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The mitochondrial dihydroorotate dehydrogenase (DHODase), the single redox reaction in the pyrimidine de novo synthetic pathway, was purified to near homogeneity by detergent solubilization and fast protein liquid chromatography (FPLC) techniques from the mature trophozoites and schizonts of Plasmodium falciparum, human malaria parasite. The purified DHODase was monofunctional protein with a M(r) of 56,000 +/- 4000, based on Superose 12 gel filtration FPLC and SDS-PAGE analyses. Polyclonal antibodies raised against the purified P. falciparum protein was cross-reacted with P. berghei, rodent malaria parasite. The optimal activity of DHODase required long chain of coenzyme Q (CoQ6-10) which were essential for electron transfer. The Km and kcat values for L-dihydroorotate were 14.4 +/- 5.9 microM and 15.0 +/- 1.4 min-1, respectively; for CoQ6, they were 22.5 +/- 6.4 microM and 21.6 +/- 3.4 min-1. L-Orotate, an enzymatic product, was a strong competitive inhibitor with Ki of 18.2 +/- 3.6 microM. The 5-substituted L-orotates having antimalarial activities against P. falciparum in vitro were found to be competitive inhibitors. The inhibitory effect by these 5-substituted L-orotates on the malarial DHODase was different from the mammalian enzyme. Various benzoquinones and naphthoquinones were found to inhibit the purified DHODase activity at a different degree. Mitochondria from erythrocytic cycle of P. falciparum were purified, using differential centrifugation and followed by Percoll density gradient separation, with purifications of 13-fold and overall yields of 33%. The double-membraned mitochondria had a few tubular-like cristae structure as what found in many protozoan parasites. DHODase was localized inside the mitochondria as probed by immunogold labeling with the polyclonal antibodies and selective solubilization by digitonin.

Fatty liver in the goose results from an increased hepatic lipogenesis in response to overfeeding, together with a deficient secretion of triacylglycerol as very-low-density lipoproteins (VLDL). Orotic acid and estrogen, which both modify lipid metabolism in the liver, were used in male geese as tools to understand the alterations of liver lipids and plasma lipoproteins during the induction of liver steatosis. Liver lipids were analyzed after solvent extraction and plasma lipoproteins after separation by density gradient ultracentrifugation. Contrary to what is known in the rat, orotic acid (1% in food for 2 weeks) failed to induce liver steatosis. In force-fed geese, liver weight increased from approximately 100 g to approximately 800 g in 2 weeks, as a consequence of a specific accumulation of triacylglycerol. In both groups, VLDL contained less triacylglycerol (35%) than normal. Such an uncoupling of triacylglycerol synthesis and secretion, of which the precise reason is still unknown, may facilitate their accumulation when force-feeding increases hepatic lipogenesis. As with force-feeding, triacylglycerol synthesis was enhanced by estrogen, but their secretion as VLDL was very efficient and prevented liver steatosis almost completely. Since HDL concentrations were considerably decreased by estrogen, VLDL were the main lipoprotein species, with 48 g/l and 62% triacylglycerol. Where estrogen-treated geese were force-fed concomitantly, VLDL concentration was even higher (62 g/l), but triacylglycerol secretion could not prevent liver steatosis (liver weight 640 g). The data are discussed in relation to in vitro studies showing that channelling of triacylglycerol towards secretion as VLDL or hepatic storage depends on their residence time in the different intracellular compartments.

Transplanted Morris hepatomas in Buffalo-strain rats were found to be resistant to the changes in ribonucleotide levels in rat liver caused by a high-orotate diet or an arginine-deficient diet. The increase in UTP levels and decrease in ATP levels seen in the livers of rats on a 1%-orotate diet were less marked in the livers of BUB- and DBA-strain mice on this diet. Although the changes were less than in rat liver, there was a 2-3-fold increase in UTP concentration in the livers of mice on the high-orotate diet. However, there was a similar response in nucleotide levels in the two species when the animals were maintained on an arginine-deficient diet, and there was a greater than 10-fold increase in the UTP level in the livers of both rats and mice. These diets had much less effect on the levels of deoxyribonucleotides than of ribonucleotides. In contrast to the insensitivity of hepatomas to dietary modulation of nucleotide levels, treatment of hepatoma-bearing rats with carbamoylating agents (sodium cyanate and 2-chloroethyl isocyanate) caused decreases in the levels of nucleotides in the tumors which were generally greater than in host livers. For example, 2-chloroethyl isocyanate depressed ATP levels in the Morris hepatomas 5123C and 20 under conditions in which there was no significant effect on host liver ATP. The data revealed selective modulation of nucleotide levels in normal and neoplastic liver which may be achieved by either dietary modification or drug treatment.

In rats fed orotic acid, the incorporation in liver subcellular fractions of sugars injected intraperitonealy is altered only for mannose, but not for fucose or galactose. Direct determinations of several glycosyltransferases are done in smooth and rough microsomes: fucosyl-, glactosyl-, N-acetylglucosaminyltransferase activities are at quite similar levels in normal and fatty livers. By contrast, sialyltransferase activity is increased (+50%) in smooth microsomes of fatty livers, while mannosyltransferase activity is inhibited by 30%. These alterations are not caused by interfering reactions (pyrophosphatases or proteases). For the mannosyltransferase activity, the inhibition is found in the dolichylphosphorylmannose intermediates. Kinetic studies suggest that there is deficiency of both enzyme and endogenous dolichyl phosphate.

Total nuclear RNA extracted from nuclei of rat liver cells by phenol/chloroform in the presence of sodium dodecyl sulphate was separated by combined gel filtration on Sepharose 4 B and affinity chromatography on poly(U) Sepharose into fractions differing in their molecular weights and contents of poly(A) sequences. The poly(A)-containing 45-S RNA became labelled most rapidly if rats were administered [3H] orotic acid. This fraction showed a high template activity when added to postmitochondrial supernatants of the Krebs ascites tumour. Fractions of nRNA, free of poly(A) sequences, had no stimulating effect on protein synthesis in this system. The 45-S RNA-containing poly(A) was readily bound to crude polyribosomes from rat liver at 0 degrees C and both ATP and GTP were necessary for this reaction. Sucrose gradient analyses provided evidence that this RNA species is bound predominantly to 80-S ribosomes. No binding was obtained with polyribosomes washed with 0.5 M KCl. The binding ability of washed polyribosomes was restored by the addition of the ribosomal wash fraction or rat liver cytosol. Crude polyribosomes bound significantly lower quantities of nRNA species free of poly(A) when compared with poly(A)-45-S RNA. The label was scattered through the whole ribosomal sedimentation pattern with no predominant peaks and the binding reaction required neither soluble factors nor nucleotide cofactors. The labelling kinetics and high template activity of poly(A)-45-S nRNA indicate that this fraction contains precursors of cytoplasmic mRNA. Requirements for soluble factors and nucleotide cofactors in the binding of this RNA species to 80-S ribosomes suggest that this binding, unlike that of other nRNA species, has a specific mechanism resembling that of mRNA binding during peptide initiation.

The objectives of this study were to measure intestinal very-low-density lipoprotein (VLDL) production in obese Zucker rats and to assess an eventual effect of a high-fat diet. VLDL secretion was specifically inhibited by orotic acid, and intestinal VLDL output was measured following the Triton WR-1339 method. After a control diet, total VLDL secretion (without orotic acid) was 4.8 +/- 0.3 and 1.4 +/- 0.1 mg triacylglycerol/ml in obese and lean rats, respectively, decreasing by 30% in obese rats after fat-feeding. Intestinal VLDL production was similar in obese and lean rats fed the control diet (0.32 +/- 0.05 and 0.27 +/- 0.05 mg triacylglycerol/ml, respectively), increasing 2.5-fold after fat-feeding in both genotypes. Thus, intestine contributed 21 and 60% of total VLDL in lean but only 7 and 24% in obese rats with the control and high-fat diets, respectively. These results show that the intestine of obese Zucker rats does not contribute to their hypertriglyceridemia, suggesting that it originates solely from liver. Moreover, their intestinal VLDL production was stimulated by fat-feeding to the same extent as in lean animals.

1. The effect of a high-fat diet (30% fat by wt.) on intestinal very low density lipoprotein (VLDL) secretion was studied in male rats after specific inhibition of hepatic VLDL secretion by dietary orotic acid. Total VLDL secretion (from liver and intestine) was measured in animals not receiving orotic acid. 2. Fat-feeding resulted in a 32% decreased post-Triton secretion of total serum VLDL triacylglycerols as compared to a control (low fat) diet. Concomitantly, a large stimulation of post-Triton intestinal VLDL triacylglycerols secretion was measured in fat-fed rats. Thus, the major part (64%) of circulating triacylglycerols transported as VLDL originated from the intestine in these animals, leading presumably to an increased secretion of intestinal apolipoproteins. 3. Intestinal VLDL and chylomicron secretion rates increased with the amount of fat in the diet (7, 13, 20 or 30% fat by wt.). Whereas the chylomicron secretion was linearly related to the dietary fat content, the relationship between intestinal VLDL secretion and fat content of the diet was sigmoidal. The highest stimulation of intestinal VLDL formation was observed within a narrow range of dietary fat content (between 10 and 20%).