Subcellular sites of enzymes catalyzing the phosphorylation-dephosphorylation of dolichol in the central nervous system

The subcellular locations of several enzymes involved in dolichyl monophosphate (Dol-P) metabolism in brain have been investigated. Dolichol kinase is highly enriched in a heavy microsomal fraction from calf brain, while 71% of the Dol-P phosphatase activity was recovered with the light microsomes. Lower amounts of the phosphatase activity were also found in the heavy microsomal, mitochondrial-lysosomal, and synaptic plasma membrane fractions. Since the light microsomal fraction also contained substantial acetylcholinesterase activity, an axon plasma membrane marker, an axolemma-enriched fraction, was prepared from rat brain by a second procedure. A comparison with microsomal and mitochondrial-lysosomal fractions revealed that the axolemma-enriched fraction contained the highest specific activity of Dol-P phosphatase, indicating that the enzyme was present in the axon plasma membrane. The tunicamycin-sensitive UDP-N-acetylglucosamine:Dol-P N- acetylglucosaminylphosphotransferase , glucosyl- phosphoryldolichol (Glc-P-Dol) synthase, Glc-P-Dol:oligosaccharide glucosyltransferase, and the oligosaccharyltransferase were all found predominantly in the heavy microsomes. These results indicate that the enzymes responsible for the initiation and termination of biosynthesis, as well as the transfer of dolichol-linked oligosaccharides, reside in the rough endoplasmic reticulum (ER) of central nervous tissue. Evidence that at least some Dol-P molecules formed by dolichol kinase are accessible to multiple glycosyltransferases in the rough ER of brain is also presented.     

Localization and solubilization of a rat liver microsomal carnitine acetyltransferase.

Carnitine acetyltransferase activity had been previously shown to occur in peroxisomes, mitochondria, and a membranous fraction of rat and pig hepatocytes. When components of this third subcellular fraction (plasma membranes, components of the Golgi apparatus, and microsomes) were further separated, carnitine acetyltransferase fractionated with the microsomes. Microsomes isolated by three different methods (isopycnic sucrose density zonal centrifugation, high-speed differential centrifugation, and aggregation with Ca²⁺ followed by low-speed differential centrifugation) all contained carnitine acetyltransferase activity. The lability of carnitine acetyltransferase in microsomes isolated by different methods and in different isolation media is reported.When total microsomes were subfractionated into rough and smooth components, carnitine acetyltransferase activity was found to the same extent in both and was tightly associated with the microsomal membrane. The microsomal enzyme was rapidly inactivated in 0.25 m sucrose or 0.1 m phosphate, but was stable for at least 2 weeks in 0.4 m KCl. Extensive treatment with high ionic strength salt solutions, 1% Triton X-100, or a combination of the two was used to solubilize microsomal carnitine acetyltransferase activity.Carnitine octanoyltransferase activity was also found in the microsomal fractions isolated by three different methods, but no carnitine palmitoyltransferase was detected in the microsomal fractions. It is proposed that microsomal carnitine acetyl- and octanoyltransferases could be involved in the transfer of acyl groups across the microsomal membrane, thereby providing a source of acetyl and other acyl CoA's at sites of acetylation reactions and synthesis.     

Golgi-enriched membrane fractions from rat brain and liver contain long-chain polyisoprenyl pyrophosphate phosphatase activity.

The subcellular distribution of polyisoprenyl pyrophosphate phosphatase activity has been examined in rat brain by assaying the release of 32Pi from [beta-32P]dolichyl pyrophosphate (Dol-P-P) as described previously (Scher,M.G. and Waechter, C.J. (1984) J. Biol. Chem., 259, 14580-14585). The highest specific activities of Dol-P-P phosphatase in rat brain were found in the Golgi-enriched light microsomal, synaptic plasma membrane and heavy microsomal fractions. A comparative analysis of the distribution of galactosyltransferase and dolichol kinase reveals that Dol-P-P phosphatase activity co-fractionates with galactosyltransferase activity, and that the high level found in the Golgi-enriched fraction is not due to cross-contamination with heavy microsomes. When beta-labelled C95 Dol-P-P and the C95 allylic polyisoprenyl pyrophosphate (Poly-P-P) were compared as substrates for the Golgi-enriched light microsomal and heavy microsomal fractions, similar Km values were calculated for the two pyrophosphorylated substrates for each membrane fraction. Based on these kinetic analyses, the enzyme(s) catalysing this reaction do not distinguish between substrates containing saturated or allylic alpha-isoprene units. When Dol-P-P phosphatase activity was assessed in submicrosomal fractions obtained from rat liver by two separate procedures, the highest specific activity was also detected in the Golgi-enriched fraction. While the specific activities for Dol-P-P phosphatase and sialyltransferase were in the relative order of Golgi greater than smooth endoplasmic reticulum (ER) greater than rough ER, the relative order of dolichol kinase was rough ER greater than smooth ER greater than Golgi.(ABSTRACT TRUNCATED AT 250 WORDS)     

High affinity specific antiestrogen binding sites are concentrated in rough microsomal membranes of rat liver

Saturation and competitive binding analyses demonstrated the presence of a high affinity (K_D = 0.92 nM), specific antiestrogen binding site (AEBS) in rat liver microsomes and at least 75% of total liver AEBS was recovered in this fraction. When microsomes were further separated into smooth and rough fractions, AEBS was concentrated in the latter. Subsequent dissociation of ribosomes from the rough membranes revealed that AEBS was associated with the membrane and not the ribosomal fraction. Antiestrogen binding activity could not be extracted from membranes with 1 M KCl or 0.5 M acetic acid but could be solubilized with sodium cholate. These data indicate that AEBS is an integral membrane component of the rough microsomal fraction of rat liver.     

Saponin permeabilization of rough microsomes from rat liver reveals a novel prothrombin pool

Saponin permeabilization of rough microsomes in the presence of high salt revealed a novel pool of prothrombin associated by ionic interactions to the microsomal membrane. The lumenal content was obtained by treating rough microsomes with 0.32% saponin in a low salt (0.05 M KCl) buffer. By a subsequent treatment with 0.32% saponin in a slightly alkaline high salt buffer a fraction of peripherally associated membrane prothrombin was released from rough microsomes. Finally, the membrane-bound fraction was solubilized with 2.5% Triton X-100. The lumenal content fraction, the peripherally membrane-associated and the membrane-bound fraction from normal rats contained 55%, 29% and 16% of the total rough microsomal prothrombin, respectively. The corresponding fractions from warfarin-treated rats contained 86%, 5% and 9% of the total prothrombin. Following ¹⁴C-γ-carboxylation of intact microsomes for 30 min, the novel membrane-associated and the membrane-bound pool contained 42% and 33%, respectively, of labeled prothrombin. A similar distribution was found with warfarin-treated rats.     

Variation in microsomal subfractions obtained from normal animal tissues and transformed cells following cell disruption by nitrogen cavitation

Summary Microsomal membranes were obtained from MPC-11 cells, L-cells, Krebs II ascites cells and various normal animal tissues following cell disruption by nitrogen cavitation. Membrane preparations were applied to discontinuous sucrose gradients designed to separate three fractions — heavy rough (HR), light rough (LR) and smooth (S) microsomes. In each of the transformed cell lines all three fractions were found whilst in the normal tissues tested the HR fraction was absent. Of the normal tissues liver and pancreas were rich in both LR and S microsomes, the presence of large amounts of LR indicating a rich protein synthesizing activity on membrane-bound polysomes. Kidney also contained appreciable LR but much less than both liver and pancreas. Both heart and lung contained virtually only S microsomal material — a reflection of low protein synthetic activity on membrane-bound polysomes. Attempts to promote the appearance of the HR fraction in liver, kidney and pancreas by incubation in tissue culture medium, or, in the case of pancreas, by cholecystokinin/pancreozymin/secretin, stimulation bothin vivo andin vitro were unsuccessful.     

Exchange of phospholipids between mitochondria and rough or smooth microsomes in vitro.

Phospholipids in mitochondria can be exchanged with those in two microsomal fractions from rough endoplasmic reticulum (rough microsomes) and smooth endoplasmic reticulum (smooth microsomes) in vitro in the presence of cell supernatant. The amounts of phospholipids transferred from each submicrosomal fraction to nitochondria were slightly different. The compositions of the phospholipids transferred to mitochondria from both microsomal fractions were the same, though these two fractions actually had different phospholipid compositions.     

Molecular composition of the submicrosomal membrane lipid of rat brain

Rough-surfaced and light and heavy smooth-surfaced microsomes were isolated from rat brain by means of discontinuous sucrose gradient centrifugation. Electron microscopically, the rough-surfaced microsomes were characterized by vesicles with ribosomes and the light and heavy smooth-surfaced microsomes by fairly homogeneous membrane features without ribosomes. The rough-surfaced microsomal membranes were distinguished by the absence of glycolipids, such as ganglioside, cerebroside, and sulfatide. Cerebroside was exclusively recovered in the light smooth-surfaced microsomal membranes. Ganglioside and Na,K-ATPase were contained in larger amounts in the heavy smooth-surfaced microsomal membranes than in the light smooth-surfaced microsomal membranes in terms of protein content. Among the three submicrosomal membranes, cholesterol and phospholipid were found in the largest amounts in the light smooth-surfaced microsomal membranes, where the molar ratio of cerebroside-cholesterol-phospholipid was about 1:10:10. The membranes of rough- and smooth-surfaced microsomes were very similar in regards to the composition of phospholipid classes, although the fatty acid composition of the former contained a greater proportion of unsaturated fatty acids than that of the latter. When the membrane proteins were analyzed by sodium dodecyl sulfate gel electrophoresis, some differences were observed between the light and heavy smooth-surfaced microsomal membranes.     

The phospholipid composition of embryonic chick liver microsomes

1. The phospholipid composition of hepatic microsomal fractions from different developmental stages of embryonic chick was established. The major components were phosphatidylcholine (approx. 66%), phosphatidylethanolamine plus phosphatidylserine (approx. 21%) and sphingomyelin (approx. 9%). 2. There were no significant changes in the phospholipid composition during embryonic development from 9 to 20 days. 3. When microsomal subfractions were prepared it was found that the smooth-microsomal fractions (Ia and Ib) had a significantly greater sphingomyelin content than the rough-microsomal fraction (II). This was compensated by a lower phosphatidylcholine content in fractions Ia and Ib and an increase of phosphatidylcholine in fraction II. 4. The significance of the differences in the phospholipid composition of smooth and rough microsomes is discussed with particular reference to the origin and interrelation of smooth and rough endoplasmic reticulum.     

Localization of ATP-dependent calcium transport activity in mouse pancreatic microsomes

Electron-dense deposits representing calcium oxalate crystals which result from ATP-dependent calcium uptake have been localized within vesicles of of a heavy microsomal fraction prepared from mouse pancreatic acini. In the absence of either ATP or oxalate, no electron-dense deposits could be observed. By subfractionation of microsomes on discontinuous sucrose gradients, it could be shown that the highest energy-dependent calcium transport activity was associated with the rough endoplasmic reticulum. In rough microsomes, the 45Ca2+-uptake measured was 7 times greater than that of smooth microsomes in the presence of ATP and oxalate and about 3 times greater in he presence of ATP alone. When ribosomes were released from the rough endoplasmic reticulum vesicles by treatment with KCl in the presence of puromycin, the stripped microsomes showed a 40% increase in the specific 45Ca2+-uptake activity measured in he presence of ATP and oxalate and an increase of 80 to 90% in the presence of ATP alone. From these results it can be concluded that the calcium transport activity of microsomes prepared from mouse pancreatic acini is located predominantly in the rough endoplasmic reticulum membrane.     

Intramicrosomal location in intestine of enzymes which synthesize complex lipid.

Smooth and rough microsomal fractions were prepared from hamster intestinal mucosa and assayed for RNA, diglyceride acyltransferase, cholinephosphotransferase and lysolecithin acyltransferase. The specific activity of cholinephosphotransferase was 4-fold more in the rough than in the smooth microsomal fractions, whereas diglyceride and lysolecithin acyltransferases were respectively 26 and 57% more active in the rough microsomal fraction. The two acyltransferases were similarly located in the microsomes and more closely corresponded to the location of dietary lipid than cholinephosphotransferase.     

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.     

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.     

Biogenesis of the mitochondrial matrix enzyme, glutamate dehydrogenase, in rat liver cells. II. Significance of binding of glutamate dehydrogenase to microsomal membrane

1. Glutamate dehydrogenase and malate dehydrogenase solubilized from liver microsomes were able to rebind to microsomal vesicles while the corresponding dehydrogenases extracted from mitochondria showed no affinity for microsomes. 2. Competition was noticed between microsomal glutamate dehydrogenase and microsomal malate dehydrogenase in the binding to microsomal membranes. Mitochondrial malate dehydrogenase or bovine serum albumin did not inhibit the binding of microsomal glutamate dehydrogenase to microsomes. 3. Binding of microsomal glutamate dehydrogenase to microsomal membranes decreased when microsomes was preincubated with trypsin. 4. Rough microsomal glutamate dehydrogenase was more efficiently bound to rough microsomes than smooth microsomes. Conversely, smooth microsomal glutamate dehydrogenase had higher affinity for smooth microsomes than for rough microsomes. 5. A difference was noticed among the glutamate dehydrogenase isolated from rough and smooth microsomes, and from mitochondria, which suggested the possibility of minor post-translational modification of enzyme molecules in the transport from the site of synthesis to mitochondria.     

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.     

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.     

A study of intracellular transport of secretory glycoproteins in rat liver

To study the transport of secretory glycoproteins in the endoplasmic reticulum of rat liver, the distribution of nascent glycoproteins in the membrane and luminal fraction of rough and smooth microsomes has been examined after a short-time incorporation of radioactive glucosamine in vivo. 50--60% of the radioactivity was associated with the membranes of rough and smooth microsomes, whereas about 10% of the serum albumin was found in the same fractions. The relative amount of radioactivity in the membranes was the same whether the luminal content of the microsomal vesicles was released by sonication, French press, Triton X-100, Brij 35 or sodium deoxycholate. The distribution of labeled glycoproteins between the membrane and luminal fraction of rough and smooth microsomes did not change during the time interval of 15--120 min after administration of the isotope. The similarity of the labeling patterns obtained after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis indicated that the same set of glycoproteins were located in the lumen and the membrane of rough and smooth microsomes. A specific precipitation of nascent glycoproteins from both the membrane and luminal fractions of rough and smooth microsomes were obtained with rabbit antiserum against rat serum. The nascent glycoproteins associated with the membranes were not released by high ionic strength or treatment with mercaptoethanol. A slow exchange between [14C]glucosamine-labeled glycoproteins in the lumen and membrane fraction was, however, found.     

Effect of carbachol or histamine stimulation on rat gastric membranes enriched in (H+-K+)-ATPase

We have examined histamine- or carbachol-induced changes in rat gastric membranes enriched in K+-stimulated ATPase. Stimulation of secretion by both secretagogues in vivo produced a class of microsomal membranes which exhibited valinomycin-independent, KCl-dependent H+ transport. In contrast, membrane vesicles isolated from cimetidine inhibited resting mucosa exhibited largely the ionophore-dependent H+ transport. In addition, only in the carbachol-stimulated membranes a portion of the ionophore-independent H+ transport was refractory to cimetidine pretreatment. The gastric microsomal membranes were resolved into light and heavy fractions by centrifugation over isotonic 2H2O media. The ionophore-independent H+ transport was almost exclusively associated with the heavy microsomal fraction while the ionophore-dependent H+ transport was detected in the light fraction. Also, these fractions were considerably different from each other in their appearance in electron micrographs and SDS gel electrophoresis patterns. Secretagogue stimulation increased the population of the heavy microsomal membrane vesicles exhibiting the valinomycin-independent, K+-dependent H+ transport and their overall content of K+-stimulated ATPase. Cimetidine treatment, on the other hand, increased the ATPase activity associated with the light microsomes, and produced the heavy microsomal membranes showing only a marginal degree of the ionophore independent H+ accumulation, even though they were very similar to the carbachol-stimulated heavy membranes in the specific activity of K+-stimulated ATPase. SDS gel patterns and appearance in electron micrograph. These observations suggest that activation of secretion involves at least two distinctive events; transformation of the light to the heavy gastric membranes containing a K+-dependent H+ pump and an increased KCl permeability in the latter.     

Collagen lysyl hydroxylation occurs within the cisternae of the rough endoplasmic reticulum.

Resolution of the heavy microsomal fraction of lung tissue by Ficoll density gradient centrifugation yielded a rough endoplasmic reticulum microsomal fraction containing the highest specific activity of detergent-released lysyl hydroxylase. This same microsomal fraction was previously shown to contain the highest specific activity of detergent-released prolyl hydroxylase activity. When hydroxylation was inhibited during the biosynthesis of collagen, this microsomal fraction contained lysine-rich, hydroxylysine-deficient, collagenase-digestible substrate that could be hydroxylated in the absence of detergent. The results indicate coordinate localization of both prolyl and lysyl hydroxylation reactions within the cisternae of the rough endoplasmic reticulum.