Biosynthesis of thyroglobulin. Incorporation of [1-14C]galactose, [1-14C]mannose and [4,5-3H2]leucine into soluble proteins by rat thyroids in vitro

1. Rat thyroid lobes were incubated for various periods of time in Krebs–Ringer bicarbonate containing [³H]leucine and either [1-¹⁴C]galactose or [1-¹⁴C]mannose. Radioactivity in soluble proteins was determined after their separation by sucrose-gradient centrifugation. 2. The time-course of incorporation of label from [¹⁴C]-mannose into soluble thyroid proteins was parallel to that observed for [³H]leucine. There was a lag of at least 30min. before either label appeared in non-iodinated thyroglobulin (protein 17–18s). During this time both labels were detected in two fractions known to contain subunit precursors of thyroglobulin (fractions 12s and 3–8s). Radioactivity from double-labelled fractions 12s and 3–8s was transferred to protein 17–18s during subsequent incubation in an unlabelled medium. 3. In contrast, most of the [¹⁴C]galactose was immediately incorporated into protein 17–18s. 4. During the first hour of incubation, puromycin almost completely inhibited the incorporation of label from [³H]leucine and [¹⁴C]mannose into all protein fractions, but had little effect on the incorporation of [¹⁴C]galactose into protein 17–18s. 5. These results indicate that mannose is incorporated into the carbohydrate groups of protein 17–18s at an earlier stage in its formation than galactose. It is suggested that the synthesis of the carbohydrate groups of ghyroglobulin begins soon after formation of the polypeptide components, more than 30min. before these are aggregated to protein 17–18s; carbohydrate synthesis then proceeds in a stepwise manner, galactose being incorporated at about the time of aggregation of subunits to protein 17–18s. Most, if not all, the carbohydrate is added to thyroglobulin before it is iodinated.     

Incorporation of l-[C]leucine into egg proteins by liver slices from cod

1. Liver slices from cod (Gadus morhua L.) were incubated with l-[¹⁴C]leucine and the incorporation of label into total protein, precipitated with trichloroacetic acid, and into egg proteins, precipitated with an antibody after addition of carrier egg proteins, was measured. 2. Liver slices from immature male or female cod, and from male fish with developing testes, did not incorporate significant amounts of l-[¹⁴C]leucine into egg proteins, whereas with slices from female cod with developing ovaries the rate of incorporation into egg proteins was 8% of the rate of incorporation into total protein. 3. Liver slices from immature male or female fish that had received an intramuscular injection of oestradiol benzoate (1mg/kg) 5–8 days previously incorporated l-[¹⁴C]leucine into egg proteins at about 26% of the rate of incorporation into total protein. 4. Incorporation into total protein and into egg proteins was inhibited by puromycin, and 1.2 and 0.13μg of puromycin/mg of tissue protein, respectively, gave 50% inhibition.     

Biosynthesis of liver membranes. Incorporation of [3H]leucine into proteins and of [14C]glucosamine into proteins and lipids of liver microsomal and plasma-membrane fractions

1. The smooth-and rough-microsomal and the light and heavy plasma-membrane fractions of mouse liver homogenates were prepared and characterized by using biochemical markers. 2. The hexosamine/protein ratio was threefold higher in the plasma membranes than in the smooth-microsomal fraction. Glucosamine was bound only to protein, and galactosamine was attached mainly to lipids. 3. [(3)H]-Leucine and [(14)C]glucosamine were injected into animals and the rates of incorporation of radioactivity into the fractions were determined. Both precursors were rapidly incorporated into the microsomal fractions, but plasma membranes showed a slower rate of synthesis which reached a maximum at 2-4h after intravenous administration. 4. The light- and heavy-plasma-membrane fractions showed similar patterns of incorporation, and therefore a precursor-product relationship appears unlikely. 5. Plasma membranes, especially the light subfraction, showed appreciable incorporation of hexosamine into chloroform-methanol-soluble components which were shown to be mainly glycolipids. 6. The results indicate that liver plasma-membrane proteins and glycoproteins are synthesized at similar rates. However, glycolipid synthesis in plasma membranes occurred more rapidly.     

Incorporation of [14C]glucosamine into synaptosomes in vitro

Abstract— Synaptosomes isolated from rat cerebral cortex by zonal centrifugation in-corporated radioactive glucosamine into macromolecules in vitro as glucosamine, galactosamine, N-acetylneuraminic acid, and glucuronic acid. The largest percentage of incorporated radioactivity was recovered in the particulate fraction in which radioactive carbohydrates were bound in covalent linkage requiring acid hydrolysis or enzymatic digestion for release. Less than 20 per cent of the particulate radioactivity represented incorporation into gangliosides. Some 20 per cent of the radioactivity was incorporated into proteins as glucosamine, identified in hydrolysates by paper chromatography and by the amino acid analyser. After incubation, radioactivity was demonstrable in the proteins as sialic acid by paper chromatography and specific enzymic digestion; and as glucuronic acid by chromatography, electrophoresis, and digestion with hyaluronidase. Incorporation of carbohydrate was stimulated by sodium and potassium at concentrations demonstrated to enhance incorporation of amino acids, and involved the macro-molecules of all subsynaptosomal fractions. Significant incorporation of radioactivity was found in the synaptic plasma membrane. The synthesis of glycoproteins was suggested by simultaneous incorporation of [¹⁴C]glucosamine and [³H]leucine into glycopeptides subsequently hydrolysed and subjected to polyacrylamide gel electrophoresis and two-dimensional paper chromatography and electrophoresis. Such studies demonstrated that amino acids and carbohydrates may be incorporated into glycoproteins of the synaptic membrane and suggest the possibility of local synthesis as well as modification of material brought to the nerve ending by axoplasmic flow.     

Incorporation of [14C] ethanolamine and [3H] Methionine into phospholipids of rat brain and liver in vivo and in vitro

Comparative studies were undertaken on the in vivo and in vitro incorporation of [¹⁴C] ethanolamine, [³H] methionine and [¹⁴C] S-adenosyl-methionine into phosphatidylethanolamine (PhE) and phosphatidylcholine (PhC) of rat liver and brain. It was observed that brain can synthesize de novo PhC from PhE via the transmethylation pathway, however synthesis rates were (1) markedly lower than those of liver and (2) decreased significantly with age. In the choline-containing lipids more than 95% of the radioactivity was found in PhC. Studies on the localization of the radioactivity in PhC following the intracranial injection of [³H] methionine or [¹⁴C] ethanolamine revealed that both precursors are incorporated almost exclusively into the choline moiety of this phospholipid. There was significant labeling of PhC only when the precursors were administered intracranially and much less incorporation was observed with the systemic routes. Thus following the intravenous administration of [¹⁴C] ethanolamine, the specific radioactivities of liver PhE and PhC were up to 75 times as high as those of brain and 4 to 5 times as high in the organs of the 20-day old as those of the adult. In contrast, when this precursor was administered intracranially the specific radioactivities of both phospholipids in liver were only twice as high as those of brain. Although the short-and long-term time-course studies on the in vivo incorporation of [¹⁴C] ethanolamine and [³H] methionine into PhC of both organs could suggest a precursor-product relationship between the biosynthesis of this phospholipid in liver and brain, this apparent relationship could also be due to the high turnover of PhE in liver, with half-life of 2.87 hr, and its low turnover in brain, with half-life of 10.7 days. The present findings on the low rate of formation of PhC from PhE in brain coupled with the fact that this conversion declines sharply with age, especially when the isotopes are administered systemically, could explain the observation of previous investigators that the brain cannot synthesize its own choline and thus it must derive its choline from exogenous sources such as lipid-choline. It was concluded that the brain can synthesize its own choline; however it remains also dependent on liver and dietary choline which are probably transported into the brain as free choline.     

Incorporation of [3H]palmitate and [14C]choline into disaturated phosphatidylcholines in rat alveolar macrophages

We studied the synthesis of disaturated phosphatidylcholines in rat alveolar macrophages and, in some cases, compared it with that which occurs in isolated alveolar type II cells. Alveolar macrophages suspended in phosphate-buffered medium incorporate palmitate, choline and glycerol into disaturated phosphatidylcholines. The time-course for incorporation of palmitate into disaturated phosphatidylcholines is linear for 20-30 min and reaches a maximum in 2-3 h. Incorporation is dependent on extracellular palmitate with a Vmax (at 1 mM) of 1.53 nmol palmitate incorporated into disaturated phosphatidylcholines per 5 X 10(5) cells per 2 h and a K 1/2 of 0.19 mM palmitate. Exposure of the cells to zymosan particles increases incorporation of palmitate disaturated phosphatidylcholines by almost 2-fold, while cholinergic and beta-adrenergic agonists have no effect. On a per cell basis, alveolar macrophages incorporate only one-third to one-half as much palmitate into disaturated phosphatidylcholines as do type II cells isolated by centrifugal elutriation. The following results suggest there is extensive remodeling of disaturated phosphatidylcholines in alveolar macrophages: (1) palmitate- and choline-labeled disaturated phosphatidylcholines are catabolized by the cells; (2) the products of catabolism are palmitate and water-soluble choline products; (3) addition of unlabeled palmitate and choline to the medium enhances catabolism of the labeled phospholipid. Addition of oleate also enhances catabolism, suggesting that modification of phospholipids is not specific for the saturated variety. Some of the recently labeled disaturated phosphatidylcholines is released from alveolar macrophages into the extracellular space. Several possible functions of alveolar macrophage disaturated phosphatidylcholines are discussed.     

Incorporation of [14C]-glutamate into proteins and chlorophylls inDunaliella tertiolecta,a marine chlorophyte

Incorporation of [¹⁴C]-glutamate into soluble proteins accounted for about 14% of the total glutamate uptake, while < 1% of the ammo acid carbon was incorporated into chlorophylla. Most of the remaining 85% of the glutamate was retained in low molecular mass pools. High uptake rates appeared to be related to a “shift-up” phenomenon, resulting from the addition of fresh media. Apparent turnover times, estimated from reverse isotope dilution experiments, varied from about 1 to 3.5 h for the soluble proteins and about 3 to 5 h for chlorophyll a. Rapid changes in glutamate to glutamine ratios, resulting from changes in irradiance, suggest that the intermediary metabolism of glutamate is regulated by irradiance.     

Incorporation of dl-[2-14C]ornithine and dl-[5-14C]arginine in milk constituents by the isolated lactating sheep udder

1. Lactating mammary glands of sheep were perfused for several hours in the presence of dl-[2-(14)C]ornithine or dl-[5-(14)C]arginine and received adequate quantities of acetate, glucose and amino acids. 2. In the [(14)C]ornithine experiment 1.4% of the casein and 1% of the expired carbon dioxide came from added ornithine; 96% of the total radioactivity in casein was recovered in proline; 13% of the proline of casein originated from plasma ornithine. 3. In this experiment the results of chemical degradation of proline of casein as well as relative specific activities in the isolated products are consistent with the view that ornithine is metabolized, by way of glutamic gamma-semialdehyde, to proline or glutamic acid. 4. In the [(14)C]arginine experiments 3% of the casein and 1% of the expired carbon dioxide came from arginine; 84% of the arginine and 9% of the proline of casein originated from plasma arginine. 5. In these experiments the relative specific activities of arginine, ornithine and proline in plasma are in agreement with the view that arginine is metabolized by way of ornithine to proline. The conversion of arginine into ornithine is probably catalysed by arginase, so that arginase in mammary tissue may be involved in the process of milk synthesis.     

Incorporation of [2-14C]mevalonic acid into phytoene by isolated chloroplasts

1. Chloroplasts prepared by the non-aqueous technique will, after fragmentation by ultrasonic treatment, incorporate [2-(14)C]mevalonic acid into phytoene, the first C(40) compound formed in the biosynthetic sequence to coloured carotenoids. 2. With suspensions containing 3.5mg. of chlorophyll, the optimum amounts of cofactor required were ATP (10mumoles), magnesium chloride (20mumoles) and glutathione (20mumoles); neither NAD(+) nor NADP(+) was required. 3. Very small amounts of squalene are also formed and synthesis is stimulated by addition of NADH or NADPH. Phytoene synthesis was not affected by the presence of these cofactors and no lycopersene (the C(40) homologue of squalene) was detected. 4. The phytol side chain of chlorophyll is also labelled under these conditions. 5. Preparations of developing chloroplasts are more active than preparations of mature chloroplasts.