concanavalin-a and retinol-palmitate

Previous work demonstrated both acid and neutral, bile salt-independent retinyl ester hydrolase activities in rat liver homogenates. Here we present the purification, identification, and characterization of an acid retinyl ester hydrolase activity from solubilized rat liver microsomes. Purification to homogeneity was achieved by sequential chromatography using SP-Sepharose cation exchange, phenyl-Sepharose hydrophobic interaction, concanavalin A-Sepharose affinity and Superose 12 gel filtration chromatography. The isolated protein had a monomer molecular mass of approximately 62 kDa, as measured by mass spectrometry. Gel filtration chromatography of the purified protein revealed a native molecular mass of approximately 176 kDa, indicating that the protein exists as a homotrimeric complex in solution. The purified protein was identified as carboxylesterase ES-10 (EC 3.1.1.1) by N-terminal Edman sequencing and extensive LC-MS/MS sequence analysis and cross-reaction with an anti-ES-10 antibody. Glycosylation analysis revealed that only one of two potential N-linked glycosylation sites is occupied by a high mannose-type carbohydrate structure. Using retinyl palmitate in a micellar assay system the enzyme was active over a broad pH range and displayed Michaelis-Menten kinetics with a K(m) of 86 microm. Substrate specificity studies showed that ES-10 is also able to catalyze hydrolysis of triolein. Cholesteryl oleate was not a substrate for ES-10 under these assay conditions. Real time reverse transcriptase-PCR and Western blot analysis revealed that ES-10 is highly expressed in liver and lung. Lower levels of ES-10 mRNA were also found in kidney, testis, and heart. A comparison of mRNA expression levels in liver demonstrated that ES-10, ES-4, and ES-3 were expressed at significantly higher levels than ES-2, an enzyme previously thought to play a major role in retinyl ester metabolism in liver. Taken together these data indicate that carboxylesterase ES-10 plays a major role in the hydrolysis of newly-endocytosed, chylomicron retinyl esters in both neutral and acidic membrane compartments of liver cells.

Topics: Animals; Binding Sites; Blotting, Western; Carboxylesterase; Carboxylic Ester Hydrolases; Chromatography, Gel; Chromatography, Ion Exchange; Concanavalin A; Diterpenes; DNA Primers; DNA, Complementary; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Glycosylation; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Liver; Male; Mass Spectrometry; Micelles; Microsomes, Liver; Protein Structure, Tertiary; Rats; Rats, Sprague-Dawley; Retinyl Esters; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sepharose; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tissue Distribution; Vitamin A

Retinyl esters are a major endogenous storage source of vitamin A in vertebrates and their hydrolysis to retinol is a key step in the regulation of the supply of retinoids to all tissues. Some members of nonspecific carboxylesterase family (EC 3.1.1.1) have been shown to hydrolyze retinyl esters. However, the number of different isoenzymes that are expressed in the liver and their retinyl palmitate hydrolase activity is not known. Six different carboxylesterases were identified and purified from rat liver microsomal extracts. Each isoenzyme was identified by mass spectrometry of its tryptic peptides. In addition to previously characterized rat liver carboxylesterases ES10, ES4, ES3, the protein products for two cloned genes, AB010635 and D50580 (GenBank accession numbers), were also identified. The sixth isoenzyme was a novel carboxylesterase and its complete cDNA was cloned and sequenced (AY034877). Three isoenzymes, ES10, ES4 and ES3, account for more than 95% of rat liver microsomal carboxylesterase activity. They obey Michaelis-Menten kinetics for hydrolysis of retinyl palmitate with Km values of about 1 micro m and specific activities between 3 and 8 nmol.min-1.mg-1 protein. D50580 and AY034877 also hydrolyzed retinyl palmitate. Gene-specific oligonucleotide probing of multiple-tissue Northern blot indicates differential expression in various tissues. Multiple genes are highly expressed in liver and small intestine, important tissues for retinoid metabolism. The level of expression of any one of the six different carboxylesterase isoenzymes will regulate the metabolism of retinyl palmitate in specific rat cells and tissues.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Carboxylic Ester Hydrolases; Chromatography, Affinity; Chromatography, Liquid; Concanavalin A; Diterpenes; DNA; Electrophoresis, Polyacrylamide Gel; Isoelectric Point; Isoenzymes; Liver; Microsomes, Liver; Molecular Sequence Data; Organ Specificity; Rats; Retinyl Esters; Sequence Analysis, DNA; Sequence Analysis, Protein; Spectrometry, Mass, Electrospray Ionization; Vitamin A

A study was conducted on the activity exerted by prolonged dietary supplementation with progressive amounts of retinoids on cell-mediated immune responses and the growth of transplantable tumors in mice. A few groups of BALB/c mice received 0 (group C), 50 (group A 50), 200 (group A 200), 500 (group A 500), and 1,000 (group A 1000) IU retinol palmitate/mouse/day in drinking water for 150 days. At progressive intervals mice from each group were tested for proliferative responses to concanavalin A (Con A), Escherichia coli lipopolysaccharide, interleukin-2, and interferon-gamma release to Con A. Ten mice from each group were also challenged with the 90-100% tumor-inducing dose of 3 distinct transplantable tumors. At the end of the experiment the principal organs were histologically examined, and the accumulation of vitamin A was evaluated. In groups A 200, A 500, and A 1000, an increase in the proliferative responses and production of lymphokines as compared to those in group C occurred after 60-90 days, but vanished after 150 days. The takes of the 3 tumors were impaired when the challenges were performed on days 75 and 150. This enhancement of distinct functions of cellular reactivity and resistance to transplantable tumors showed a linear relationship with the amount of supplemental retinol palmitate for the first 60-90 days. After 150 days, however, these enhancement effects vanished or tended to decrease.

Topics: Animals; Concanavalin A; Diet; Diterpenes; Dose-Response Relationship, Drug; Female; Fibrosarcoma; Immunity, Cellular; Interferon-gamma; Interleukin-2; Lipopolysaccharides; Lymphocyte Activation; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Neoplasms, Experimental; Retinoids; Retinyl Esters; Vitamin A

The vitamin A analogs retinoic acid and retinol caused a significant increase in concanavalin A-induced agglutination of human erythrocytes, while its esters, retinyl acetate and retinyl palmitate, were found to be ineffective. The effect of membrane labilizers and stabilizers on the enhancement of agglutination as well as the properties of the model system employed showed that the action of vitamin A is due to a direct action on cell membrane and is not mediated by the release of lysosomal proteases into the medium, a hypothesis proposed by earlier workers.

Topics: Concanavalin A; Diterpenes; Erythrocytes; Hemagglutination; Humans; Retinyl Esters; Tretinoin; Vitamin A

The behaviour of subcultures of vitamin A-treated human embryo fibroblasts was studied from the point of view of morphology, growth characteristics, karyology and agglutinability at subsequent passages after treatment. Evidence was obtained that vitamin A-induced rupture of lysosomes may lead to transient alterations in the pattern of growth, structure of the membranes and morphology in cell generations deriving from the cells injured by the action of the vitamin.

Topics: Acid Phosphatase; Cell Aggregation; Cell Division; Cell Line; Chromosomes, Human; Concanavalin A; Diterpenes; Fibroblasts; Humans; Lysosomes; Palmitates; Retinyl Esters; Vitamin A; Vitamin D