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, beta-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. IV. Biochemical, physical, and morphological modifications of microsomal components induced by digitonin, EDTA, and pyrophosphate.

Isopycnic equilibration and sedimentation rate studies of rat liver microsomes led previously to the assignment of microsomal constituents into group a1 (monoamine oxidase), group a2 (5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and cholesterol), group a3 (galactosyltransferase), group b (NADH cytochrome c reductase, NADPH cytochrome c reductase, aminopyrine demethylase, cytochrome b(5) and P 450), and group c (glucose 6-phosphatase, esterase, nucleoside diphosphatase, beta-glucuronidase and glucuronyltransferase). Confirmation and extension of the assignment into groups has been obtained by studying the differential effect of the reagents digitonin, EDTA, and PPi. Digitonin specifically affected the equilibrium density only of the group a2 and (to a lesser extent) group a3, and not of groups b and c under conditions which preserved the structure-linked latency of nucleoside diphosphatase and galactosyltransferase. Within experimental error the rate of sedimentation of all microsomal constituents was unaffected. The morphological appearance under the electron microscope was indistinguishable from that of nondigitonin-treated microsomes, except that a few smooth membranes (< 10%) exhibited broken-looking profiles. Treatment of microsomes with EDTA or PPi detached a substantial part of RNA and released protein in excess over the amount accountable for by detachment of ribosome constituents. This detachment was confirmed by electron microscopy. EDTA and PPi decreased markedly the equilibrium density and the density dispersion of groups b and c, due mainly to the uncoating of rough elements. EDTA and PPi shifted slightly the distribution profiles of groups a towards lower densities, possibly as a result of the release of adsorbed proteins. The combination of EDTA and digitonin, used subsequently, rendered the average equilibrium density of group a2 higher than that of groups b and c. Dense subfractions were thus enriched in constituents of group a2 and showed mainly broken-looking vesicles under the electron microscope. The import of our results on the biochemical and enzymic properties of the subcellular components of the microsome fractions is discussed.

Analytical study of microsomes and isolated subcellular membranes from rat liver VIII. Subfractionation of preparations enriched with plasma membranes, outer mitochondrial membranes, or Golgi complex membranes.

Preparations enriched with plasmalemmal, outer mitochondrial, or Golgi complex membranes from rat liver were subfractionated by isopycnic centrifugation, without or after treatment with digitonin, to establish the subcellular distribution of a variety of enzymes. The typical plasmalemmal enzymes 5'-nucleotidase, alkaline phosphodiesterase I, and alkaline phosphatase were markedly shifted by digitonin toward higher densities in all three preparations. Three glycosyltransferases, highly purified in the Golgi fraction, were moderately shifted by digitonin in both this Golgi complex preparation and the microsomal fraction. The outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the out mitochondrial membrane preparation, in agreement wit its behavior in microsomes. With the exception of NADH cytochrome c reductase (which was concentrated in the outer mitochondrial membrane preparation), typical microsomal enzymes (glucose-6-phosphatase, esterase, and NADPH cytochrome c reductase) displayed low specific activities in the three preparations; except for part of the glucose-6-phosphatase activity in the plasma membrane preparation, their density distributions were insensitive to digitonin, as they were in microsomes. The influence of digitonin on equilibrium densities was correlated with its morphological effects. Digitonin induced pseudofenestrations in plasma membranes. In Golgi and outer mitochondrial membrane preparations, a few similarly altered membranes were detected in subfractions enriched with 5'-nucleotidase and alkaline phosphodiesterase I. The alterations of Golgi membranes were less obvious and seemingly restricted to some elements in the Golgi preparation. No morphological modification was detected in digitonin-treated outer mitochondrial membranes. These results indicate that each enzyme is associated with the same membrane entity in all membrane preparations and support the view that there is little overlap in the enzymatic equipment of the various types of cytomembranes.

Analytical study of microsomes and isolated subcellular membranes from rat liver. I. Biochemical methods.

The series introduced by this paper reports the results of a detailed analysis of the microsomal fraction from rat liver by density gradient centrifugation. The biochemical methods used throughout this work for the determination of monoamine oxidase, NADH cytochrome c reductase, NADPH cytochrome c reductase, cytochrome oxidase, catalase, aminopyrine demethylase, cytochromes b(5) and P 450, glucuronyltransferase, galactosyltransferase, esterase, alkaline and acid phosphatases, 5'-nucleotidase, glucose 6-phosphatase, alkaline phosphodiesterase I, N-acetyl-beta-glucosaminidase, beta-glucuronidase, nucleoside diphosphatase, aldolase, fumarase, glutamine synthetase, protein, phospholipid, cholesterol, and RNA are described and justified when necessary.

Analytical study of microsomes and isolated subcellular membranes from rat liver. II. Preparation and composition of the microsomal fraction.

Liver homogenates have been submitted to quantitative fractionation by differential centrifugation. Three particulate fractions: N (nuclear), ML (large granules), and P (microsomes), and a final supernate (S) have been obtained. The biochemical composition of the microsomal fraction has been established from the assay and distribution pattern of 25 enzymatic and chemical constituents. These included marker enzymes for mitochondria (cytochrome oxidase), lysosomes (acid phosphatase and N-acetyl-beta-glucosaminidase), and peroxisomes (catalase). The microsomal preparations were characterized by a moderate contamination with large cytoplasmic granules (only 6.2% of microsomal protein) and by a high yield in microsomal components. Enzymes such as glucose 6-phosphatase, nucleoside diphosphatase, esterase, glucuronyltransferase, NADPH cytochrome c reductase, aminopyrine demethylase, and galactosyltransferase were recovered in the microsomes to the extent of 70% or more. Another typical behavior was shown by 5'-nucleotidase, alkaline phosphatase, alkaline phosphodiesterase I, and cholesterol, which exhibited a "nucleomicrosomal" distribution. Other complex distributions were obtained for several constituents recovered in significant amount in the microsomes and in the ML or in the S fraction.

Rat liver plasma membrane Ca2+- or Mg2+-activated ATPase. Evidence for proton movement in reconstituted vesicles.

The Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane was partly purified by treatments with sodium cholate and lysophosphatidylcholine, and by isopycnic centrifugation on sucrose gradients. The ATPase activity had high sensitivity to detergents, poor nucleotide specificity and broad tolerance for divalent cations. It was insensitive to mitochondrial ATPase inhibitors such as oligomycin and to transport ATPase inhibitors such as vanadate and ouabain. Using the cholate dialysis procedure, the partly purified enzyme was incorporated into asolectin vesicles. Upon addition of Mg2+-ATP, fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) was observed. The quenching was abolished by a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Asolectin vesicles or purified ATPase alone failed to promote quenching. These data suggest that the Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane is able of H+-translocation coupled to ATP hydrolysis.

Colocalization of stomatin (band 7.2b) and actin microfilaments in UAC epithelial cells.

Cytolocalization of stomatin, an integral membrane protein also called erythrocyte band 7.2b, was investigated in a human epithelial cell line in which the expression of this protein is up-regulated after treatment with interleukin-6 and dexamethasone. A monoclonal antibody against stomatin was used to perform immunofluorescence and immunoelectron microscopy. The data show that stomatin concentrates preferentially in small plasma membrane protrusions. It is also found in abundance in a juxtanuclear structure possibly derived from the Golgi apparatus. Fluorescent double staining using the anti-stomatin antibody and the actin binding drug phalloidin shows a significant degree of colocalization of stomatin and cortical actin microfilaments. This association remains after actin filament disruption disruption by cytochalasin D treatment indicating a strong connection between stomatin and the membrane-associated cytoskeleton.

Properties of HIV membrane reconstituted from its recombinant gp160 envelope glycoprotein.

Human immunodeficiency virus (HIV) membrane has been reconstituted from the recombinant envelope glycoprotein precursor (gp160) by a detergent dialysis technique. Electron microscopy shows that gp160-virosomes are spherical vesicles with a mean diameter identical to that of viral particles. Enzyme-linked immunosorbent assay and immunogold labeling demonstrate efficient association of gp160 with lipid vesicles and proteolysis treatment reveals an asymmetric insertion with about 90% of glycoproteins having their gp120-moiety pointing outside. Glycoproteins are organized as dimers and tetramers and gp160 retains its ability to specifically bind CD4 receptor after reconstitution into virosome.

A three-dimensional study of organelle interrelationships in regenerating rat liver. 2. Transient connections with luminal continuity between thin- and thick-membraned organelles.

Luminal continuities can be established between thin- and thick-membraned structures, and serve the transfer of their contents. The abrupt change in membrane thickness at the site of connection suggests that exchange of membrane constituents is very limited. This enables retrograde separation of the connected organelles. sER (endoplasmic space) makes up such continuities with exoplasmic structures (mature Golgi cisterna, GERL-system, occasionally MVB). Transfer tubules, possessing a thiny membrane but nevertheless belonging to the exoplasmic space, take up endocytized material from large coated vesicles or from coated plasma membrane loops and carry it to MVB. They offer the possibility to recycle the internalized plasma membrane.

Purification and characterization of a heterogeneous glycosylated invertase from the rumen holotrich ciliate Isotricha prostoma.

The invertase (beta-fructofuranosidase, EC 3.2.1.26) of the rumen holotrich ciliate Isotricha prostoma has been purified. This is the first report of an enzyme purification from a known species of rumen protozoon. Cells were disrupted by ultrasonic treatment and the enzyme was purified from the cell-free extract by three successive liquid column chromatographies (Sepharose CL4B/octyl-Sepharose CL4B, DE52 DEAE-cellulose and concanavalin A-Sepharose 4B). This resulted in a 160-fold purification and a 15% yield. The major form of the purified enzyme was a tetramer with Mr about 350,000 that was readily dissociated by electrophoresis. The invertase was heterogeneous, as five types of monomers were shown by SDS/polyacrylamide-gel electrophoresis after denaturation. Part of this heterogeneity was due to different glycosylated forms of one of the polypeptides present in the purified enzyme. Isotricha prostoma invertase exhibited maximum activity at pH 5.5-6.0 and 50 degrees C. The kinetic properties of the purified enzyme were very similar to those of invertases from other sources such as yeast or plants (substrate and product inhibition, transferase activity).

A three-dimensional study of organelle interrelationships in regenerating rat liver. 6. Golgi apparatus.

Luminal materials from the ER are supplied to the Golgi apparatus (GA) either via precisternal elements (PE) detaching from the ER and building up a first cisterna, or via direct luminal continuities with the sER. Membrane supply is believed to follow only the first of these two pathways. It is further argued that during the processing of luminal materials and membrane - two Golgi activities that proceed at a different rate - bits of membrane carrying the differentiation enzymes are recycled to less differentiated membranes in the form of Golgi vesicles (GV).

A three-dimensional study of organelle interrelationships in regenerating rat liver. 5. Autophagy.

ER, mature Golgi, plasma and lysosomal membranes are all able to sequester bits of cytosol and eventually some organelles. These four modes to provide in autophagy are complementary to each other. Apart from the digestion of the sequestered material, the implicated membranes (respectively ER, Golgi, plasma or lysosomal membranes) are partially degraded. This may counterbalance the continuous new formation of these membranes. It is proposed that the thin outer membrane of ER-derived autophagosomes is gradually converted into thick lysosomal membrane through consecutive fusions with different types of Golgi vesicles and with small dense bodies.

A three-dimensional study of organelle interrelationships in regenerating rat liver. 7. Size, shape and dynamism of secondary lysosomes.

Digestion of macromolecules inside secondary lysosomes results in small molecules leaving the lysosome by permeation. This may cause a drop in osmotic pressure and a consequent shrinkage of the lysosome. A membrane invagination may be formed which becomes internalized and then digested. This digestion causes new shrinkage. Minimal-sized lysosomes may ensue. The later are again engaged in digestive events when fusing with larger lysosomes. Through consecutive shrinkage and fusion cycles of many lysosomes residues slowly accumulate. Shrinkage may be counteracted by adding digestible material without increasing the lysosomal membrane surface. Transfer tubules can do so. They deliver their content to multivesicular bodies. The other lysosome types, however, are subject to shrinkage since they fuse with organelles (phagosomes, primary lysosomes) which insert at least part of their membrane.

A three-dimensional study of organelle interrelationships in regenerating rat liver. 3. Organelles related to bile.

A number of cell structures are described which show a morphological relationship to the bile canaliculi. Two types of peribiliary vesicles are identified: osmication positive ones occurring between the bile canaliculi and the osmicated immature Golgi cisternae and probably deriving from the latter, and osmication negative ones related to MVB, on which they appear as buds. Small coated vesicles are seen attached to this second type. Large lacunae may originate from MVB, as suggested by the MVB-like internal vesicles they may contain. Some stay in luminal continuity with the bile canaliculi. Canalicular coated vesicles are seen as parts of the canalicular plasma membrane and free in the cytoplasm.

A three-dimensional study of organelle interrelationships in regenerating rat liver. 4. Multivesicular bodies.

Double-membraned plasma-membrane-loops are formed from the plasma membrane and released into the cytoplasm. The vesiculation of mainly their inner membrane may transform them into MVB. The so-formed internal vesicles contain cytosol. In many MVB the surface of all their internal vesicles to gether corresponds well with the surface of their bordering membrane. This correlation may be the result of a dynamic equilibrium whereby degradation (and partial recyclage) of internal vesicles is compensated for by the formation of new internal vesicles. When this equilibrium becomes disturbed, MVB may show much less internal vesicles. Such 'depleted' MVB are often producing peribiliary vesicles and may themselves transform into lacunate emptying into the bile canaliculi.

PIF1: a DNA helicase in yeast mitochondria.

The PIF1 gene is involved in repair and recombination of mitochondrial DNA (mtDNA). In this study, the PIF1 gene product, which cannot be identified in normal yeast cells, has been overproduced from the GALI promoter to detectable protein levels. Location of PIF1 in mitochondria has been shown by immunoelectron microscopy and in vivo import experiments using ts mas1 mutants deficient in the mitochondrial matrix-localized processing protease. Overproduction of PIF1 protein in pif1 mutants restores mtDNA recombination proficiency but is toxic to yeast cells as observed by slower growth. The overproduced PIF1 protein, which is firmly associated with insoluble mitochondrial structures, has been partially purified in a mitochondrial nuclease deficient nuc1 strain by a procedure including solubilization by urea and renaturation by dialysis at alkaline pH. PIF1 is a single-stranded (ss) DNA-dependent ATPase and a DNA helicase which unwinds partially DNA duplexes in a 5' to 3' direction with respect to the ss DNA on which it binds first.

A three-dimensional study of organelle interrelationships in regenerating rat liver. 1. The Gerl-system.

Arguments are put together indicating that the GERL-system, or at least its thick membrane and part of its content are derived from the mature Golgi cisterna. Just like every Golgi cisterna does, the GERL shows direct continuities with ER, from which a complement of products may be supplied. Rigid lamellae are interpreted as intermediate forms in the transformation of the mature Golgi cisterna into the GERL-system. In hepatocytes the GERL produces two types of lysosomes: dense bodies, which may either contain lipoprotein granules or not, and small coated vesicles. The probable meaning of lipoprotein digestion (crinophagy) is discussed.

Proliferation of intracellular structures upon overexpression of the PMA2 ATPase in Saccharomyces cerevisiae.

The PMA2 gene is a presumed isogene of the PMA1 gene, encoding the major yeast plasma membrane H(+)-ATPase. When controlled by its own promoter, PMA2 in multiple copies does not complement a deficient PMA1 gene. Under the control of the PMA1 promoter, however, and expressed on a centromeric plasmid in yeast strains specially designed for stable expression, the PMA2 gene replaces the PMA1 gene to some extent, allowing growth on standard medium but not on acidic media. Plasma membranes of cells expressing only the PMA2 enzyme display low ATPase activity correlating with low amounts of PMA2 protein. This low activity is maintained throughout growth and does not increase when overexpression is favored by increased gene dosage. Immunoelectron microscopy reveals a dramatic proliferation of intracellular structures (probably endoplasmic reticulum) in which overexpressed PMA2 protein accumulates. Overexpression of PMA1 ATPase causes a similar phenomenon, but quantitative effects are lower compared to PMA2. These results indicate that the PMA2 gene encodes a functional plasma membrane H(+)-ATPase and suggest a specific control of the intracellular traffic of plasma membrane ATPase.