Identification and characterization of mammalian mitochondrial tRNA nucleotidyltransferases.

The CCA-adding enzyme (ATP:tRNA adenylyltransferase or CTP:tRNA cytidylyltransferase (EC )) generates the conserved CCA sequence responsible for the attachment of amino acid at the 3' terminus of tRNA molecules. It was shown that enzymes from various organisms strictly recognize the elbow region of tRNA formed by the conserved D- and T-loops. However, most of the mammalian mitochondrial (mt) tRNAs lack consensus sequences in both D- and T-loops. To characterize the mammalian mt CCA-adding enzymes, we have partially purified the enzyme from bovine liver mitochondria and determined cDNA sequences from human and mouse dbESTs by mass spectrometric analysis. The identified sequences contained typical amino-terminal peptides for mitochondrial protein import and had characteristics of the class II nucleotidyltransferase superfamily that includes eukaryotic and eubacterial CCA-adding enzymes. The human recombinant enzyme was overexpressed in Escherichia coli, and its CCA-adding activity was characterized using several mt tRNAs as substrates. The results clearly show that the human mt CCA-adding enzyme can efficiently repair mt tRNAs that are poor substrates for the E. coli enzyme although both enzymes work equally well on cytoplasmic tRNAs. This suggests that the mammalian mt enzymes have evolved so as to recognize mt tRNAs with unusual structures.

Cell-free translation reconstituted with purified components.

We have developed a protein-synthesizing system reconstituted from recombinant tagged protein factors purified to homogeneity. The system was able to produce protein at a rate of about 160 microg/ml/h in a batch mode without the need for any supplementary apparatus. The protein products were easily purified within 1 h using affinity chromatography to remove the tagged protein factors. Moreover, omission of a release factor allowed efficient incorporation of an unnatural amino acid using suppressor transfer RNA (tRNA).

The role of tightly bound ATP in Escherichia coli tRNA nucleotidyltransferase.

BACKGROUND: The CCA-adding enzyme [ATP(CTP): tRNA nucleotidyltransferase (EC. 2.7.7.25)] catalyses the addition of the conserved CCA sequence to the 3'-terminus of tRNAs. All CCA-adding enzymes are classified into the nucleotidyltransferase superfamily. In the absence of ATP, the Escherichia coli CCA-adding enzyme displays anomalous poly(C) polymerase activity. RESULTS: We show that CCA-adding enzyme over-expressed in E. coli exists in an ATP-bound form. The affinities of ATP and CTP towards the enzyme were estimated by several methods, and the dissociation constants for ATP and CTP were determined to be 6.3 and 188 microM, respectively. AMP-incorporation terminated the nucleotidyltransferase reaction, while in the absence of ATP, the enzyme continued poly(C) polymerization. In the case of a tRNA substrate with a mutation in the T-loop region, normal CC was added at a much slower rate compared with the wild-type, but anomalous poly(C) polymerization occurred at the same rate as in the wild-type. CONCLUSION: Based on the findings outlined above, we concluded that the E. coli CCA-adding enzyme possesses at least two distinct nucleotide binding sites, one responsible for ATP binding and the other(s) for CTP binding. The addition of ATP from the tight ATP binding site terminates nucleotide incorporation, thus limiting poly(C) polymerization to CCA. It is also suggested that during anomalous poly(C) polymerization, tRNA translocates from the tRNA binding site upon the third C addition.

TMP-153, a novel ACAT inhibitor, lowers plasma cholesterol through its hepatic action in golden hamsters.

The mechanism of the hypocholesterolemic action of N-[4-(2-chlorophenyl)-6,7-dimethyl-3-quinolyl]-N'-(2, 4-difluorophenyl) urea (TMP-153), a potent acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, was studies in Golden hamsters. TMP-153 (0.5-1.5 mg/kg) dose-dependently reduced plasma total- and low density lipoprotein (LDL)-cholesterol without affecting high density lipoprotein (HDL)-cholesterol. TMP-153 markedly reduced the cholesterol influx into the plasma upon intravenous injection of Triton WR-1339. The compound also decreased cholesterol absorption calculated from dietary intake, biliary secretion and the absorption co-efficient. Hepatic cholesterol secretion was calculated by substracting the cholesterol absorption from the cholesterol influx. In hamsters, the liver accounted for 92% of the cholesterol influx with the remaining 8% coming from the intestine, and both were markedly decreased by TMP-153. Thus, it is likely that TMP-153 lowers plasma cholesterol through its hepatic action. In the liver, the compound significantly reduced the unesterified cholesterol content in addition to markedly reducing the content of esterified cholesterol. In accordance with this reduction, the half-life time of [125I]-LDL was significantly shortened by the compound, suggesting an increase in LDL receptors. However, the hepatic cholesterogenesis from [14C]acetate was decreased by TMP-153 treatment. This effect seems to be secondary, since the compound did not inhibit cholesterogenesis from [14C]acetate in HepG2 cells. From the data described above, the contribution of hepatic secretion and intestinal absorption of cholesterol to the plasma cholesterol level in Golden hamsters are discussed.

Effects of hypophysectomy on TRH and its related peptides concentrations in various rat organs.

The effects of hypophysectomy on thyrotropin releasing hormone (TRH), TRH-glycine (TRH-Gly) and pre-pro-TRH (178-199) concentrations in the rat hypothalamus, cerebrum, cerebellum, brain stem, stomach and retina were studied 7 days after the operation. The hypophysectomized rats were administered a single i.p. injection of T4 (500 micrograms/kg), T3 (100 micrograms/kg) or bovine TSH (1.25 IU/kg), and 5 rats of each subgroup were decapitated at 4 hours later. After hypophysectomy, TRH-Gly and pre-pro-TRH (178-199) concentrations in the hypothalamus increased significantly and TRH concentrations decreased after hypophysectomy. After the injection of T4, T3, TRH or TSH TRH-Gly and pre-pro-TRH (178-199) concentration in the hypothalamus of hypophysectomized rats decreased significantly, while that of TRH significantly increased. No changes in TRH, TRH-Gly and pre-pro-TRH (178-199) concentration in other tissues were observed after hypophysectomy or hormone treatment. The findings suggest that hypophysectomy stimulated TRH synthesis and release in the hypothalamus, that TRH, TSH, T3 and T4 regulate hypothalamic TRH levels, and that pro-TRH synthesis in the tissues except the hypothalamus may not be regulated by thyroid hormone.

The regulation of gene expression by insulin is differentially impaired in the liver of the genetically obese-hyperglycemic Wistar fatty rat.

The regulation by insulin and carbohydrates of the gene expression of three key enzymes involved in glucose metabolism was studied in the liver of the Wistar fatty rat, a model of obese non-insulin-dependent diabetes mellitus. A high glucose or fructose diet, or insulin administration caused a similar magnitude of increase in the level of L-type pyruvate kinase mRNA in the liver of Wistar fatty rats and their lean littermates. However, the induction of glucokinase mRNA and repression of phosphoenolpyruvate carboxykinase mRNA by dietary glucose or insulin were impaired in the fatty rats, whereas fructose caused a similar decrease in phosphoenolpyruvate carboxykinase mRNA in both types of rats. These results indicate that the regulation of gene expression of glucokinase and phosphoenolpyruvate carboxykinase, but not of L-type pyruvate kinase, by insulin is impaired in the liver of the Wistar fatty rat.

Lymphatic absorption of structured glycerolipids containing medium-chain fatty acids and linoleic acid, and their effect on cholesterol absorption in rats.

The effects of various structured triglycerides containing medium-chain (caprylic or capric acids) and long-chain (linoleic acid) fatty acids on fatty acid and cholesterol absorption were studied in lymph-cannulated rats. A considerable portion of capric and caprylic acid was absorbed through the lymph duct, although to a lesser extent than was linoleic acid. Capric and linoleic acid located at the 2-position of 2-decanoyl-1,3-dilinoleoyl-glycerol (18:2/10:0/18:2) and 2-linoleoyl-1,3-didecanoyl-glycerol (10:0/18:2/10:0), respectively, tended to be absorbed more efficiently than those located at the 1- and 3-position or those from tricaprin (10:0/10:0/10:0) or trilinolein (18:2/18:2/18:2). A similar trend was observed when the medium-chain fatty acid was caprylic acid instead of capric acid. Caprylic acid absorption from 2-octanoyl-1,3-dilinoleoyl-glycerol (18:2/8:0/18:2) was significantly greater (p less than 0.05) than from 2-linoleoyl-1,3-dioctanoyl-glycerol (8:0/18:2/8:0) or tricaprylin (8:0/8:0/8:0). Preferential absorption of caprylic and linoleic acid was not observed when the 1 to 2 and the 2 to 1 mixtures of 8:0/8:0/8:0 and 18:2/18:2/18:2, respectively, were administered. The structured lipids did not affect the lymphatic absorption of cholesterol. The results suggest that structured triglycerides composed of medium-chain fatty acids and linoleic acid may be more useful for the treatment of lipid malabsorption than are mixtures of medium-chain triglyceride (MCT) and long-chain triglyceride (LCT).

Interrelated effects of dietary fiber and fat on lymphatic cholesterol and triglyceride absorption in rats.

Lymph cannulated rats were administered intragastrically a test emulsion containing 25 mg of [14C]cholesterol, 50 mg of either guar gum, cellulose or chitosan, and 200 mg of either safflower, high-oleic safflower or palm oil, and the absorption of labeled cholesterol and fatty acids was measured. The type of both dietary fiber (P less than 0.001) and fat (P less than 0.05) significantly influenced cholesterol absorption. A significant interaction of fiber and fat on cholesterol absorption (P less than 0.05) was also observed. Chitosan effectively lowered cholesterol absorption more than did guar gum or cellulose, and this effect was more significant when given with safflower or high-oleic safflower oil than with palm oil. When guar gum was the source of dietary fiber, dietary fats did not modify cholesterol absorption. Dietary fiber also significantly affected triglyceride absorption (P less than 0.05). Absorption tended to be low in the chitosan, high in the cellulose and intermediate in the guar gum group. Absorption of safflower and high-oleic safflower oils tended to be higher than that of palm oil when cellulose or guar gum was fed. Guar gum, as compared with the other fibers, altered the absorption pattern of both cholesterol and triglyceride. The results showed that the type of dietary fat significantly influenced the effect that dietary fiber exerted on lipid absorption.

Effects of short-term cholestyramine feeding on cholesterol metabolism in differently aged rats.

The effects of short-term (7 days) feeding of a diet containing cholestyramine (5%) on the cholesterol metabolism were studied in male Sprague-Dawley rats at ages of 5 weeks (young) and 9-10 months (adult). Cholestyramine significantly enhanced the activities of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7 alpha-hydroxylase both in young and adult rats; however, the absolute values were significantly higher in the former. The time-courses of changes in the activities of these enzymes after cessation of cholestyramine were comparable in both groups of rats. The rate of incorporation of mevalonate into sterol was also higher in young than in adult rats, while the stimulating effect of cholestyramine was markedly greater in adult rats. Hepatic acyl-CoA:cholesterol acyltransferase activity was comparable, but cholestyramine significantly decreased it only in adult rats. In adult rats hepatic cholesterol was decreased significantly by the resin while it remained uninfluenced in young rats. The serum cholesterol level tended to be higher in adult rats regardless of the dietary manipulation. The results indicate an appreciable age-dependent change in the hepatic cholesterol metabolism in response to the interruption of enterohepatic circulation of bile acids.