CoA- and non-CoA-dependent retinol esterification in retinal pigment epithelium

Washed, buffered microsomes from bovine retinal pigment epithelium catalyze retinyl ester synthesis from retinol in the absence of an exogenous acyl donor. A plot of retinyl ester synthesis versus time reaches a plateau at 123 +/- 26 nmol of retinyl ester mg-1 microsomal protein, providing a minimum value of the concentration of the endogenous acyl donor. Fatty acyl-CoA analysis by three different methods employing high performance liquid chromatography resulted in the detection of less than 1 nmol mg-1 protein of acyl-CoA, indicating that fatty acyl-CoA is not the endogenous acyl donor. Stimulation of the rate of retinyl ester synthesis by palmitoyl-CoA or ATP, CoA, and palmitate is observed following its addition at the beginning of the reaction or after the endogenous acyl source has been exhausted by 20 min of reaction with retinol. Palmitate from [14C]palmitoyl-CoA is incorporated into retinyl ester at a rate similar to that for the incorporation of [3H] retinol, demonstrating the presence of an apparent acyl-CoA:retinol acyl transferase activity. The acyl group from palmitoyl-CoA can be transferred initially to a component of the microsomes and subsequently to retinol. The product of retinyl ester synthesis from all-trans-retinol and palmitoyl-CoA is all-trans-retinyl palmitate, indicating that the stereochemical configuration is retained during esterification. The kinetic parameters for the esterification of 11-cis-retinol and all-trans-retinol are similar.     

Vitamin A metabolism in the human intestinal Caco-2 cell line

The human intestinal Caco-2 cell line, described as enterocyte-like in a number of studies, was examined for its ability to carry out the metabolism of vitamin A normally required in the absorptive process. Caco-2 cells contained cellular retinol-binding protein II, a protein which is abundant in human villus-associated enterocytes and may play an important role in the absorption of vitamin A. Microsomal preparations from Caco-2 cells contained retinal reductase, acyl-CoA-retinol acyltransferase (ARAT), and lecithin-retinol acyltransferase (LRAT) activities, which have previously been proposed to be involved in the metabolism of dietary vitamin A in the enterocyte. When intact Caco-2 cells were provided with beta-carotene, retinyl acetate, or retinol, synthesis of retinyl palmitoleate, oleate, palmitate, and small amounts of stearate resulted. However, exogenous retinyl palmitate or stearate was not used by Caco-2 cells as a source of retinol for ester synthesis. While there was a disproportionate synthesis of monoenoic fatty acid esters of retinol in Caco-2 cells compared to the retinyl esters typically found in human chylomicrons or the esters normally synthesized in rat intestine, the pattern was consistent with the substantial amount of unsaturated fatty acids, particularly 18:1 and 16:1, found in the sn-1 position of Caco-2 microsomal phosphatidylcholine, the fatty acyl donor for LRAT. Both ARAT and LRAT have been proposed to be responsible for retinyl ester synthesis in the enterocyte.(ABSTRACT TRUNCATED AT 250 WORDS)     

The molecular basis of retinoid absorption: a genetic dissection

The intestine and other tissues are able to synthesize retinyl esters in an acyl-CoA-dependent manner involving an acyl-CoA:retinol acyltransferase (ARAT). However, the molecular identity of this ARAT has not been established. Recent studies of lecithin:retinol acyltransferase (LRAT)-deficient mice indicate that LRAT is responsible for the preponderance of retinyl ester synthesis in the body, aside from in the intestine and adipose tissue. Our present studies, employing a number of mutant mouse models, identify diacylglycerol acyltransferase 1 (DGAT1) as an important intestinal ARAT in vivo. The contribution that DGAT1 makes to intestinal retinyl ester synthesis becomes greater when a large pharmacologic dose of retinol is administered by gavage to mice. Moreover, when large retinol doses are administered another intestinal enzyme(s) with ARAT activity becomes apparent. Surprisingly, although DGAT1 is expressed in adipose tissue, DGAT1 does not catalyze retinyl ester synthesis in adipose tissue in vivo. Our data also establish that cellular retinol-binding protein, type II (CRBPII), which is expressed solely in the adult intestine, in vivo channels retinol to LRAT for retinyl ester synthesis. Contrary to what has been proposed in the literature based on in vitro studies, CRBPII does not directly prevent retinol from being acted upon by DGAT1 or other intestinal ARATs in vivo.     

Modulation by retinyl acetate of microfilament bundle formation in C3H/10T1/2 cells

Retinyl acetate has been previously shown to inhibit carcinogen-induced neoplastic transformation in 10T1/2 cells and to accentuate many aspects of the nontransformed phenotype. Scanning electron microscopy of logarithmic phase 10T1/2 cells treated for 3 days with 0.3 micrograms/ml retinyl acetate revealed that this treatment caused extensive flattening of cells to the plastic substrate. In contrast the tumor promoter tetradecanoyl phorbol acetate, which antagonizes the antineoplastic activity of retinyl acetate, caused cell rounding and completely inhibited the action of retinyl acetate on cell morphology. During this same time course, the formation of microfilament bundles was also found to be modulated by retinyl acetate. Transmission electron micrographs of unsectioned peripheral regions of flattened cells showed that while the unit density of microfilament bundles was not influenced, the thickness of bundles, particularly those with a diameter of 100 nm or more, was increased by retinyl acetate. Tetradecanoyl phorbol acetate had little effect on microfilament bundle diameters but did partially antagonize the action of retinyl acetate. To determine if this increase was associated with an increase in total actin/cell, total cell proteins, and proteins not extractable by glycerol-triton extraction, were subjected to sodium dodecylsulfate/ polyacrylamide gel electro-phoresis. It was found that while total cellular actin was not increased by retinyl acetate, the proportion of nonextractable actin (which includes microfilament bundles) increased from 65% to 88% of total actin. This increase was not inhibited by inhibitors of protein or RNA synthesis. These studies again demonstrate that retinyl acetate accentuates the nontransformed phenotype of 10T1/2 cells; it is hypothesized that these actions are related to the antineoplastic activity of retinoids.     

Studies on phospholipase activities in Neurospora crassa conidia

Cytosol retinyl ester lipoprotein complex from rat liver was capable of transferring its unesterified retinol component to serum aporetinol-binding protein. In the presence of serum albumin and aporetinol-binding protein, 68% of retinyl ester was hydrolyzed and up to 30% of unesterified retinol was transferred from cytosol retinyl ester lipoprotein complex to serum aporetinol-binding protein in 24 h at 30 °C. The reconstituted retinol-retinol-binding protein complex showed biochemical and biophysical properties similar to native retinol-retinol-binding protein. Both native and reconstituted retinol-retinol-binding proteins had identical uv, CD, and fluorescence spectra as well as binding affinity to prealbumin. Treatment of cytosol retinyl ester lipoprotein with sulfhydryl reagent, with 1 n NaCl, or with diisopropyl fluorophosphate (0.14 mm) abolished the hydrolysis of retinyl ester; however, the activity of retinol transfer from cytosol retinyl ester lipoprotein complex to serum retinol-binding protein was still unaffected. The activity of retinol transfer was proportional to the amount of retinol content in the complex and the amount of aporetinol-binding protein. These experiments suggest that the cytosol retinyl ester lipoprotein complex serves three major functions: (i) as a storage form of retinyl ester and retinol; (ii) as an enzyme for hydrolyzing its own retinyl ester ligand; and (iii) as a medium for transfer of unesterified retinol to serum retinol-binding protein.     

Hydrolysis of retinyl palmitate by enzymes of rat pancreas and liver. Differentiation of bile salt-dependent and bile salt-independent, neutral retinyl ester hydrolases in rat liver

Previous studies have demonstrated that homogenates of the livers of rats contain a neutral retinyl ester hydrolase activity that requires millimolar concentrations of bile salts for maximal in vitro activity. The enzymatic properties of this neutral, bile salt-dependent retinyl ester hydrolase activity in liver homogenates are nearly identical to those observed in the present report for the in vitro hydrolysis of retinyl palmitate by purified rat pancreatic cholesteryl ester hydrolase (EC 3.1.1.13). Moreover, anti-rat pancreatic cholesteryl ester hydrolase IgG completely inhibits the bile salt-dependent retinyl ester hydrolase activity of rat liver homogenates whereas normal rabbit IgG does not. We also show that liver homogenates contain a neutral, bile salt-independent retinyl ester hydrolase activity that differs from the bile salt-dependent activity in that 1) its absolute activity does not vary markedly among individual rats, 2) it is not inhibited by antibodies to pancreatic cholesteryl ester hydrolase, and 3) it is localized in the microsomal fraction of liver homogenates. Subfractionation of microsomes demonstrates that the neutral, bile salt-independent retinyl ester hydrolase activity is associated with liver cell plasma membranes and thus may play a role in the hydrolysis of retinyl esters delivered to the liver by chylomicron remnants.     

Acyl coenzyme A dependent retinol esterification by acyl coenzyme A:diacylglycerol acyltransferase 1

We provide biochemical evidence that enzymes involved in the synthesis of triacylglycerol, namely acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT), are capable of carrying out the acyl coenzyme A:retinol acyltransferase (ARAT) reaction. Among them, DGAT1 appears to have the highest specific activity. The apparent K_m values of recombinant DGAT1/ARAT for retinol and palmitoyl coenzyme A were determined to be 25.9 ± 2.1 μM and 13.9 ± 0.3 μM, respectively, both of which are similar to the values previously determined for ARAT in native tissues. A novel selective DGAT1 inhibitor, XP620, inhibits recombinant DGAT1/ARAT at the retinol recognition site. In the differentiated Caco-2 cell membranes, XP620 inhibits ～85% of the Caco-2/ARAT activity indicating that DGAT1/ARAT may be the major source of ARAT activity in these cells. Of the two most abundant fatty acyl retinyl esters present in the intact differentiated Caco-2 cells, XP620 selectively inhibits retinyl–oleate formation without influencing the retinyl–palmitate formation. Using this inhibitor, we estimate that ～64% of total retinyl ester formation occurs via DGAT1/ARAT. These studies suggest that DGAT1/ARAT is the major enzyme involved in retinyl ester synthesis in Caco-2 cells.     

Hepatic stellate cells retain the capacity to synthesize retinyl esters and to store neutral lipids in small lipid droplets in the absence of LRAT

Hepatic stellate cells (HSCs) play an important role in liver physiology and under healthy conditions they have a quiescent and lipid-storing phenotype. Upon liver injury, HSCs are activated and rapidly lose their retinyl ester-containing lipid droplets. To investigate the role of lecithin:retinol acyltransferase (LRAT) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) in retinyl ester synthesis and lipid droplet dynamics, we modified LC–MS/MS procedures by including multiple reaction monitoring allowing unambiguous identification and quantification of all major retinyl ester species. Quiescent primary HSCs contain predominantly retinyl palmitate. Exogenous fatty acids are a major determinant in the retinyl ester species synthesized by activated HSCs and LX-2 cells, indicating that HSCs shift their retinyl ester synthesizing capacity from LRAT to DGAT1 during activation. Quiescent LRAT^−/− HSCs retain the capacity to synthesize retinyl esters and to store neutral lipids in lipid droplets ex vivo. The median lipid droplet size in LRAT^−/− HSCs (1080 nm) is significantly smaller than in wild type HSCs (1618 nm). This is a consequence of an altered lipid droplet size distribution with 50.5 ± 9.0% small (≤ 700 nm) lipid droplets in LRAT^−/− HSCs and 25.6 ± 1.4% large (1400–2100 nm) lipid droplets in wild type HSC cells. Upon prolonged (24 h) incubation, the amounts of small (≤ 700 nm) lipid droplets strongly increased both in wild type and in LRAT^−/− HSCs, indicating a dynamic behavior in both cell types. The absence of retinyl esters and reduced number of lipid droplets in LRAT-deficient HSCs in vivo will be discussed.     

Effects of retinyl acetate on surface morphology and intramembrane particle distribution in the plasma membrane of 10T1/2 cells.

Scanning electron microscopy and freeze fracture electron microscopy were used to characterize membrane ultrastructural differences between parental, C3H/10T1/2, and carcinogen-initiated, INIT C3H/10T1/2, cells and treatments with retinyl acetate. The intramembranous particle distribution on the E-face was detected and quantitated by the methods of automated image analysis to obtain statistically meaningful numerical characteristics of intramembranous particle size and density. Subtle differences were found when no differences were apparent by light microscopy or by scanning electron microscopy. Initial retinyl acetate treatment caused a significant increase of the intramembranous particle size in parental cells. Intramembranous particle density increased for retinyl acetate treatment in parental and INIT cells and in INIT cells previously maintained but withheld from retinyl acetate. Intramembranous particle distribution analysis includes the interparticle distance of nearest neighbors and the randomness of the distribution by the differential density distribution function, which compares the observed sample to Poisson modified for particle size. These measures show that the three cell groups that have been treated with retinyl acetate have a more even distribution of intramembranous particles than was found for untreated parental cells. The relationship between the freeze fracture morphology and the biological responses to retinyl acetate treatment is discussed.     

Vitamin A metabolism in cultured somatic cells from rat testis

Sertoli and peritubular myoid cells, the somatic cells of the seminiferous tubule, support growth and differentiation of developing germ cells. This action strictly depends on the availability of in situ synthesized retinoic acid and we have previously documented the ability of Sertoli, but not peritubular cell extracts, to support the oxidation of retinol to retinoic acid. Using primary cultures of somatic cells treated with a physiological concentration of free retinol, we show here that the same is essentially true also for whole cultured cells. Sertoli cells are capable of producing not only retinoic acid, but are also the major site of retinyl ester (mainly, retinyl palmitate) formation. Compared with retinyl palmitate accumulation, retinoic acid synthesis was both faster and positively influenced by prior exposure to retinol. This increase in retinoic acid synthesis was further augmented by treatment with the retinoic acid catabolic inhibitor liarozole, thus indicating that enhanced synthesis, rather than reduced catabolism, is responsible for such an effect. Myoid cells had a higher capacity to incorporate exogenously supplied retinol, yet retinoic acid synthesis, and even more so retinyl palmitate formation, were considerably lower than in Sertoli cells. Retinoic acid synthesis in myoid cells was not only depressed, but also very little influenced by prior retinol exposure and totally insensitive to liarozole. These data further support the view that myoid cells are involved in retinol uptake from the blood and its transfer to other cells, rather than in metabolic interconversion or long-term storage of vitamin A, two processes that mainly take place in Sertoli cells.     

Retinyl esterase activity of purified rat liver retinyl ester lipoprotein complex.

Retinyl ester lipoprotein complex from rat liver was shown to possess a retinyl esterase activity toward its own ligand complement. In the presence of serum albumin the retinyl esterase activity at 30 °C was about fivefold larger than the activity at 4 °C, while higher temperatures than 30 °C led to some degradation of retinyl compounds. The pH optimum was 7.8. The esterase activity was markedly enhanced by serum albumin although the serum albumin as such had no retinyl esterase activity. In the presence of serum albumin and under optimal conditions, some 75 to 80% of the total retinyl ester complement of the lipoprotein was hydrolyzed in 24 h. The retinyl esterase activity was totally abolished by treatment with the serine esterase inhibitor diisopropyl fluorophosphate (1.4 × 10^?4 M), by treatment with sulfhydryl reagents, and by detergents (0.2% of Tween 80 and sodium deoxycholate). From this series of experiments it was concluded that the retinyl ester lipoprotein complex possesses the additional physiological function of hydrolyzing its own retinyl ester complement to unesterified retinol which may then combine with serum retinol-binding protein.     

Hydrolysis of retinyl esters by pancreatic triglyceride lipase

Previously [van Bennekum, A. M., et al. (1999) Biochemistry 38, 4150-4156] we showed that carboxyl ester lipase (CEL)-deficient (CELKO) mice have normal levels of pancreatic, bile salt-dependent retinyl ester hydrolase (REH) activity. In the present study, we further investigated this non-CEL REH activity in pancreas homogenates of CELKO and wild-type (WT) mice, and rats. REH activity was detected in both the presence and absence of tri- and dihydroxy bile salts in rats, WT mice, and CELKO mice. In contrast, pancreatic cholesteryl ester hydrolase (CEH) activity was only detected in the presence of trihydroxy bile salts and only in rats and WT mice, consistent with CEL-mediated cholesteryl ester hydrolysis. Enzyme assays of pancreatic triglyceride lipase (PTL) showed that there was a colipase-stimulated REH activity in rat and mouse (WT and CELKO) pancreas, consistent with hydrolysis of retinyl ester (RE) by PTL. Pancreatic enzyme activities related to either CEL or PTL were separated using DEAE-chromatography. In both rats and mice (WT and CELKO), REH activity could be attributed mainly to PTL, and to a much smaller extent to CEL. Finally, purified human PTL exhibited similar enzymatic characteristics for triglyceride hydrolysis as well as for retinyl ester hydrolysis, indicating that RE is a substrate for PTL in vivo. Altogether, these studies clearly show that PTL is the major pancreatic REH activity in mice, as well as in rats.     

Growth of Kluyveromyces marxianus and formation of ethyl acetate depending on temperature

Conversion of lactose into ethyl acetate by Kluyveromyces marxianus allows economic reuse of whey-borne sugar. The high volatility of ethyl acetate enables its process-integrated recovery by stripping. This stripping is governed by both the aeration rate and the partition coefficient, K _EA,L/G. Cultivation at elevated temperatures should decrease the K _EA,L/G value and thus favor stripping. K. marxianus DSM 5422 as a potent producer of ethyl acetate was cultivated aerobically in whey-borne media for studying temperature-dependent growth and ester formation. Shake flask cultivation proved thermal tolerance of this yeast growing from 7 to 47 °C with a maximum rate of 0.75 h^?1 at 40 °C. The biomass yield was 0.41 g/g at moderate temperatures while low and high temperatures caused distinct drops. The observed μ-T and Y _X/S-T dependencies were described by mathematical models. Further cultivations were done in an 1-L stirred reactor for exploring the effect of temperature on ester synthesis. Cultivation at 32 °C caused significant ester formation (Y _EA/S?=?0.197 g/g) while cultivation at 42 °C suppressed ester synthesis (Y _EA/S?=?0.002 g/g). The high temperature affected metal dissolution from the bioreactor delivering iron for yeast growth and preventing ester synthesis. Cultivation at 32 °C with a switch to 42 °C at the onset of ester synthesis allowed quick and efficient ester production (Y _EA/S?=?0.289 g/g). The high temperature lowered the K _EA,L/G value from 78 to 44 L/L which heightened the gas-phase ester concentration (favoring ester recovery) without increasing the liquid-phase concentration (avoiding product inhibition).     

Sequestration of retinyl esters is essential for retinoid signaling in the zebrafish embryo

For vertebrate development, vitamin A (all-trans retinol) is required in quantitative different amounts and spatiotemporal distribution for the production of retinoic acid, a nuclear hormone receptor ligand, and 11-cis retinal, the chromophore of visual pigments. We show here for zebrafish that embryonic retinoid homeostasis essentially depends on the activity of a leci-thin:retinol acyltransferase (Lratb). During embryogenesis, lratb is expressed in mostly non-overlapping domains opposite to retinal dehydrogenase 2 (raldh2), the key enzyme for retinoic acid synthesis. Blocking retinyl ester formation by a targeted knock down of Lratb results in significantly increased retinoic acid levels, which lead to severe embryonic patterning defects. Thus, we provide evidence that a balanced competition between Lratb and Raldh2 for yolk vitamin A defines embryonic compartments either for retinyl ester or retinoic acid synthesis. This homeostatic mechanism dynamically adjusts embryonic retinoic acid levels for gene regulation, concomitantly sequestering excess yolk vitamin A in the form of retinyl esters for the establishment of larval vision later during development.     

Dietary vitamins A and E influence retinyl ester composition and content of the retinal pigment epithelium

Experiments were conducted to determine the influence of dietary levels of vitamin A and alpha-tocopherol on the amounts and composition of retinyl esters in the retinal pigment epithelium of light-adapted albino rats. Groups of rats were fed diets containing alpha-tocopherol and either no retinyl palmitate, adequate retinyl palmitate, or excessive retinyl palmitate. Other groups of rats received diets lacking alpha-tocopherol and containing the same three levels of retinyl palmitate. Retinoic acid was added to diets lacking retinyl palmitate. After 27 weeks, the animals were light-adapted to achieve essentially total visual pigment bleaches, and the neural retinas and retinal pigment epithelium-eyecups were then dissected from each eye for vitamin A ester determinations. Almost all of the retinyl esters were found in the retinal pigment epithelium-eyecup portions of the eyes, mainly as retinyl palmitate and retinyl stearate. Maintaining rats on a vitamin A-deficient, retinoic acid-containing diet led to significant reductions in retinal pigment epithelial retinyl ester levels in rats fed both the vitamin E-supplemented and vitamin E-deficient diets; contrary to expectations, the effect of dietary vitamin A deficiency was more pronounced in the vitamin E-supplemented rats. Vitamin A deficiency in retinoic acid-maintained animals also led to significant reductions in retinyl palmitate-to-stearate ester ratios in the retinal pigment epithelia of both vitamin E-supplemented and vitamin E-deficient rats. Excessive dietary intake of vitamin A had little, if any, effect on retinal pigment epithelial retinyl ester content or composition. Vitamin E deficiency resulted in significant increases in retinal pigment epithelial retinyl palmitate content and in palmitate-to-stearate ester ratios in rats fed all three levels of vitamin A, but had little effect on retinal pigment epithelial retinyl stearate content. In other tissues, vitamin E deficiency has been shown to lower vitamin A levels, and it is widely accepted that this effect is due to autoxidative destruction of vitamin A. The increase in retinal pigment epithelial vitamin A ester levels in response to vitamin E deficiency indicates that vitamin E does not regulate vitamin A levels in this tissue primarily by acting as an antioxidant, but rather may act as an inhibitor of vitamin A uptake and/or storage. The effect of vitamin E on pigment epithelial vitamin A levels may be mediated by the vitamin E-induced change in retinyl palmitate-to-stearate ratios.     

Reestablishment of a mucociliary epithelium in tracheal organ cultures exposed to retinyl acetate: a biochemical and morphometric study

Tracheal explants derived from vitamin A-deficient rats underwent keratinizine squamous metaplasia in organ culture when grown in serum-free medium. Within 1 d after the addition of 0.1, 2, or 10 microgram retinyl acetate per ml of medium, there was a concentration-dependent increase in the uptake of [3H]glucosamine and [14C]serine into both the total mucous glycoprotein and the principal purified mucin fraction eluted from a DEAE-Sephacel column with 0.2 M NaCl. The stimulation of mucin synthesis continued throughout the 21-d exposure period in a concentration-dependent fashion. It was also found that vitamin A had a greater effect on the incorporation of [3H]glucosamine than on [14C]serine into the secreted mucins, particularly at the higher retinyl acetate concentrations. This result indicated a greater effect of the vitamin on the synthesis of the carbohydrate moiety of the mucins. Morphological analysis by light and electron microscopy demonstrated that the keratinizing squamous epithelium began to revert to a mucus-secreting tissue as early as 24 h after addition of 10 microgram retinyl acetate to the medium. The response was slower with the lower vitamin concentrations. Stereological analysis revealed that the increase in the volume fraction of the Golgi apparatus reached a stable level which could not be altered with continued exposure to retinyl acetate, but that the volume fraction of mucin droplets continually increased and apparently did not reach a maximum in the 21-d exposure period. Conversely, the volume fraction of filament bundles and the number of desmosomes decreased during the vitamin A treatment.     

Terpenoid biosynthesis in Euphorbia latex

The latex of Euphorbia lathyris can utilize acetate, pyruvate and mevalonate for triterpene synthesis in vitro. Acetyl-CoA, hydroxymethylglutarate, hydroxymethylglutaryl-CoA and isopentenyl pyrophosphate were not effective as precursors for triterpene biosynthesis. Acetate is utilized only by the terpenoid pathway and by the tricarboxylic acid cycle; it is not used for fatty acid synthesis in this system. However, phospholipids were found to be efficient acyl donors for triterpene ester synthesis. The observed selectivity of precursor utilization as well as the observed rates for product formation indicate separate sites for triterpenol and triterpene ester synthesis and that one is not precursor for the other.     

Retinyl ester hydrolases in retinal pigment epithelium

In bovine retinal pigment epithelium membranes we have found three hydrolases which were active against trans-retinyl palmitate. This was possible by assaying different subcellular fractions as a function of pH in the range 3-9. Detection of these activities has been favored by the use in the enzyme assay of Triton X-100, which has an activating effect up to a concentration of 0.03% at a detergent-protein ratio of about 1.5-3.0. Apparent kinetic parameters for the retinyl ester hydrolases have been determined after a study of the optimization of assay conditions. Vmax values for hydrolases acting at pH 4.5, 6.0, and 7.0 were, respectively, 156, 55, and 70 nmol/h/mg. To identify the subcellular site for these hydrolytic activities, assays of marker enzymes from various organelles in each subcellular preparation were carried out, demonstrating the lysosomal origin of the pH 4.5 retinyl ester hydrolase and the microsomal origin of the pH 6.0 retinyl ester hydrolase and suggesting that the pH 7.0 retinyl ester hydrolase originates from the Golgi complex.     

Production and biological function of volatile esters in Saccharomyces cerevisiae

The need to understand and control ester synthesis is driven by the fact that esters play a key role in the sensorial quality of fermented alcoholic beverages like beer, wine and sake. As esters are synthesized in yeast via several complex metabolic pathways, there is a need to gain a clear understanding of ester metabolism and its regulation. The individual genes involved, their functions and regulatory mechanisms have to be identified. In alcoholic beverages, there are two important groups of esters: the acetate esters and the medium-chain fatty acid (MCFA) ethyl esters. For acetate ester synthesis, the genes involved have already been cloned and characterized. Also the biochemical pathways and the regulation of acetate ester synthesis are well defined. With respect to the molecular basis of MCFA ethyl ester synthesis, however, significant progress has only recently been made. Next to the characterization of the biochemical pathways and regulation of ester synthesis, a new and more important question arises: what is the advantage for yeast to produce these esters? Several hypotheses have been proposed in the past, but none was satisfactorily. This paper reviews the current hypotheses of ester synthesis in yeast in relation to the complex regulation of the alcohol acetyl transferases and the different factors that allow ester formation to be controlled during fermentation.