Isolation of plasma membranes from corn roots by sucrose density gradient centrifugation: an anomalous effect of ficoll

An investigation was conducted into the isolation of plasma membrane vesicles from primary roots of corn (Zea mays L., WF9 × M14) by sucrose density gradient centrifugation. Identification of plasma membranes in cell fractions was by specific staining with the periodic-chromic-phosphotungstic acid procedure. Plasma membrane vesicles were rich in K⁺-stimulated ATPase activity at pH 6.5, and equilibrated in linear gradients of sucrose at a peak density of about 1.165 g/cc. It was necessary to remove mitochondria (equilibrium density of 1.18 g/cc) from the homogenate before density gradient centrifugation to minimize mitochondrial contamination of the plasma membrane fraction. Endoplasmic reticulum (NADH-cytochrome c reductase) and Golgi apparatus (latent IDPase) had equilibrium densities in sucrose of about 1.10 g/cc and 1.12 to 1.15 g/cc, respectively. A correlation (r = 0.975) was observed between K⁺-stimulated ATPase activity at pH 6.5 and the content of plasma membranes in various cell fractions. ATPase activity at pH 9 and cytochrome c oxidase activity were also correlated.     

A method for rapid isolation of rough and smooth microsomes and golgi apparatus from rat liver in the same sucrose gradient

A technique is presented for the rapid isolation, by centrifugation in the same sucrose gradient, of rough and smooth surfaced microsomes and a Golgi apparatus enriched fraction from rat liver. Ethanol treatment is not necessary. The purity and recovery of the isolated fractions, as judged from determinations of marker enzymes and morphological analyses, are similar to previous data obtained with more complicated procedures. The method is based on adding a 10000 g supernatant to a discontinuous sucrose gradient ranging from bottom to top (1.30 M–15 mM CsCl, 1.15 M–15 mM CsCl, 10000 g supernatant in 1.05 M–10 mM CsCl, 1.05 M sucrose and 0.3 M sucrose). The Golgi apparatus enriched fraction bands at the border line of 0.3/1.05 M sucrose. The smooth microsomes band at the bondary of 1.15/1.30 M sucrose and finally the rough microsomes are collected at the bottom of the gradient. All fractions are obtained within h.     

Isolation of dictyosomes fromEuglena gracilis

Summary A method for the isolation of dictyosomes fromEuglena gracilis Klebs strain Z (Pringsheim) is described. An extensive Golgi system, with the individual dictyosomes commonly containing ten to twenty cisternae is present. Log phase cells are broken in a French pressure cell at 105 to 120 kg/cm₂ in a breaking mix containing sucrose, sorbitol and ficoll. Addition of 0.3% of glutaraldehyde or formaldehyde to the breaking mix increases the number of stacked cisternae present in the final preparation. In addition to membrane stacks, the fractions contain numerous smooth vesicles. Swollen cisternae, which are also present, may account for these vesicles. Three dictyosome-enriched fractions are obtained by centrifugation in a discontinuous sucrose gradient. Fractions differ morphologically in the degree of stacking of cisternae. Further identification of the membrane fractions was accomplished by measuring IDPase activities in each of the fractions. Inosine diphosphatase activity is enriched 8–10-fold relative to the initial homogenate. The highest IDPase activity was present in the fraction containing the greatest number of stacked cisternae.     

Distribution of terminal glycosyltransferases in hepatic Golgi fractions

The distribution of the three glycosyltransferases synthesizing the terminal trisaccharide sialic acid yields D-galactose yields N- acetylglucosamine present in many glycoproteins was determined in Golgi fractions prepared from rat liver homogenates by a modification of the procedure of Ehrenreich et al. (1973, J. Cell Biol. 70:671--684). The enzymes were assayed with asialofetuin, ovomucoid, and Smith-degraded ovomucoid as sugar acceptors. Careful adjustment of the pH of all sucrose solutions to 7.0 +/- 0.1 prevented enzyme inactivation, and allowed quantitative recoveries at every isolation step. The three morphologically and functionally different Golgi fractions GJ1, GF2, and GF3 showed (in that order) decreasing specific activities of all three enzymes, but the relative amounts and relative specific activities of the three transferases in any given fraction were nearly identical. Two marginal fractions, one extra heavy (collected on the gradient below GF3) and the other extra light (isolated by flotation from the postmicrosomal supernate) were found to contain recognizable Golgi elements. An enrichment of any transferase over the two others was not detected in either preparation. A partial release of content from a combined GF1+2 was achieved by treatment with the nonionic detergent Triton X-100. Low Triton/phospholipid ratios (less than 2 mg/mg) led to lysis of the vesicles and cisternae and loss of very low density lipoprotein particles (ascertained by electron microscopy), but failed to separate the transferases from each other; the three enzymes sedimented together with a population of empty vesicles to a density of approximately 1.08 g/ml.     

Acute in vivo stimulation of low-Km cyclic AMP phosphodiesterase activity by insulin in rat-liver Golgi fractions

A low-Km phosphodiesterase activity, which is acutely stimulated by insulin in vivo, has been identified in plasma membranes and Golgi fractions prepared from rat liver homogenates in isotonic sucrose. Within seconds after insulin injection (25 micrograms/100 g body weight) cAMP phosphodiesterase activity increases by 30-60% in Golgi fractions and by 25% in plasma membranes; activity in crude particulate and microsomal fractions is unaffected. The increase in activity is short-lived in the light and intermediate Golgi fractions, but persists for at least 10 min in the heavy Golgi fraction. It precedes the translocation of insulin and insulin receptors to these fractions, which is maximal at 5 min. The doses of insulin required for half-maximal and maximal activation are, respectively, 7.5 micrograms/100 g and 25 micrograms/100 g body weight. Golgi-associated cAMP phosphodiesterase activity shows non-linear kinetics; a high-affinity component (Vmax, 13 pmol min-1 mg protein-1; Km, 0.35 microM) is detectable. Insulin treatment increases the Vmax 60-70%, but does not affect the Km. Unlike the low-Km cAMP phosphodiesterase associated with crude particulate fractions, the Golgi-associated activity is not easily extractable by solutions of low or high ionic strength. On analytical sucrose density gradients, low-Km cAMP phosphodiesterase associated with the total particulate fraction equilibrates at lower densities than endoplasmic reticulum and lysosomal markers, but at a higher densities than plasma membrane, Golgi markers and insulin receptors. Insulin treatment increases the specific activity of the enzyme by 20-60% at densities below 1.12 g cm-3, and by 20-40% in the density interval 1.23-1.25 g cm-3. Such treatment also causes a slight, but significant shift in the distribution of phosphodiesterase towards lower densities. It is suggested that Golgi elements or physically similar subcellular structures are a major site of localization of insulin-sensitive cAMP phosphodiesterase in rat liver. However, internalization of the insulin-receptor complex is probably not required for enzyme activation.     

GOLGI FRACTIONS PREPARED FROM RAT LIVER HOMOGENATES : II. Biochemical Characterization

The three Golgi fractions isolated from rat liver homogenates by the procedure given in the companion paper account for 6–7% of the protein of the total microsomal fraction used as starting preparation. The lightest, most homogeneous Golgi fraction (GF₁) lacks typical "microsomal" activities, e.g., glucose-6-phosphatase, NADPH-cytochrome c-reductase, and cytochrome P-450. The heaviest, most heterogeneous fraction (GF₃) is contaminated by endoplasmic reticulum membranes to the extent of ~15% of its protein. The three fractions taken together account for nearly all the UDP-galactose: N-acetyl-glucosamine galactosyltransferase of the parent microsomal fraction, and for ~70% of the activity of the original homogenate. Omission of the ethanol treatment of the animals reduces the recovery by half. The transferase activity is associated with the membranes of the Golgi elements, not with their content. Galactose is transferred not only to N-acetyl-glucosamine but also to an unidentified lipid-soluble component.     

LOCALIZATION OF ENZYMES WITHIN MICROBODIES

Microbodies from rat liver and a variety of plant tissues were osmotically shocked and subsequently centrifuged at 40,000 g for 30 min to yield supernatant and pellet fractions. From rat liver microbodies, all of the uricase activity but little glycolate oxidase or catalase activity were recovered in the pellet, which probably contained the crystalline cores as many other reports had shown. All the measured enzymes in spinach leaf microbodies were solubilized. With microbodies from potato tuber, further sucrose gradient centrifugation of the pellet yielded a fraction at density 1.28 g/cm³ which, presumably representing the crystalline cores, contained 7% of the total catalase activity but no uricase or glycolate oxidase activity. Using microbodies from castor bean endosperm (glyoxysomes), 50–60% of the malate dehydrogenase, fatty acyl CoA dehydrogenase, and crotonase and 90% of the malate synthetase and citrate synthetase were recovered in the pellet, which also contained 96% of the radioactivity when lecithin in the glyoxysomal membrane had been labeled by previous treatment of the tissue with [¹⁴C]choline. When the labeled pellet was centrifuged to equilibrium on a sucrose gradient, all the radioactivity, protein, and enzyme activities were recovered together at peak density 1.21–1.22 g/cm³, whereas the original glyoxysomes appeared at density 1.24 g/cm³. Electron microscopy showed that the fraction at 1.21–1.22 g/cm³ was comprised of intact glyoxysomal membranes. All of the membrane-bound enzymes were stripped off with 0.15 M KCl, leaving the "ghosts" still intact as revealed by electron microscopy and sucrose gradient centrifugation. It is concluded that the crystalline cores of plant microbodies contain no uricase and are not particularly enriched with catalase. Some of the enzymes in glyoxysomes are associated with the membranes and this probably has functional significance.     

Identification and characterization of Golgi equivalents fromAllomyces macrogynus

Golgi equivalents similar to those described in other fungi were identified in freeze substituted hyphae ofAllomyces macrogynus. These Golgi equivalents were composed of individual or a few loosely associated cisternae, were surrounded by vesicles, and were in a zone relatively free of ribosomes. Certain smooth cisternae in both vegetative hyphae and gametangia stained positively for the Golgi marker enzyme thiamine pyrophosphatase. Subcellular fractionation and biochemical analysis of vegetative hyphae and gametangia revealed endoplasmic reticulum and Golgi membrane fractions with average buoyant densities of 1.09 and 1.15 g/cm³, respectively. Enriched membranes obtained by differential centrifugation were further purified by ultracentrifugation on sucrose step gradients to obtain a presumptive Goldi fraction. Gel electrophoresis of both crude homogenates and fractions prepared by differential centrifugation demonstrated stage-specific glycoproteins that bind the lectin concanavalin A. The results demonstrated thatA. macrogynus has a Golgi complex composed of structurally simple Golgi equivalents and that these Golgi equivalents have physiological functions, such as glycoprotein processing, typically associated with stacked Golgi cisternae from other organisms.     

Cellular fractionation and isolation of the plasma membrane of Burkitt's lymphoma cells

A procedure for cellular fractionation and preparation of plasma membrane from a Burkitt's lymphoma cell line is described. This procedure involves homogenization with a Polytron in buffered isotonic sucrose, and separation of cellular fractions by differential and isopycnic centrifugation in sucrose. The isolated plasma membrane fraction contains 44% of the cellular cholesterol, 50% of the ouabain-sensitive (Na⁺ + K⁺)-ATPase activity, 43% of the γ-glutamyltranspeptidase activities and 16% of the phospholipid. This fraction contains only 3% of cellular protein and is contaminated with less than 4% of the total cellular activities of microsomal, lysosomal, mitochondrial, Golgi and soluble marker enzymes. The cholesterol : phospholipid molar ratio of the crude plasma membrane is 0.56. The membranes in this fraction are in the form of vesicles. Further purification of plasma membrane is achieved by sucrose density gradient centrifugation and results in a 25- to 30-fold enrichment of plasma membrane markers. Plasma membrane markers band in these gradients between 1.10 and 1.15 g/cm³.The distribution patterns in the cell fractions of 18 cellular constituents are quantitatively determined. Most constituents are found to distribute in a fashion consistent with the results obtained in other systems. Thymidine-5′-phosphodiesterase (phosphodiesterase I), esterase, nucleoside diphosphatase and glucose-6-phosphatase, however, are shown to be poor markers of membrane fractions in this system.Lactoperoxidase-catalyzed iodination was used to identify several plasma membrane proteins which are exposed at the surface. After separation of labeled polypeptides by sodium dodecyl sulfate gel electrophoresis, the predominant labeled protein was identified as the heavy chain of IgM. Several lesser labeled proteins were observed.     

Intracellular localization of N-formyl chemotactic receptor and Mg dependent ATPase in human granulocytes

Human granulocytes were disrupted by nitrogen cavitation and the lysates fractionated by sucrose density gradient centrifugation at 83 000 × g for 20 min (rate zonal) or 3.5 h (isopycnic). The distribution of marker enzymes allowed the identification of the following subcellular components: plasma membrane, Golgi, endoplasmic reticulum, azurophil granules, specific granules, mitochondria and cytosol. Examination of the gradient fractions by electron microscopy confirmed the biochemical marker analysis. The protocol permitted isolation of vesicles highly enriched in either plasma membrane or Golgi (galactosyl transferase) activities. Absolute plasma membrane yields of 40–60% were achieved with a 20–70-fold increase in specific activity of surface marker over the cells. Plasma membrane sedimented to an average density of 1.14 g·cm⁻³. Galactosyl transferase activity was bimodal in distribution. The denser peak cosedimanted with specific granules (g9 = 1.19). The lighter peak sedimented to unique position at an average density of 1.11, was enriched 18-fold over the low speed supernatant, and contained structures resembling Golgi. N-Formyl-Met-Leu-Phe binding and Mg²⁺ -ATPase activities cosedimented with the plasma membrane as well as specific granule and/or high density galactosyl transferase fractions. These findings suggest that Mg²⁺ -ATPase and N-formyl chemotactic peptide receptor activities may be localized in an internal pool of membranes as well as in the plasma membrane and that Golgi may have been a contaminant of previous granulocyte plasma membrane or specific granule preparations.     

Isolation of a Golgi rich fraction from rat ventral prostate

Unmodified procedures for isolation of fractions rich in Golgi elements from other tissues have not proved applicable to the rat ventral prostate because of the tendency of membranous material to aggregate. We have devised a new procedure whereby: 1) a Golgi rich fraction from rat ventral prostate was released by a gentle two-step homogenization and isolated by centrifugation through discontinuous sucrose density gradients; 2) the specific activity of UDP-galactose: glycoprotein galactosyltransferase increased 69-fold in this fraction; 3) the isolated Golgi fraction was reasonably free from mitochondria, lysosomes, endoplasmic reticulum and plasma membranes as shown by the relatively low activities of marker enzymes; 4) the specific activities of acid phosphatase and 5'-nucleotidase in the Golgi rich fraction was 4 times greater than that in prostate homogenate. Both enzymes are secretory products and their presence in Golgi elements is probably associated with their packaging in secretory granules.     

Isolation of plasma membranes and Golgi apparatus from a single chicken liver homogenate

Chicken liver plasma membranes, minimally contaminated with Golgi apparatus-derived vesicles, were prepared from a low-speed (400 g) pellet by means of flotation in isotonic Percoll solution, followed by a hypotonic wash and flotation in a discontinuous sucrose gradient. Based on the analysis of suitable marker enzymes, alkaline phosphatase and alkaline phosphodiesterase, two plasma membrane fractions were isolated with enrichments, depending on the equilibrium density and marker of 28-97 and with a total yield of 4-5%. Golgi apparatus fractions were prepared by flotation of microsomes, obtained from the same homogenate as the low-speed pellet, in a discontinuous sucrose gradient. The trans-Golgi marker galactosyltransferase was 27-fold enriched in a fraction of intermediate density (d=1.077-1.116 g/ml). Approximately 12% of galactosyltransferase was recovered in the membranes equilibrating d=1.031-1.148 g/ml. Contamination with plasma membrane fragments was low in the light (d=1.031-1.077 g/ml) and intermediate density Golgi vesicles. The isolation of purified plasma membranes and Golgi vesicles from one liver homogenate will enable future studies on receptor cycling between these cell organelles.     

Characterization of the Golgi Complex Cleared of Proteins in Transit and Examination of Calcium Uptake Activities

To characterize endogenous molecules and activities of the Golgi complex, proteins in transit were >99% cleared from rat hepatocytes by using cycloheximide (CHX) treatment. The loss of proteins in transit resulted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that >90% of the elements could be positively identified as Golgi stacks or cisternae. Biochemical analysis showed that the cis-, medial-, trans-, and TGN Golgi markers were enriched over the postnuclear supernatant 200- to 400-fold with and 400- to 700-fold without proteins in transit. To provide information on a mechanism for import of calcium required at the later stages of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a thapsigargin-resistant pump not resident to the Golgi complex and a thapsigargin-sensitive pump resident to the Golgi. Experiments using CHX SGF1 showed that the thapsigargin-resistant activity was a plasma membrane calcium ATPase isoform in transit to the plasma membrane and the thapsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum calcium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen.     

Effect of acute ethanolic intoxication on the neuraminidase activity of rat liver golgi apparatus

Neuraminidase and galactosyltransferase were investigated in total Golgi apparatus and in the three fractions of increasing densities (GF₁, GF₂ and GF₂) isolated from the microsomal fraction of rat liver homogenates by flotation in a discontinuous sucrose density gradient (Ehrenreich, J.H., Bergeron, J.J.M., Siekevitz, P. and Palade, G.E. (1973) J. Cell Biol. 59, 45–72). About 50% decreases in neuraminidase content (units/g liver) and specifixc activity (units/ mg protein) were observed in total Golgi as well as in the three fractions isolated at 45 min, 90 min, 180 min and 16 h after administration of a single oral dose of 50% aqueous ethanol (0.6 g/100 g body weight). Colchicine administration (intraperitoneal injection, 0.5 mg/100 g body weight) caused a similar loss of neuraminidase activity; however, the effect of ethanol plus colchicine was not additive. Golgi galactosyltransferase, on the other hand, experienced marked increases of activity following ethanol administration but, unlike the results reported by others (Gang, H., Lieber, C.S. and Rubin, E. (1973) Nat. New Biol. 243, 123–125), significant increases in total activity and specific activity were already quite evident at 90 min after ethanol ingestion. In contrast with the decreased values observed in Golgi, the total particle-bound neuraminidase was significantly elevated following ethanol administration. Ultrastructural studies revealed increased lysosomal content and detachment of polysomes from the rough endoplasmic reticulum. A model, which takes into account these enzymological and ultrastructural findings and their biological significance, is proposed.     

Presence of nadph-cytochrome P-450 reductase in rat liver golgi membranes: Evidence obtained by immunoadsorption method

Light Golgi fractions (GF(1+2)) prepared from rat liver homogenates by a modification of the Ehrenreich et al. procedure (J. Cell Biol. 59:45) had significant NADPH-cytochrome P(450) reductase (NADPH-cyt c reductase) activity if assayed immediately after their isolation. An antibody raised in rabbits against purified microsomal and Golgi fractions. To find out whether this activity is located in bona fide Golgi elements or in contaminating microsomal vesicles, we used the following 3-step immunoadsorption procedure: (a) antirabbit IgG (raised in goats) was conjugated to small (2-5 μm) polycrylamide (PA) beads; (b) rabbit anti NADPH-cyt c reductase was immunoadsorbed to the antibody-coated beads; and (c) GF(1+2) was reacted with the beads carrying the two successive layers of antibodies. The beads were then recovered by centrifugation, and were washed, fixed, embedded in agarose, and processed for transmission electromicroscopy. Antireductase- coated beads absorbed 60 percent of the NADPH-cyt c reductase (and comparable fractions of NADH-cyt c reductase and glucose-6-phosphatase) but only 20 percent of the galactosyltransferase activity of the input GF(1+2). Differential vesicle counts showed that approximately 72 percent of the immunoadsorbed vesicles were morphologically recognizable Golgi elements (vesicles with very low density lipoprotein [VLDL] clusters or Golgi cisternae); vesicles with single VLDL and smooth surfaced microsome-like vesicles were too few (approximately 25 percent) to account for the activity. It is concluded that NADPH-cytochrome P(450) reductase is a Golgi membrane enzyme of probably uneven distribution among the elements of the Golgi complex.     

Hydrodynamic parameters and isolation of mitochondria, microperoxisomes and microsomes of rat heart

1. Analytical differential centrifugation of rat heart homogenates revealed a single population of mitochondria and microperoxisomes. Using cytochorme c oxidase, malate dehydrogenase and amine oxidase as mitochondrial marker enzymes, the s̄-value of mitochondria was estimated to s̄ = 10326 ± 406 S (average for the three marker enzymes). The −s-value of microperoxisomes was found to be −s = 1381 ± 40 S using catalase as the marker enzyme. The −s-value for the two orgenelles did not change significantly when the isoosmotic sucrose medium was substituted by an isoosmotic mannitol medium. 2. Analytical differential centrifugation revealed a polydispercity of the microsomal fraction using glucose-6-phosphatase and NADPH-cytochrome c reductase as the marker enzymes. The s̄-values were found to be −s_H1 = 1569 ± 412 S (NADPH-cytochrome c reductase), (glucose-6-phosphatase) and (NADPH-cytochrome c reductase and glucose-6-phosphatase). The recovery of marker enzymes in the isolated subcellular fractions was in the range of 84–94%. 3. When the mitochondrial and microperoxisomal fractions were subjected to isopycnic gradient centrifugation, using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.25 M sucrose medium, buoyant densities of 1.10 g/cm³ (main fraction of mitochondria) and 1.06 g/cm³ (main fraction of microperixosomes) were obtained. The density gradient centrifugation separated microperoxisomes from contaminating lysosomes of high specific activity in acid phosphatase. A value 1.04 g/cm³ was foung for the density of the microsomal fraction. 4. Based on the estimated -values, an optimal procedure is described for the isolattion of mitochondrial and microperoxisomal fractions from rat heart muscle.     

ISOLATION OF THE GOLGI APPARATUS FROM PLANT CELLS

A method for the isolation of the Golgi apparatus from stem tissues of onion is described. Preparations that consisted mainly of morphologically identifiable Golgi apparatus have been obtained. The best preparations were obtained from tissue homogenized under conditions of minimum shear, and in the presence of sucrose and certain additives which aid in preservation of the integrity of the Golgi membranes. Those additives, which had a pronounced stabilizing effect on the isolated apparatus, included both monovalent and divalent ions (sodium and calcium) and dextran. A large portion of the Golgi apparatus did not appear to change microscopic appearance upon isolation, but were observed to fuse into large aggregate structures not unlike those occurring naturally in certain animal or insect cells (12). Fusion occurred both at the edges of the cisternae and in register, but the integrity of the individual cisternae was not destroyed. The major contaminants of the Golgi apparatus fraction were numerous small and large spherical vesicles. At least some of these vesicles appeared to have been derived from the Golgi apparatus; others may have been fragments of the cell membrane, the endoplasmic reticulum, or other cell debris. By utilizing this procedure, it has been possible to obtain fractions of Golgi apparatus from plant tissues other than onion stem. However, at the present time it is only with onion that the Golgi apparatus has been isolated in a form that would warrant further purification for biochemical analysis.     

Albumin secreted by rat liver bypasses Golgi apparatus cisternae

Albumin was isolated immunologically from various subcellular fractions from livers of adult male rats receiving an intraperitoneal injection of [³H]leucine to investigate the kinetics and pathway of subcellular transfer of newly synthesized albumin during secretion. At appropriate time intervals, livers were excised and fractionated into endoplasmic reticulum and Golgi apparatus. Golgi apparatus were further subfractionated into cisternae and secretory vesicles. In endoplasmic reticulum fractions, labeled albumin appeared within 7.5 min of injection of isotope, followed by a rapid decline in specific activity. Albumin in Golgi apparatus was labeled and concentrated in secretory vesicles over 25 min. The radioactivity in albumin per mg total protein was highest in secretory vesicles and insignificant in the cisternal fraction. Labeled albumin was present in serum by 30 min and radioactivity in serum albumin reached a plateau within 60–90 min after injection of isotope. Results provide evidence for the migration of albumin from its site of synthesis on endoplasmic reticulum membrane-bound polyribosomes to its site of secretion into the circulation via the Golgi apparatus. The pathway of albumin transport to secretory vesicles is suggested to involve peripheral elemenst of the Golgi apparatus. Secretory vesicle formation and maturation required 20 to 30 min for completion, via a mechanism whereby the inner spaces of the central saccules may be bypassed.     

EVIDENCE FOR THE PARTICIPATION OF THE GOLGI APPARATUS IN THE INTRACELLULAR TRANSPORT OF NASCENT ALBUMIN IN THE LIVER CELL

A comparative biochemical and radioautographic in vivo study was performed to identify the site of synthesis and route of migration of albumin in the parenchymal liver cell after labeling with leucine-¹⁴C or leucine-³H via the portal vein. Free cytoplasmic ribosomes, membrane-bound ribosomes, rough- and smooth-surfaced microsomes, and Golgi membranes were isolated. The purity of the Golgi fraction was examined morphologically and biochemically. After administration of leucine-¹⁴C, labeled albumin was extracted, and the sequence of transport was followed from one fraction to the other. Approximately 2 min after the intravenous injection, bound ribosomes displayed a maximal rate of leucine-¹⁴C incorporation into albumin. 4 min later, a peak was reached for rough microsomes. Corresponding maximal activities for smooth microsomes were recorded at 15 min, and for the Golgi apparatus at ~20 min. The relative amount of albumin, calculated on a membrane protein basis, was higher in the Golgi fraction than in the microsomes. By radioautography the silver grains were preferentially localized over the rough-surfaced endoplasmic reticulum at the 5 min interval. Apparent activity in the Golgi zone was noted 9 min after the injection; at 15 and 20 min, the majority of the grains were found in this location. Many of the grains associated with the Golgi apparatus were located over Golgi vacuoles containing 300–800 A electron-opaque bodies. It is concluded that albumin is synthesized on bound ribosomes, subsequently is transferred to the cavities of rough-surfaced endoplasmic reticulum, and then undergoes migration to the smooth-surfaced endoplasmic reticulum and the Golgi apparatus. In the latter organelle, albumin can be expected to be segregated together with very low density lipoprotein in vacuoles known to move toward the sinusoidal portion of the cell and release their content to the blood.     

The submicrosomal localization of acyl-coenzyme A–cholesterol acyltransferase and its substrate, and of cholesteryl esters in rat liver

To determine the submicrosomal distribution of acyl-CoA–cholesterol acyltransferase and of cholesteryl esters, the microsomal fraction and the digitonin-treated microsomal preparation of rat liver were subjected to analytical centrifugation on sucrose density gradients. With untreated microsomal fractions the distribution profile and the median density of acyl-CoA–cholesterol acyltransferase were very similar to those of RNA. This is in contrast with hydroxymethylglutaryl-CoA reductase and cholesterol 7α-hydroxylase, which are confined to endoplasmic reticulum membranes with low ribosomal coating. In digitonin-treated microsomal preparations activity of acyl-CoA–cholesterol acyltransferase was not detectable. The labelling of untreated microsomal fractions with trace amounts of [¹⁴C]cholesterol followed by subfractionation of the labelled microsomal fraction showed that the specific radioactivity of cholesteryl esters obtained in vitro by the various subfractions was similar with all subfractions but different from the specific radioactivity of the 7α-hydroxycholesterol obtained in vitro by the same subfraction. These results demonstrate the existence of two pools of cholesterol confined to membranes from the endoplasmic reticulum, one acting as substrate for cholesterol 7α-hydroxylase and the other acting as substrate for acyl-CoA–cholesterol acyltransferase. The major part of cholesteryl esters present in both untreated and digitonin-treated microsomal fractions was distributed at densities similar to those of membranes from the smooth endoplasmic reticulum and at densities lower than those of smooth membranes from Golgi apparatus. The ratio of the concentrations of non-esterified to esterified cholesterol in the subfractions from both untreated and digitonin-treated microsomal fractions was highest at the maximum distribution of plasma membranes.