Secretion of β-glucosidase by Ochromonas danica

-Glucosidase released by the phytoflagellate Ochromonas danica was the result of secretion; this was adduced from the following: (1) The enzyme was released during growth, including early log phase. (2) The amount released was calculated to be much more than could be attributed to cell lysis. (3) -Glucosidase was released by cells during short term incubation in a dilute salt solution; this release was nearly linear for at least 24 h. (4) Release occurred while cell counts remained nearly constant and cells remained viable. (5) Control experiments excluded cell damage resulting from incubation and cell manipulation as a source of the exoenzyme. (6) No alkaline phosphatase was released and 5 times less phosphoglucose isomerase than glucosidase was released while the cells contained 7 times more phosphoglucose isomerase. (7) The kinetics of release of nonspecific protein and UV absorbing material was markedly different from glucosidase release. (8) Glucosidase release was temperature and energy dependent; anaerobiosis decreased enzyme release. (9) Release was inhibited by cycloheximide. (10) Cells incubated with ³H-leucine synthesized labeled protein which was secreted linearly for at least 24h. Cycloheximide inhibited incorporation of ³H-leucine into protein and the secretion of the labeled protein.Non-Standard Abbreviations CHI cycloheximide - DNP 2,4-dinitrophenol - IAA iodoacetic acid - PGI phosphoglucose isomerase - SIS salt incubation solution     

Effect of dexamethasone, indomethacin, and lipopolysaccharide on the secretion of interstitial collagenase and type V collagenase by cultured macrophages

The rabbit alveolar macrophage secretes at least two collagenolytic metalloproteinases in vitro including an interstitial collagenase and a type V collagenase. Using assays previously shown to discriminate between these two activities, the secretion of these two enzyme activities was investigated. Both enzyme activities accumulated in culture over 11 days and the release of both were similarly inhibited by cycloheximide. Collagenolytic activity was negligible in cell lysates. The interstitial collagenase was found in a latent form but the type V collagenase activity was active in the culture medium. When cultured in the presence of dexamethasone, the secretion of both the enzymes were identically inhibited in a dose-dependent manner. Indomethacin was an effective inhibitor of secretion of both collagenases at a concentration of 10(-5) M but not at lower concentrations. Finally, bacterial lipopolysaccharide stimulated the secretion of both type V and interstitial collagenase by these cells. These studies indicate that, like the interstitial collagenase, the type V collagenase is released from the cell as synthesized and is not stored intracellularly. Protein synthesis is necessary for the release of both these collagenases. Furthermore, the release of type V collagenase responded to dexamethasone, indomethacin, and lipopolysaccharide in a manner identical to the secretion of the interstitial collagenase suggesting that synthesis and secretion of these two enzymes are regulated in a similar manner.     

Probucol inactivates ABCA1 in the plasma membrane with respect to its mediation of apolipoprotein binding and high density lipoprotein assembly and to its proteolytic degradation

Probucol has been shown to inhibit the release of cellular lipid by helical apolipoprotein and thereby to reduce plasma high density lipoprotein. We attempted to explore the underlying mechanism for this effect in human fibroblast WI-38. Probucol inhibited the apoA-I-mediated cellular lipid release and binding of apoA-I to the cells in a dose-dependent manner. It did not influence cellular uptake of low density lipoprotein, transport of cholesterol to the cell surface whether de novo synthesized or delivered as low density lipoprotein, and overall cellular content of cholesterol, although biosynthesis of lipids from acetate was somewhat increased. Probucol did not affect the mRNA level of ABCA1, and ABCA1 was recovered along with marker proteins for plasma membrane regardless of the presence of probucol. However, the protein level of ABCA1 increased, and the rate of its decay in the presence of cycloheximide was slower in the probucol-treated cells. ABCA1 in the probucol-treated cells was resistant to digestion by calpain but not by trypsin. We concluded that probucol inactivates ABCA1 in the plasma membrane with respect to its function in mediating binding of and lipid release by apolipoprotein and with respect to proteolytic degradation by calpain.     

Hormonal and pharmacological alteration of angiotensinogen secretion from rat hepatocytes

Isolated hepatocytes were prepared from rat liver by collagenase perfusion and density gradient centrifugation. The hepatocyte preparation released angiotensinogen at a basal rate of 50-120 pmol/g wet weight per h. Release was linear with time for at least 4 h. Angiotensinogen secretion was reduced in the presence of actinomycin D, and inhibited by cycloheximide, puromycin, colchicine and vinblastine. In the presence of tunicamycin, an inhibitor of N-glycosylation, the secretion of angiotensinogen as well as total protein and albumin secretion were diminished. Hepatocytes from nephrectomized rats exhibit an increased secretion rate of angiotensinogen, whereas total protein secretion was unaltered. Preincubation of hepatocytes with hydrocortisone (0.1 mM) or angiotensin II (10 nM) induced an increase of angiotensinogen release. There was no concomitant increase of total protein or albumin secretion, indicating that these effects are not the expression of a general stimulation of protein synthesis and secretion.     

Synthesis of glycoprotein, glycolipid, protein, and lipid in synchronized L5178Y cells

Synthesis of four macromolecular classes found in membranes—glycoprotein, glycolipid, protein, and lipid—was measured as a function of time of the cell cycle in synchronized L5178Y cells. Incorporation of leucine, choline, fucose, glucosamine, or thymidine into the cells, protein, nucleic acid, or lipid was measured by pulse-labeling for ½ hr at ½ hr intervals after release from the mitotic block. The amount of protein, lipid, glycoprotein, or glycolipid released or secreted into the medium by the L5178Y cells was also measured as a function of time of the cell cycle. Cellular protein was found to be synthesized throughout the cell cycle, with the highest synthesis occurring in the S period; synthesis was depressed in the M period. Cellular glycoprotein was synthesized at approximately the same times as protein, except that the rates of glycoprotein synthesis in the S period relative to other periods were much greater than for protein. Secreted protein was synthesized throughout the cell cycle without any general pattern, except that secretion was elevated in the late S and G₂ periods. Secreted glycoprotein was similar to secreted protein. Cellular lipid and cellular glycolipid were synthesized almost exclusively in the G₂ and M periods; there was no synthesis in the G₁ and S periods. Release or secretion of glycolipid and lipid also occurred in the G₂ and M periods.     

Microsomal triglyceride transfer protein activity is not required for the initiation of apolipoprotein B-containing lipoprotein assembly in McA-RH7777 cells

We previously demonstrated that the N-terminal 1000 amino acid residues of human apolipoprotein (apo) B (designated apoB:1000) are competent to fold into a three-sided lipovitellin-like lipid binding cavity to form the apoB "lipid pocket" without a structural requirement for microsomal triglyceride transfer protein (MTP). Our results established that this primordial apoB-containing particle is phospholipid-rich (Manchekar, M., Richardson, P. E., Forte, T. M., Datta, G., Segrest, J. P., and Dashti, N. (2004) J. Biol. Chem. 279, 39757-39766). In this study we have investigated the putative functional role of MTP in the initial lipidation of apoB:1000 in stable transformants of McA-RH7777 cells. Inhibition of MTP lipid transfer activity by 0.1 microm BMS-197636 and 5, 10, and 20 microm of BMS-200150 had no detectable effect on the synthesis, lipidation, and secretion of apoB:1000-containing particles. Under identical experimental conditions, the synthesis, lipidation, and secretion of endogenous apoB100-containing particles in HepG2 and parental untransfected McA-RH7777 cells were inhibited by 86-94%. BMS-200150 at 40 microm nearly abolished the secretion of endogenous apoB100-containing particles in HepG2 and parental McA-RH cells but caused only 15-20% inhibition in the secretion of apoB: 1000-containing particles. This modest decrease was attributable to the nonspecific effect of a high concentration of this compound on hepatic protein synthesis, as reflected in a similar (20-25%) reduction in albumin secretion. Suppression of MTP gene expression in stable transformants of McA-RH7777 cells by micro-interfering RNA led to 60-70% decrease in MTP mRNA and protein levels, but it had no detectable effect on the secretion of apoB:1000. Our results provide a compelling argument that the initial addition of phospholipids to apoB:1000 and initiation of apoB-containing lipoprotein assembly occur independently of MTP lipid transfer activity.     

Products of Cultured Neuroglial Cells. III. Release of an 85,000 Molecular Weight Glycoprotein by C6 Glioma Cells In Vitro

Abstract: With [³H]fucose as a marker, C6 glioma cells in culture released an 85,000 molecular weight molecule into the medium as the major extracellular glycoprotein. The quantity and extracellularkytoplasmic ratio of this glycoprotein suggest that its cellular processing is different from that of five other released glycoproteins of molecular weights 55,000, 115,000, 130,000, 150,000, and 170,000. Nearly 40% of newly synthesized glycoproteins in the cells was released into the culture medium. Major glycoproteins retained by the cells migrated electrophoretically to molecular weight positions of 82,000, 110,000, 120,000, 140,000, and 160,000, and approximately one-third of these retained glycoproteins were labile to trypsinization. Both synthesis and release of these macromolecules were inhibited more than 95% with cycloheximide treatment, demonstrating that nearly all fucosylation was linked to protein synthesis. Since 40% of all glycoproteins was released under conditions of more than 99% cellular viability, it is likely that these extracellular glycoproteins are physiological products of membrane turnover and secretion, but not of cell lysis. The results provide a basis for the further study of glial differentiation and of shed glioma antigens.     

Astrocytes produce and secrete FGF-1, which promotes the production of apoE-HDL in a manner of autocrine action

The astrocytes prepared by 1 week secondary culture after 1 month primary culture of rat brain cells (M/W cells) synthesized and secreted apolipoprotein E (apoE) and cholesterol more than the astrocytes prepared by conventional 1 week primary and 1 week secondary culture (W/W cells) (Ueno, S., J. Ito, Y. Nagayasu, T. Furukawa, and S. Yokoyama. 2002. An acidic fibroblast growth factor-like factor secreted into the brain cell culture medium upregulates apoE synthesis, HDL secretion and cholesterol metabolism in rat astrocytes. Biochim. Biophys. Acta. 1589: 261-272). M/W cells also highly expressed fibroblast growth factor-1 (FGF-1) mRNA. FGF-1 was identified in the cell lysate of both cell types, but M/W cells released more of it into the medium. Immunostaining of FGF-1 and apoE revealed that both localized in the cells that produce glial fibrillary acidic protein. The conditioned media of M/W cells and FGF-1 stimulated W/W cells to release apoE and cholesterol to generate more HDL. Pretreatment with a goat anti-FGF-1 antibody or heparin depleted the stimulatory activity of M/W cell-conditioned medium. The presence of the anti-FGF-1 antibody in the medium suppressed apoE secretion by M/W cells. Differential inhibition of signaling pathways suggested that FGF-1 stimulates apoE synthesis via the phosphoinositide 3-OH kinase for PI3K/Akt pathway. Thus, astrocytes release FGF-1, which promotes apoE-HDL production by an autocrine mechanism. These results are consistent with our in vivo observation that astrocytes produce FGF-1 before the increase of apoE in the postinjury lesion of the mouse brain (Tada, T., J. Ito, M. Asai, and S. Yokoyama. 2004. Fibroblast growth factor 1 is produced prior to apolipoprotein E in the astrocytes after cryo-injury of mouse brain. Neurochem. Int. 45: 23-30).     

Coupled induction of exocrine proteins and intracellular compartments involved in the secretory pathway in AR4-2J cells by glucocorticoids

Treatment of AR4-2J cells with dexamethasone at 10 nM for 96 h inhibited cell replication by 75% and increased cell size (30%), protein content (1.6-fold) and protein synthesis (2-fold). The increase in protein synthesis was largely due to a 5 to 10-fold increase in the synthesis of secretory proteins. Amylase activity increased 20 to 30-fold in cellular homogenates and 10 to 20-fold in culture medium. Both in the presence and absence of dexamethasone AR4-2J cells release their secretory proteins by constitutive secretion. The proportion of newly synthesized amylase retained by the cells over the 14 h labeling period increased from 15 to 30% with hormone treatment. As judged by comigration on polyacrylamide gels and Western blots analyzed by immunospecific sera, AR4-2J cells synthesize and secrete the majority of known pancreatic secretory proteins. Dexamethasone increased the synthesis of trypsinogen 12 to 16-fold, chymotrypsinogen 4.5 to 6-fold, the group of procarboxypeptidases 6-fold, and amylase 7 to 10-fold. Messenger RNA levels for trypsinogen, amylase and lipase were each increased 4 to 5-fold. At the ultrastructural level dexamethasone led to significant increases in rough endoplasmic reticulum (RER) (30-fold) and Golgi elements (1.5-fold) and to the de novo appearance of electron-opaque granules (0.1-0.5 microns) which were shown to contain amylase by immunolocalization techniques employing protein A-gold. Dexamethasone also led to the formation of gap junctions between AR4-2J cells. These findings indicate that AR4-2J cells provide a model for differentiation of pancreatic acinar cells which should also be studied for the differentiation markers for the regulated secretory pathway.     

Osmotic regulation of invertase formation and secretion by protoplasts of Saccharomyces

Osmotic regulation of invertase formation and secretion by protoplasts of Saccharomyces was examined using sorbitol, KCl, NaCl, or magnesium sulfate as the osmotic support. The synthesis and secretion of the enzyme is remarkably sensitive to the osmolarity of the supporting medium irrespective of the particular support employed. Invertase formation was inhibited at high osmolarity and was maximal at 0.65 to 0.75 osmolal, even though some leakage of the intracellular enzyme alpha-glucosidase and of ultraviolet (UV)-absorbing materials occurred under these conditions. The reduction of invertase formation and secretion due to high osmolarity was eliminated promptly when protoplasts were transferred into a medium of lower osmolarity. The rate of fructose uptake and of threonine incorporation into protein was decreased by high osmolarity; also reduction of invertase formation could be partially reversed by increasing the level of sugar supplied as energy source. Thus changes in the permeability of the plasma membrane (and presumably also in its structure) are important factors in the response of protoplasts to high osmolarity, though certainly not the complete explanation. Protoplasts suspended in 0.8 m sorbitol, with 10mm fructose as the energy source, increased their invertase level 5- to 10-fold during a 2-hr incubation without substantial release of alpha-glucosidase or UV-absorbing materials. Both the large and small forms of invertase were present in the protoplasts, but only the large form was released into the medium when enzyme was being actively synthesized. Formation and secretion of newly formed invertase and the release of enzyme initially present were inhibited by cycloheximide.     

Assembly and secretion of lipophorin by the larval fat body of the southwestern corn borer,Diatraea grandiosella: An in vitro study

The synthesis, processing, and secretion of lipophorin by the larval fat body of the southwestern corn borer, Diatraea grandiosella, was examined using in vitro techniques. Pulse-labeling of lipophorin with [³⁵S]methionine showed that apolipophorin-I and -II were each synthesized and secreted from the fat body into Grace's medium with an intracellular transit time of about 45 min. Secretion of the apolipoproteins from the fat body became insensitive to the presence of monensin, which disrupts protein processing in the Golgi complex, at 30 min, indicating that most of the pulse-labeled apolipoprotein has transited the Golgi complex by this time. Three inhibitors of protein processing, carbonylcyanide m-chlorophenyl hydrazone, monensin, and brefeldin A, inhibited secretion of lipophorin into medium. Puromycin treatment did not appear to result in the secretion into the medium of lipophorin particles containing incomplete translation products of apolipophorin-I or -II. Incubation of fat bodies with [³H]oleate resulted in the secretion of lipophorin containing [³H]glycerides, a process that was inhibited by cycloheximide, puromycin, and monensin, indicating that apolipoprotein synthesis is required for secretion of [³H]glyceride on nascent lipophorin particles. In contrast, suramin, which has been shown to block the binding of lipophorin to plasma membrane receptors, inhibited the synthesis and secretion of lipophorin, but it did not appear to inhibit the transfer of [³H]lipid from the fat body to lipophorin. Inhibitors of protein synthesis and processing, therefore, can be used to distinguish between secretion of lipophorin-associated lipids and secretion of lipids mediated by the lipid-transfer particle outside the plasma membrane of the fat body.     

Citrate transporters play an important role in regulating aluminum-induced citrate secretion in Glycine max

To further understand the process of Al-induced citrate secretion from soybean roots, the effect of protein synthesis inhibitor, anion channel blockers, and citrate carrier inhibitors on Al-induced citrate exudation was investigated in Al-resistant soybean cultivar PI 416937. Citrate exudation from roots increased with the increase of Al concentration from 10 to 50 μM and initiated after 4 h of Al exposure. Protein synthesis inhibitor, cycloheximide (CHM; 25 μM) completely inhibited Al-induced citrate secretion during 12-h exposure, suggesting that novel protein synthesis was necessary in Al-induced citrate efflux. Also both anion channel blocker anthracene-9-carboxylic acid (A-9-C) and citrate carrier inhibitor mersalyl acid (Mersalyl) significantly reduced citrate secretion, suggesting that both anion channels in plasma membrane and citrate carriers in mitochondria membrane were the rate limiting factors of Al dependent citrate release. However, Al-induced citrate secretion was insensitive to anion channel blockers phenylglyoxal (PG), 4,4′-diisothiocyanostibene-2,2′-disulfonat (DIDS) and citrate carrier inhibitor pyridoxal 5′-P (PP).     

Hormonal regulation during secretion of alpha-fetoprotein in hepatoma cells grown in synthetic medium

The variant cell line of H4-II-E-C3 cells derived from the Reuber H-35 hepatoma cells has been established using protein- and lipid-free synthetic medium. This H4-II-E-C3-V line can synthesize and secrete considerable amounts of alpha-fetoprotein (AFP) and albumin. The addition of 5 X 10(-7) M dexamethasone to the medium stimulated the excretion of AFP without increasing total AFP synthesis, whereas 8.7 X 10(-8) M insulin inhibited the excretion of AFP without a significant inhibition of intracellular AFP synthesis. However, neither dexamethasone nor insulin altered either the cellular or secreted levels of albumin. Cells were pulse labeled with [35S]methionine and then chased after addition of excess unlabeled methionine. AFP appeared in the medium after 10 min, and 50% of the protein was secreted after 110 min. The rate of secretion of AFP was much slower than that of albumin, 50% of which was secreted after 25 min. Dexamethasone, 5 X 10(-7) M, caused a marked enhancement in the rate of AFP secretion, with 50% released after 75 min. Insulin, 8.7 X 10(-8) M, by contrast, caused a marked delay in AFP secretion with only 20% released after 180 min and then a plateau was approached. Since the intracellular AFP was excreted 55% after 180 min the remaining 25% of newly made AFP was suggested to be degraded during secretion. The kinetics of movement of AFP during secretion and endoglycosidase H treatment of intracellular and secreted AFP suggested that insulin impeded the transport of AFP from the rough endoplasmic reticulum to the Golgi apparatus.     

Verapamil: a novel probe of surfactant secretion from rat type II pneumocytes

Surfactant from type II pneumocytes prevents the alveolar atelectasis found in both the neonatal and adult forms of respiratory distress syndrome. We have found that verapamil, a phenylalkene with calcium channel and alpha 1-receptor binding properties, has a multiphasic concentration effect on surfactant secretion from [3H]choline-labeled rat type II pneumocytes in culture. Verapamil (10(-8) M) caused a 24% stimulation of surfactant secretion, whereas an 8% inhibition was found at 10(-6) M and a 70% stimulation was found at 10(-4) M. Lactate dehydrogenase release occurred at 5 x 10(-4) M verapamil. Verapamil (10(-4) M) also produced a 100% increase in adenosine 3',5'-cyclic monophosphate (cAMP) in comparison with concentrations of less than or equal to 10(-6) M, an effect that could not be blocked by propranolol (10(-4) M). Verapamil (10(-6) M) increased the total formation of inositol phosphates (IP) by 23% in comparison with IP formation in control cells. Calcium influx was inhibited 15% by 10(-8) M verapamil and 37% by 10(-4) M verapamil. Calcium efflux was stimulated 44% by 10(-5) M verapamil. In combination with 50% effective concentrations (EC50) of terbutaline, phorbol ester, and ATP, the respective effects of verapamil (10(-4) M) on surfactant secretion were approximately additive. We conclude that verapamil has a novel multiphasic concentration effect on surfactant secretion, which appears to involve several signal transduction pathways including cAMP formation, IP formation, inhibition of calcium influx, and stimulation of calcium efflux.     

Accumulation of lysophosphatidylinositol in RAW 264.7 macrophage tumor cells stimulated by lipid A precursors

N2,O3-Diacylglucosamine 1-phosphate (lipid X), a monosaccharide precursor of Escherichia coli lipid A, was used to stimulate RAW 264.7 macrophage tumor cells, and the effects on macrophage phospholipid metabolism were examined. The addition of E. coli lipid X to the medium of cells that had been uniformly labeled with 32Pi resulted in a 4-8-fold increase in the level of lysophosphatidylinositol. This effect was maximal at 5 microM lipid X. Lysophosphatidylinositol levels reached a maximum 45 min after stimulation, followed by a gradual decline to near normal levels within 2 h. The formation of lysophosphatidylinositol was dependent upon extracellular calcium and was almost completely inhibited when cycloheximide was added at the time of stimulation. The addition of the disaccharide lipid A precursor IVA, commercial lipopolysaccharide (1 microgram/ml), phorbol 12-myristate 13-acetate (10(-7) M), or calcium ionophore A23187 (10(-6) M) to these cells resulted in a similar increase in lysophosphatidylinositol levels, but phosphatidic acid was inactive. The stimulation by IVA and phorbol myristate acetate was blocked by cycloheximide, but the stimulation by lipopolysaccharide was only partially blocked. The stimulation by A23187 was unaffected by cycloheximide. The increase in lysophosphatidylinositol levels might be related to the stimulation of arachidonate release and prostaglandin synthesis that is also observed in cells treated with lipid A precursors. The disaccharide precursor, IVA, was at least 100 times more effective than lipid X at stimulating lysophosphatidylinositol formation and prostaglandin release. The relative ability of lipid X and IVA to stimulate these cells correlated well with their effects on other lipopolysaccharide-responsive systems. Macrophage tumor cells also had the ability to inactivate lipid X by dephosphorylating it.     

Molecular and cellular mechanisms of adipose secretion: comparison of leptin and angiotensinogen

Besides their function of lipid storage, the adipose cells secrete a number of proteins of physiopathological importance. To get further insights into this function, which remains poorly characterized, we sought to compare the mechanisms and regulation of secretion of two individual proteins in the same cells. Leptin and angiotensinogen were chosen and assessed by radioimmunoassay and quantitative immunoblotting, respectively, in primary culture of epididymal adipose cells from young obese Zucker rats. Leptin was secreted at a steady rate of 4 ng/10(6) cells/h over 2-6 h. Despite secretion, leptin cellular content remained stable at 3 ng/10(6) cells. In contrast, the rate of angiotensinogen secretion decreased regularly from 45 arbitrary units/10(6) cells/h at 2 h, to half this value at 6 h, although cell content remained constant at 100 arbitrary units/10(6) cells. Inhibition of protein synthesis by cycloheximide depleted the cells from leptin, but not from angiotensinogen for up to 6 h. Insulin increased leptin secretion (+75%) and cell content (+70 %), without affecting angiotensinogen. Secretion of both proteins was inhibited by Golgi-disturbing agents, brefeldin A and monensin. The presence of brefeldin A led to a specific rise in leptin cell content, an effect inhibited by cycloheximide and enhanced by insulin (+80%). These data show that leptin and angiotensinogen are both secreted through Golgi-dependent pathways and that their respective intracellular pool exhibit distinct turn-over rate and insulin sensitivity. These characteristics might account for the differential response of these adipose proteins to variations in the systemic environment.     

Binding properties of high-density lipoprotein subfractions and low-density lipoproteins to rabbit hepatocytes

Primary cultures of rabbit hepatocytes which were preincubated for 20 h in a medium containing lipoprotein-deficient serum subsequently bound, internalized and degraded 125I-labeled high-density lipoproteins2 (HDL2). The rate of degradation of HDL2 was constant in incubations from 3 to 25 h. As the concentration of HDL2 in the incubation medium was increased, binding reached saturation. At 37 degrees C, half-maximal binding (Km) was achieved at a concentration of 7.3 micrograms of HDL2 protein/ml (4.06 X 10(-8)M) and the maximum amount bound was 476 ng of HDL2 protein/mg of cell protein. At 4 degrees C, HDL2 had a Km of 18.6 micrograms protein/ml (1.03 X 10(-7)M). Unlabeled low-density lipoproteins (LDL) inhibited only at low concentrations of 125I-labeled HDL2. Quantification of 125I-labeled HDL2 binding to a specific receptor (based on incubation of cells at 4 degrees C with and without a 50-fold excess of unlabeled HDL) yielded a dissociation constant of 1.45 X 10(-7)M. Excess HDL2 inhibited the binding of both 125I-labeled HDL2 and 125I-labeled HDL3, but excess HDL3 did not affect the binding of 125I-labeled HDL3. Preincubation of hepatocytes in the presence of HDL resulted in only a 40% reduction in specific HDL2 receptors, whereas preincubation with LDL largely suppressed LDL receptors. HDL2 and LDL from control and hypercholesterolemic rabbits inhibited the degradation of 125I-labeled HDL2, but HDL3 did not. Treatment of HDL2 and LDL with cyclohexanedione eliminated their capacity to inhibit 125I-labeled HDL2 degradation, suggesting that apolipoprotein E plays a critical role in triggering the degradative process. The effect of incubation with HDL on subsequent 125I-labeled LDL binding was time-dependent: a 20 h preincubation with HDL reduced the amount of 125I-labeled LDL binding by 40%; there was a similar effect on LDL bound in 6 h but not on LDL bound in 3 h. The binding of 125I-labeled LDL to isolated liver cellular membranes demonstrated saturation kinetics at 4 degrees C and was inhibited by EDTA or excess LDL. The binding of 125I-labeled HDL2 was much lower than that of 125I-labeled LDL and was less inhibited by unlabeled lipoproteins. The binding of 125I-labeled HDL3 was not inhibited by any unlabeled lipoproteins. EDTA did not affect the binding of either HDL2 or HDL3 to isolated liver membranes. Hepatocytes incubated with [2-14C]acetate in the absence of lipoproteins incorporated more label into cellular cholesterol, nonsaponifiable lipids and total cellular lipid than hepatocytes incubated with [2-14C]acetate in the presence of any lipoprotein fraction. However, the level of 14C-labeled lipids released into the medium was higher in the presence of medium lipoproteins, indicating that the effect of those lipoproteins was on the rate of release of cellular lipids rather than on the rate of synthesis.     

Release of acetylcholinesterase by cultured spinal cord cells

The release of acetylcholinesterase from neurons was studied using cultured chick-embryo spinal-cord cells. Cells dissociated from 12-day-old chick-embryo spinal cords were grown in culture for 10-12 days. Numerous well differentiated spinal neurons were found after 7-10 days in culture. Acetylcholinesterase activity per dish increased by 60-fold from days 2-12. Acetylcholinesterase was released into the surrounding media by the cells when they were incubated either in the standard culture medium or the serum-free medium. Acetylcholinesterase release was significantly reduced when protein synthesis and microtubules were disrupted by cycloheximide and colchicine, respectively. Histochemical localization of acetylcholinesterase indicated that the synthesis and relase of acetylcholinesterase are attributable to neurons. Cultured chick-embryo brain and neuroblastoma cells also released acetylcholinesterase into the media. These results are discussed with regard to possible physiological roles for acetylcholinesterase secretion from neurons.     

The Relationship Between Protein Synthesis,ATP and Adenylate Energy Charge in Isolated Mung Bean Axes during Imbibition

The increase in ATP and E.C. in the mung bean axes during imbibi- tion was accompanied by an increase in the rate of protein synthesis. When the axes were treated with 5×l0-5 M, and 5×10-4M 2,4-Dinitrophenol at the first 4 hours of imbibition respectively, the production of ATP was inhibited, and the E.C. value decreased; at the same time, the incorporation of 3H-leucine into the trichloroacetic acidinsoluble protein was inhibited also. CCCP (1×10-5M and 1×10-4M) had a similar effect as DNP on mung bean axes. Incubated with 0.2 μg. ml-1 cycloheximide for 4 hours, the protein synthsized reduced by 69% compared to the control, the ATP and E. C. were slightly higher than the untreated one; while incubated with 1 μg and 5 μg cycloheximide, the protein synthesis almost stopped, the content of ATP decreased slightly, and E. C. value remained constant. When the mung bean axes were incubated with 1 μg, and 10 μg. ml-1 of actinomycin D for 4 hours, the protein synthesis was inhibited 23%, and 48% respectively. On the other hand, ATP, E. C. and the adenylate pool were not affected. These results showed that protein synthesis in mung bean axes during im- bibition was highly sensitive to the changes of ATP level and E. C. value. In contrast, adenylate pool was not affected by the actinomycin D.