The effect of cholesterol on ubiquinone and tetrahymanol biosynthesis in Tetrahymena pyriformis.

The biosynthesis of ubiquinone-8 from radioactive mevalonate by cultures of Tetrahymena pyriformis is demonstrated. Under normal conditions the incorporation of this radioactive precursor into ubiquinone and the triterpenoid alcohol tetrahymanol reflects the amounts of these two compounds in the cell. Growth of T. pyriformis in the presence of cholesterol results in a complete inhibition of incorporation of radioactive mevalonate into tetrahymanol while there is a corresponding increase of radioactive incorporation into ubiquinone. This increased incorporation of mevalonic acid into ubiquinone must reflect a reduced level of mevalonic acid in the cell under these conditions and is not due to increased ubiquinone biosynthesis, indicating tight regulation of the pathway prior to mevalonate formation.     

The effect of excess mevalonic acid on ubiquinone and tetrahymanol biosynthesis in Tetrahymena pyriformis.

When T. pyriformis is grown in the presence of 10(-2)M-mevalonic acid, the uptake exceeds the cell's requirement for this biosynthetic intermediate. The majority of the excess mevalonic acid is diverted into ubiquinone-8 biosynthesis whereas the biosynthesis of tetrahymanol, the major product of the mevalonic acid pathway, is unchanged. In the presence of excess external mevalonic acid, the biosynthesis of mevalonic acid by the cell is inhibited. It is proposed that ubiquinone biosynthesis is normally regulated by mevalonic acid availability, whereas tetrahymanol biosynthesis is regulated primarily at a later point in the pathway.     

Polyterpenoids as cholesterol and tetrahymanol surrogates in the ciliate Tetrahymena pyriformis

The tetracyclic sterol precursors, cyclolaudenol, cycloartenol and lanosterol, inhibit efficiently the tetrahymanol biosynthesis in the ciliate Tetrahymena pyriformis, as reported earlier for cholesterol and other sterols. The prokaryotic bacteriohopanetetrols have little effect, and diplopterol, another hopanoid, as well as the carotenoid, canthaxanthin, have no effect. In the presence of triparanol, a hypocholesterolemic drug inhibiting the squalene cyclase of T. pyriformis and modifying the fatty acid metabolism, the cells do not grow further, but growth can be restored by the addition to the culture medium of suitable polyterpenoids. Thus, growth in presence of triparanol (13 microM) is almost normal after addition of a sterol such as sitosterol and cyclolaudenol, and longer lag times and lower absorbances than those of untreated cultures are observed in presence of cyclartenol, lanosterol, euphenol (a lanosterol isomer), bacteriohopanetetrols and three carotenoids. No growth at all is observed in the presence of tetrahymanol and diplopterol, although these triterpenoids are the normal reinforcers of the ciliate, probably because of a poor bioavailability. Thus, structurally different polyterpenoids are (at least partially) functionally equivalent and capable of replacing tetrahymanol or sterols and might act as membrane reinforcers in T. pyriformis cells.     

Respiratory inhibition by copper in Tetrahymena pyriformis GL

An excess of copper incorporated into Tetrahymena cells was mainly distributed in mitochondria, and inhibited oxygen uptake of Tetrahymena cells. The inhibition of oxygen uptake was clearly to copper uptake in mitochondria. Succinate was most favorable as a substrate stimulating oxygen uptake in mitochondria, and oxygen uptake was most strongly inhibited by copper (0.1 mM) in the presence of succinate among various substrates. The copper incorporated into mitochondria was in the fraction with the inner membranes. Succinate dehydrogenase (SDH) was inhibited at the lowest copper concentration (0.1 mM) among respiratory related enzymes. The redox potential of respiratory components was raised by copper. These results suggest that respiratory inhibition of Tetrahymena cells by copper may be mainly cause by inhibition of SDH as a FAD-protein and oxidation of electron carriers. At higher copper concentrations, MDH, cytochrome c reductase, and ATP synthesis were also inhibited. Growth inhibition may be due to these effects of copper in mitochondria. Mercury affected both oxygen uptake and SDH more strongly than copper. Zinc (0.1 mM) also affected oxygen uptake in mitochondria and a little in whole cells, however, it did not inhibit SDH. Cobalt, manganese, and nickel affected both oxygen uptake and SDH only a little at the same concentration (0.1 mM) as copper.     

The role of glucolipid in the biosynthesis of glycoprotein in Tetrahymena pyriformis

Cell-free preparations from Tetrahymena pyriformis catalyze the incorporation of glucose from UDP-glucose into a glucolipid having properties which are identical to those of other dolichyl phosphoryl sugar derivatives. Kinetic and other experiments have provided evidence that this glucolipid serves as glucose donor for two other types of glucosylated substances, one of which has been tentatively identified as an oligosaccharide lipid and the other a glycoprotein or glycoproteins. In addition, the partially purified glucolipid served as a glucosyl donor to these cell components, suggesting that in this protozoan, at least part of the glycoprotein is synthesized by reactions involving lipid-linked sugars in a manner analogous to that which has been observed in glycoprotein synthesis in mammalian cells.     

The presence of acyl-CoA: cholesterol acyltransferase in Tetrahymena pyriformis W

1. The esterification of cholesterol was studied in Tetrahymena pyriformis an organism which does not synthesize sterols nor are sterols required for growth. 2. Microsomes catalyzed the esterification of cholesterol in the presence of oleoyl-CoA but not oleic acid or lecithin. 3. The enzyme has a similar sterol substrate specificity to that of mammalian acyl-CoA: cholesterol acyltransferase (ACAT) and was inhibited by the specific ACAT inhibitor 58-035. 4. The enzyme is constitutive since activity was observed in cells grown in sterol-free medium when cholesterol was added to the in vitro assay.