Formation of retinoylated proteins from retinoyl-CoA in rat tissues

Retinoylation (acylation of proteins by retinoic acid) is considered as one mechanism of retinoic acid (RA) action occurring in cells in vitro and in vivo. Previously, our studies showed that in rat tissues the formation of retinoyl-CoA from RA, the first step of retinoylation, required ATP, CoA and MgCl(2). In the current study, we examined whether the transfer of retinoyl-CoA into proteins, the second step of retinoylation, occurs in rat tissues. [(3)H]-Labeled-retinoyl-CoA bound covalently to proteins in rat liver, kidney, testis, and brain. The levels of incorporation of retinoyl-CoA into proteins were higher in vitamin A-deficient rats than in normal ones. The formation of retinoylated proteins depended on the incubation time, and the concentrations of retinoyl-CoA and homogenate. The reaction was suppressed by fatty acyl-CoAs and palmitic acid, but not by arachidonic acid. The Vmax and Km values for retinoyl-CoA in the formation of retinoylated proteins using a crude liver extract were estimated to be 2,597.3 pmol/min/mg protein and 9.5 x 10(-5) M, respectively. Retinoylated proteins formed from retinoyl-CoA, including a 17 kDa protein exhibiting high radioactivity, disappeared in the presence of 2-mercaptoethanol, indicating that RA was linked to the proteins through a thioester bond. These results demonstrate that retinoylation in rat tissues occurs via retinoyl-CoA formed from RA. This process may play a significant physiological role in cells.     

Formation of retinoyl-CoA in rat tissues

Retinoylation (retinoic acid acylation) is a posttranslational modification of proteins occurring in a variety of cell types in vitro and in tissues in vivo. The widespread occurrence of retinoylation suggests that it may play a role in many effects of retinoic acid (RA) on cells. One metabolic pathway for retinoylation involves the intermediate formation of retinoyl-CoA and subsequent transfer and covalent binding of the retinoyl moiety to protein. However, such reactions are not well known. To gain further insight into retinoylation, we studied the synthesis of retinoyl-CoA, the first step in this multi-stage process. The formation of [(3)H]-retinoyl-CoA was determined in incubation mixtures containing rat liver extract, [(3)H]-RA, ATP, CoA, and MgCl(2). No retinoyl-CoA was formed in the presence of boiled extract, or in the absence of ATP, CoA, or MgCl(2) (a divalent cation). A greater amount of retinoyl-CoA was obtained from microsomal fractions of rat liver than from other subfractions. The presence of retinoyl-CoA was also detected in extracts prepared from rat testis, kidney, brain, spleen, and pancreas. The level of retinoylation in various tissue extracts was related directly to the amount of retinoyl-CoA formed. V(max) and K(m) values for RA in the formation of liver retinoyl-CoA were estimated to be 1.0 x 10(-4) micromol/min/mg protein and 24 nM, respectively. Synthesis of retinoyl-CoA was suppressed by fatty acids and fatty acyl-CoAs. These results indicate that ATP-dependent generation of retinoyl-CoA occurs in rat tissues and may play a significant physiological role in RA actions mediated by retinoylation.     

Formation of retinoic acid from retinol in the rat

1. The formation in vivo of retinoic acid from microgram quantities of intrajugularly administered [15-(14)C]retinol was demonstrated in the rat. 2. Endogenously formed retinoic acid (about 0.1mug./rat) was found in liver, and to a much smaller extent in intestine, 12hr. after retinol administration. 3. Excretion of some of the endogenously formed retinoic acid occurred in the bile of bile-duct-cannulated rats. 4. Excretion of unaltered retinoic acid in the urine of intact rats did not occur even after the intrajugular administration of preformed retinoic acid.     

Formation of taurine and amino acid pools in rat tissues upon stimulation of NAD synthesis

A single intraperitoneal injection of nicotinamide (500 mg/kg) to mongrel albino rats causes a 6-hour increase in the 2-oxoglutarate level and the free NAD+/NADH ratio in liver mitochondria. The levels of taurine and taurocholates as well the activity of cysteine oxidase in liver tissues remains thereby unchanged, whereas the cysteine transaminase activity diminishes. In the heart and brain of experimental animals the activity of both enzymes is decreased. In the liver, blood plasma and heart of experimental animals, the Ala and Ser levels are low, whereas the taurine content is elevated both in blood plasma and brain. Nicotinamide administration eliminates positive correlations between the levels of taurine, its precursors and metabolically bound amino acids. In the liver the negative correlations between the activities of cysteine oxidase and cysteine transaminase observed in the control group disappear in the experimental group. Apparently, one of regulatory mechanisms of the taurine pool formation in the liver is the ratio of activities of the both enzymes as well as their competition at the substrate level. This emphasizes the importance of the transamination reactions in the metabolism of sulphur-containing amino acids.     

Simultaneous degradation of the herbicides 2,4-dichlorophenoxyacetic acid and 2-(2-methyl-4-chlorophenoxy)propionic acid by mixed bacterial cultures

The simultaneous degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-(2-methyl-4-chlorophenoxy)propionic acid (mecoprop) was achieved by two mixed cultures in the absence of any additional carbon or energy substrates. Mecoprop was not completely degraded by either of the two cultures, nor did addition of 2,4-D affect the degradation of mecoprop. The cultures completely degraded 2,4-D, and the degradation was uninfluenced by the addition of mecoprop. Nearly complete dechlorination of the mixture of two herbicides was achieved by both cultures, on the basis of the total amount of the two herbicides degraded. During the course of the reaction, however, the expected values of chloride were not met. Cell growth continued after the degradation of the parent substrates ceased. Although the mecoprop degradation did not continue to completion, spectral and growth data indicated that the metabolites which had accumulated during the reaction were degraded upon further incubation.     

Three-dimensional structure of a fluorescein-Fab complex crystallized in 2-methyl-2,4-pentanediol

The crystal structure of a fluorescein-Fab (4-4-20) complex was determined at 2.7 A resolution by molecular replacement methods. The starting model was the refined 2.7 A structure of unliganded Fab from an autoantibody (BV04-01) with specificity for single-stranded DNA. In the 4-4-20 complex fluorescein fits tightly into a relatively deep slot formed by a network of tryptophan and tyrosine side chains. The planar xanthonyl ring of the hapten is accommodated at the bottom of the slot while the phenylcarboxyl group interfaces with solvent. Tyrosine 37 (light chain) and tryptophan 33 (heavy chain) flank the xanthonyl group and tryptophan 101 (light chain) provides the floor of the combining site. Tyrosine 103 (heavy chain) is situated near the phenyl ring of the hapten and tyrosine 102 (heavy chain) forms part of the boundary of the slot. Histidine 31 and arginine 39 of the light chain are located in positions adjacent to the two enolic groups at opposite ends of the xanthonyl ring, and thus account for neutralization of one of two negative charges in the haptenic dianion. Formation of an enol-arginine ion pair in a region of low dielectric constant may account for an incremental increase in affinity of 2-3 orders of magnitude in the 4-4-20 molecule relative to other members of an idiotypic family of monoclonal antifluorescyl antibodies. The phenyl carboxyl group of fluorescein appears to be hydrogen bonded to the phenolic hydroxyl group of tyrosine 37 of the light chain. A molecule of 2-methyl-2,4-pentanediol (MPD), trapped in the interface of the variable domains just below the fluorescein binding site, may be partly responsible for the decrease in affinity for the hapten in MPD.     

Fluorometric determination of alpha-ketosuccinamic acid in rat tissues

A method for the fluorometric determination of alpha-ketosuccinamic acid, the alpha-keto acid analog of asparagine, is described. The procedure involves the hydrolysis of alpha-ketosuccinamate to oxaloacetate by omega-amidase followed by NADH-dependent reduction of oxaloacetate to malate by malate dehydrogenase. A correction for endogenous oxaloacetate is made by using control samples lacking omega-amidase. Of the rat tissues investigated, liver contained the highest concentration, followed by kidney (53 +/- 6 (n = 11) and 18 +/- 3 (n = 3) mumol/kg wet wt, respectively). alpha-Ketosuccinamate was not detected in brain (less than 8 mumol/kg wet wt). Some chemical properties of alpha-ketosuccinamate were investigated. Concentrated solutions of sodium alpha-ketosuccinamate frozen for extended periods and the solid sodium salt of alpha-ketosuccinamate dimer heated to 130 degrees C are converted to at least 10 products by processes involving dimerization, dehydration, and decarboxylation. Isobutane chemical ionization mass spectral analysis (170-230 degrees C) of the free acid monomer yielded similar products. Many of the breakdown products were identified as di- and monoheterocyclic compounds, some of which are known to be of biological importance.     

Metabolism of free malonic acid in rat tissues

It is found that the liver skeletal muscle and brain utilize free malonic acid in lipogenesis reactions with different rate. It is shown that the inclusion of malonic acid to lipid biosynthesis is connected with its decarboxylation by the first carbon atom, with the next condensation of the formed intermediate with coenzyme A and further transformations of acetyl-coenzyme A.     

Formation of 2,4-Diarylallophanate from Arylthionocarbamate in an Iodine-DMSO System

The structures of cotylenins, leaf growth substances produced by a fungus, have been assigned on the basis of degradative and spectroscopic evidence. They are novel glycosides with a common aglycone, cotylenol: cotylenins A and C have unusual sugar moieties consisting of an 6-O-methyl-α-d-glucosyl derivative with an oxygenated C₅-isoprene unit.     

Formation of malate from glyoxylate in animal tissues

1. Incubation of rat liver homogenate with [1-(14)C]glyoxylate, ATP and acetate shows a rapid sequential incorporation of radioactivity into malate, oxaloacetate and citrate. 2. In liver from normal rats the rate of the formation of each substance in question is higher than that in liver from thiamin-deficient rats. 3. The net accumulation of malate is greater with liver from thiamin-deficient rats. Its further metabolism is retarded, it is suggested, by inhibitors formed by a condensation of glyoxylate and oxaloacetate.     

Stimulation of polysaccharide formation by 2,4-dichlorophenoxyacetic acid in callus tissues of Arabidopsis thaliana

A relatively high concentration of 2,4-dichlorophenoxyacetic acid (45 μ M ) in solid culture medium stimulated the formation and secretion of mucilage polysaccharides by callus tissues of Arabidopsis thaliana L. Heynh. (line Estland). The mucilage was composed of at least two polysaccharides as revealed by gel chromatography on Sepharose 4B: the major component (87%) eluted in the void volume (molecular weight 2 × 10⁶ or greater) and the minor component (13%) eluted in the molecular weight range from 2 × 10⁴ to 4 × 10⁵. Both polysaccharide components contained small amounts of uronic acids. The major polysaccharide consisted mostly of galactose (49%), arabinose (28%) and fucose (10%), whereas the minor one consisted of galactose (44%), xylose (18%), arabinose (14%) and rhamnose (14%). One of the components of the secreted mucilage seems to be an arabinogalactan.