Proteins Associated with Adaptation of Cultured Tobacco Cells to NaCl

Cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) adapted to grow in medium containing high levels of NaCl or polyethylene glycol (PEG) produce several new or enhanced polypeptide bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The intensities of some of the polypeptide bands (molecular weights of 58, 37, 35.5, 34, 26, 21, 19.5, and 18 kilodaltons) increase with increasing levels of NaCl adaptation, while the intensities of other polypeptide bands (54, 52, 17.5, and 16.5 kilodaltons) are reduced. Enhanced levels of 43- and 26-kilodalton polypeptides are present in both NaCl and PEG-induced water stress adapted cells but are not detectable in unadapted cells. In addition, PEG adapted cells have enhanced levels of 29-, 17.5-, 16.5-, and 11-kilodalton polypeptides and reduced levels of 58-, 54-, 52-, 37-, 35.5-, 34-, 21-, 19.5-, and 18-kilodalton polypeptide bands.

Synthesis of 26-kilodalton polypeptide(s) occurs at two different periods during culture growth of NaCl adapted cells. Unadapted cells also incorporate ³⁵S into a 26-kilodalton polypeptide during the later stage of culture growth beginning at midlog phase. The 26-kilodalton polypeptides from adapted and unadapted cells have similar partial proteolysis peptide maps and are immunologically cross-reactive. During adaptation to NaCl, unadapted cells synthesize and accumulate a major 26-kilodalton polypeptide, and the beginning of synthesis corresponds to the period of osmotic adjustment and culture growth. From our results, we suggest an involvement of the 26-kilodalton polypeptide in the adaptation of cultured tobacco cells to NaCl and water stress.

     

Osmotic Effects on Membrane Permeability in a Marine Bacterium

When cells of Alteromonas haloplanktis 214 (ATCC 19855) were preloaded with α-[¹⁴C]aminoisobutyric acid or the K⁺ in the cells was labeled with ⁴²K by incubation in a buffered salt solution containing 0.05 M MgSO₄, 0.01 M KCl, and 0.3 M NaCl, the cells retained their radioactivity when resuspended in the same salt solution. When NaCl was omitted from the solution, 80 to 90% of the radioactivity was lost from the cells. Cells suspended at intermediate concentrations of NaCl also lost radioactivity. New steady-state levels of the intracellular solutes were established within 15 s of suspending the cells; the percentage of radioactivity retained at each level decreased proportionately as the osmolality of the NaCl in the suspending solution decreased. With minor variations in effectiveness, MgCl₂, LiCl, and sucrose could substitute for NaCl on an equiosmolal basis for the retention of radioactivity by the cells. KCl, RbCl, and CsCl were appreciably less effective as replacements for NaCl, particularly when their osmolalities in the suspending solutions were low. The amount of α-[¹⁴C]aminoisobutyric acid taken up by the cells at the steady-state level increased to a maximum as the NaCl concentration in the suspending medium increased to 0.3 M. At suboptimal levels of NaCl, either LiCl or sucrose could substitute for NaCl in increasing the steady-state levels. The results obtained indicate that the porosity of the cytoplasmic membrane of this organism is determined by the difference between the osmotic pressure of the cytoplasm and the suspending medium. The lesser effectiveness of K⁺, Rb⁺, and Cs⁺ than Na⁺, Li, or Mg²⁺ in permitting the retention of solutes by the cells is attributed to the greater penetrability of the hydrated ions of the former group through the dilated pores of a stretched cytoplasmic membrane.     

Efflux of sphingolipids metabolically labeled with [1-3H]sphingosine, L-[3-3H]serine and [9,10-3H]palmitic acid from normal cells in culture

The membrane complex lipids of human fibroblasts and differentiated rat cerebellar granule cells in culture were metabolically radiolabeled with [1-³H]sphingosine, L-[3-³H]serine and [9,10-³H]palmitic acid. A relevant efflux of radioactive sphingolipids and phosphatidylcholine was observed from cells to the culture medium in the presence of fetal calf serum. This event was independent of the concentration and structure of the metabolic precursor administered to cells, and it was linearly time-dependent. The radioactive lipid patterns present in the medium were different from those present in the cells. Radioactive sphingomyelin and ganglioside GM3 containing short acyl chains were the main species present in the medium from human fibroblasts, while sphingomyelin and GD3 ganglioside in that from neuronal cells. In the absence of proteins in the culture medium, the efflux of complex lipids was much lower than in the presence of serum, and the patterns of released molecules were again different from those of cells. This work was supported by COFIN-PRIN, Consiglio Nazionale delle Ricerche (PF Biotechnology), Italy.     

Direct interaction,instrumental for signaling processes,between LacCer and Lyn in the lipid rafts of neutrophil-like cells

Lactosylceramide [LacCer; β-Gal-(1-4)-β-Glc-(1-1)-Cer] has been shown to contain very long fatty acids that specifically modulate neutrophil properties. The interactions between LacCer and proteins and their role in cell signaling processes were assessed by synthesizing two molecular species of azide-photoactivable tritium-labeled LacCer having acyl chains of different lengths. The lengths of the two acyl chains corresponded to those of a short/medium and very long fatty acid, comparable to the lengths of stearic and lignoceric acids, respectively. These derivatives, designated C18-[³H]LacCer-(N₃) and C24-[³H]LacCer-(N₃), were incorporated into the lipid rafts of plasma membranes of neutrophilic differentiated HL-60 (D-HL-60) cells. C24-[³H]LacCer-(N₃), but not C18-[³H]LacCer-(N₃), induced the phosphorylation of Lyn and promoted phagocytosis. Incorporation of C24-[³H]LacCer-(N₃) into plasma membranes, followed by illumination, resulted in the formation of several tritium-labeled LacCer-protein complexes, including the LacCer-Lyn complex, into plasma membrane lipid rafts. Administration of C18-[³H]LacCer-(N₃) to cells, however, did not result in the formation of the LacCer-Lyn complex. These results suggest that LacCer derivatives mimic the biological properties of natural LacCer species and can be utilized as tools to study LacCer-protein interactions, and confirm a specific direct interaction between LacCer species containing very long fatty acids, and Lyn protein, associated with the cytoplasmic layer via myristic/palmitic chains.     

Relationships between heat resistance and phospholipid fatty acid composition of Vibrio parahaemolyticus.

Vibrio parahaemolyticus was grown in tryptic soy broth (TSB) containing NaCl levels of 0.5, 3.0, and 7.5% (wt/vol). Cultures incubated at 21, 29, and 37 C were harvested in late exponential phases and thermal death times at 47 C (D47 c; time at 47 C required to reduce the viable population by 90%) were determined in phosphate buffer containing 0.5, 3.0, and 7.5% NaCl. At a given NaCl concentration in the growth medium, D47 c values increased with elevated incubation temperatures and with elevated levels of NaCl in the heating menstrua. Differences in thermal resistance of cells cultured at a particular temperature were greater between those grown in TSB containing 0.5 and 3.0% NaCl than between those grown in TSB containing 3.0 and 7.5% NaCl. D47c values ranged from 0.8 min (grown at 21 C in TSB with 0.5% NaCl) to 6.5 min (grown at 37 C in TSB with 7.5%, heated in 7.5% NaCl buffer). Methyl esters of major phospholipid fatty acids extracted from cells were quantitated. The ratio of saturated to unsaturated fatty acids in cells grown at a given NaCl concentration increased with elevated incubation temperature. At a particular growth temperature, however, saturated to unsaturated fatty acids ratios were lowest for cells grown in TSB containing 3.0% NaCl.     

Photoactive covalent labeling of membrane components from within the lipid core

Radioactive 1-Azidonaphthalene and 1-Azido-4-iodobenzene added to liposomes and sarcoplasmic reticulum membranes, are able to reach the liquid hydrocarbon like core, as demonstrated by encounter quenching of perylene fluorescence. Photoactivation converts the azides into the reactive nitrenes which label covalently the fatty acyl chains of the phospholipids. In addition, approximately half of the radioactivity is associated with membrane proteins. Only some 25% is released by exhaustive pronase treatment. Gel electrophoresis shows that the label is located in the Ca⁺⁺-stimulated Adenosine triphosphatase and in a high molecular weight polypeptide. These results are discussed in terms of possible labeling of biological membranes from within the lipid core.     

Release of phospholipid acyl groups from fetal human fibroblasts

Human diploid fibroblasts incorporate [1-¹⁴C]oleate primarily into phospholipids; this radioactivity is gradually lost by cells of adult origin, but retained, after an initial loss, by fetal cells during growth in unlabeled unchanged medium. [1-¹⁴C]oleate is released from fetal cells after each subsequent medium change even when conditioned medium is used. Unlabeled cells readily incorporate medium radioactivity previously released by labeled cells. Raising the serum concentration of the medium increases acyl group release. The apparent retention of acyl groups by fetal cells in the absence of medium changes results from a dynamic equilibrium of fatty acid moieties between cellular phospholipids and the culture medium.     

Identification of acyl donors and acceptor proteins for fatty acid acylation in BHK cells infected with Semliki Forest virus

The modification of viral glycoproteins through the covalent attachment of fatty acids was studied in baby hamster kidney (BHK) cells infected with Semliki Forest virus (SFV). Comparative pulse-chase experiments with [3H]palmitic acid and [35S]methionine revealed that a precursor polypeptide, designated p62, of the structural SFV glycoprotein and E1 serve as the primary acceptors of acyl chains. Acylation of p62 occurs immediately prior to its proteolytical cleavage to E2 and E3 emphasizing the post-translational and specific nature of this hydrophobic modification. To trace the acyl donor(s) for protein acylation the covalent attachment of fatty acids to p62 was studied after extremely short labeling periods with [3H]palmitic acid and correlated to the metabolism of the exogenous tritiated fatty acid. The shortest possible labeling time, a 10 s pulse with [3H]palmitic acid, was sufficient to acylate SFV p62. Analysis of the labeled lipids extracted from the same cells revealed that palmitoyl-CoA and phosphatidic acid showed the highest specific radioactivity among the tritiated lipid species. Out of these lipid species palmitoyl-CoA was identified as the functional acyl donor lipid in a cell-free system for the acylation of polypeptides.     

Effects of lipid composition on the membrane activity and lipid phase behaviour of Vibrio sp. DSM14379 cells grown at various NaCl concentrations

The membrane lipid composition of living cells generally adjusts to the prevailing environmental and physiological conditions. In this study, membrane activity and lipid composition of the Gram-negative bacterium Vibrio sp. DSM14379, grown aerobically in a peptone-yeast extract medium supplemented with 0.5, 1.76, 3, 5 or 10% (w/v) NaCl, was determined. The ability of the membrane to reduce a spin label was studied by EPR spectroscopy under different salt concentrations in cell suspensions labeled with TEMPON. For lipid composition studies, cells were harvested in a late exponential phase and lipids were extracted with chloroform-methanol-water, 1:2:0.8 (v/v). The lipid polar head group and acyl chain compositions were determined by thin-layer and gas-liquid chromatographies. ³¹P-NMR spectroscopy was used to study the phase behaviour of the cell lipid extracts with 20 wt.% water contents in a temperature range from −10 to 50 °C. The results indicate that the ability of the membrane to reduce the spin label was highest at optimal salt concentrations. The composition of both polar head groups and acyl chains changed markedly with increasing salinity. The fractions of 16:0, 16:1 and 18:0 acyl chains increased while the fraction of 18:1 acyl chains decreased with increasing salinity. The phosphatidylethanolamine fraction correlated inversely with the lysophosphatidylethanolamine fraction, with phosphatidylethanolamine exhibiting a minimum, and lysophosphatidylethanolamine a maximum, at the optimum growth rate. The fraction of lysophosphatidylethanolamine was surprisingly high in the lipid extracts. This lipid can form normal micellar and hexagonal phases and it was found that all lipid extracts form a mixture of lamellar and normal isotropic liquid crystalline phases. This is an anomalous behaviour since the nonlamellar phases formed by total lipid extracts are generally of the reversed type.     

Studies on the characterization of the sodium-potassium transport adenosinetriphosphatase. XII. Evidence for interaction of the acyl phosphate at the active site with neighboring groups

The sodium-potassium adenosinetriphosphatase (NaK ATPase), partially purified from beef brain, has been phosphorylated with [γ-³²P]ATP in the presence of Na and Mg and digested with pronase. A single ³²P-labeled peptide spot has been identified on paper electrophoresis, accounting for 60% of the radioactivity in the ³²P-labeled enzyme, the remainder of the radioactivity being [³²P]-orthophosphate resulting from breakdown of the highly labile acyl phosphate during pronase digestion. The ³²P in the pronase peptide was released as [³²P]-orthophosphate by N-propylhydroxylamine—as to be expected of an acyl phosphate compound. The pH stability of the acyl phosphate in the denatured phosphorylated NaK ATPase, in the pronase peptide and in acetyl phosphate were quite different. The phosphorylated protein had the lowest stability of higher pHs, acetyl phosphate had the highest stability, and the pronase peptide had an intermediate stability. These results indicate that the neighboring groups in the polypeptide chain containing the acyl phosphate residue influence the stability of the acyl phosphate bond.     

Differences in the incorporation of [3H]-glucosamine into nascent polypeptide chains on free polysomes and two fractions of membrane-bound polysomes in mouse myeloma cells

The incorporation of [³H]-glucosamine into polypeptides of three fractions of polysomes in MPC-11 cells was studied. After short term incubation greatest incorporation was observed in a fraction of membrane-bound polysomes, which after nitrogen cavitation of cells, remained bound to the endoplasmic reticulum (ER) associated with the nucleus (fraction 2). Polypeptide chains on membrane-bound polysomes in the microsomal fraction (fraction 1) and free polysomes contained much less radioactivity. Since nascent polypeptide chains contained within membrane-bound polysomes of fraction 2 are glycosylated at an earlier stage than those in fraction 1 it is likely that this represents a difference in type of proteins synthesized in the respective fractions of ER.     

Identification of a 51-kilodalton polypeptide fatty acyl chain acceptor in soluble extracts from mouse cardiac tissue

We have identified a protein in the soluble fraction from mouse cardiac tissue extracts which is rapidly and selectively acylated by myristyl CoA. This protein was partially purified by anion-exchange chromatography and gel filtration, and the acylation reaction was measured using [3H]myristyl CoA as substrate, followed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis to resolve [3H]fatty acyl polypeptides. The [3H]acyl protein migrated as heterogeneous bands corresponding to relative masses (MrS) of 42,000-51,000 under nonreducing conditions or as a single polypeptide of Mr 51,000 in the presence of reducing agents. Fatty acyl chain incorporation into protein was very rapid and already maximum after 30 s of incubation, whereas no acylation was detected using heat-denatured samples or when the reaction was stopped immediately after initiation. Only the acyl CoA served as fatty acyl chain donor. No incorporation into protein occurred when myristyl CoA was substituted by myristic acid, ATP, and CoA. A time-dependent reduction in the level of [3H]fatty acyl polypeptide was observed upon addition of excess unlabeled myristyl CoA, indicating the ability of the labeled acyl moiety of the protein to turn over during incubation. The saturated C10:0, C14:0, and C16:0 acyl CoAs were more effective to chase the label from the [3H]acyl polypeptide than the C18:0 and C18:1 acyl CoAs. These results provide evidence for a 51-kilodalton polypeptide which serves as an acceptor for fatty acyl chains and could represent an important intermediate in fatty acyl chain transfer reactions in cardiac tissue.     

Changes in the sulfation extent of membrane-associated proteoglycans produced by sertoli cells in culture

Sertoli cells in culture synthesize two different membrane-associated proteoglycans (MA-PG): a proteoglycan containing heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycan (GAG) chains and a CS-PG containing only CS-GAG chains. The structure of these molecules is regulated by the presence of fetal calf serum (FCS) in the culture medium. Changes in the concentration of FCS resulted in changes in the total ³⁵SO₄ incorporation into MA-PG and a shift in the elution profile of each component subjected to ion-exchange chromatography. Thus, without FCS, the incorporation was low, while in 1% and 10% FCS, the uptake of the precursor was 1.7 and 4.5 times higher, respectively. MA-PG synthesized by Sertoli cells cultured in 10% FCS eluted from DEAE-Sephacel columns at higher salt concentration than the MA-PG synthesized by cells cultured in 0% or 1% FCS. Double-labeled experiments showed that the ³⁵SO_4/3H-glucosamine ratio incorporated into MA-PG produced by Sertoli cells, increased from 17.6 to 23.6 and 50.9 in cells cultured at 0, 1, and 10% FCS, respectively. However, the presence of FCS affected neither the hydrodynamic size nor the chemical nature of GAG chains of MA-PG. These results show that changes in the FCS concentration promote changes in the sulfation extent of MA-PG molecules produced by Sertoli cells.     

Processing and secretion of the Yarrowia lipolytica RNase.

Secretion of the extracellular RNase from the yeast Yarrowia lipolytica was studied in pulse-chase and immunoprecipitation experiments. A polypeptide of 45,000 daltons was immunoprecipitated from [35S]methionine-labeled cell extracts and supernatant medium by rabbit anti-RNase antiserum. The RNase was secreted rapidly; the time between synthesis and appearance in the extracellular medium was about 5 min. In pulse-chase experiments, about 50% of the RNase was still cell associated 30 min after labeling. A polypeptide of 73,000 daltons whose immunoprecipitation was blocked by an excess of purified RNase was also detected. It broke down to a polypeptide with the same mobility and same peptide map as the mature RNase. Peptide maps of the undegraded 73-kilodalton polypeptide and the intracellular mature RNase contained several peptides of identical mobility. Immunoprecipitates from cells labeled in the presence of tunicamycin contained 66- and 45-kilodalton polypeptides. Endoglycosidase H treatment of the 73-kilodalton polypeptide converted it to a 66-kilodalton form, but did not change the apparent molecular weight of the mature form of the RNase. Labeling kinetics from pulse-chase experiments did not clearly support a precursor-product relationship between the 73-kilodalton polypeptide and the intracellular 45-kilodalton form of the RNase, and other relationships between the two polypeptides are possible.     

Appearance of newly synthesized protein in myelin of young rats.

Abstract— Seventeen-day-old rats were injected intracranially with [³H]leucine, then sacrificed between 1 and 24 h. Myelin was prepared from the brains on discontinuous sucrose gradients and the proteins were separated by discontinuous gel electrophoresis in buffers containing sodium dodecyl sulphate. Proteins were stained with acid Fast Green and the distribution was quantitated by densitometry. The gels were then sliced and the radioactivity in each slice was determined. Between 1 and 24 h, the radioactivity in proteolipid protein increased from 18% to 37% of the total radioactivity in the proteins of isolated myelin. During this same period, the per cent distribution of radioactivity in basic and Wolfgram proteins remained constant while that in the remaining high molecular weight proteins decreased. Similar results were also obtained with [³H]glycine as a precursor. The relative specific activity of all of the myelin proteins increased between 1 and 6 h, then remained constant between 6 and 24 h. At 1 h, proteolipid protein reached only 25% of its maximal (6 h) relative specific radioactivity, while the other two proteins reached 50% of maximum. These results indicate a lag in the appearance of labelled amino acids in proteolipid protein relative to the other myelin proteins.     

Effect of salinity on the lipid and fatty acid composition of the halophyteNavicula sp.: potential in mariculture

Navicula sp. (cf.N. tenelloides) was isolated from a salt marsh in Kuwait. The alga grew best with 0.5M NaCl, but abundant growth still occurred up to 2.5M NaCl. The total lipid content and the carotene to chlorophyll ratio of the cells increased with increasing salinity of the medium from 0.5 to 1.7M NaCl, but declined with 2.5M NaCl. Irrespective of the medium salinity, the major lipid class was that of triacylglycerols. The predominant fatty acids in the total lipids of cells grown at different NaCl concentrations were palmitic (16:0) and palmitoleic (16:1) acids; eicosapentaenoic acid (20:5) made up 8–9% of the total fatty acids. The fatty acid composition of the individual lipid classes of cells grown at different salinities is described. The highest concentration of 20:5 occurred in monogalactosyldiacylglycerols and digalactosyldiacylglycerols. In view of the rather small size of this diatom, its halotolerance and its fair content of 20:5, it is suggested as a potential food source for the mariculture industry.     

Biosynthesis of a 42-kD Polypeptide in the Cytoplasmic Membrane of the Cyanobacterium Anacystis nidulans Strain R2 during Adaptation to Low CO(2) Concentration

When cells of Anacystis nidulans strain R2 grown under high CO₂ conditions (3%) were transferred to low CO₂ conditions (0.05%), their ability to accumulate inorganic carbon (C_i) increased up to 8 times. Cytoplasmic membranes (plasmalemma) isolated at various stages of low CO₂ adaptation were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was a marked increase of a 42-kilodalton polypeptide in the cytoplasmic membrane during adaptation; a linear relationship existed between the amount of this polypeptide and the C_i-accumulating capability of the cells. No significant changes were observed during this process in the amount of other polypeptides in the cytoplasmic membranes or in the polypeptide profiles of the thylakoid membranes, cell walls, and soluble fractions. Spectinomycin, an inhibitor of protein biosynthesis, inhibited both the increase of the 42-kilodalton polypeptide and the induction of high C_i-accumulating capability. The incorporation of [³⁵S]sulfate into membrane proteins was greatly reduced during low CO₂ adaptation. Radioautograms of the ³⁵S-labeled membrane proteins revealed that synthesis of the 42-kilodalton polypeptide in the cytoplasmic membrane was specifically activated during the adaptation, while that of most other proteins was greatly suppressed. These results suggested that the 42-kilodalton polypeptide in the cytoplasmic membrane is involved in the active C_i transport by A. nidulans strain R2 and its synthesis under low CO₂ conditions leads to high C_i-transporting activity.     

Protein acylation in Tetrahymena

Examination of exhaustively delipidated Tetrahymena mimbres cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of several protein bands containing covalently linked fatty acids. Palmitic (16:0) and stearic (18:0) acids together accounted for approximately 90% of the protein-linked acyl chains, with myristic acid (14:0) comprising most of the remainder. Each of these three fatty acids was present mainly in alkali-stable linkage, indicating that unlike most other systems examined, fatty acids are attached to proteins of Tetrahymena principally by amide bonds. Smaller proportions of the acyl chains were susceptible to release by hydroxylaminolysis or by alkaline hydrolysis as would be expected from an ester linkage. The protein-bound acyl chains accounted for 0.3% of the cells' total fatty acids. They closely resembled in composition the highly saturated free fatty acid pool but not the vast pool of glycerolipid-associated fatty acids, which were mainly unsaturated. Cells subjected to thermal stress by rapid chilling from 39 to 15 degrees C responded by sharply increasing the ratio of palmitate to stearate in covalent association with proteins.     

Preparation and characterization of a cell-free system from Chinese hamster ovary cells that translates natural messenger ribonucleic acid and analysis of intermediary reactions

A cell-free system from cultured Chinese hamster ovary cells has been developed, which translates endogenous mRNAs, exogenous natural mRNAs, and synthetic polynucleotide templates. The analysis of most of the reactions involved in initiation, elongation, and termination of protein synthesis can be carried out in this system. The postmitochondrial fraction, containing ribosomal 40 and 60 S subunits, 80 S ribosomes, polysomes, and cytosol proteins, incorporates amino acids into protein. The preparation is capable of recycling endogenous mRNA by initiating protein synthesis on polysomal mRNA, and of initiating protein synthesis on exogenous templates. When endogenous mRNA is degraded with micrococcal nuclease, polysomes are no longer evident and protein synthesis is markedly depended on added mRNA, ATP, GTP, and a nucleoside triphosphate-generating system. Amino acid incorporation is linear for over 2 h, polysomes containing nascent polypeptide chains are reformed and, with time, most of the protein synthesized is released into the media. Gel electrophoretic analysis of the product formed in response to globin mRNA indicates that most of the radioactivity migrates as a single peak, in the region corresponding to globin. Comparison of the electrophoretic pattern obtained from labeled Chinese hamster ovary cells with that from incubations of cell extract and Chinese hamster ovary mRNA indicates that essentially all of the polypeptides formed by the intact cell are synthesized by the cell-free system. Sucrose gradient centrifugation of incubations containing mRNA-depleted extract and [³⁵S]methionine, in the absence of added mRNA, is used to detect initiation intermediates in the formation of the [40 S Met-tRNA_f] complex and, with added natural mRNA plus cycloheximide, to detect intermediates in the formation of the 80 S initiation complex. Chain elongation reactions are measured by the incorporation of [³H]phenylalanine into polyphenylalanine in extracts supplemented with poly(U), or by the formation of nascent polypeptide chains on polysomes with natural mRNA. Chain termination is measured by analyzing the amount of radioactive protein released into the cytosol.     

Reduction of opioid binding in neuroblastoma x glioma cells grown in medium containing unsaturated fatty acids

Neuroblastoma x glioma cells NG108-15 were cultured in lipid-free medium supplemented with fatty acids of various chain length and unsaturation. Binding of ³H-labelled [DAla²]-[Dleu⁵]-enkephalin by membranes of cells grown in saturation fatty acids of different chain length was not significantly different from that of the control. On the other hand, a proportional decrease of binding capacity with no change in residual receptor affinity was noticed when cells were cultured in medium containing fatty acids of increasing unsaturation. This decrease was time dependent and reached a maximum at about 48 h. Binding of [³H]dihydromorphine and [³H]naloxone was similarly affected. In contrast, when membranes of cells grown in normal medium were preincubated up to 3 h with unsaturated fatty acid and tested for opioid binding, no significant reduction was observed. Examination of the fatty acid composition of phospholipid from cells grown in linolenate indicated that a significant alteration of the acyl composition has occurred. To wval;uate the underlying cause of this type of inhibition, the effect of linolenic acid on cell growth and protein synthesis was examined. When cells were cultured in 100 μM of this fatty acid, both growth and protein synthesis were retarded by 28% and 19%, respectively. Since opiate receptors are proteineous in nature, a reduction of protein synthesis may partially account for the loss of opioid binding activity. On the other hand, an increase of membrane fluidity is known to affect a number of cellular functions, including ligan-receptor recognition. Whether this can offer a satisfactory explanation for our obervations remains to be established.