Association of sialic acid with microsomal membrane structures in rat liver

The amount of sialic acid on phospholipid basis increases from rough, through smooth II and smooth I microsomes, to Golgi membranes, all of them free from most of the adsorbed and luminal protein. The incorporation rate of glucosamine-³H into sialic acid also follows a similar order. Deoxycholate removes phospholipid and sialic acid to an identical extent, and a significant part of the latter remains after trypsin and neuraminidase treatment. The sialic acid/phospholipid ratio decreases in phenobarbital-induced smooth but not in rough membranes, while the incorporation rate of glycosamine-³H into sialic acid decreases in both subfractions.     

Membranes of Tetrahymena. IV. Isolation and characterization of temperature-responsive smooth and rough microsomal subfractions.

Temperature-responsive microsomes of the ciliate protozoan Tetrahymena have been originally fractionated by step centrifugation on two-layered, Mg2+-containing sucrose gradients. Three fractions have been obtained, which are termed smooth I, smooth II and rough according to the appearance of the membrane vesicles upon electron-microscopy. Smooth I, smooth II, and rough microsomes exhibit RNA/protein ratios of 0.09, 0.20, and 0.34; their phospholipid/protein ratios and their neutral lipid/phospholipid ratios were 0.52, 0.43 and 0.25, and 0.17, 0.18 and 0.13, respectively. All three fractions contain equivalent, low succinic dehydrogenase and 5'-nucleotidase activities. Glucose-6-phosphatase and acid phosphatase are more concentrated in smooth I membranes than in rough membranes. The reverse is true for ATPase. The smooth II membranes occupy an intermediate position except that their ATPase activity is the lowest of the three fractions. The specific activities of these enzymes of the three microsomal fractions are compared to those of homogenates of whole cells. Thin-layer chromatography reveals a very similar polar and nonpolar lipid pattern of the three microsomal fractions. The major phospholipid compounds are phosphatidlethanolamine, glycerideaminoethylphosphonate and phosphatidylcholine, while diglycerides, an unknown NL-compound, and triglycerides are the major apolar lipids. Gas liquid chromatography shows that the fatty acids are mainly even-numbered ranging between C12 and C18. The smooth I, smooth II and rough membranes contain 65.2, 69.3 and 72.7% unsaturated fatty acids in their polar lipids, whereas only 52.7, 49.7 and 48.3% unsaturated acids are found in their apolar lipids, respectively. The fatty acids are more unevenly distributed among the individual polar lipids than in the apolar ones.     

COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG : II. Lipids

The lipid composition of rough and smooth microsomal membranes, zymogen granule membranes, and a plasmalemmal fraction from the guinea pig pancreatic exocrine cell has been determined. As a group, membranes of the smooth variety (i.e., smooth microsomes, zymogen granule membranes, and the plasmalemma) were similar in their content of phospholipids, cholesterol and neutral lipids, and in the ratio of total lipids to membrane proteins. In contrast, rough microsomal membranes contained much less sphingomyelin and cholesterol and possessed a smaller lipid/protein ratio. All membrane fractions were unusually high in their content of lysolecithin (up to ~20% of the total phospholipids) and of neutral lipids, especially fatty acids. The lysolecithin content was shown to be due to the hydrolysis of membrane lecithin by pancreatic lipase; the fatty acids, liberated by the action of lipase on endogenous triglyceride stores, are apparently scavenged by the membranes from the suspending media. Similar artifactually high levels of lysolecithin and fatty acids were noted in hepatic microsomes incubated with pancreatic postmicrosomal supernatant. E 600, an inhibitor of lipase, largely prevented the appearance of lysolecithin and fatty acids in pancreatic microsomes and in liver microsomes treated with pancreatic supernatant.     

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.     

Phosphatidylcholine synthesis for incorporation into membranes or for secretion as plasma lipoproteins by Golgi membranes of rat liver

Phosphatidylcholine, the major phospholipid of very low density lipoproteins, is packaged with triglyceride in the Golgi cisternae. CTP-phosphocholine cytidyltransferase and CDP-choline phosphotransferase activities of Golgi subfractions were higher than those of rough or smooth microsomes measured under the same conditions, indicating that phosphatidylcholine synthesis can occur in Golgi membranes. Consistent with this, the specific activity of phosphatidylcholine of Golgi membranes rose more rapidly than that of rough and smooth microsomes after injection of [14C]choline in vivo. The specific activity of the Golgi content phosphatidylcholine (non-membrane fraction) remained low. The S-adenosylmethionine phosphatidylethanolamine methyltransferase activity of Golgi subfractions was also higher than that of rough or smooth microsomes. After injection of [3H]methyl-labeled methionine in vivo, the specific activity of phosphatidylcholine of the Golgi membranes rose in parallel with that of the rough and smooth microsomes. The specific activity of the Golgi content phosphatidylcholine rose above that of the Golgi membranes and exhibited a different pattern, suggesting that this pathway may selectively label phosphatidylcholine which is secreted as lipoproteins. These observations indicate that the Golgi membranes have the enzymes necessary for synthesis of phosphatidylcholine, and incorporation of lipid precursors indicates that synthesis of phosphatidylcholine by Golgi membranes occurs in vivo.     

Distribution of protein-bound sugar residues in microsomal subfractions and Golgi membranes

Liver microsomal subfractions and Golgi membranes free from adsorbed and secretory proteins have a characteristic sugar composition. The ratio of mannose to galactose is largest in rough microsomes, smaller in smooth I microsomes, still smaller in smooth II microsomes, and smallest in Golgi membranes. There is about twice as much glucosamine in Golgi membranes and 3 times as much in smooth II microsomes as in the other microsomal subfractions. Golgi membranes are rich in sialic acid in comparison to rough microsomes and it is present at even higher levels in the two smooth microsomal subfractions. Increasing concentrations of deoxycholate preferentially remove protein-bound mannose and glucosamine, while releasing significantly less galactose. About half of the microsomal mannose and galactose can be liberated from the surface of intact microsomal vesicles by treatment with trypsin. When trypsin is added to permeable vesicles where the inside surface can be also attacked, an additional 20% of the total mannose but no additional galactose is liberated.     

Distribution of protein-bound sugar residues in microsomal subfractions and Golgi membranes.

Liver microsomal subfractions and Golgi membranes free from adsorbed and secretory proteins have a characteristic sugar composition. The ratio of mannose to galactose is largest in rough microsomes, smaller in smooth I microsomes, still smaller in smooth II microsomes, and smallest in Golgi membranes. There is about twice as much glucosamine in Golgi membranes and 3 times as much in smooth II microsomes as in the other microsomal subfractions. Golgi membranes are rich in sialic acid in comparison to rough microsomes and it is present at even higher levels in the two smooth microsomal subfractions. Increasing concentrations of deoxycholate preferentially remove protein-bound mannose and glucosamine, while releasing significantly less galactose. About half of the microsomal mannose and galactose can be liberated from the surface of intact microsomal vesicles by treatment with trypsin. When trypsin is added to permeable vesicles where the inside surface can be also attacked, an additional 20% of the total mannose but no additional galactose is liberated.     

Fatty-acid composition of lipids and physicochemical characteristics of membrane fractions and the membrane of endoplasmic reticulum of thymus and Pliss' lymphosarcoma

The paper is concerned with composition of neutral lipids and phospholipids, the regularity of lipid bilayer and structural reorganization of plasma membranes, and membranes of smooth and rough cell reticulum of thymus and Pliss lymphosarcoma are studied at linear and stationary growth phase. No qualitative differences are found in the fatty-acid composition of lipid membranes in normal and tumour cells. In plasma membranes of phospholipids and in membranes of smooth reticulum of tumour cells the unsaturated lipid component increases in the process of growth, the cholesterin/phospholipids ratio decreases, fluidity of the lipid bilayer diminishes and structural heterogeneity of these membranes rises while in membranes of rough reticulum the saturation of lipids increases, but the cholesterin/phospholipids ratio does not change. The temperatures of structural reorganization also does not change, which evidences for a less structural lability of membranes of rough reticulum as compared with other membranes.     

Lipid composition of subcellular particles of human blood platelets

Human platelets can be fractionated into three main subcellular components: granules, membranes, and a soluble fraction. In this study we determined the phospholipid and neutral lipid content of the granules and membranes. Quantitative relationships between lipids and protein were examined. The fatty acid and aldehyde composition of individual phospholipids and neutral lipids was also determined. Whole platelets had a lower lipid to protein ratio than did the subcellular particles, but the basic lipid composition of the granules, membranes, and platelets was similar. The phospholipid composition of platelets and subcellular fractions was found to differ only in that granules had a lower percentage of lecithin. Each of the phospholipid classes displayed a distinctive fatty acid pattern which was the same in all fractions and in whole platelets. The major neutral lipid was free cholesterol. Cholesteryl esters, triglycerides, and free fatty acids were minor components. The molar ratio of cholesterol to phospholipid in the platelet membranes was lower than that of brain myelin and erythrocyte ghosts. Some differences in fatty acid composition of the neutral lipids of platelet fractions were found. A special lipid composition or constituent that would correlate with platelet function has not been found.     

Lipid Composition of Purified Vesicular Stomatitis Viruses

Methods are described for the production of vesicular stomatitis (VS) virus of sufficient purity for reliable chemical analysis. VS virions released from infected cells were concentrated and purified at least 150-fold by sequential steps of precipitation with polyethylene glycol, column chromatography, rate zonal centrifugation, and equilibrium centrifugation. The Indiana serotype (VS(Ind) virus) propagated in L-cells was found to contain 3% ribonucleic acid, 64% protein, 13% carbohydrate, and 20% lipid; the molar ratio of cholesterol to phospholipid was 0.6 or greater. Thin-layer chromatography revealed no unusual neutral lipids or phospholipids and gas-liquid chromatography revealed no unusual fatty acids incorporated into VS virions. The antigenically distinct New Jersey serotype (VS(NJ) virus) grown in L-cells showed a similar lipid profile except that the proportion of neutral lipids was larger than in VS(Ind) virus also grown in L-cells. This differences was less pronounced when the lipid composition of VS(Ind) and VS(NJ) viruses grown in chick embryo cells was compared, but VS(NJ) virus grown in either cell type always contained larger amounts of neutral lipids other than cholesterol than did VS(Ind) virus. The lipid composition of both VS(Ind) and VS(NJ) viruses grown in L-cells or chick embryo cells more closely resembled that of plasma membrane than of whole cells. A consistent finding was the relatively large amounts of phosphatidylethanolamine and sphingomyelin and the relatively small amounts of phosphatidylcholine in both VS viruses compared with uninfected whole L-cells and chick embryo cells or their plasma membranes. The methods available for isolation of plasma membranes were inadequate for conclusive comparison of the lipids of VS virions with the lipids of the plasma membranes of their host cells. Nevertheless, the data obtained are consistent with two hypotheses: (i) the lipid composition of VS viruses primarily reflects their membrane site of maturation, and (ii) the newly synthesized viral proteins inserted into cell membranes influence the proportions of phospholipids and neutral lipids selected for incorporation into the viral membrane.     

Liver ischemia increases the molecular order of microsomal membranes by increasing the cholesterol-to-phospholipid ratio

An accelerated degradation of phospholipid is the likely basis of irreversible cell injury in ischemia, and the membranes of the endoplasmic reticulum of the liver are a convenient system with which to study the effect of such a disturbance on the structure and function of cellular membranes. In the present report, electron spin resonance spectroscopy has been used to evaluate changes in the molecular ordering of microsomal membrane phospholipids in the attempt to relate the loss of lipid to alterations in membrane structure. The order parameter, S, was calculated from spectra reflecting the anisotropic motion of 12-doxyl stearic acid incorporated into normal and 3-h ischemic microsomal membranes. Over the temperature range 4-40 degrees C, the molecular order (S) of ischemic membranes was increased by 8-10%. This increase was reproduced in the ordering of the phospholipids in liposomes prepared from total lipid extracts of the same membranes. In contrast, after removal of the neutral lipids, liposomes prepared from phospholipids of ischemic and control membranes had the same molecular order. There were no differences in the phospholipid species of control and ischemic membranes or in the fatty acid composition of the phospholipids. In the neutral lipid fraction of ischemic membranes, however, triglycerides and cholesterol were increased compared to control preparations. There were no free fatty acids. The total cholesterol content of the liver was unchanged after 3 h of ischemia. The cholesterol-to-phospholipid ratio of ischemic membranes, however, was increased by 22% from 0.258 to 0.315 as a consequence of the loss of phospholipid. Addition of cholesterol to the control total lipid extracts to give a cholesterol-to-phospholipid ratio the same as in ischemic membranes resulted in liposomes with order parameters similar to those of liposomes prepared from ischemic total lipids. It is concluded that the degradation of the phospholipids of the microsomal membrane results in a relative increase in the cholesterol-to-phospholipid ratio. This is accompanied, in turn, by an increased molecular order of the residual membrane phospholipids.     

Study of electrophoretic mobility of cellular membranes isolated from rat liver

Plasma membranes as well as mitochondrial and microsomal subfractions were subjected to zone electrophoresis. Treatment with neuraminidase, phospholipase A or C does not influence the movement of plasma membranes and smooth microsomes. Trypsin increases mobility of plasma membranes and smooth by about 20%, and further treatment with phospholipase C decreases mobility of plasma membranes, total smooth and smooth I microsomes, which, however, is not the case with smooth II microsomes. Low concentrations of trypsin also solubilize enzyme proteins of smooth microsomes from phenobarbital-treated rat liver, but electrophoretic mobility is not increased, indicating structural differences in induced membranes. The mobility of the outer and inner mitochondrial membranes is significantly higher than that of submitochondrial particles. For microsomes the negative surface charge density occurs in the decreasing order of: ribosomes — rough — smooth I — smooth II. A 10 mM CsCl gradient decreases the mobility of rough microsomes by 40% and of ribosomes by 20% but has no effect on total smooth microsomes. On the other hand, 5 mM MgCl₂ decreased the mobility of all three fractions. EDTA-treated rough and EDTA-treated smooth microsomes have the same electrophoretic mobilities. However, the mobilities of non-treated rough and smooth microsomes differ significantly from each other.     

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.     

Characterization of microsomal subfractions from porcine endometrium cells

Crude microsomes from porcine endometrium and three subfractions obtained by a modification of Rothschild's technique were characterized by RNA/protein ratio, marker enzyme activities and morphological appearance. The microsomes were devoid of glucose-6-phosphatase activity. They contained approximately 10% of arylesterase-, approximately 30% of both NADPH-cytochrome reductase- and UDPgalactose-N-acetyl-glucosamine beta-D-galactosyltransferase- and approximately 60% of 5'-nucleotidase activities present in the homogenates. Subfraction I (smooth membranes) had twice the galactosyltransferase activity of Subfraction II (smooth and rough membranes + free ribosomes); both subfractions were rich in 5'-nucleotidase and cytochrome reductase activities. Subfraction III (rough membranes) had very low marker activities but exhibited the highest RNA/protein ratio, which was lowest in I.     

Schistosoma mansoni: synthesis and release of phospholipids, lysophospholipids, and neutral lipids by schistosomula

Lipids in the two surface membranes of Schistosoma mansoni may play an important role in the parasite's defense against host immunity. In particular, lysophosphatidylcholine lyses erythrocytes attached to the parasite and alters the lateral mobilities of their membrane proteins and lipids (Golan et al. 1986). Here, we have studied the incorporation of radiolabeled precursors into the major lipid classes of schistosomula as well as into lipids released by schistosomula into the medium. Radiolabeled polar head groups (choline and ethanolamine) and fatty acid precursors (palmitate and oleate) were linearly incorporated into parasite phospholipids. Fatty acids were differentially incorporated into the various phospholipid classes, principally into phosphatidylcholine and, to a lesser extent, into phosphatidylethanolamine, lysophosphatidylcholine, and phosphatidylserine. The major neutral lipid class labeled, triglycerides, had a decrease in specific activity with time after pulse labeling and the specific activity of the phospholipids increased with time. Thus, triglycerides may provide acyl chains for phospholipid synthesis. Choline was incorporated into phosphatidylcholine and lysophosphatidylcholine, and ethanolamine into phosphatidylethanolamine and lysophosphatidylethanolamine. No evidence was found for phospholipid methylation or demethylation in schistosomula. Labeled lipids were linearly and selectively released into the medium. Triglycerides were released at the highest rate with measurable quantities of phosphatidylcholine, lysophosphatidylcholine, and phosphatidylethanolamine also observed. Monopalmitoylphosphatidylcholine was the only lysophosphatidylcholine present in the medium as demonstrated by reverse-phase chromatography of released choline-labeled lysophosphatidylcholine. These studies demonstrate that schistosomula synthesize phospholipids and neutral lipids and release some of them into the culture medium. In particular, they release a single molecular species of a potent biologically active molecule, monopalmitoylphosphatidylcholine, that may play a role in the parasite's evasion of the immune response.     

Incorporation and distribution of dihomo-gamma-linolenic acid, arachidonic acid, and eicosapentaenoic acid in cultured human keratinocytes

Human keratinocytes in culture were labelled with 14C-dihomo-gamma-linolenic acid, 14C-arachidonic acid or 14C-eicosapentaenoic acid. All three eicosanoid precursor fatty acids were effectively incorporated into the cells. In phospholipids most of the radioactivity was recovered, in neutral lipids a substantial amount, and as free unesterified fatty acids only a minor amount. The most of the radioactivity was found in phosphatidylethanolamine which was also the major phospholipid as measured by phosphorous assay. The incorporation of dihomo-gamma-linolenic acid and arachidonic acid into lipid subfractions was essentially similar. Eicosapentaenoic acid was, however, much less effectively incorporated into phosphatidylinositol + phosphatidylserine and, correspondingly, more effectively into triacylglycerols as compared to the two other precursor fatty acids. Once incorporated, the distribution of all three precursor fatty acids was relatively stable, and only minor amounts of fatty acids were released into the culture medium during short term culture (two days). Our study demonstrates that eicosanoid precursor fatty acids are avidly taken up by human keratinocytes and esterified into membrane lipids. The clinical implication of this finding is that dietary manipulations might be employed to cause changes in the fatty acid composition of keratinocytes.     

Study of electrophoretic mobility of cellular membranes isolated from rat liver.

Plasma membranes as well as mitochondrial and microsomal subfractions were subjected to zone electrophoresis. Treatment with neuraminidase, phospholipase A or C does not influence the movement of plasma membranes and smooth microsomes. Trypsin increases mobility of plasma membranes and smooth by about 20%, and further treatment with phospholipase C decreases mobility of plasma membranes, total smooth and smooth I microsomes, which, however, is not the case with smooth II microsomes. Low concentrations of trypsin also solubilize enzyme proteins of smooth microsomes from phenobarbital-treated rat liver, but electrophoretic mobility is not increased, indicating structural differences in induced membranes. The mobility of the outer and inner mitochondrial membranes is significantly higher than that of submitochondrial particles. For microsomes the negative surface charge density occurs in the decreasing order of: ribosomes--rough--smooth I--smooth II. A 10 mM CsCl gradient decreases the mobility of rough microsomes by 40% and of ribosomes by 20% but has no effect on total smooth micromes. On the other hand, 5mM MgCl2 decreased the mobility of all three fractions. EDTA-treated rough and EDTA-treated smooth microsomes have the same electrophoretic mobilities. However, the mobilities of non-treated rough and smooth microsomes differ significantly from each other.     

Incorporation of 2-14C-acetate into isolated nuclei and cytosolic lipids of rat thymus cells

It is generally recognized nowadays that active lipid metabolism takes place in the nucleus of a mammalian cell. Experimental data testify to the biosynthesis of polyphosphoinositides and phosphatidylcholine and reveal corresponding enzymes within nuclei of mammalian cells. These findings suggest that lipidmediated signaling pathways in nuclei operate independently of lipid-mediated regulatory mechanisms functioning in membranes and cytosol. To explore the pathways of intranuclear lipid biosynthesis, we studied incorporation of 2-¹⁴C-acetate into lipids of cytosol and isolated nuclei of rat thymus cells after separate and combined incubation with the labeled precursor. The most efficient incorporation of 2-¹⁴C-acetate into lipids (cholesterol, free fatty acids, and phospholipids) was observed in a reaction mixture containing cytosol. When the reaction mixture contained only nuclei, incorporation of the radioactive precursor into lipids also took place, but specific radioactivity of the lipids was essentially lower than in the cytosol. In both cases, 2-¹⁴C-acetate incorporated into phosphatidylethanolamine, sphingomyelin, phosphatidylserine, phosphatidylinositol, and cardiolipin. Phosphatidylcholine, the most abundant membrane phospholipid, demonstrated the lowest radioactivity, which was significantly lower than that of phosphatidylethanolamine. Incorporation of newly synthesized free fatty acids in nuclear phospholipids was inhibited, if nuclei were incubated with cytosol. As a result, radioactive free fatty acids were accumulated in nuclei, while in cytosol they were efficiently incorporated into phospholipids. The levels of phospholipids and cholesterol remained constant regardless of incubation protocol, while the overall yield of free fatty acids decreased after combined incubation of nuclear and cytosolic fractions or after incubation of cytosol without nuclei. Putative mechanisms underlying the appearance of radioactive lipids in isolated nuclei of thymus cells are discussed.     

Lipid composition and metabolism in testicular and ejaculated ram spermatozoa

1. Spermatozoa collected directly from the testis of the conscious ram contain 25% more phospholipid than ejaculated spermatozoa. The concentration of lecithin, phosphatidylethanolamine and ethanolamine plasmalogen was greater in testicular spermatozoa; little difference was observed in choline plasmalogen. Both types of spermatozoa had significant amounts of cardiolipin and alkyl ether phospholipid. 2. The fatty acids in the phospholipid extracted from testicular spermatozoa have a very high content of palmitic acid. The phospholipids of ejaculated spermatozoa contained less palmitic acid, but more myristic acid. 3. Ejaculated spermatozoa contained less acyl ester and cholesterol. It is suggested that lipids are a source of substrate for spermatozoa during their passage through the epididymis. 4. Testicular spermatozoa when incubated with [U-¹⁴C]glucose incorporated more radioactivity into the glycerol part of the phospholipid and neutral lipid fractions than did ejaculated cells. The distribution of radioactivity in the individual phospholipids and neutral lipids was similar for both cell types. No radioactivity was detected in choline plasmalogen, which accounted for approx. 40% of the total phospholipid. 5. Testicular spermatozoa incorporated more radioactivity from glucose into formate than into acetate, whereas a higher proportion of radioactivity was found in acetate in ejaculated cells. 6. The implications of these lipid changes in the process of spermatozoal maturation are discussed.     

Recovery of ribophorins and ribosomes in "inverted rough" vesicles derived from rat liver rough microsomes

Treatment of rat liver rough microsomes (3.5 mg of protein/ml) with sublytical concentrations (0.08%) of the neutral detergent Triton X-100 caused a lateral displacement of bound ribosomes and the formation of ribosomal aggregates on the microsomal surface. At slightly higher detergent concentrations (0.12-0.16%) membrane areas bearing ribosomal aggregates invaginated into the microsomal lumen and separated from the rest of the membrane. Two distinct classes of vesicles could be isolated by density gradient centrifugation from microsomes treated with 0.16% Triton X-100: one with ribosomes bound to the inner membrane surfaces ("inverted rough" vesicles) and another with no ribosomes attached to the membranes. Analysis of the fractions showed that approximately 30% of the phospholipids and 20-30% of the total membrane protein were released from the membranes by this treatment. Labeling with avidin-ferritin conjugates demonstrated that concanavalin A binding sites, which in native rough microsomes are found in the luminal face of the membranes, were present on the outer surface of the inverted rough vesicles. Freeze-fracture electron microscopy showed that both fracture faces had similar concentrations of intramembrane particles. SDS PAGE analysis of the two vesicle subfractions demonstrated that, of all the integral microsomal membrane proteins, only ribophorins I and II were found exclusively in the inverted rough vesicles bearing ribosomes. These observations are consistent with the proposal that ribophorins are associated with the ribosomal binding sites characteristic of rough microsomal membranes.