Lipid composition and turnover of rough and smooth microsomal membranes in rat liver

Subfractions of rat liver microsomes (rough, smooth I, and smooth II), isolated in a cation-containing sucrose gradient system, were analyzed. After removal of adsorbed and luminal protein, these subfractions had the same phospholipid/protein ratio, about 0.40. Both the classes and the relative amounts of phospholipids were similar in the three subfractions, but the relative amounts of neutral lipids (predominantly free cholesterol and triglycerides) were higher in smooth I and especially in smooth II than in rough microsomes. Various pieces of evidence indicate that the neutral lipids are tightly bound to the membranes. Glycerol-(3)H was incorporated into the phospholipids of the rough and smooth I microsomes significantly faster than into those of the smooth II membranes; (32)P incorporation followed a similar but less pronounced pattern. Acetate-(3)H was incorporated into the free cholesterol of smooth I microsomes only half as fast as into the other two subfractions. Injection of phenobarbital increased the cellular phospholipid and neutral lipid content in the rough and smooth I, but not in the smooth II microsomes. Consequently, the neutral lipid/phospholipid ratio of all three subfractions remained unchanged after phenobarbital treatment. It is concluded that the membranes of the rough and the two smooth microsomal subfractions from rat liver have a similar phospholipid composition, but are dissimilar in their neutral lipid content and in the incorporation rate of precursors into membrane lipids.     

Glycoprotein synthesis in the adult rat pancreas: IV. Subcellular distribution of membrane glycoproteins

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5–10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50–100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and $\text{N-}\text{acetylglucosamine}$. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes < smooth microsomes < zymogen granules.Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptide was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [³H]glucosamine or [³H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules.Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB³H₄. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.     

Biogenesis of microsomal membrane glycoproteins in rat liver. I. Presence of glycoproteins in microsomes and cytosol

The glycoproteins of microsomes and cytosol were studied. Various washing procedures did not release the proteins from the microsomes, and immunological tests demonstrated that the sialoproteins are not serum components. Low concentrations of deoxycholate and incubation in 0.25 M sucrose solution liberated a small amount of microsomal sialoprotein and this fraction exhibited a high degree of labeling of protein-bound N-acetylneuraminic acid. A part of the glycoprotein fraction could not be solubilized, even with a high concentration of the detergent. Thoroughly perfused rat liver contained sialoproteins in the particle-free supernate. The level of sialoprotein present could not be due to contamination with serum or broken organelles. The high in vivo incorporation of [3H]glucosamine into protein-bound sialic acid of Golgi membranes and cytosol was paralleled by a delayed and lesser rate of incorporation into the rough and smooth microsomal membranes. This incorporation pattern suggests the possibility that the glycoproteins of cytosol and Golgi may later be incorporated into the membrane of the endoplasmic reticulum.     

Maintenance of epithelial surface membrane lipid polarity: A role for differing phospholipid translocation rates

Summary Large differences in lipid composition of apical and basolateral membranes from epithelial cells exist. To determine the responsible mechanism(s), rat renal cortical brush border and basolateral membrane phospholipids were labeled using³²P and either [³H]-glycerol or [2-³H] acetate for incorporation and degradation studies, respectively. Brush border and basolateral membrane fractions were isolated simultaneously from the same cortical homogenate. Different phospholipid classes were degraded at variable rates with phosphatidylcholine having the fastest decay rate. Decay rates for individual phospholipid classes were, however, similar in both brush border and basolateral membrane fractions. In phospholipid incorporation studies again, large variations existed between individual phospholipid classes with phosphatidylcholine and phosphatidylinositol showing the most rapid rates of incorporation. Sphingomyelin and phosphatidylserine showed extremely slow incorporation rates and did not enter into the isotopic decay phase for 48 hr. In contrast to degradation studies, however, the same phospholipid class labeled the two surface membrane domains at highly variable rates. The difference in these rates, with the exception of phosphatidylinositol, were identical to the differences in phospholipid compositions between the two membranes. For example, phosphatidylcholine was incorporated into the basolateral membrane 2.5 × faster than into the brush border membrane and its relative composition was 2.5 × greater in the basolateral membrane. The opposite was true for sphingomyelin. These results indicate incorporation and not degradation rates of individual phospholipids play a major role in regulating the differing phospholipid composition of brush border and basolateral membranes.     

Sialic acid incorporation into gangliosides and glycoproteins of the fish brain

—The concentration of lipid- and non-lipid-bound sialic acid in the optic nerve tract and tectum and in whole brain of fish was estimated. The incorporation of sialic acid into gangliosides and non-lipid components was studied in fish by intracranial or intraocular application of N-[³H]acetylmannosamine or N-[³H]acetylglucosamine. After intracranial injection of N-[³H]acetylmannosamine autoradiography showed lipid- and non-lipid-bound radioactivity in the tectum opticum evenly distributed over regions of nerve fibres or perikarya indicating an ubiquitous incorporation of label. Sialic acid incorporation into glycoproteins after intracranial injection of N-acetylmannosamine always exceeded that into gangliosides. TCA-precipitable non-lipid material is labelled from intracranially applied N-acetylmannosamine in the sialic acid portion and also in nonsialic acid components, whereby the percentage of label in sialic acid increases reaching 90 per cent of the total radioactivity after 90 min. After intraocular application of N-[³H]acetylmannosamine, sialic acid in gangliosides was generally found to be more highly labelled than in glycoproteins. The ratio of radioactivity in gangliosides and glycoproteins increased with time of incubation and the distance from the eye. TCA-soluble radioactivity was translocated by fast axonal transport. Cycloheximide inhibited incorporation of N-acetylmannosamine-derived radioactivity into gangliosides and proteins but not the transport of TCA-soluble material, which accumulates in the tectum. After intraocular application of N-[³H]acetylglucosamine, TCA-soluble label arrives later in the optic tectum than radioactivity of high molecular weight components. The ratio of lipid to non-lipid-bound radioactivity does not change considerably with the time after injection or the distance from the eye. There was no accumulation of TCA-soluble radioactivity after the inhibition of incorporation into high molecular weight components.     

Biogenesis of endoplasmic reticulum membranes. I. Structural and chemical differentiation in developing rat hepatocyte

The development of the endoplasmic reticulum of rat hepatocytes was studied during a period of rapid cell differentiation, i.e., from 3 days before to 8 days after birth. Before birth, the ER increases in volume, remaining predominantly rough surfaced; after birth, the increase continues but affects mainly the smooth-surfaced part of the system. These changes are reflected in variations of the RNA/protein and PLP/protein ratios of microsomal fractions: the first decreases, while the second increases, with age. The analysis of microsomal membranes and of microsomal lipids indicates that the PLP/protein ratio, the distribution of phospholipids, and the rate of P³² incorporation into these phospholipids show little variation over the period examined and are comparable to values found in adult liver. Fatty acid composition of total phosphatides undergoes, however, drastic changes after birth. During the period of rapid ER development in vivo incorporation of leucine-C¹⁴ and glycerol-C¹⁴ into the proteins and lipids of microsomal membranes is higher in the rough-than in the smooth-surfaced microsomes, for the first hours after the injection of the label; later on (~10 hr) the situation is reversed. These results strongly suggest that new membrane is synthesized in the rough ER and subsequently transferred to the smooth ER.     

Developmental Changes in the Biosynthesis of Sialoglycoprotein Substrates for Intrinsic Synaptic Sialidase

Abstract: N′-Acetyl-d -[6-³H]mannosamine was administered to 13- and 28-day-old rats by intraventricular injection. At various time intervals following the injection, synaptic membranes were prepared and the incorporation of radiolabel into sialic acid residues released from endogenous glycoproteins and gangliosides by intrinsic sialidase determined. Radiolabel was incorporated into synaptic membrane gangliosides and glycoproteins, and at all times tested, >90% of the label was associated with sialic acid. Sialic acid released from endogenous glycoproteins by intrinsic sialidase present in 28-day membranes incorporated only 20–25% as much radiolabel per nmole as sialic acid released by mild acid hydrolysis or by exogenous neuraminidase. In contrast, sialic acid released from glycoproteins present in 13-day-old membranes by intrinsic sialidase, mild acid hydrolysis, or exogenous neuraminidase all were similarly labelled. At both ages the specific radioactivity (cpm/nmol) of sialic acid released from gangliosides by the intrinsic enzyme was similar to the total ganglioside sialic acid released by mild acid hydrolysis. The results identify glycoprotein substrates for intrinsic synaptic membrane sialidase as a distinct metabolic class in the mature brain and suggest the occurrence of a developmentally related change in the metabolism of these glycoproteins.     

STUDIES ON THE BIOGENESIS OF SMOOTH ENDOPLASMIC RETICULUM MEMBRANES IN HEPATOCYTES OF PHENOBARBITAL-TREATED RATS : II. The Site of Phospholipid Synthesis in the Initial Phase of Membrane Proliferation

The specific activity of the acyltransferases of smooth microsomes of rat liver rose threefold by 12 h after injection of phenobarbital, while the activity of the acyltransferases of the rough microsomes rose slightly to peak at 3–4 h, and subsequently fell. The latter rise was abolished by treatment of the animal with actinomycin D or puromycin, while that of the smooth microsomes was unaffected. Incorporation of [¹⁴C]glycerol into phospholipid of smooth microsomes was elevated 100% by phenobarbital, while that of the rough microsomes was elevated 15%, and this could be accounted for by exchange between the microsomal phospholipids. The phospholipid/protein ratio of the smooth microsomes rose 1.5 times 3–4 h after injection of phenobarbital, while that of the rough microsomes fell slightly. The specific activity of NADPH cytochrome c reductase and NADPH diaphorase rose first in the rough microsomes, and subsequently in the smooth microsomes at a time coinciding with the return of the phospholipid/protein ratio to the control level. The rise in phospholipid/protein ratio was unaffected by actinomycin D or puromycin. These results indicate that the proliferating smooth membranes are the site of phospholipid synthesis, and that the phospholipid/protein ratio of these membranes may change independently.     

Modulation of carbachol-stimulated inositol phospholipid hydrolysis in rat cerebral cortex

Cortical slices from rat brain were used to study carbachol-stimulated inositol phospholipid hydrolysis. Omission of calcium during incubation of slices with [³H]inositol increased its incorporation into receptor-coupled phospholipids. Carbachol-stimulated hydrolysis of [³H]inositol phospholipids in slices was dose-dependent, was affected by the concentrations of calcium and lithium present and resulted in the accumulation of mostly [³H]inositol-l-phosphate. Incubation of slices withN-ethylmaleimide or a phorbol ester reduced the response to carbachol. Membranes prepared from cortical slices labeled with [³H]inositol retained the receptor-stimulated inositol phospholipid hydrolysis reaction. The basal rate of inositol phospholipid hydrolysis was higher than in slices and addition of carbachol further stimulated the process. Addition of GTP stimulated inositol phospholipid hydrolysis, suggesting the presence of a guanine nucleotide-binding protein coupled to phospholipase C. Carbachol and GTP-stimulated inositol phospholipid hydrolysis in membranes was detectable following a 3 min assay period. In contrast to slices, increased levels of inositol bisphosphate and inositol trisphosphate were detected following incubation of membranes with carbachol. These results demonstrate that agonist-responsive receptors are present in cortical membranes, that the receptors may be coupled to phosphatidylinositol 4,5-bisphosphate, rather than phosphatidylinositol, hydrolysis and that a guanine nucleotide-binding protein may mediate the coupling of receptor activation to inositol phospholipid hydrolysis in brain.     

PRODUCTION OF MEMBRANE WHORLS IN RAT LIVER BY SOME INHIBITORS OF PROTEIN SYNTHESIS

Inhibitors of protein synthesis capable of differential effects on nascent peptide synthesis on membrane-bound and free polyribosomes were employed to investigate the structure and function of cellular membranes of liver. The formation of membranous whorls in the cytoplasm and distension of nuclear membranes were induced by inhibitors of protein synthesis (i.e., cycloheximide and emetine) which predominantly interfere with nascent peptide synthesis on membrane-bound polyribosomes in situ. Other inhibitors of protein synthesis such as puromycin and fusidic acid, which inhibit nascent peptide synthesis on both free and membrane-bound polyribosomes, and chloramphenicol, which inhibits mitochondrial protein synthesis, did not induce these alterations. Cycloheximide, puromycin, and chloramphenicol produce some common cellular lesions as reflected by similar alterations in morphology, such as swelling of mitochondria, degranulation of rough endoplasmic reticulum, and aggregation of free ribosomes. The process of whorl formation in the cytoplasm, the incorporation of [³H]leucine and of [³H]choline into endoplasmic reticulum and the total NADPH-cytochrome c reductase activity of the endoplasmic reticulum were determined. During maximum formation of membranous whorls, [³H]leucine incorporation into cytoplasmic membranes was inhibited, while [³H]choline incorporation into these structures was increased; maximum inhibition of protein synthesis and stimulation of choline incorporation into endoplasmic reticulum, however, preceded whorl formation. Cycloheximide decreased the activity of NADPH-cytochrome c reductase of rough endoplasmic reticulum, but increased NADPH-cytochrome c reductase activity of smooth endoplasmic reticulum. In addition, cycloheximide decreased the content of hemoprotein in both the microsomal and mitochondrial fractions of rat liver, and the activities of mixed function oxidase and of oxidative phosphorylation were impaired to different degrees. Succinate-stimulated microsomal oxidation was also inhibited. The possible mechanisms involved in the formation of membranous whorls, as well as their functions, are discussed.     

Evidence for the Involvement of Docosahexaenoic Acid in Cholinergic Stimulated Signal Transduction at the Synapse

[4,5-³H]Docosahexaenoic acid ([³H]DHA) or [9,10-³H]palmitic acid ([³H]PAM) was infused intravenously for 5 min to awake, adult male rats before and after treatment with arecoline (15 mg/kg, i.p.), a cholinergic agonist. Animals were killed 15 min post-infusion, the brains were rapidly removed and subcellular fractions were obtained after sucrose density centrifugation. In control animals, [³H]DHA and [³H]PAM were incorporated into the synaptosomal fractions, representing 50%–60% of total membrane label. Most remaining membrane label (30%–40%) was in the microsomal fraction. Both fractions contained the synaptic marker synaptophysin. The remaining 10% of radioactivity was in the myelin and mitochondrial fractions. Arecoline significantly increased [³H]DHA entry into the synaptosomal fractions by 100% and into the microsomal fraction by 50%. In these fractions 60%–65% of the [³H]DHA was in phospholipid, the rest corresponding to free fatty acid and diacylglycerol. In contrast, arecoline did not change [³H]PAM incorporation into any brain fraction. These results demonstrate that plasma [³H]DHA incorporation is selectively increased into synaptic membrane phospholipids of the rat brain in response to cholinergic activation. The increased incorporation of DHA but not of PAM into synaptic membranes in response to cholinergic stimulation indicates a primary role for DHA in phospholipid mediated signal transduction at the synapse involving activation of phospholipase A₂ and/or C.     

The repair of the surface structure of animal cells

Experiments were performed to determine if animal cells in culture possess specific mechanisms to repair surface molecules damaged by enzymes. The surface membranes of a primary cell culture, chick fibroblasts, a permanent hamster cell line, BHK₂₁/C₁₃, and its virally transformed counterpart, C₁₃/B₄ were damaged by exposure to trypsin or to neuraminidase. Following digestion with trypsin, the incorporation of radioactive amino acids or sugars into purified surface membrane of cells was monitored. No differences were noted in rates of incorporation when control and trypsin-damaged cells were compared. Neuraminidase damage to the surface of BHK₂₁/C₁₃ and C₁₃/B₄ cells was evidenced by altered gel filtration profiles of surface glycopeptides, i.e., delayed elution because of reduction in size. By labelling cells with ¹⁴C-L-fucose prior to neuraminidase treatment and following the incorporation of ³H-L-fucose into cell surface glycopeptides after neuraminidase digestion, we were able to monitor the synthesis and turnover of fucose-containing glycopeptides in the same cells. Gel filtration profiles indicated that little or no desialylated glycoproteins were resialylated (repaired) by specific replacement of sialic acid. Comparing neuraminidase-digested and control cells we observed no difference in rates of ³H-L-fucose incorporation or of ¹⁴C-L-fucose loss from these cells; nor did we find differences in the rate of incorporation of isotopic glucosamine into sialic acid. Neuraminidase treatment failed to alter the rate of cell growth or the pattern of isotopic incorporation into various cell surface components. These results support the suggestion that return of sialic acid (repair) was effected by turnover which serves as a non-specific repair mechanism to replace damaged cell surface molecules (Warren and Glick '68; Warren, '69).     

Perturbational effects of inorganic cations on human erythrocyte membranes

Summary The perturbational effects of monovalent and divalent cations on human erythrocyte membranes were analyzed by examining their influence on kinetic and structural characteristics of trinitrobenzenesulfonic acid (TNBS) incorporation into the amino groups of protein and phospholipid structural components. The stimulatory effects of monovalent cations on TNBS incorporation, which were size-independent and attributed to nonspecific membrane alterations resulting from ionic strength factors, contrasted with the more pronounced stimulatory properties of divalent cations which were markedly size-dependent. These stimulatory effects of cations on TNBS incorporation were associated with alterations not only in rate but also in activation energy of incorporation. Changes in activation energy produced by divalent cations paralleled their ability to perturb membrane protein components and probably reflected changes in probe permeation. The rate of TNBS incorporation exhibited a dependence on divalent cation ionic radius which paralleled ion-induced perturbations in the labelling of the membrane amino phospholipid phosphatidylethanolamine. Divalent cations differed both in the relative extent and in the characteristics of protein and phospholipid perturbation. Alkaline earth cations behaved as a rather homogeneous group while Ni⁺⁺, Co⁺⁺ and Mn⁺⁺ constituted a second heterogeneous group. The influence of monovalent and divalent cations on the hemolytic behavior of intact erythrocytes paralleled their effects on TNBS incorporation into isolated membranes rather closely. It is suggested that TNBS incorporation may provide a valuable means of analyzing functionally relevant cation-induced alterations in biological membranes in general.     

Incorporation of galactose from UDP-galactose into microsomal and golgi membranes of rat liver

Rough and smooth microsomes and Golgi membranes were incubated with UDP[¹⁴C]galactose and the incorporation of radioactivity into the lipid extract and into endogenous protein acceptors were measured. Antagonistic pyrophosphatases were inhibited with ATP and interference from β-galactosidase activity was greatly decreased by carrying out the incubation at pH 7.8. After incubation the particles were centrifuged to remove free oligosaccharide residues. Radioactivity was found in the lipid extract from Golgi membranes but not from rough and smooth microsomes. This radioactivity, however, was not associated with dolichol or retinyl phosphates. The incorporation of radioactivity into proteins of the Golgi fraction was more than double than that of the microsomal fractions. In addition, the transferases in these two types of particles exhibited different properties. Trypsin treatment of intact rough microsomal vesicles, smooth vesicles and Golgi membranes removed about 5, 15 and 50%, respectively, of newly incorporated protein-bound galactose, indicating that the proportion of the newly galactosylated proteins, which are localized at the cytoplasmic surface of the membrane, is lowest in rough microsomes, intermediate in smooth, and highest in Golgi membranes.     

Differential distribution and metabolism of arachidonic acid and docosahexaenoic acid by human placental choriocarcinoma (BeWo) cells

The time course of incorporation of [¹⁴C]arachidonic acid and [³H]docosahexaenoic acid into various lipid fractions in placental choriocarcinoma (BeWo) cells was investigated. BeWo cells were found to rapidly incorporate exogenous [¹⁴C]arachidonic acid and [³H] docosahexaenoic acid into the total cellular lipid pool. The extent of docosahexaenoic acid esterification was more rapid than for arachidonic acid, although this difference abated with time to leave only a small percentage of the fatty acids in their unesterified form. Furthermore, uptake was found to be saturable. In the cellular lipids these fatty acids were mainly esterified into the phospholipid (PL) and the triacyglycerol (TAG) fractions. Smaller amounts were also detected in the diacylglycerol and cholesterol ester fractions. Almost 60% of the total amount of [3H]Docosahexaenoic acid taken up by the cells was esterified into TAG whereas 37% was in PL fractions. For arachidonic acid the reverse was true, 60% of the total uptake was incorporated into PL fractions whereas less than 35% was in TAG. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes. The higher incorporation of docosahexaenoic acid and arachidonic acid was found in PC and PE, respectively. The greater cellular uptake of docosahexaenoic acid and its preferential incorporation in TAG suggests that both uptake and transport modes of this fatty acid by the placenta to fetus is different from that of arachidonic acid.     

Involvement of phospholipids in triglyceride biosynthesis by developing soybean cotyledons

The incorporation of phospholipids specifically labeled with glycerol-2³H and acyl-¹⁴C by whole cell tissues of developing soybean cotyledons (Glycine max L.) reveals that phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, N-acylphosphatidylethanolamine, and phosphatidic acid can be metabolized to diglyceride. The diglyceride formed may be recylced into phospholipid or acylated to triglyceride. Diglyceride from phosphatidic acid and phosphatidylethanolamine is used readily in triglyceride biosynthesis compared to the other phospholipids. Incorporation of N-acylphosphatidylethanolamine having [9-10-³H(N)]oleic acid esterified at sn-3 in cotyledons shows rapid acyltransfer of ³H into triglyceride and therefore N-acylphosphatidylethanolamine appears to participate in triglyceride biosynthesis as an acyl donor. These studies emphasize phospholipid metabolism in developing soybean cotyledons is a dynamic process which plays a key role in triglyceride formation.     

Tissue distribution of EDTA encapsulated within liposomes containing glycolipids or brain phospholipids

Multilamellar liposomes were prepared with various asialoglycolipids, gangliosides, sialic acid, or brain phospholipids in the liposome membrane and with ethylenediaminetetraacetic acid (EDTA) encapsulated in the aqueous compartments. The liposomes containing glycolipids or sialic acid were prepared from a mixture of phosphatidylcholine, cholesterol, and one of the following test substances: galactocerebroside, glucocerebroside, galactocerebroside sulfate, mixed gangliosides, monosialoganglioside G_M1, monosialoganglioside G_M2, monosialoganglioside G_M3, disialoganglioside G_D1a, or sialic acid. The liposomes containing brain phospholipids were mixtures of either sphingomyelin and cholesterol or a brain total phospholipid extract and cholesterol. Distribution of ¹⁴C-labeled EDTA were determined in mouse tissues from 15 min to 6 h or 12 h after a single injection of liposome prepartion. Liver uptake of encapsulated EDTA was lowest from all liposome preparations containing sialic acid or sialogangliosides regardless of the amount of sialic acid moiety present or the identity of the particular ganglioside; highest uptake of encapsulated EDTA by liver was from the liposomes containing galactocerebroside or brain phospholipids. Lungs and brain took up the largest amounts of EDTA from liposomes containing sphingomyelin and lesser amounts from liposomes containing G_D1a. Use of mouse brain phospholipid extract to prepare liposomes did not increase uptake of encapsulated EDTA by the brain. EDTA in liposomes containing monosialogangliosides, brain phospholipids, galactocerebroside, or sialic acid was taken up well by spleen and marrow. Highest thymus uptake of encapsulated EDTA was from liposomes containing G_D1a. These results demonstrate that inclusion of sialogangliosides in liposome membranes decreases uptake of liposomes by liver, thus making direction of encapsulated drugs to other organs more feasible. Liposomes containing glycolipids also have potential uses as probes of cell surface receptors.     

ONTOGENETIC CHANGES OF PROTEINS OF ENDOPLASMIC RETICULUM

The proteins of the smooth and rough endoplasmic reticulum from fetal, immature, and adult male rats were compared after incorporation of two radioactively labeled precursors, ¹⁴C-labeled amino acids and δ-aminolevulinic acid-³H by means of gel electrophoresis. The labeling patterns indicated that protein components present in two major electrophoretic bands underwent significant synthesis in fetal tissue while three actively incorporating protein bands were noted in adult tissue. Although the uptake of the amino acids-¹⁴C decreased for the smooth and rough elements of the endoplasmic reticulum as a whole during liver development, the qualitative patterns were not significantly different in adult and fetal livers. The over-all incorporation (disintegrations per minute per milligram protein) of the heme precursor into the smooth and rough elements also did not change with development. However, a change was noted in the distributional electrophoretic patterns with development. The estimation of molecular weight (by disc electrophoresis) and the incorporation of the heme precursor suggested the similarity of the two major protein bands to cytochrome P-450 and cytochrome b₅, components of the endoplasmic reticulum, thought to be involved in the mixed-function oxidase system. The evidence indicated that in fetal liver, at a time when the oxidase capability was low, the amino acid incorporation into these two protein groups was the same as in the adult. The incorporation of the heme moiety, however, was different, decreasing in the cytochrome b₅ region and increasing in the cytochrome P-450 region during development. These results correlate with the increase in oxidase activity associated with liver development.     

Phospholipid metabolism of stimulated lymphocytes: Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [³H]arachidonate and [¹⁴C]oleate or [³H]arachidonate and [¹⁴C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3–7-fold) and phosphatidylethanolamine (2–3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.     

Effects of exogenous phospholipases on brain membrane phospholipid perturbation, (Na+ + K+)-ATPase activity and cellular swelling of brain slices

Rat brain membranes were incubated with bee venom phospholipase A₂ (PLA₂) or phospholipase C (PLC) from Clostridium perfringens. PLA₂ caused a significant increase in free polyunsaturated fatty acids concomitant with membrane phospholipid degradation as monitored by HPLC and by gas chromatography. Equal concentrations of PLC had a much lesser effect than PLA₂. Divergent and differential effects were shown on deacylation and incorporation of [³H]arachidonic acid in membrane phospholipids. The incorporation of [³H]arachidonic acid into various phospholipids was greatly reduced by PLA₂ (0.018 units/ml) whereas PLC at identical concentration was not effective. PLA₂ inhibited (Na⁺ + K⁺)-ATPase but was not effective on p-nitrophenyl-phosphatase activity whereas PLC stimulated both enzymes. PLA₂ induced swelling of cortical brain slices whereas PLC was not effective. Thus, the severity of the perturbation of membrane integrity, and the inhibition of (Na⁺ + K⁺)-ATPase in brain membranes may play an important role in cellular swelling of brain slices induced by PLA₂.