Analytical study of microsomes and isolated subcellular membranes from rat liver. 3. Subfractionation of the microsomal fraction by isopycnic and differential centrifugation in density gradients

Rat liver microsomal fractions have been equilibrated in various types of linear density gradients. 15 fractions were collected and assayed for 27 constituents. As a result of this analysis microsomal constituents have been classified, in the order of increasing median density, into four groups labeled a, b, c, and d. Group a includes: monoamine oxidase, galactosyltransferase, 5''-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and cholesterol; group b: NADH cytochrome c reductase, NADPH cytochrome c reductase, aminopyrine demethylase, cytochrome b ₅, and cytochrome P 450; group c: glucose 6-phosphatase, nucleoside diphosphatase, esterase, β-glucuronidase, and glucuronyltransferase; group d: RNA, membrane-bound ribosomes, and some enzymes probably adsorbed on ribosomes: fumarase, aldolase, and glutamine synthetase. Analysis of the microsomal fraction by differential centrifugation in density gradient has further dissociated group a into constituents which sediment more slowly (monoamine oxidase and galactosyltransferase) than those of groups b and c, and 5''-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and the bulk of cholesterol which sediment more rapidly (group a2). The microsomal monoamine oxidase is attributed, at least partially, to detached fragments of external mitochondrial membrane. Galactosyltransferase belongs to the Golgi complex. Group a2 constituents are related to plasma membranes. Constituents of groups b and c and RNA belong to microsomal vesicles derived from the endoplasmic reticulum. These latter exhibit a noticeable biochemical heterogeneity and represent at the most 80% of microsomal protein, the rest being accounted for by particles bearing the constituents of groups a and some contaminating mitochondria, lysosomes, and peroxisomes. Attention is called to the operational meaning of microsomal subfractions and to their cytological complexity.     

Analytical study of microsomes and isolated subcellular membranes from rat liver. VII. Distribution of protein-bound sialic acid

Detailed investigations by quantitative centrifugal fractionation were conducted to determine the subcellular distribution of protein-bound sialic acid in rat liver. Homogenates obtained from perfused livers were fractionated by differential centrifugation into nuclear fraction, large granules, microsomes, and final supernate fraction, or were used to isolate membrane preparations enriched in either plasma membranes or Golgi complex elements. Large granule fractions, microsome fractions, and plasma membrane preparations were subfractionated by density equilibration in linear gradients of sucrose. In some experiments, microsomes or plasma membrane preparations were treated with digitonin before isopycnic centrifugation to better distinguish subcellular elements related to the plasma membrane or the Golgi complex from the other cell components; in other experiments, large granule fractions were obtained from Triton WR-1339-loaded livers, which effectively resolve lysosomes from mitochondria and peroxisomes in density gradient analysis. Protein-bound sialic acid and marker enzymes were assayed in the various subcellular fractions. The distributions obtained show that sialoglycoprotein is restricted to some particular domains of the cell, which include the plasma membrane, phagolysosomes, and possibly the Golgi complex. Although sialoglycoprotein is largely recovered in the microsome fraction, it has not been detected in the endoplasmic reticulum-derived elements of this subcellular fraction. In addition, it has not been detected either in mitochondria or in peroxisomes. Because the sialyltransferase activities are associated with the Golgi complex, the cytoplasm appears compartmentalized into components which biogenetically involve the Golgi apparatus and components which do not.     

Analytical study of microsomes and isolated subcellular membranes from rat liver. IX. Nicotinamide adenine dinucleotide glycohydrolase: a plasma membrane enzyme prominently found in Kupffer cells

The distribution of nicotinamide adenine dinucleotide (NAD) glycohydrolase in rat liver was investigated by subcellular fractionation and by isolation of hepatocytes and sinusoidal cells. The behavior of NAD glycohydrolase in subcellular fractionation was peculiar because, although the enzyme was mainly microsomal, plasma membrane preparations contained distinctly more NAD glycohydrolase than could be accounted for by their content in elements derived from the endoplasmic reticulum or the Golgi complex identified by glucose-6-phosphatase and galactosyltransferase, respectively. When microsomal and plasmalemmal preparations were brought to equilibrium in a linear-density gradient, NAD glycohydrolase differed from these enzymes and behaved like 5'-nucleotidase and alkaline phosphodiesterase I. NAD glycohydrolase was markedly displaced towards higher densities after treatment with digitonin. This behavior in density-gradient centrifugation strongly suggests that NAD glycohydrolase is an exclusive enzyme of the plasma membrane. NAD glycohydrolase differed clearly from other plasmalemmal enzymes when the liver was fractionated into hepatocytes and sinusoidal cells; its specific activity was considerably greater in sinusoidal cell than in hepatocyte preparations. Further subfractionation of sinusoidal cell preparations into endothelial and Kupffer cells by counterflow elutriation showed that NAD glycohydrolase is more active in Kupffer cells. We estimate that the specific activity of NAD glycohydrolase activity is at least 65-fold higher at the periphery of Kupffer cells than at the periphery of hepatocytes. As the enzyme shows not structure-linked latency and is an exclusive constituent of the plasma membranes, we conclude that it is an ectoenzyme that cannot lead to a rapid turnover of the cytosolic pyridine nucleotides.     

Glycine and serine rich peptides isolated from rat liver preparations enriched with smooth endoplasmic membranes

Three electrochromatographic homogeneous peptides belonging to acidic, basic and neutral electrophoretic glycopeptide fractions isolated from rat liver preparations enriched with smooth endoplasmic membranes contain a very high level of glycine and serine and only a slight level of phenylalanine but do not contain tyrosine. Their amino acid compositions differ chiefly in the contents of basic and acidic amino acids.     

Analytical study of microsomes and isolated subcellular membranes from rat liver. VI. Electron microscope examination of microsomes for cytochrome b5 by means of a ferritin-labeled antibody

The distribution of cytochrome b5 in rat liver microsomes, and in two microsomal subfractions isolated by density equilibration in a linear sucrose gradient, was studied under the electron microscope by means of a ferritin-labeled hybrid anti-cytochrome b5/anti-ferritin antibody. Results of this study show that cytochrome b5 is present in essentially all microsomal vesicles derived from endoplasmic reticulum (ER), whether rough or smooth. Thus, the dissociation of ER constituents into two groups (b and c), achieved by subfractionating microsomes by isopycnic centrifugation (Beaufay, H., A. Amar-Costesec, D. Thines- Sempoux, M. Wibo, M. Robbi, and J. Berthet. 1974. J. Cell Biol. 61:213- 231), does not reflect the association of each group with distinct microsomal particles but reflects rather an enzymatic heterogeneity of the ER: the ratio of group c to group b enzymes increasing with the density and ribosome load of the particles.     

Analytical study of microsomes and isolated subcellular membranes from rat liver. V. Immunological localization of cytochrome b5 by electron microscopy: methodology and application to various subcellular fractions

The localization of cytochrome b5 on the membranes of various subcellular organelles of rat liver was studied by a cytoimmunological procedure using anti-cytochrome b5/anti-ferritin hybrid antibodies and ferritin as label. For this study, highly purified and biochemically characterized membrane preparations were employed. Outer mitochondrial membranes were found to be heavily labeled by the hybrid antibodies whereas Golgi and plasma membranes were not marked by the reagent. Peroxisome membranes were moderately labeled by the hybrid antibodies, suggesting that they may contain some cytochrome b5. The preparation and purification of hybrid antibodies without peptic digestion is described and an analysis made of the composition of the final reagent product.     

Subfractionation of rat liver microsomes by immunoprecipitation and immunoadsorption methods.

Rabbit antisera were prepared against cytochrome b5 and NADPH-cytochrome c reductase [EC 1.6.2.4] purified from rat liver microsomes, and utilized in examining the distribution of these and other membrane-bound enzymes among the vesicles of rat liver microsomal preparations by immunoprecipitation and immunoadsorption methods. Smooth microsomes with an average vesicular size of 200 nm (diameter) and sonicated smooth microsomes with an average diameter of 40-60 nm were used in subfractionation experiments. Immunoprecipitation of microsomal vesicles with anti-cytochrome b5 immunoglobulin failed to show any separation of the microsomes into fractions having different enzyme compositions. Cytochrome b5 was apparently distributed among all vesicles even when sonicated microsomes were used. When the antibody against NADPH-cytochrome c reductase was used, however, immunoadsorption of microsomes on Sepharose-bound antibody produced some separation of NADPH-cytochrome c reductase and cytochrome P-450 from NADH-cytochrome b5 reductase and cytochrome b5. The separation was more pronounced when sonicated microsomes were used. These results indicate microheterogeneity of the microsomal membrane, and suggest the clustering of NADPH-cytochrome c reductase and cytochrome P-450 molecules in the membrane.     

Hydrodynamic properties of porin isolated from outer membranes of rat liver mitochondria

The reaction of gamma-glutamyltranspeptidase with phenobarbital or with thiobarbituric acid resulted in a irreversible loss of its enzymatic activity. The inactivation followed pseudo-first-order kinetics. Half-maximal velocity of inactivation (Ki) at 37 degrees C in the presence of phenobarbital or thiobarbituric acid was calculated to be 43 mM and 20 mM, respectively. The inactivation of the enzyme activity by both these inhibitors was prevented by serine borate, a known competitive inhibitor, and by the substrate, reduced glutathione, suggesting an active-site-directed nature of the these inhibitors. Maleate provided slight protection against inactivation by thiobarbituric acid. Complete inactivation of the enzyme with tritium-labeled phenobarbital resulted in a stoichiometric incorporation of radioactivity into the enzyme protein. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis of tritium-labeled phenobarbital-enzyme complex, nearly all the radioactivity was found to be associated with the small subunit (Mr = 22 000) of the enzyme, indicating that the catalytic component of the enzyme is on the small subunits.     

NADH-activated cell-free transfer between Golgi apparatus and plasma membranes of rat liver.

This report concerns development of a cell-free system from rat liver to study transport of membrane constituents from the Golgi apparatus to the plasma membrane. Highly purified Golgi apparatus as donor and a mixture of sheets and vesicles as plasma membrane acceptor fractions were combined to analyze requirements for lipid and protein transport. In the reconstituted system, the Golgi apparatus donor was in suspension. To measure transfer, membrane constituents of the donor membranes were radiolabeled with [3H]acetate (lipids) or [3H]leucine (proteins). The plasma membrane vesicles were used as the acceptor and were unlabeled and immobilized on nitrocellulose for ease of recovery and analysis. The reconstituted cell-free transfer was dependent on temperature, but even at 37 degrees C, the amount of transfer did not increase with added ATP, was not specific for any particular membrane fraction or subfraction nor was it facilitated by cytosol. ATP was without effect both in the presence or absence of a cytosolic fraction capable of the support of cell-free transfer in other systems. In contrast to results with ATP, NADH added to the reconstituted system resulted in an increased amount of transfer. A further increase in transfer was obtained with NADH plus a mixture of ascorbate and dehydroascorbate to generate ascorbate free radical. The transfer of labeled membrane constituents from the Golgi apparatus to the plasma membrane supported by NADH plus ascorbate radical was stimulated by a cytosol fraction enriched in less than 10 kDa components. This was without effect in the absence of NADH/ascorbate radical or with ATP as the energy source. Specific transfer was inhibited by both N-ethylmaleimide and GTP gamma S. The findings point to the possibility of redox activities associated with the trans region of the Golgi apparatus as potentially involved in the transport of membrane vesicles from the Golgi apparatus to the cytoplasmic surface of the plasma membrane.     

Glycosyltransferases with endogenous acceptor activity in plasma membranes isolated from rat liver

Plasma membrane fractions from rat liver exhibited glycosyltransferase activity with endogenous membrane-associated acceptors and either UDP-galactose, UDPglucose, UDP-N-acetylglucosamine, or GDPmannose donors. Of these, incorporation into non-lipid acceptors was most active with UDP-galactose and only with UDPgalactose and UDPmannose was there incorporation into endogenous lipid acceptors. CMP-N-acetylneuraminic acid was inactive as a donor with the isolated plasma membranes. In order to demonstrate transferase activity, low concentrations of substrate sugar nucleotides and short incubation times were used as well as sulfhydryl protectants and a phosphatase inhibitor (NaF) in the reaction mixtures. The findings support the concept of surface localization of at least a galactosyl transferase in cells of rat liver.     

Sphingomyelin and ceramide-phosphoethanolamine synthesis by microsomes and plasma membranes from rat liver and brain

Pulse-chase experiments showed that phosphatidylethanolamine (PE) was the direct precursor for ceramide-phosphoethanolamine, a sphingomyelin analogue, in the same way as phosphatidylcholine was for sphingomyelin. Ceramide-phosphoethanolamine could be identified by incorporation of radioactivity from labeled PE, as well as by its stability in alkaline methanolysis and its ability to be methylated by S-adenosyl-methionine. Ceramide-phosphoethanolamine synthesis from labeled exogenous PE seemed to be independent of exogenous ceramide; it was proportional to the amount of incubated membrane, when taking into account the isotopic dilution of labeled precursor by endogenous PE. Sphingomyelin synthesis, which was demonstrated using natural PC as a substrate, was not possible using dipalmitoyl-PC. The formation of sphingomyelin and ceramide-phosphoethanolamine was demonstrated in microsomes and plasma membranes from rat brain and liver.     

Interaction of the Golgi membranes isolated from rabbit liver with microtubules in vitro.

We have developed a reconstituted model system to study the interaction of the Golgi membranes isolated from rabbit liver with taxol-stabilized bovine-brain microtubules without microtubule-associated proteins (MAPs). The Golgi membranes are associated with microtubules. The sheets of vesicles and the membranous tubules are observed along microtubules by direct visualization using differential-interference-contrast, dark field, or fluorescence microscopy. The monoclonal antibody against Golgi membranes suggests that the Golgi membranes, but not the contaminating vesicles, are interacting with microtubules. The degree of association is assayed quantitatively using rhodamine-labeled microtubules after separation of the complex from unbound microtubules by centrifugation upon sucrose gradient. The association is inhibited by crude MAPs, purified MAP2, or 1.0 mM ATP. However, the association neither requires the cytosol from rat liver or bovine brain nor N-ethylmaleimide, brefeldin A, or GTP-gamma-S. The association is mediated by trypsin-sensitive peripheral protein(s) on the Golgi membranes.     

Localization of particulate guanylate cyclase in plasma membranes and microsomes of rat liver.

The subcellular localization of guanylate cyclase was examined in rat liver. About 80% of the enzyme activity of homogenates was found in the soluble fraction. Particulate guanylate cyclase was localized in plasma membranes and microsomes. Crude nuclear and microsomal fractions were applied to discontinuous sucrose gradients, and the resulting fractions were examined for guanylate cyclase, various enzyme markers of cell components, and electron microscopy. Purified plasma membrane fractions obtained from either preparation had the highest specific activity of guanylate cyclase, 30 to 80 pmol/min/mg of protein, and the recovery and relative specific activity of guanylate cyclase paralleled that of 5'-nucleotidase and adenylate cyclase in these fractions. Significant amounts of guanylate cyclase, adenylate cyclase, 5'-nucleotidase, and glucose-6-phosphatase were recovered in purified preparation of microsomes. We cannot exclude the presence of guanylate cyclase in other cell components such as Golgi. The electron microscopic studies of fractions supported the biochemical studies with enzyme markers. Soluble guanylate cyclase had typical Michaelis-Menten kinetics with respect to GTP and had an apparent Km for GTP of 35 muM. Ca-2+ stimulated the soluble activity in the presence of low concentrations of Mn-2+. The properties of guanylate cyclase in plasma membranes and microsomes were similar except that Ca-2+ inhibited the activity associated with plasma membranes and had no effect on that of microsomes. Both particulate enzymes were allosteric in nature; double reciprocal plots of velocity versus GTP were not linear, and Hill coefficients for preparations of plasma membranes and microsomes were calculated to be 1.60 and 1.58, respectively. The soluble and particulate enzymes were inhibited by ATP, and inhibition of the soluble enzyme was slightly greater. While Mg-2+ was less effective than Mn-2+ as a sole cation, all enzyme fractions were markedly stimulated with Mg-2+ in the presence of a low concentration of Mn-2+. Triton X-100 increased the activity of particulate fractions about 3- to 10-fold and increased the soluble activity 50 to 100%.