The role of cholesteryl 14-methylhexadecanoate in peptide elongation reactions

1. Peptide-elongation factors were purified from rat liver and human tonsils and the contents of cholesteryl 14-methylhexadecanoate were determined in fractions obtained during enzyme purification. The relative contents of this compound in purified enzyme preparations was several times higher than that in the crude starting material. Elongation factors from human tonsils contained a significantly larger quantity of the cholesteryl ester than enzyme from rat liver. 2. Transfer enzymes extracted with various organic solvents showed variable decreased activities in both binding and peptidization assay. The decrease of enzymic activity was proportional to the amount of cholesteryl 14-methylhexadecanoate extracted from a given enzymic preparation. In systems containing both extracted elongation factors the polyphenylalanine synthesis was limited by the residual activity of the less active transfer factor. 3. The original enzymic activity of extracted transferases was fully recovered by the addition of pure cholesteryl 14-methylhexadecanoate in quantities corresponding to those extracted. 4. Increase of the relative contents of this cholesteryl ester during enzyme purification, decrease of the enzymic activity after the extraction and its recovery by the addition of this compound indicates that the presence of this ester in elongation factors is essential for the normal function of these enzymes.     

Comparison of cell-free protein synthesis by different regions of chicken brain

The cell-free protein synthesis by the postmitochondrial supernatant from chicken cerebrum was twofold greater than protein synthesis by the cerebellum or optic lobes. Ribosomal aggregation of mRNA and ribonuclease activity of the postmitochondrial supernatant from the three brain regions was not statistically different. The higher protein synthetic activity of the cerebral postmitochondrial supernatant was associated with both the postribosomal supernatant (cell sap) and microsomal fractions. Cerebral monomeric ribosomes were more active in polyuridylic acid directed polyphenylalanine synthesis than monomeric ribosomes from either the cerebellum or optic lobes. The ability of cerebral cell sap to support polyuridylic acid directed polyphenylalanine synthesis was 1.6 to 2 times greater than cell sap from the other two regions. Cell sap factors other than tRNA^phe or phenylalanyl-tRNA synthetases appear to be responsible for the higher protein synthetic activity of the cbr cell sap.     

The role of multiply phosphorylated S6 in ribosome degradation

Rat liver ribosomes, prepared 1–24 h after intraperitoneal cortisol injection, contain multiple phosphorylated S6 consisting of four distinct derivatives in addition to the original non-phosphorylated S6. 25 h following the hormone injection the extent of S6 phosphorylation, as judged by its electrophoretic pattern in two-dimensional gels, resembles that of untreated rats. Ribosomal subunits with gradually increased degree of S6 phosphorylation, isolated at different time intervals after cortisol injection, exhibit polyphenylalanine polymerization levels inversely proportional to the extent of S6 phosphorylation. In addition, they show an elevated misincorporation of leucine in a poly(U)-programmed in vitro system. The lower amount of polyphenylalanine synthesized by multiple phosphorylated ribosomes in vitro is likely due to an enhanced susceptibility of nascent polypeptide chains synthesized in the in vitro system to proteinases present in the pH 5 and S-100 fractions. Liver polysomes derived from cortisol-treated animals lose their highly phosphorylated derivatives when exposed to S-100 enzymes. The loss can be prevented by concomitant action of proteinase and RNAase inhibitors (phenylmethylsulfonyl fluoride and heparin) but not by an inhibitor of phosphatase (sodium fluoride). In the absence of RNAase and proteinase inhibitors only degradation of old 40 S subunits can be demonstrated. 25 h after the cortisol treatment degradation of liver ribosomes occurs simultaneously with S6 dephosphorylation and is preceded by polysomal breakdown.     

Effect of cholesteryl 14-methylhexadecanoate on the activity of some amino acid-transfer ribonucleic acid ligases from mammalian tissues

1. l-Tyrosine-, l-alanine-, l-tryptophan- and l-threonine-tRNA ligases (where tRNA is transfer RNA) were purified from mammalian tissues and the relative contents of cholesteryl 14-methylhexadecanoate were determined in fractions obtained during the isolation. Purified enzymes were extracted with various organic solvents. 2. Cholesteryl 14-methylhexadecanoate contents in purified ligases were up to 210-fold that in the starting material. Different enzymes showed different contents of this cholesteryl ester. 3. Extracted enzymes lost in most cases their ability to catalyse formation of the aminoacylhydroxamate and aminoacyl-tRNA complexes. Enzymes extracted with various solvents showed a variable decreased activity. 4. The original activity could be restored to 70-100% by the addition of cholesteryl 14-methylhexadecanoate. Cholesteryl palmitate, cholesteryl margarate and cholesteryl stearate were inactive in this respect. 5. Incubation mixtures of extracted enzymes with cholesteryl 14-methylhexadecanoate added showed an initial delay in the time-course of both reactions assayed. 6. It is concluded that the effect of cholesteryl 14-methylhexadecanoate on the activity of amino acid-tRNA ligases seems to be specific and that this compound may play some role in the function of these enzymes.     

Intermediate reactions in the binding of aminoacyl-transfer ribonucleic acid to rat liver ribosomes. The interaction of cholesteryl 14-methylhexadecanoate

1. Transferase I from rat liver extracted with iso-octane binds significantly less aminoacyl-tRNA than the non-extracted enzyme. The original activity can be fully restored by the addition of cholesteryl 14-methylhexadecanoate. The binding capacity for GTP is not affected by the extraction. 2. In the presence of extracted transferase I the binding of aminoacyl-tRNA to ribosomes is decreased to 11-26% and the simultaneous binding of GTP to 32-43%. Cholesteryl 14-methylhexadecanoate induces a full reactivation of the extracted enzyme in both respects. 3. Extracted complexes A (aminoacyl-tRNA-GTP-transferase I) become bound to ribosomes to the same extent as the corresponding non-extracted preparations. 4. It is concluded that cholesteryl 14-methylhexadecanoate interacts with the binding site of transferase I for aminoacyl-tRNA and secondarily with that for GTP. It does not affect the binding site for ribosomes.     

Inhibition of protein synthesis by ricin: experiments with rat liver mitochondria and nuclei and with ribosomes from Escherichia coli (Short Communication)

1. Ricin, a toxic protein from the seeds of Ricinus communis which inhibits poly(U)-directed polyphenylalanine synthesis by rat liver ribosomes (Montanaro et al., 1973), does not affect protein synthesis by isolated rat liver mitochondria. 2. The toxin is ineffective also on poly(U)-directed polyphenylalanine synthesis in reconstituted systems with ribosomes isolated from rat liver mitochondria or from Escherichia coli. 3. Ricin inhibits protein synthesis by isolated rat liver nuclei, but at concentrations much higher than those affecting rat liver ribosomes.     

The preferential uptake of very-low-density lipoprotein cholesteryl ester by rat liver in vivo.

The removal from the blood and the uptake by the liver of injected very-low-density lipoprotein (VLDL) preparations that had been radiolabelled in their apoprotein and cholesteryl ester moieties was studied in lactating rats. Radiolabelled cholesteryl ester was removed from the blood and taken up by the liver more rapidly than sucrose-radiolabelled apoprotein. Near-maximum cholesteryl ester uptake by the liver occurred within 5 min of the injection of the VLDL. At this time, apoprotein B uptake by the liver was only about 25% of the maximum. Maximum uptake of the injected VLDL apoprotein B label was not achieved until at least 15 min after injection, by which time the total uptakes of cholesteryl ester and apoprotein B label were very similar. The results suggest that preferential uptake of the lipoprotein cholesteryl ester by the liver occurred before endocytosis of the entire lipoprotein complex. The fate of the injected VLDL cholesteryl ester after its uptake by the liver was also monitored. Radiolabel associated with the hepatic cholesteryl ester fraction fell steadily from its early maximum level, the rate of fall being faster and more extensive when the fatty acid, rather than the cholesterol, moiety of the ester was labelled. By 30 min after the injection of VLDL containing [3H]cholesteryl ester, over one-third of the injected label was already present as [3H]cholesterol in the liver. When VLDL containing cholesteryl [14C]oleate was injected, a substantial proportion (about 25%) of the injected radiolabelled fatty acid appeared in the hepatic triacylglycerol fraction within 60 min: very little was present in the plasma triacylglycerol fraction at this time.     

The role of cholesteryl 14-methylhexadecanoate in the function of eukaryotic peptide elongation factor 1

The binding of [3H]cholesteryl 14-methylhexadecanoate by a highly purified peptide elongation factor 1 from rabbit reticulocytes is significantly enhanced by GTP and CTP, much less by guanosine 5'-[beta, gamma-methylene]-triphosphate and not at all by ATP or UTP. Removal of endogenous cholesteryl 14-methylhexadecanoate present in the molecule of the factor [Hradec, J. et al. (1971) Biochem. J. 123, 959-966] by digestion with immobilized cholesterol esterase resulted in an almost complete loss of GTPase activity and this could be restored to nearly normal values by the addition of the ester. The same holds true for the GTP-dependent autophosphorylation of the protein-synthesis factor. Cholesteryl 14-methylhexadecanoate was bound only by the beta subunit of the factor. Addition of the alpha subunit, which was inactive on its own, stimulated the binding of the ester to the beta subunit in a sigmoid dependence. The binding of the ester was significantly stimulated by aminoacyl-tRNA but this effect was fully abolished by sodium fluoride, indicating a relation of cholesteryl 14-methylhexadecanoate to the dephosphorylation of the peptide elongation factor. Treatment of the factor with cholesterol esterase decreased its activity in the poly(U)-dependent binding of phenylalanyl-tRNA to ribosome and this activity was again restored by the addition of cholesteryl 14-methylhexadecanoate. The ester thus interacts with the GTP-dependent autophosphorylation of peptide elongation factor 1 and in this way modulates the activity of the factor. A putative scheme is presented explaining the mode of action of cholesteryl 14-methylhexadecanoate.     

THE METABOLISM OF CHYLOMICRON CHOLESTERYL ESTER IN RAT LIVER : A Combined Radioautographic-Electron Microscopic and Biochemical Study

Chylomicrons containing labeled cholesterol, mainly (70%) present as cholesteryl ester, were injected intravenously into intact rats, and samples of liver were obtained 27–210 min later. Most (58–75%) of the injected label was recovered in the liver after 27–75 min. Hepatic uptake occurred without hydrolysis of the labeled cholesteryl ester. In separate experiments, in vitro perfusion of livers of similarly treated rats for 30–35 min washed out only 3–9% of the labeled sterol. Samples of liver and small intestine were prepared for electron microscopy with Aquon as the dehydrating agent. Good retention (70% or more) of labeled cholesterol and satisfactory preservation of ultrastructure were obtained. After 30 min, the radioautographic reaction was localized mainly over the region of the cell boundary of the parenchymal liver cells, with fewer grains being present over intracellular organelles. At later time intervals, when considerable hydrolysis of the labeled cholesteryl ester had occurred, the radioautographic reaction was more evenly distributed. Phagocytosed labeled lipid was seen in Kupffer cells after the larger lipid load; phagocytosis by parenchymal cells was not seen. In other experiments, cholesteryl ester hydrolase activity was found in all subcellular fractions, the microsome and plasma membrane fractions showing the highest activity per mg protein. The mechanism of cholesteryl ester transport into the liver cell may involve: (1) hydrolysis at the cell surface; or (2) slow entry of intact molecules followed by intracellular hydrolysis of the ester bond.     

Hepatic processing of the cholesteryl ester from low density lipoprotein in the rat

Human low density lipoprotein (LDL), radiolabeled in the cholesteryl ester moiety, was injected into estrogen-treated and -untreated rats. The hepatic and extrahepatic distribution and biliary secretion of [3H]cholesteryl esters were determined at various times after injection. In order to follow the intrahepatic metabolism of the cholesteryl esters of LDL in vivo, the liver was subfractioned into parenchymal and Kupffer cells by a low temperature cell isolation procedure. In control rats, the LDL cholesteryl esters were mainly taken up by the Kupffer cells. After uptake, the [3H]cholesteryl esters are rapidly hydrolyzed, followed by release of [3H]cholesterol from the cells to other sites in the body. Up to 24 h after injection of LDL, only 9% of the radioactivity appeared in the bile, whereas after 72 h, this value was 30%. Hepatic and especially the parenchymal cell uptake of [3H]cholesteryl esters from LDL was strongly increased upon 17 alpha-ethinylestradiol treatment (3 days, 5 mg/kg). After rapid hydrolysis of the esters, [3H]cholesterol was both secreted into bile (28% of the injected dose in the first 24 h) as well as stored inside the cells as re-esterified cholesterol ester. It is concluded that uptake of human LDL by the liver in untreated rats is not efficiently coupled to biliary secretion of cholesterol (derivatives), which might be due to the anatomical localization of the principal uptake site, the Kupffer cells. In contrast, uptake of LDL cholesterol ester by liver hepatocytes is tightly coupled to bile excretion. The Kupffer cell uptake of LDL might be necessary in order to convert LDL cholesterol (esters) into a less toxic form. This activity can be functional in animals with low receptor activity on hepatocytes, as observed in untreated rats, or after diet-induced down-regulation of hepatocyte LDL receptors in other animals.     

Influence of cholesteryl 14-methylhexadecanoate on some ribosomal functions required for peptide elongation

1. Polyribosomes and ribosomal subunits from rat liver were adsorbed on a cellulosic ion-exchange adsorbent, freeze-dried and extracted with organic solvents. The activity of extracted particles in peptide elongation was tested in the presence of purified peptideelongation factors. 2. Chloroform-methanol mixture (2:1, v/v) extracted 1.87+/-0.15 pmol of cholesteryl 14-methylhexadecanoate/pmol of the smaller ribosomal subunit and 0.92+/-0.11 pmol/pmol of the larger subunit. 3. In the presence of transferase I, extracted polyribosomes and 40S subunits bound more phenylalanyl-tRNA than did control non-extracted particles. The same binding as in control mixtures was obtained with extracted particles supplemented with cholesteryl 14-methylhexadecanoate in quantities corresponding to those extracted. 4. The polymerization of phenylalanine was greatly decreased with extracted polyribosomes and subunits and addition of the cholesteryl ester could not fully restore the original activity. 5. Extraction significantly decreased the activity of the P site of peptidyl transferase and normal activity was recovered after the addition of the ester. The A site of peptidyl transferase in extracted polyribosomes showed an increased activity when compared with non-extracted polyribosomes. 6. Cholesteryl 14-methylhexadecanoate apparently affects the function of the ribosomal A site and peptidyl transferase site and probably also that of the guanosine triphosphatase site and P site. The presence of different amounts of the ester in polyribosomes may be one of the mechanisms modulating peptide elongation at the ribosomal level.     

Mammalian mitochondrial ribosomes. Studies on the exchangeability of polypeptide chain elongation factors from bacterial and mitochondrial systems

Mammalian mitochondrial ribosomes from rat liver synthesised poly(phenylalanine) from [14C]-Phe-tRNA in the presence of a homologous 10(5) X gav supernatent fraction. The activity depended on the addition of synthetic template and was resistant to cycloheximide. The polyanion spermidine had a stimulatory effect on peptide synthesis in vitro. In contrast to Escherichia coli ribosomes, which also functioned with heterologous supernatant fractions, 55-S mitochondrial ribosomes were inactive when supplemented with heterologous supernatant fractions from E. coli or with purified bacterial elongation factors. EF-T slightly stimulated polyphenylalanine synthesis when added in combination with mitochondrial supernatant fractions. Two-dimensional electrophoretic analysis of the protein content of both supernatant fractions revealed considerable differences in the distribution of the species-specific proteins according to their isoelectric points. The mitochondrial supernatant proteins were in general more basic, and the few acidic proteins did not co-migrate with EF-Tu or EF-G from E. coli.     

Ability of six different lipoprotein fractions to regulate the rate of hepatic cholesterogenesis in vivo.

Two in vivo assay procedures were used to study the inhibitory activity of cholesterol carried in three intestinal lymph and three serum lipoprotein fractions on the rate of cholesterol synthesis in the liver. In the first preparation, different lipoproteins were injected intravenously as a bolus into rats at the mid-light phase of the diurnal light cycle, following which they were killed 12 hours later in the mid-dark phase of the cycle. Using this assay, three intestinal lymph lipoprotein fractions of varying Sf values all produced a similar degree of inhibition which averaged approximately 11%/mg of cholesterol injected. The serum lipoprotein fractions caused only about one-third this amount of inhibition. Detailed analysis of events occurring within the liver during this 12-hour assay period revealed that there were marked differences in the rate of net cholesterol uptake into the liver and in the rate of new removal of cholesterol esters from the liver following injection of each of these different lipoprotein fractions. The amount of inhibition of sterol synthesis produced by any fraction was proportional to the product of the incremental increase in hepatic cholesterol ester content and the time over which this increase in esters occurred. In the second type of assay where the lipoprotein fractions were administered to the animals as a continuous intravenous infusion over 24 hours the largest increase in hepatic cholesterol ester content and the greatest inhibition of cholesterol synthesis was found with intestinal lipoproteins having Sf values larger than 8000. Intestinal lipoprotein fractions with lower Sf values and all serum lipoprotein fractions were significantly less effective in bringing about an increase in hepatic cholesterol ester content and in producing inhibition of cholesterol synthesis by the liver. These studies emphasize the primary role of cholesterol carried in lipoproteins of intestinal origin in regulating hepatic sterol synthesis. The inhibitory activity of these fractions appears to correlate with the ability of these lipoproteins to bring about a maximal increase in hepatic cholesterol ester content which, in turn, appears to relate to the capacity of these fractions to transfer cholesterol rapidly into the hepatocyte while, at the same time, slowing the rate of cholesterol mobilization from the liver.     

Effects of injecting exogenous lipid transfer protein into rats

Rats were injected intravenously with preparations of partially purified lipid transfer protein isolated from human plasma. Cholesteryl ester transfer activity disappeared from the plasma of recipient rats with a t1/2 of about 10 h and after 24 h had fallen to a level comparable to that in human plasma. By contrast there was no measurable cholesteryl ester transfer activity in the plasma of control rats. Plasma collected from rats 24 h after the injection was subjected to ultracentrifugation at 1.225 g/ml; lipoproteins in the 1.225 g/ml supernatant were subsequently separated by both gel filtration chromatography and gradient gel electrophoresis. The major change in the treated animals was a total loss of the large, cholesteryl ester-rich, apolipoprotein E-rich high-density lipoproteins, HDL1, which are prominent in the plasma of control rats. This loss of HDL1 unmasked an obvious peak of low-density lipoproteins that had been obscured in the control rats. Other changes in the treated rats included an increase in the relative cholesteryl ester content of very-low-density lipoproteins and the emergence of a peak of triacylglycerol in the high-density lipoproteins.     

Rapid transport of fatty acids from rat liver endothelial to parenchymal cells after uptake of cholesteryl ester-labeled acetylated LDL

Acetylated low-density lipoprotein (acetyl-LDL) radiolabeled in the oleate moiety of cholesteryloleate was injected into rats. Isolation of the various liver cell types at different times after acetyl-LDL injection by a low-temperature procedure allowed the intrahepatic metabolism of the oleate moiety to be followed in vivo. The cholesteryloleate radioactivity is rapidly cleared from the circulation and at 5 min after injection recovered into parenchymal and endothelial liver cells, mainly as cholesteryloleate ester. At longer time intervals after injection, the amount of cholesteryl esters associated with the endothelial cells was sharply decreased and the [14C]oleate was redistributed within the liver and mainly recovered in the parenchymal cells. The cholesteryl ester initially directly taken up by the parenchymal cells was also rapidly hydrolysed but, in contrast to the endothelial cells, the [14C]oleate remained inside the cells and was incorporated into triacylglycerols and phospholipids. The 14C radioactivity in parenchymal cells taken up between 5 and 30 min after injection of the cholesteryl [14C]oleate-labeled acetyl-LDL (transported as oleate from endothelial cells), followed a similar metabolic route as the amount which was directly associated to parenchymal cells. The data indicate that the liver and, in particular, the liver endothelial cell has the full capacity to rapidly catabolize modified lipoproteins. In this catabolism, the liver functions as an integrated organ in which fatty acids, formed from cholesteryl esters in endothelial cells, are rapidly transported to parenchymal cells, indicating the concept of metabolic cooperation between the various liver cell types.     

Metabolism of HDL-cholesteryl ester in the rat, studied with a nonhydrolyzable analog, cholesteryl linoleyl ether

Intralipid was sonicated with [3H]cholesteryl linoleyl ether (a nonhydrolyzable analog of cholesteryl linoleate) and incubated with rat HDL and d greater than 1.21 fraction of rabbit serum at a ratio of 0.012 mg triacylglycerol to 1 mg HDL protein. 25% of [3H]cholesteryl linoleyl ether was transferred to HDL. The labeled HDL was injected into donor rats and was screened for 4 h. [125I]HDL was subjected to the same protocol as the 3H-labeled HDL, including screening. The screened, labeled sera were injected into acceptor rats and the disappearance of radioactivity from the circulation was compared. The t1/2 in the circulation of [125I]HDL was about 10.5 h, while that of [3H]cholesteryl linoleyl ether-HDL was about 8 h. The liver and carcass were the major sites of uptake of [3H]cholesteryl linoleyl ether-HDL and accounted for 29-41% (liver) and 30% (carcass) of the injected label. Maximal recovery of [3H]cholesteryl linoleyl ether in the liver was seen 48 h after injection, and thereafter there was a progressive decline of radioactivity, which reached 7.8% after 28 days. The maximal recovery of [125I]HDL in the liver was about 9%. Pretreatment of the acceptor rats with estradiol for 5 days resulted in a 20% increase in the hepatic uptake of [3H]cholesteryl linoleyl ether-HDL and a 5-fold increase in adrenal uptake. The present findings indicate that in the rat the liver is the major site of uptake of HDL cholesteryl ester and that part of the HDL cholesteryl ester may be cleared from the circulation separately from the protein moiety. On the basis of our previous findings (Stein, Y., Kleinman Y, Halperin, G., and Stein, O. (1983) Biochim. Biophys. Acta 750, 300-305) the loss of the [3H]cholesteryl linoleyl ether from the liver after 14-28 days was interpreted to indicate that the labeled [3H]cholesteryl linoleyl ether had been taken up by hepatocytes.     

Transfer of esterified cholesterol from serum lipoproteins to the liver.

The fate of cholesteryl esters of the serum lipoproteins was studied in intact rats and in isolated perfused rat livers. The lipoproteins of fasting rat serum were labeled in vitro with [3H]cholesteryl oleate. Following intravenous injection, it was found that the majority of the radioactive ester was rapidly taken up by the liver where hydrolysis of the ester bond occurred. At 5 min, 58% of the injected material was recovered in the liver, 85% of which was still in the ester form, while at 30 min only 22% of the liver radioactivity was in cholesteryl esters. There was very little difference in the rate at which radioactivity was taken up from the different lipoprotein classes. Similar phenomena were observed in the perfused liver, but it was found that although the radioactive esters were being taken up, there was no change in the concentrations of free or esterified cholesterol in the perfusing medium, indicating that the lipoprotein cholesteryl ester was gaining access to the liver through an exchange of molecules. After uptake, cell fractionation experiments showed that the plasma membranes had the greatest relative amounts of radioactivity, suggesting that this is the site of exchange. Small amounts of radioactivity were recovered in the bile, demonstrating that serum lipoproteins can serve as precursors of at least some of the bile steroids.     

The effect of calcium antagonists on cholesterol metabolism in human aortal intima cells and macrophage lines]

Effects of two Ca-antagonists, verapamil and nifedipine, on the total cellular cholesterol content and accumulation, as well as on the synthesis and hydrolysis of cholesteryl esters in human aortic intimal smooth muscle cells and P388D1 cell line have been studied. Verapamil and nifedipine used at 10(-6) M and higher concentrations decreased the total cellular cholesterol content (by 25-40%) in intimal cells isolated from atherosclerotic lesions without any effect on the cholesterol content in normal intimal cells or P388D1 cells. At 2 x 10(-5) M verapamil and nifedipine prevented the accumulation of cholesterol induced by atherogenic blood serum or atherogenic low density lipoproteins in both types of cells. At 10(-5) M and higher concentrations verapamil and nifedipine inhibited (2-3-fold) cholesteryl ester synthesis in intimal cells and, used at 10(-6) M and higher doses, in P388D1 cells as well. Verapamil and nifedipine (2 x 10(-5) M) enhanced the hydrolysis of cholesteryl esters in both types of cells. The Ca-channel agonist Bay K8644 had no effect on cholesteryl ester synthesis, nor did it suppress its inhibition by Ca-antagonist. The beta-receptor blocker propranolol induced the accumulation of cholesterol in intimal cells and inhibited the synthesis and hydrolysis of cholesterol esters in these cells. The data obtained suggest that the antiatherosclerotic action of Ca-blockers is determined by their ability to reduce the cellular cholesterol content which is suggested to be the result of enhanced hydrolysis of cellular cholesteryl esters.     

The protein-synthesizing activity of ribosomes isolated from the mammary gland of lactating and pregnant guinea pigs

1. Polyribosome preparations were made from the deoxycholate-treated post-nuclear fractions obtained by the disruption of mammary glands from lactating and pregnant guinea pigs. 2. A high proportion of large polyribosomes was obtained from the glands of lactating animals whereas mainly small polyribosomes were obtained from the glands of pregnant animals. The isolated preparations incorporated [(14)C]phenylalanine into protein. The polyribosomes from the glands of pregnant animals were less active than those from the glands of lactating animals but the activity of the former was stimulated more by poly(U) than was the latter. 3. The ribosomes from mammary gland could be dissociated into subunits after incubation, under conditions necessary for protein synthesis, in the presence of puromycin. The subunits could be recombined to give a preparation that actively polymerized [(14)C]phenylalanine in the presence of poly(U). The subunits from guinea-pig mammary gland could be combined with subunits from liver of either guinea pig or rat. Hybrid ribosomes were also formed from subunits derived from glands of pregnant and lactating animals. The hybrids were as active as were the ribosomes formed by reassociation of subunits from the same tissue, suggesting that in this respect the ribosomes from pregnant animals were not defective. 4. Polyribosomes from mammary glands of lactating animals when incubated with cell sap from the same source were tested for their ability to synthesize alpha-lactalbumin. The polyribosomes were incubated in the presence of [(3)H]leucine and alpha-lactalbumin was isolated from the supernatant. The protein was finally treated with cyanogen bromide and the C-terminal and N-terminal fragments were separated and their radioactivity was determined. Both fragments were radioactive consistent with the synthesis of alpha-lactalbumin. 5. The results are discussed in relation to protein synthesis in the mammary gland after parturition.     

Effect of apolipoprotein E-free high density lipoproteins on cholesterol metabolism in cultured pig hepatocytes

We studied cholesterol synthesis from [14C]acetate, cholesterol esterification from [14C]oleate, and cellular cholesterol and cholesteryl ester levels after incubating cells with apoE-free high density lipoproteins (HDL) or low density lipoproteins (LDL). LDL suppressed synthesis by up to 60%, stimulated esterification by up to 280%, and increased cell cholesteryl ester content about 4-fold. Esterification increased within 2 h, but synthesis was not suppressed until after 6 h. ApoE-free HDL suppressed esterification by about 50% within 2 h. Cholesterol synthesis was changed very little within 6 h, unless esterification was maximally suppressed; synthesis was then stimulated about 4-fold. HDL lowered cellular unesterified cholesterol by 13-20% within 2 h and promoted the removal of newly synthesized cholesterol and cholesteryl esters. These changes were transient; by 24 h, both esterification and cellular unesterified cholesterol returned to control levels, and cholesteryl esters increased 2-3-fold. HDL core lipid was taken up selectively from 125I-labeled [3H]cholesteryl ester- and ether-labeled HDL. LDL core lipid uptake was proportional to LDL apoprotein uptake. The findings suggest that 1) the cells respond initially to HDL or LDL with changes in esterification, and 2) HDL mediates both the removal of free cholesterol from the cell and the delivery of HDL cholesteryl esters to the cell.