14C] Alanine incorporation into proteins and their contents in the tissue of intact and adrenalectomized rabbits after hydrocortisone and cortitropin administration

A single administration of hydrocortisone to intact rabbits increases the incorporation of [14C] alanine into proteins of the brain and liver tissue homogenates and soluble fractions as well as in blood plasma proteins and reduces the label incorporation into the brain tissue proteins and reduces its incorporation into the blood plasma proteins. Adrenalcetomy is followed by an increase in the incorporation of [14C] alanine into proteins of the brain and muscle tissue homogenates and soluble fraction and into proteins of blood plasma and liver tissue homogenates as well as by reducing the label incorporation into the spleen soluble fraction proteins. ACTH administered to adrenalectomized rabbits reduces incorporation of [14C] alanine into the brain and muscle tissue proteins, total proteins of liver tissue homogenate and increases it into the proteins of the spleen tissue soluble fraction. Multiple administration of the soluble fraction hormones both to intact and adrenalectomized rabbits inhibits the label incorporation into the studied tissue proteins. Parallel with the change in [14C] alanine incorporation into proteins under the hormones effect certain shifts in their contents were also established.     

In vitro synthesis of myelin proteolipid protein from rat brain using a total homogenate system

In vitro synthesis of myelin proteolipid protein (PLP) was explored at different ages using rat brain total homogenates, incubated for 30 min with [³H]glycine. Total proteolipids, extracted from the incubated samples, were separated by SDSPAGE and the radioactivity was measured in the band corresponding to myelin PLP. The incorporation into PLP in relation to the incorporation into brain total proteins increased from 0.04% at 10 days of age to 0.63% at 20 days, and declined slowly thereafter. Time course experiments were carried out using brain homogenates obtained from rats of 20 days of age (i.e. at the period of maximal synthesis of PLP). Labeled PLP molecules were already found at 2.5 min of incubation and the incorporation of the label into this protein, relative to the incorporation into total proteins, did not vary throughout the entire incubation time (30 min). Pulsechase experiments using a similar system and adding cycloheximide at different incubation times showed that the appearance of label into mature PLP was immediately blocked by the inhibitor of protein synthesis. These data suggest that PLP is synthesized as such and not as a pre-protein which is subsequently processed to render mature PLP.     

Cooperative activity of phospholipid-N-methyltransferases localized in different membranes

The possibility that the phospholipid-N-methyltransferases from yeast are capable of acting upon a phospholipid substrate, localized in a different membrane than in which the enzymes reside ('trans-catalysis' hypothesis), was investigated using cho2 and opi3 gene disruptant strains, which are defective in phosphatidylethanolamine transferase (PEMT) and phospholipid methyltransferase (PLMT), respectively. When cell homogenates or microsomes of the two disruptant strains are mixed, the combined methyltransferase activity, measured as the incorporation of [(3)H]methyl label from S-adenosyl methionine, exceeds that expected based on the separate activities of PEMT and PLMT. The increased incorporation implies that monomethylphosphatidylethanolamine generated by PEMT becomes available for PLMT, as evidenced by increased synthesis of dimethylphosphatidylethanolamine and phosphatidylcholine. The kinetics of the cooperativity suggest a collision-based process, enabling either transport of substrate or 'trans-catalysis'.     

Biosynthesis of retinal phospholipids: incorporation of radioactivity from labeled phosphorylcholine and cytidine diphosphate choline

Phosphorylcholine-1,2-(14)C and choline-1,2-(14)C-labeled cytidine diphosphate choline are incorporated into lecithin by whole homogenates and particulate fractions of rat retina with optimal incorporation of label by the microsomal fraction. The soluble fraction contains a factor(s) which stimulates incorporation of label with release of inorganic phosphate. Mg(++) is required for optimal incorporation of intermediates into lecithin in the presence of added diglycerides; without added diglycerides, incorporation of phosphorylcholine or cytidine diphosphate choline was moderately stimulated by preincubating the system in the absence of Mg(++) with added phosphatidic acid and by adding this mixture to fresh enzyme and the complete incubation mixture (including Mg(++)). The results show that the retina is capable of de novo synthesis of phosphatides and suggest that the rod outer segments depend on the pigment epithelium and(or) the inner rod segments for a source of phospholipids. Coenzyme A and ATP added to whole homogenate of retina did not significantly increase the incorporation of CDP-choline-1,2-(14)C into lecithin but slightly increased the radioactivity found in lysolecithin and sphingomyelin. Rats with hereditary retinitis pigmentosa have an abnormally high lipid phosphorus content of the retina, but they do not incorporate labeled CDP-choline into lecithin of retina at a higher rate than do normal animals.     

Catecholamine-induced phosphorylation of cardiac muscle proteins

The catecholamine-induced phosphorylation of cardiac muscle protein was investigated using a rat ventricular muscle slice preparation. Slices 0.5 mm thick and weighing 40–50 mg were incubated for 40 min in oxygenated bathing medium containing ³²P to partially label intracellular ATP. Subsequent addition of 10^?5 M isoproterenol for 10 min resulted in a 44–63% (based on protein) or a 63–70% (based on inorganic phosphate) increase in ³²P incorporation into 100 000 × g particulate and 100 000 × g supernatant (soluble) fractions without an increase into homogenates, 1000 and 29 000 × g particulate fractions prepared from the slices. The catecholamines also produced a 93% increase in ³²P incorporation ans a 27% increase in inorganic phosphate in trichloroacetic acid-insoluble protein that was obtained from ventricular slice homogenates. A significant increase in the incorporation of ³²P occurred in the 100 000 × g particulate and supernatant fractions and the acid-insoluble protein within 2 and 1 min, respectively. While the β-adrenergic blocking agent propanolol had no effect by itself on ³²P incorporation, it prevented the isoproterenol-induced incorporation of ³²P into the 100 000 × g particulate and supernatant fractions and the acid-insoluble protein. Removal of isoproterenol from the bathing medium eliminated the differences in ³²P incorporation, indicating that the effects of the catecholamine were reversible. Norepinephrine and ipinephrine at 10^?5 M caused phosphorylation effects similar to that of isoproterenol. When the slices were bathed under anoxic conditions isoproterenol failed to enhance the incorporation of ³²P into proteins of the 100 000 ×g particulate and supernatant fractions or acid-insoluble protein. SDS gel eloectrophoresis of ventricular slice homogenates revealed that isoproterenol enhanced the ³²P incorporation into several myocardial proteins having molecular weights of 155, 94 (glycogen phosphorylase), 79, 68–77, and 54–59 · 10³ and decreased the incorporation into a 30 · 10³ dalton protein(s). These results are consistent with the notion that catecholamines may increase the phosphorylation of myocardial proteins in the intact myocardium which in turn may play a role in catecholamine-induced glycogenolysis and augmentation of contractility.     

Effect of formate on 14C incorporation from various precursors into rat liver proteins and lipids

Experiments on the rat liver homogenates and slices show that formate stimulates carbon incorporation from [1-14C] lysine and [2-14C] acetate into proteins and from [2-14C] acetate into lipids. The stimulating effect depends on both the formate concentration and nature of the labelled precursor. The in vitro experiments demonstrate the highest stimulating effect on the metabolism of both proteins and lipids under administration of 2 microM formate per 100 g of animal mass. Determination of the label incorporation rate at different time after formate administration showed that the latter evokes an intensified synthesis of protein with rate of its decay remaining the same.     

Inhibition of fatty acid synthesis by RMI 14,514 (5-tetradecyloxy-2-furoic acid)

RMI 14,514 strongly inhibited the incorporation of label from [1-¹⁴C]acetyl-CoA into fatty acids by rat liver homogenates. No inhibition was observed when [2-¹⁴C]malonyl-CoA was used as the labeled fatty acid precursor. These results suggest that the drug inhibits $\text{de novo}$ fatty acid biosynthesis at the step mediated by acetyl-CoA carboxylase. The data presented in this communication support earlier reports that RMI 14,514 probablyexerts its hypolipidemic effects by inhibition of fatty acid biosynthesis.     

Effects of dolichol monophosphate on galactose incorporation into glycoconjugates of cell cultures.

Cultured-cell homogenates catalysed the incorporation of galactose from UDP-galactose into protein and sphingolipid acceptors. Dolichol monophosphate stimulated the incorporation of galactose into glycoproteins, but it did not affect the rate of glycosylation of either exogenous or endogenous glycosphingolipids. It is proposed that, under certain conditions, galactose may be incorporated into glycoproteins via polyisoprenol intermediates, as is the case with N-acetylglucosamine and mannose.     

Incorporation of 35S-sulfate into yolk platelets of Xenopus laevis embryos. A study using electron microscope autoradiography.

Inorganic 35S-sulfate was injected into Xenopus laevis embryos before first cleavage to study incorporation of the label into the yolk platelets in order to localize glycosaminoglycan synthesis. Electron microscope autoradiography of embryonic thin sections from blastulae and gastrulae revealed that the primary site of label incorporation is at the edge of the yolk platelets, and, to a lesser extent, in their interiors. Autoradiography of isolated yolk platelets, lacking unit membranes, indicated the absence of label. Thus, edge associated label comes from the yolk platelets membrane, and interior label is solubilized in the glycerol-water gradient during yolk platelets isolation. Ruthenium red staining of yolk platelet in situ shows haavy deposits of the dye on the yolk platelet membrane surface facing the cytoplasmic surface. The crystalline main body of isolated yolk platelets does not take up the dye. It appears that continuous synthesis or sulfation of glycosaminoglycan occurs primarily at the outer surface yolk platelet membranes during early development, providing a novel site for this process.     

Incorporation of 35S-Sulfate into Yolk Platelets of Xenopus laevis Embryos

Inorganic ³⁵S-sulfate was injected into Xenopus laevis embryos before first cleavage to study incorporation of the label into the yolk platelets in order to localize glycosaminoglycan synthesis. Electron microscope autoradiography of embryonic thin sections from blastulae and gastrulae revealed that the primary site of label incorporation is at the edge of the yolk platelets, and, to a lesser extent, in their interiors. Autoradiography of isolated yolk platelets, lacking unit membranes, indicated the absence of label. Thus, edge associated label comes from the yolk platelets membrane, and interior label is solubilized in the glycerol-water gradient during yolk platelets isolation.
Ruthenium red staining of yolk platelet in situ shows heavy deposits of the dye on the yolk platelet membrane surface facing the cytoplasmic surface. The crystalline main body of isolated yolk platelets does not take up the dye.
It appears that continuous synthesis or sulfation of glycosaminoglycan occurs primarily at the outer surface yolk platelet membranes during early development, providing a novel site for this process.     

Investigation of human lymphocyte plasma membrane associated nucleic acid.

Plasma membranes were prepared from the human lymphocyte cell line WIL23A by hypotonic swelling, Dounce homogenization, differential and equilibrium centrifugation. The resulting vesiculated membrane fragments were found to have densities of 1.10 and 1.17 g/ml, and were defined by lactoperoxidase mediated whole cell iodination, L-[3H] fucose incorporation, 5'-nucleotidase activity (EC 3.1.3.5) and electron micrographic visualization. Recovery of plasma membrane from whole cell homogenates was estimated to be approximately 30-35% as judged by the recovery of 125I-labeled cell surface protein. When plasma membranes were prepared from cells which had been incubated for 18 h in the presence of 0.5 muCi/ml [3H] thymidine such that greater than 10(9) acid insoluble counts could be demonstrated in the whole cell homogenates, no [3H] thymidine label and presumably, therefore, no DNA, could be shown to be coincident with either the 1.10 or 1.17 density. Similar experiments with [3H] uridine suggested that 90% of the plasma membranes did not contain RNA, while 10% remained questionable.     

Acetoacetate is a cholesterogenic precursor for myelinating rat brain and spinal cord. Incorporation of label from [3-14C]acetoacetate, [14C]glucose and 3H2O

Rat pups, 3 weeks old, were injected i.p. with combinations of 3H2O and either [3-14C]acetoacetate or [14C]glucose. 3H/14C incorporation ratios were measured in lipid fractions of homogenates and myelin prepared from whole brain and spinal cord. Spinal cord synthesized at least twice as much fatty acids and 3-fold more sterols than whole brain. Both tissues used acetoacetate preferentially for sterol synthesis, whereas label from [14C]glucose was distributed between fatty acids and sterols in the same way as 3H from 3H2O. The relative contributions of acetoacetate to sterol synthesis in whole tissue and in the purified myelin fraction were about the same, both for the cerebrum and for the spinal cord.     

Lipid metabolism in Aspergillus niger under conditions of heat shock

The processes of lipid synthesis and decomposition in Aspergillus niger under conditions of heat shock (HS) were studied in a pulse-chase experiment with ¹⁴C-labeled sodium acetate. HS (60 min) resulted in the synthesis of phospholipids and sphingolipids intensified compared to the control, as was evident from incorporation of the labeled substrate. The same pattern was observed for neutral lipids, especially for triacylglycerides, while incorporation of the label into sterols remained almost the same. Further cultivation for 3 h in the medium without the labeled substrate resulted in a significant decrease of the label content in the membrane lipids of both the control and the experiment, although under HS conditions this decrease was much more pronounced, especially for phosphatidylcholines and phosphatidylethanolamines. A threefold increase of the label content in phosphatidic acids was observed only under HS conditions. These results indicate more intense metabolism of the membrane lipids under heat shock and suggest the degradation of the major cell phospholipids as the factor responsible for the increased level of phosphatidic acids in A. niger mycelium.     

Trypsin is the primary mechanism by which the (18)O isotopic label is lost in quantitative proteomic studies

Labeling with (18)O is currently one of the most commonly used methods for incorporating a stable isotopic label into samples for comparative proteomic studies. In this approach, isotopic labeling involves the enzymatic digestion, typically performed with trypsin, of a protein population in (18)O-water, which incorporates the stable isotope into the C termini of the newly formed peptides. Although trypsin is often used to facilitate isotopic incorporation after digestion, it is typically overlooked that this same mechanism can lead to isotopic loss even under conditions such as low pH where it is assumed that trypsin is inactive. To examine the role that trypsin plays in isotopic loss, several experiments were performed on the rate of delabeling under conditions relevant to multidimensional proteomic experiments. Results from these studies demonstrate that enzyme-facilitated exchange of (18)O in the peptide with (16)O in the aqueous solvent was the major process by which the label is removed from the peptides, even under conditions of low pH and temperature where trypsin is thought to be inactive. This study brings the rapid, tryptic-facilitated exchange to the attention of laboratories using this scheme to prevent inaccuracies in quantitative labeling due to loss of the isotopic label.     

Decrease in ADP-ribosylation of HeLa non-histone proteins from interphase to metaphase

Variations for non-histones in the ADP-ribosylating activities of interphase and metaphase cells were investigated. 32P-Labeled nicotinamide adenine dinucleotide ([32P]NAD), the specific precursor for the modification, was used to radioactively label proteins. Permeabilized interphase and mitotic cells, as well as isolated nuclei and chromosomes, were incubated with the label. One-dimensional and two-dimensional gels of the proteins of total nuclei and chromatin labeled with [32P]NAD showed more than 100 modified species. Changing the labeling conditions resulted in generally similar patterns of modified proteins, though the overall levels of incorporation and the distributions of label among species were significantly affected. A less complex pattern was found for nuclear scaffolds. The major ADP-ribosylated proteins included the lamins and poly(ADP-ribose) polymerase. Inhibitors of ADP-ribosylation were effective in preventing the incorporation of label by most non-histones. Snake venom phosphodiesterase readily removed protein-bound 32P radioactivity. A fundamentally different distribution of label from that of interphase nuclei and chromatin was found for metaphase chromosome non-histones. Instead of 100 or more species, the only major acceptor of label was poly(ADP-ribose) polymerase. This profound change during mitosis may indicate a structural role for ADP-ribosylation of non-histone proteins.     

The mechanism of hepatic iron uptake from native and denatured transferrin and its subcellular metabolism in the liver cell.

Hepatic iron uptake and metabolism were studied by subcellular fractionation of rat liver homogenates after injection of rats with a purified preparation of either native or denatured rat transferrin labelled with 125I and 59Fe. (1) With native transferrin, hepatic 125I content was maximal 5 min after injection and then fell. Hepatic 59Fe content reached maximum by 16 h after injection and remained constant for 14 days. Neither label appeared in the mitochondrial or lysosomal fractions. 59Fe appeared first in the supernatant and, with time, was detectable as ferritin in fractions sedimented with increasingly lower g forces. (2) With denatured transferrin, hepatic content of both 125I and 59Fe reached maximum by 30 min. Both appeared initially in the lysosomal fraction. With time, they passed into the supernatant and 59Fe became incorporated into ferritin. The study suggests that hepatic iron uptake from native transferrin does not involve endocytosis. However, endocytosis of denatured transferrin does occur. After the uptake process, iron is gradually incorporated into ferritin molecules, which subsequently polymerize; there is no incorporation into other structures over 14 days.     

Ketone-body utilization and lipid synthesis by developing rat brain—a comparison between in vivo and in vitro experiments

The distribution of ketone bodies between oxidation and lipid synthesis was analysed in homogenates of developing rat brain. The capacity for lipid synthesis of homogenized or minced brain preparations was compared with rates of lipid synthesis in vivo, assessed by incorporation of ³H from ³H₂O into fatty acids and cholesterol. Brain homogenates of suckling rats (but not those of adults) incorporated label from [3-¹⁴C]ketone bodies into lipids, but this process was slow as compared to ¹⁴CO₂ production (< 5%) and much slower than the total rate of ketone-body utilization (< 0.5%). Study of ³H₂O incorporation demonstrated that the rates of lipogenesis and cholesterogenesis are at least one order of magnitude higher in vivo than in vitro. Maximal rates of ³H incorporation into fatty acids (3 μmol/g brain . h) and into cholesterol (0.6 μmol/g brain . h) were found during the third postnatal week. Adult rats still incorporated ³H into brain fatty acids at an appreciable rate (1 μmol/g brain . h), whereas cholesterogenesis was very low. It is concluded that in vitro measurements of lipid synthesis severely underestimate the rates that occur in developing rat brain in vivo. The high rate of ³H incorporation into lipids by developing and adult rat brain as compared to the amounts of these lipids present in the brain suggests an important contribution of endogenous lipid synthesis during brain development and an appreciable rate of fatty acid turnover during brain growth, but also in the adult brain.     

14C-Metabolism in cultured cotyledon explants of radiata pine

Excised cotyledons of radiata pine ( Pinus radiata D. Don), cultured under shootforming (plus cytokinin) and elongating (minus cytokinin) conditions, were incubated in ¹⁴C-glucose, ¹⁴C-acetate or ¹⁴C-bicarbonate at different stages of growth and differentiation. ¹⁴CO₂ was produced when the cotyledons were fed ¹⁴C-glucose and ¹⁴C-acetate (no measurement was made for ¹⁴C-bicarbonate feeding). Label from these precursors was incorporated into ethanol-soluble and -insoluble fractions. The largest percentage of radioactivity was associated with the ethanol-soluble portion, which was further fractionated into lipids, amino acids, organic acids and sugars. The amount of label and the pattern of labelling associated with each of the above classes of metabolites varied with time in culture and morphogenetic behaviour of the cotyledons. In general, there was a tendency towards a high rate of incorporation of label in elongating cotyledons during the period of rapid elongation. On the other hand, a high rate of incorporation of label in shoot-forming cotyledons coincided with the period of meristematic tissue formation. The data obtained support the hypothesis that organized development in vitro involves a shift in metabolism, which precedes and is coincident with the initiation of the process.     

Stimulation of adenyl cyclase in pupal wing epidermis by -ecdysone

The injection of β-ecdysone into chilled Hyalophora gloveri pupae resulted in the stimulation of adenyl cyclase activity in the wing epidermis as measured by the incorporation of label into cyclic AMP from a prelabeled endogenous pool. Stimulation was also obtained in pupal wings in vitro and in wing epidermal homogenates. Although the sequence of responses to β-ecdysone in vitro depended on the composition of the incubation medium, the stimulation of cyclic AMP synthesis always preceded increases in the rates of RNA and protein synthesis. The increase in adenyl cyclase activity is the earliest metabolic event thus far discerned as a result of β-ecdysone action. It is suggested that β-ecdysone stimulates adenyl cyclase (and guanyl cyclase) but that the hormone also exerts effects on target cells independent of the cyclic AMP system.