed 9-cis and 13-cis retinal oxidation competitively with inhibition constants of 0.60 and 0.32, respectively, but had no effect on RALDH3 activity. The divalent cation MgCl₂ activated 13-cis retinal oxidation by RALDH4 by 3-fold, did not significantly influence 9-cis retinal oxidation, and slightly activated RALDH3 activity.

Conclusions

These data extend the kinetic characterization of RALDH3 and 4, providing their specificities for retinal isomer substrates.

General significance

The kinetic characterization of RALDHs should give useful information in determining amino acid residues that are involved in the specificity for retinal isomers and on the role of these enzymes in the synthesis of RAs in specific tissues.     

Key enzymes of the retinoid (visual) cycle in vertebrate retina

A major goal in vision research over the past few decades has been to understand the molecular details of retinoid processing within the retinoid (visual) cycle. This includes the consequences of side reactions that result from delayed all-trans-retinal clearance and condensation with phospholipids that characterize a variety of serious retinal diseases. Knowledge of the basic retinoid biochemistry involved in these diseases is essential for development of effective therapeutics. Photoisomerization of the 11-cis-retinal chromophore of rhodopsin triggers a complex set of metabolic transformations collectively termed phototransduction that ultimately lead to light perception. Continuity of vision depends on continuous conversion of all-trans-retinal back to the 11-cis-retinal isomer. This process takes place in a series of reactions known as the retinoid cycle, which occur in photoreceptor and RPE cells. All-trans-retinal, the initial substrate of this cycle, is a chemically reactive aldehyde that can form toxic conjugates with proteins and lipids. Therefore, much experimental effort has been devoted to elucidate molecular mechanisms of the retinoid cycle and all-trans-retinal-mediated retinal degeneration, resulting in delineation of many key steps involved in regenerating 11-cis-retinal. Three particularly important reactions are catalyzed by enzymes broadly classified as acyltransferases, short-chain dehydrogenases/reductases and carotenoid/retinoid isomerases/oxygenases. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.     

马肝醇脱氢酶基因的克隆及其在大肠杆菌中的表达

利用RT-PCR技术从马肝扩增HLADH-E和HLADH-S基因,通过基因工程方法构建表达质粒pLY115E和pLY115S,在大肠杆菌中表达,并利用Ni柱分离纯化。利用紫外检测辅酶NADH在340nm的吸光值,来考察表达产物转化环己醇的活性。试验结果证明马肝醇脱氢酶HLADH-E和HLADH-S基因均能在大肠杆菌中表达,并且可溶性表达产物都具有氧化环己醇的活性,为马肝醇脱氢酶的进一步研究开发奠定了基础。     

Tissue-specific and embryonic expression of the retinoid X receptors in Sebastiscus marmoratus

Retinoid X receptors (RXRs) are highly conserved members of the nuclear receptor family and mediate various physiological processes in vertebrates. Most studies on RXRs have concentrated on their structure and function in mammals and their characterization and developmental expression in Danio rerio. However, there is little information concerning the distribution of RXRs in teleost tissues. In the present study, we cloned partial sequences of three RXR subtypes (RXRa, -b, -g) from Sebastiscus marmoratus by RACE PCR and analyzed the phylogeny of the teleost and the tetrapod RXR genes, and identified some inconsistencies with previous studies. The tissue-specific and embryonic expression profiles of each RXR gene were explored using real time quantitative PCR. This analysis demonstrated that these RXRs were expressed in all test tissues indicating their participation in many physiological processes. However, we found a great difference in the distribution of RXRg between teleosts and mammals. Furthermore, we followed expression of the three subtypes through various embryo developmental stages and found that the RXRa orthologues of teleosts might be involved in the development of the anterior hindbrain, tailbud and neural crest and in the formation of the pharynx and fin, that RXRb played ubiquitous roles in fish early development, and that RXRg probably played a role in brain and nervous system development and function.     

The maize cytosolic glyceraldehyde-3-phosphate dehydrogenase gene family: organ-specific expression and genetic analysis

Summary The distribution of the cytosolic glyceraldehyde-3-phosphate dehydrogenase gene family (Gpc) in the maize genome was investigated; a genetic variant of glyceraldehyde-3-phosphate dehydrogenase activity is also described. Restriction fragment length polymorphism analysis of an F₂ population shows that the variant is not linked to the three known Gpc genes. However, this trait is linked to one of two genomic DNA fragments that hybridize to a fragment of the Gpc3 coding region, implying the existence of a fourth Gpc gene. Antibodies and cDNA clones were used to investigate the organ-specific expression of the Gpc genes. Results were compared with the expression of the alcohol dehydrogenase 1 (Adh1) gene. RNA and protein levels were examined in seedling roots and shoots, as well as the leaves, developing endosperm and embryo, and the aleurone. In general, it was found that Gpc3 expression behaves in parallel with Adh1 in these organs, and protein levels closely parallel that of RNA for each gene examined. Both Gpc3 and Adh1 show a marked increase in expression during endosperm development, reaching a maximum 15 days after pollination, but no expression is detected in the leaf. Gpc1 expression is similar to that of Gpc2, with an overall decrease in the level of RNA during endosperm development. This expression is discussed in terms of the common sequences found upstream of genes expressed in the developing maize seed.     

Circadian variations in the activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in the liver of control and streptozotocin-induced diabetic rats

The aim of this study was to examine: the 24 h variation of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase activities, key enzymes for the maintenance of intracellular NADPH concentration, in rat liver in control and streptozotocin-induced diabetic animals. Adult male rats were fed ad libitum and synchronized on a 12:12 h light-dark cycle (lights on 08:00 h). One group of animals was treated with streptozotocin (STZ, 55 mg/kg, intraperitoneal) to induce experimental diabetes. Eight weeks after STZ injection, the animals were sacrificed at six different times of day—1, 5, 9, 13, 17 and 21 Hours After Lights On (HALO)—and livers were obtained. Enzyme activities were determined spectrophotometrically in triplicate in liver homogenates and expressed as units per mg protein. 6-phosphogluconate dehydrogenase activity was measured by substituting 6-phosphogluconate as substrate. Glucose-6-phosphate dehydrogenase activity was determined by monitoring NADPH production. Treatment, circadian time, and interaction between treatment and circadian time factors were tested by either one or two way analysis of variance (ANOVA). Two-way ANOVA revealed that 6-phosphogluconate dehydrogenase activity significantly depended on both the treatment and time of sacrifice. 6-phosphogluconate dehydrogenase activity was higher in control than diabetic animals; whereas, glucose-6-phosphate dehydrogenase activity did not vary over the 24 h in animals made diabetic by STZ treatment. Circadian variation in the activity of 6-phosphogluconate dehydrogenase was also detected in both the control and STZ treatment groups (one-way ANOVA). Time-dependent variation in glucose-6-phosphate dehydrogenase activity during the 24 h was detected in control but not in diabetic rats. No significant interaction was detected between STZ-treatment and time of sacrifice for both hepatic enzyme activities. These results suggest that the activities of NADPH-generating enzymes exhibit 24 h variation, which is not influenced by diabetes.     

Regulation of the murine alpha(2)(I) collagen promoter by retinoic acid and retinoid X receptors

Retinoic acid decreases collagen production by hepatic stellate cells. This study investigated the effects of retinoic acid receptor beta (RARbeta) and retinoid X receptor alpha (RXRalpha) on the regulation of the alpha(2)(I) collagen promoter. Retinoic acid and the RARbeta and RXRalpha expression vectors suppressed the promoter in transfected stellate cells with maximal suppression obtained when combined. Mutation of the retinoic acid response element (RARE) at -879 to -874 (site 1) enhanced promoter activity and diminished but did not eliminate the suppression by RARbeta and RXRalpha. Mutation of another RARE site (site 2), at -930 to -911, resulted in low activity that was inhibited by retinoic acid. Mutation of the AP-2-binding site enhanced promoter activity that was inhibited by retinoic acid. This study shows that the suppressive effect of retinoic acid on the promoter is maximal with a combination of RARbeta and RXRalpha and occurs at more than one RARE site. The effect of retinoic acid is not mediated by AP-2.     

Identification and characterization of glucocorticoid receptors in liver of nude mice

Glucocorticoids regulate the expression of many liver-specific genes via glucocorticoid receptors. The presence of glucocorticoid receptors in liver has been reported in many mammalian species but not in nude mice. In the present study, we demonstrate the presence of specific glucocorticoid receptors in nude mouse liver. The binding of ligands to these receptors could be completely inhibited by RU486, and partially blocked by hydrocortisone and progesterone, whereas estrogen and testosterone had no effect. Hydrocortisone down-regulated the level of glucocorticoid receptors in livers of nude mice and correspondingly enhanced the activities of tyrosine aminotransferase and -glutamyltransferase. Our results indicate that glucocorticoid receptors in nude mouse liver are specific, fully functional, and present at levels 28.5-fold higher than in the liver of normal inbred mice. We suggest that the nude mouse is a valuable model for studies of hepatic glucocorticoid action and may provide a clue to a putative hepatic-thymic interaction.     

Enriched uranium affects the expression of vitamin D receptor and retinoid X receptor in rat kidney

An increasing awareness of the radiological impact of the nuclear power industry and other nuclear technologies is observed nowadays on general population. This led to renew interest to assess the health impact of the use of enriched uranium (EU). The aim of this work was to investigate in vivo the effects of a chronic exposure to EU on vitamin D(3) metabolism, a hormone essential in mineral and bone homeostasis. Rats were exposed to EU in their drinking water for 9 months at a concentration of 40 mg l(-1) (1mg/rat day). The contamination did not change vitamin D plasma level. Vitamin D receptor (vdr) and retinoid X receptor alpha (rxralpha), encoding nuclear receptors involved in the biological activities of vitamin D, showed a lower expression in kidney, while their protein levels were paradoxically increased. Gene expression of vitamin D target genes, epithelial Ca(2+) channel 1 (ecac1) and Calbindin-D28k (cabp-d28k), involved in renal calcium transport were decreased. Among the vitamin D target organs examined, these molecular modifications occurred exclusively in the kidney, which confirms that this organ is highly sensitive to uranium exposure. In conclusion, this study showed that a chronic exposure to EU affects both mRNA and protein expressions of renal nuclear receptors involved in vitamin D metabolism, without any modification of the circulating vitamin D.     

Development of a tightly regulated and highly inducible ecdysone receptor gene switch for plants through the use of retinoid X receptor chimeras

Chemical inducible gene regulation systems provide essential tools for the precise regulation of transgene expression in plants and animals. Recent development of a two-hybrid ecdysone receptor (EcR) gene regulation system has solved some of the drawbacks that were associated with the monopartate gene switch. To further improve the versatility of the two-hybrid EcR gene switch for wide spread use in plants, chimeras between Homo sapiens retinoid X receptor (HsRXR) and insect, Locusta migratoria RXR (LmRXR) were tested in tobacco protoplasts as partners with Choristoneura fumiferana EcR (CfEcR) in inducing expression of the luciferase reporter gene. The RXR chimera 9 (CH9) along with CfEcR, in a two-hybrid format gave the best results in terms of low-background expression levels in the absence of ligand and high-induced expression levels of the reporter gene in the presence of nanomolar concentrations of the methoxyfenozide ligand. The performance of CH9 was further tested in corn and soybean protoplasts and the data obtained was compared with the other EcR switches that contained the wild-type LmRXR or HsRXR as EcR partners. In both transient expression studies and stable transformation experiments, the fold induction values obtained with the CH9 switch were several times higher than the values obtained with the other EcR switches containing LmRXR or HsRXR. The new CfEcR two-hybrid gene switch that uses the RXR CH9 as a partner in inducing reporter gene expression provides an efficient, ligand-sensitive and tightly regulated gene switch for plants.     

Cloning and expression of Thiobacillus versutus aspartate-semialdehyde dehydrogenase gene in Escherichia coli

The Thiobacillus versutus asd gene coding for aspartate-semialdehyde dehydrogenase was cloned in Escherichia coli cells using pBR322 as a vector. The gene was expressed independently of its orientation, suggesting that E. coli RNA polymerase recognized T. versutus promoter sequence. The T. versutus DNA coded protein, of the molecular weight 44,000, was identified by the analysis of the proteins produced by minicells.     

Morphological changes in the zonula adhaerens during embryonic development of chick retinal pigment epithelial cells

Summary Retinal pigment epithelial cells from chicks at various stages of development were examined by transmission electron microscopy to determine how the adult form of the zonula adhaerens, composed of subunits termed zonula adhaerens complexes, is acquired. During early stages of development, between embryonic day 4 and embryonic day 7, the intermembrane discs of zonula adhaerens complexes appear to be formed from material already present between the junctional membranes of the zonulae adhaerentes. In contrast, the cytoplasmic plaque material of the zonulae adhaerentes is difficult to detect before hatching; it is seen as a dense band along the junctional membranes at hatching and as individual subunits in register with the intermembrane discs in adult retinal pigment epithelial cells. After embryonic day 16, when the zonulae adhaerentes increase dramatically in size, single zonula adhaerens complexes are also present basal to the zonulae adhaerentes along the lateral cell membrane. This suggests that, during later stages of development, the junctions grow in size and/or turn over by the addition of pre-assembled zonula adhaerens complexes.Abbreviations CMB Circumferential microfilament bundle - ZA Zonula adhaerens - ZAC Zonula adhaerens complex - RPE Retinal pigment epithelium     

Seasonal changes in gene expression of steroidogenic enzymes,androgen and estrogen receptors in frog testis

The anuran amphibian Pelophylax esculentus shows an annual cycle of sexual steroid production and spermatogenesis. To more thoroughly comprehend the steroidogenic pathways that govern the seasonal reproductive cycle, we investigated the mRNA expression of key enzymes involved in the androgenic and oestrogenic biosynthesis pathways in the testis of frogs taken in the reproductive and postreproductive period. Furthermore, we also analysed androgen and oestrogen levels and their own receptor gene expressions. Our findings showed that during the reproductive period, 3β‐hydroxysteroid dehydrogenase, 17β‐hydroxysteroid dehydrogenase and 5α‐reductase mRNA levels were higher than those during the postreproductive period. High testosterone and 5α‐dihydrotestosterone titres as well as the expression levels of androgen receptors in the reproductive testis strongly confirmed that the androgenic pathway is necessary for spermatogenesis activation. Conversely, during the postreproductive period, the highest P450 aromatase, estrogen receptor α and β mRNA levels, paralleling with oestradiol titres, indicated that the oestrogenic pathway is essential for the interruption of the reproductive processes. Our findings demonstrated, for the first time in amphibians, that testicular endocrine cyclic activity could be modulated by the up‐regulation of key steroidogenic enzyme gene expressions. This in turn determines the activation of the androgenic pathway in reproductive phase and the oestrogenic one in postreproductive phase.