Incorporation of mevalonate into squalene, lanosterol and cholesterol by different neonatal chick tissues

The role of neonatal chick liver and kidneys in the incorporation of mevalonic acid into squalene, lanosterol and cholesterol was studied. Differences between the synthesizing ability of these and other tissues and the influence of the in vivo or in vitro conditions were also examined. In the in vivo experiments, distribution of radioactivity among the nonsaponifiable lipids was not dependent of the doses of mevalonic acid injected. About 80-95% of radioactivity was recovered as cholesterol in liver and brain, whereas in kidneys this percentage was only about 35%. Squalene and lanosterol were formed by kidneys in a high percentage, higher than in liver and other tissues. 12 hr after mevalonate injection, the percentage of cholesterol formed by kidneys increased until more than 50%. In the in vitro experiments carried out in the presence of 0.045-4.0 mM mevalonate, cholesterol was also the main nonsaponifiable identified, but in a lesser percentage than in vivo. In the same conditions, the incorporation of mevalonic acid by kidneys was maximal into squalene. After in vitro incubations for 2 hr, the percentage of cholesterol in kidneys also increased.     

Incorporation of squalene into rod outer segments

We have reported previously that squalene is the major radiolabeled nonsaponifiable lipid product derived from [3H]acetate in short term incubations of frog retinas (Keller, R. K., Fliesler, S. J., and Nellis, S. W. (1988) J. Biol. Chem. 263, 2250-2254). In the present study, we demonstrate that newly synthesized squalene is incorporated into rod outer segments under similar in vitro conditions. We show further that squalene is an endogenous constituent of frog rod outer segment membranes; its concentration is approximately 9.5 nmol/mumol of phospholipid or about 9% of the level of cholesterol. Pulse-chase experiments with radiolabeled precursors revealed no metabolism of outer segment squalene to sterols in up to 20 h of chase. Taken together with our previous absolute rate studies (Keller, R. K., Fliesler, S. J., and Nellis, S. W. (1988) J. Biol. Chem. 263, 2250-2254), these results suggest that most, if not all, of the squalene synthesized by the frog retina is transported to rod outer segments. Synthesis of protein is not required for squalene transport since puromycin had no effect on squalene incorporation into outer segments. Conversely, inhibition of isoprenoid synthesis with mevinolin had no effect on the incorporation of opsin into the outer segment. These latter results support the conclusion that the de novo synthesis and subsequent intracellular trafficking of opsin and isoprenoid lipids destined for the outer segment occur via independent mechanisms.     

Biosynthesis of squalene and sterols by rat aorta

The synthesis of nonsaponifiable compounds from radioactive mevalonate by segments of adult rat aorta was studied in vitro. The labeled products consisted largely of substances with the chromatographic and chemical behavior of squalene, lanosterol, lathosterol, and cholesterol. Even after 3 or 4 hr of incubation, the incorporation of mevalonate into squalene was higher than its incorporation into C(27) sterols; cholesterol contained less than 20% of the radioactivity in the total sterols. Lanosterol was the most highly labeled sterol. The level of radioactivity in lathosterol was comparable to the level in cholesterol. Small amounts of radioactivity were found in other sterols. Material with the same mobility on TLC as 7-dehydrocholesterol had less radioactivity than cholesterol, but more than sterols with the mobility of desmosterol. The results of measurements made after short periods of incubation showed that squalene and lanosterol became labeled before the other nonsaponifiable compounds.     

The biosynthesis of squalene and sterols by the adipose tissue of rat, sheep and man

1. The subcutaneous and omental adipose tissue of man, the epididymal fat pads of the rat and the fat tail of the Syrian sheep incorporate mevalonic acid into non-saponifiable lipids. 2. Time studies showed that the rates of decarboxylation of mevalonic acid and synthesis of non-saponifiable lipids slightly decline after 20min. but subsequently remain linear for 6hr. 3. About one-half of the incorporated radioactivity in the non-saponifiable lipids was in squalene, 20% in lanosterol and cholesterol, and the remainder in unidentified substances.     

Protein synthesis in tomato-fruit locule tissue: Incorporation of amino acids into protein by aseptic cell-free systems

1. Osmotically disrupted protoplasts and isolated plastids from tomato-fruit locule tissue were found capable of incorporating (14)C-labelled amino acids under aseptic conditions into an exhaustively washed trichloroacetic acid-insoluble protein fraction. 2. The disrupted protoplast system incorporated 20-45mumumoles of amino acid/mg. of protein in 10min. The isolated plastid system incorporated 10-20mumumoles of amino acid/mg. of protein; 40-150mumug. of carbon/mg. of protein was incorporated in 10min. from (14)C-labelled amino acid mixture. 3. Incorporation is stimulated by added ATP in the dark, but no added ATP is required when the system is illuminated. The cell-free plastid system is to some extent self-sufficient and does not normally require an added supernatant fraction or unlabelled amino acids. 4. Amino acid incorporation by plastids is inhibited by chloramphenicol, puromycin, actinomycin D, ribonuclease and deoxyribonuclease. It is suggested that the mechanism of protein synthesis in the cell-free plastids, and in the tissue generally, is basically the same as established for bacteria. Ribosomes and highspeed supernatant from this tissue were to some extent interchangeable with Escherichia coli ribosomes and supernatant in cell-free incubations. 5. Incorporation of amino acids by isolated plastids was stimulated by indol-3-ylacetic acid and kinetin, and, whereas incorporation normally proceeds for only 10-20min., the time-course was extended in the presence of these growth substances. It is suggested that hormones may be involved in the regulation of protein synthesis in plants.     

Incorporation of amino acids into protein by cell-free extracts of penicillium chrysogenum

Summary The preparation of ribosomal particles from P. chrysogenum has been described. After correction to 20°C and extrapolation to infinite dilution, they had sedimentation coefficients of approximately 80S, 60 S, and 40 S. The washed ribosomes plus purified supernatant fraction, KCl and sucrose incorporated C¹⁴-l-amino acids into protein. There was no stimulation of incorporation by ATP, GTP, CTP, UTP, or Mg ions. Incorporation was inhibited by streptomycin and chloramphenicol but not by ribonuclease.The amino acid incorporating system from P. chrysogenum did not resemble bacterial and yeast systems in many respects and reasons for this are discussed.     

Incorporation of mevalonate into dolichol and other isoprenoids during estrogen-induced chick oviduct differentiation

Incorporation of [14C]mevalonate into dolichol and other isoprenoid compounds by chick oviduct explants has been studied. A reliable assay of dolichol biosynthesis employing several chromatographic procedures, including two-dimentional TLC, was developed. Incorporation of [14C]mevalonate into dolichol by oviduct explants was linear for at least 6 h. The effect of estrogen-induced differentiation was studied by incubation of explants obtained from chicks treated for various periods of time with diethylstilbestrol. Mevalonate incorporation into dolichol, when expressed as cpm per g of tissue, was not affected by estrogen treatment, but since the oviduct increased about 100-fold in mass during differentiation, each oviduct synthesizes about 100-fold more dolichol. In most tissues, the major product of mevalonate incorporation is cholesterol. However, although approx. 90% of the non-saponifiable 14C-labeled compounds were in the so-called 'cholesterol fraction', oviduct explants from estrogenized chicks synthesized little, if any, cholesterol. A number of cholesterol biosynthetic intermediates were observed, with compounds comigrating with squalene and lanosterol accounting for about 50% of the total. Since the estrogenized chick has serum cholesterol levels in the range of 800-900 mg/dl, these results suggest that oviduct has secondary control points which allow it to inhibit cholesterol synthesis when mevalonate is used as the precursor. In support of this hypothesis is the observation that explants from untreated chicks can incorporate mevalonate into cholesterol.