Radioimmunolocalization of murine mammary tumor virus proteins in gels

Nycodenz is a new nonionic iodinated gradient medium which readily dissolves in water to give nontoxic, autoclavable solutions. This paper describes the use of diffusion techniques to prepare isotonic Nycodenz gradients with maximum densities up to 1.15 g/ml, which is sufficient to band most types of cells.     

Rapid subcellular fractionation of the rat liver endocytic compartments involved in transcytosis of polymeric immunoglobulin A and endocytosis of asialofetuin.

The distributions of two endocytosed radiolabelled ligands (polymeric immunoglobulin A and asialofetuin) in rat liver endocytic compartments were investigated by using rapid subcellular fractionation of post-mitochondrial supernatants on vertical density gradients of Ficoll or Nycodenz. Two endocytic compartments were identified, both ligands being initially associated with a light endocytic-vesicle fraction on Ficoll gradients, asialofetuin then accumulating in denser endosomes before transfer to lysosomes for degradation.     

Isopycnic centrifugation of mammalian metaphase chromosomes in Nycodenz

Separation of chromosomes on the basis of buoyant density has proved difficult due to their high density and sensitivity to high ionic strength. Nycodenz is a new non-ionic gradient medium which offers significant advantages over previously described media for separation of chromosomes. The purpose of this study was to examine the banding characteristics of isolated mammalian metaphase chromosomes in Nycodenz gradients. The results indicate that chromosomes can be efficiently concentrated and separated from nuclei using Nycodenz gradients. Furthermore, chromosomes density-labeled with bromodeoxyuridine (BrdU) can be separated from unlabeled chromosomes in Nycodenz gradients. Nycodenz does not appear to alter chromosome morphology or protein complement. The introduction of Nycodenz represents a significant new tool for use in chromosome separation and purification.     

Buoyant densities of macromolecules, macromolecular complexes, and cell organelles in Nycodenz gradients

Nycodenz is a new nonionic iodinated density gradient medium which has several advantages over metrizamide. Although, overall, biological samples band at similar densities in Nycodenz and metrizamide gradients, a number of significant differences were found. As compared with metrizamide, not only does Nycodenz appear to interact less with proteins but also the buoyant density of chromatin is less affected by the amount loaded onto the gradient. A high degree of resolution is obtainable using Nycodenz gradients; thus, it is possible to separate density-labeled DNA and to subfractionate subcellular membrane fractions.     

Effect of ligand binding on the buoyant density of DNA in Nycodenz gradients.

The effect of ligand binding upon the buoyant density of DNA in Nycodenz gradients has been studied using DNAs of differing base compositions. The effect of both intercalating ligands (ethidium bromide and proflavin) and non-intercalating ligands (distamycin A, DAPI and netropsin) has been studied. The binding of intercalating ligands to DNA has essentially no effect on the buoyant density of DNA in Nycodenz gradients. The non-intercalating ligands were found to increase the buoyant density of DNA in a base specific manner. The increase in buoyant density can be interpreted in terms of disruption of the hydration shell of the DNA molecule caused by the binding of the ligand along the minor groove of the DNA helix.     

Subcellular distribution of superoxide dismutases (SOD) in rat liver: Cu,Zn-SOD in mitochondria

Rat liver was homogenized in isotonic buffer, fractionated by differential centrifugation, and then subfractionated by equilibrium sedimentation in Nycodenz gradients. Fractions were assayed for both Cu,Zn-superoxide dismutase (SOD) and Mn-SOD by exploiting the cyanide sensitivity of the former activity and by the use of specific antibodies. As expected, the cytosol and lysosomal fractions contained Cu,Zn-SOD; while the mitochondrial matrix contained Mn-SOD. In mitochondria, Cu,Zn-SOD was found in the intermembrane space and Mn-SOD in the matrix and also on the inner membrane. The Mn-SOD associated with the inner membrane was solubilized by 0.5 m NaCl. Surprisingly the intracellular membrane fraction (microsomes) contained bound Cu,Zn-SOD that could be solubilized with a detergent, and to lesser degree with 0.5 m NaCl. Both the cytosolic and mitochondrial Cu,Zn-SODs were isolated and compared. They have identical molecular mass, cyanide sensitivity, SDS sensitivity, heat stability, and chloroform + ethanol stability. Tissue from Cu,Zn-SOD knockout mice was entirely devoid of Cu,Zn-SOD; indicating that the cytosolic and the intermembrane space Cu,Zn-SODs are coded for by the same gene. The significance of this distribution of the SODs is discussed.     

The Preparation of Subcellular Organelles from Mouse Liver in Self-Generated Gradients of Iodixanol

This paper reports the use of a new density gradient compound, Iodixanol, for the resolution of the major organelles from mouse liver. A major advantage of Iodixanol over other iodinated density gradient media is its ready ability to form self-generated gradients. Gradient-forming conditions have been modulated to provide optimal recoveries of Golgi membranes, lysosomes, mitochondria, and peroxisomes. The organelles were isolated in high yield (80-90% of gradient input) and high purity. Nycodenz and Iodixanol were compared using preformed gradients. Iodixanol provided resolution superior to that of Nycodenz, notably of peroxisomes and mitochondria and the separation of lysosomes from endoplasmic reticulum. Because Iodixanol does not interfere significantly with marker enzyme activities, gradient fractions can be analyzed without removal of the gradient medium.     

Isolation of the major subcellular organelles from mouse liver using Nycodenz gradients without the use of an ultracentrifuge

Commonly, subcellular organelles such as nuclei, mitochondria, lysosomes, and Golgi membranes are isolated first by differential centrifugation in low-speed or high-speed centrifuges and then purified by gradient centrifugation in ultracentrifuges. We have prepared these organelles using a new high-speed centrifuge (28,000 rpm max) which allows the generation of higher radial centrifugal forces (rcfs) than are available in standard machines. We have shown that most subcellular organelles can be purified by using low-viscosity Nycodenz gradients at rcfs lower than those normally used in ultracentrifuges, without increasing the time of centrifugation. Use of Nycodenz also allows rapid harvesting of material from gradients and we have adapted a number of enzyme assays to facilitate gradient analysis.     

Evidence for a role of the hepatic endocytic compartment in the modulation of the extracellular matrix

The presence of fibronectin in rat liver endocytic compartment was investigated using biochemical and immunological approaches. Three endosome subfractions were separated from postmitochondrial lysosome supernatants using sucrose and isoosmotic Nycodenz gradients. Using these endosomes the presence of fibronectin in the "early" and "late" endosome subfractions and in a receptor-enriched fraction was demonstrated by Western blot analysis. Furthermore, immunofluorescence studies using an anti-rat fibronectin antiserum and an anti-rat endosome antiserum showed similar patterns of staining in frozen liver sections. The results indicate that the components of the extracellular matrix extend into the endocytic compartment and suggest that fibronectin is internalised in a manner similar to that of some plasma membrane proteins.     

SEPARATION OF CATECHOLAMINE STORING SYNAPTOSOMES IN COLLOIDAL SILICA DENSITY GRADIENTS

Abstract— Catecholamine storing particles mainly from rat brain hypothalamus and corpus striatum have been isolated by isopycnic centrifugation in density gradients made of colloidal silica. As markers, tritium-labelled noradrenaline, endogenous noradrenaline and dopamine were measured. Cytochrome oxidase was determined as an indicator of mitochondria.
Two distinct populations of amine containing particles were recognized with densities of 1 , 03–1.04 g/ml and 1 , 045–1.065 g/ml in continuous isotonic gradients made of silica sol and a polymer. The light fraction was assumed to contain myelin fragments, light synaptosomes and possibly also catecholamine storage vesicles, while the other one was probably a heavy population of synaptosomes containing more mitochondria. Free mitochondria were found in a band at a density of 1 , 09–1.11.
The distribution pattern in isotonic gradients was compared with that in density gradients made of silica sol and sucrose or sucrose alone. The heavy population of the catecholamine particles was found to have a higher density in hypertonic gradients. Furthermore these synaptosomes seemed to lose more mitochondria and catecholamines than those in isotonic gradients probably due to the hypertonicity.
The present results confirm similar findings by other workers separating brain sub- cellular particles in isotonic gradients of Ficoll and sucrose.
Colloidal silica solutions might be of value for analytical centrifugation of brain sub-cellular particles, since it has a lower tonicity than sucrose, lower viscosity than Ficoll and furthermore it is very easy to handle. The silica sol is inexpensive and allows large scale work.     

Buoyant density of Escherichia coli is determined solely by the osmolarity of the culture medium

In previous studies, we had shown that the buoyant density ofEscherichia coli is determined by the osmolarity of the growth medium by varying the osmolarity of the medium with NaCl or sucrose. However, the buoyant density of the cells always exceeded that of the growth medium. Here we determined the effect of medium with a buoyant density greater than the expected buoyant density of cells by adding Nycodenz to Luria broth. Percoll gradients of cells were analyzed by laser light scattering. The buoyant density for 125- and 375-mOsM-grown cells was 0.002 g/ml and 0.003 g/ml more, respectively, for cells grown in the presence of Nycodenz than those grown without Nycodenz, while the buoyant density of 250-mOsM-grown cells was 0.005 g/ml less for cells grown in the presence of Nycodenz than those grown without Nycodenz. Cells grown in 500-mOsM medium with or without Nycodenz had the same buoyant density. the buoyant density of cultures grown in defined medium was the same as those grown in rich medium, with only the medium osmolarity correlating to buoyant density. We conclude from these experiments that neither buoyant density nor chemical make-up of the medium determines the buoyant density of cells grown in that medium. Only the medium osmolarity determines cell buoyant density, suggesting thatE. coli has no mechanisms to sense buoyant density.     

Subfractionation of hepatic endosomes in Nycodenz gradients and by free-flow electrophoresis. Separation of ligand-transporting and receptor-enriched membranes.

The complexity of rat liver endosome fractions containing internalized radioiodinated asialotransferrin, asialo-(alkaline phosphatase), insulin and prolactin was investigated by using free-flow electrophoresis and isopycnic centrifugation in Nycodenz gradients. Two subfractions were separated by free-flow electrophoresis. Both subfractions contained receptors for asialoglycoprotein and insulin. Glycosyltransferase activities were associated with the more electronegative vesicles, whereas 5'-nucleotidase and alkaline phosphodiesterase activities were associated with the less electronegative vesicles. Three subfractions were separated on Nycodenz gradients. Two subfractions, previously shown to become acidified in vitro, contained the ligands. At short intervals after uptake (1-2 min), ligands were mainly in subfraction DN-2 (density 1.115 g/cm3), but movement into subfraction DN-1 (density 1.090 g/cm3) had occurred 10-15 min after internalization. Low amounts of glycosyltransferase activities were associated with subfraction DN-2, and 5'-nucleotidase and alkaline phosphodiesterase activities were mainly located in subfraction DN-1. The binding sites for asialoglycoproteins and insulin were distributed towards the higher density range in the Nycodenz gradients, thus indicating a segregation of receptor-enriched vesicles and those vesicles containing the various ligands 10-15 min after internalization. Electron microscopy of the subfractions separated on Nycodenz gradients indicated that whereas the ligand-transporting fractions consisted mainly of empty vesicles (average diameter 100-150 nm), the receptor-enriched component was more granular and smaller (average diameter 70-95 nm). The properties of the endosome subfraction are used to assign their origin to the regions of the endocytic compartment where ligand-receptor dissociation and separation occur.     

Mitochondrial and Peroxisomal β-Oxidation of Stearic and Lignoceric Acids by Rat Brain

Crude subcellular fractions were prepared from adult rat brains by differential centrifugation of brain homogenates. Greater than 98% of the cellular mitochondrial marker enzyme activity sedimented in the heavy and light mitochondrial pellets, and less than 1% of the activity sedimented in microsomal pellets. Lysosomal marker enzyme activities mainly (71-78% of cellular activity) sedimented in the heavy and light mitochondrial pellets. Significant amounts of the lysosomal marker enzyme activity also sedimented in the crude microsomal pellets (9-13% of total) and high-speed supernatants (14-16% of total). The specific activities of microsomal and peroxisomal marker enzyme activities were highest in the crude microsomal pellets. Fractionation of the crude microsomal pellets on Nycodenz gradients resulted in the separation of the bulk of the remaining mitochondrial, lysosomal, and microsomal enzyme activities from peroxisomes. Fatty acyl-CoA synthetase activities separated on Nycodenz gradients as two distinct peaks, and the minor peak of the activities was in the peroxisomal enriched fraction. Fatty acid beta-oxidation activities also separated as two distinct peaks, and the activities were highest in the peroxisomal enriched fractions. Mitochondria were purified from the heavy mitochondrial pellets by Percoll density gradients. Fatty acyl-CoA synthetase and fatty acid beta-oxidation activities were present in both the purified mitochondrial and peroxisomal enriched fractions. Stearoyl-CoA synthetase activities were severalfold greater compared to lignoceroyl-CoA synthetase, and stearic acid beta-oxidation was severalfold greater compared to lignoceric acid beta-oxidation in purified mitochondrial and peroxisomal enriched fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and partial characterization of the luminal plasmalemma of microvascular endothelium from rat lungs.

This paper describes a procedure for isolating in high yield and at a high degree of purity the endothelial luminal plasmalemma from the microvasculature of the rat lung. The procedure relies on the modification of the density of the luminal plasmalemma obtained by coating it by perfusion in situ first, with cationized colloidal silica and then with Na polyacrylate. These steps generate a strongly adhering coat to the luminal plasmalemma that resists tissue homogenization to yield, upon repeated centrifugation through Nycodenz density gradients, a nearly homogeneous fraction of coated luminal plasmalemmal fragments still carrying their associated plasmalemmal vesicles. The fraction is enriched in the luminal plasmalemmal antigen, angiotensin converting enzyme, contains gp60, an antigen expected to occur on both plasmalemmal domains, is not enriched in either alkaline phosphatase or 5'-nucleotidase activity and is free of the mitochondrial and endoplasmic reticulum antigens so far tested. This procedure, that can be extended--in principle--to any vascular bed, obviates the use of cultured cells for studying the biochemistry of the endothelium, at least as far as the luminal endothelial plasmalemma is concerned.     

A rapid method for the isolation of peroxisomes from rat liver

A preparative method for the isolation of peroxisomes from the liver of normal, untreated rats is described. The peroxisome-enriched "light mitochondrial" fraction is layered on a 30% Nycodenz (5-[N-2,3-dihydroxypropylacetamido]-2,4,6-triiodo-N,N'-bis[2, 3-dihydroxypropyl]isophthalamide) solution containing 1 mM tetrasodium EDTA and then centrifuged in an angular rotor for 1 h at 130,000gavg. Peroxisomes are sedimented to the bottom leaving other organelles at the top of the tube. On the basis of morphological and biochemical studies, it is found that the peroxisomes (marker-enzymes catalase and urate oxidase) obtained in this method are not contaminated with lysosomes (marker-enzyme acid phosphatase) and contained very few mitochondria (marker-enzyme succinate-cytochrome c reductase) and microsomal vesicles (marker-enzyme glucose-6-phosphatase).     

Effect of a capsid-stabilizing pyridazinamine, R 78206, on the eclipse and intracellular location of poliovirus.

R 78206 (a pyridazinamine derivative) inhibits the formation of poliovirus eclipse particles. Its effect on the intracellular location of poliovirus was studied by separating subcellular fractions in iso-osmotic Nycodenz gradients. The compound did not inhibit internalization of intact virus into small lipid vesicles, but it did inhibit the release of virus from these vesicles and its entry into lysosomes.     

Highly purified bile-canalicular vesicles and lateral plasma membranes isolated from rat liver on Nycodenz gradients. Biochemical and immunolocalization studies.

1. A liver canalicular plasma-membrane fraction enriched 115-155-fold in five marker enzymes relative to the tissue homogenate was obtained by sonication of liver plasma membranes followed by fractionation in iso-osmotic Nycodenz gradients. 2. Two lateral-plasma membrane fractions were also collected by this procedure; the lighter-density fraction was still associated with canalicular membranes, as assessed by enzymic and polypeptide analysis. 3. The polypeptide composition of the domain-defined plasma-membrane fractions was evaluated. It was demonstrated by immunoblotting that the 41 kDa alpha-subunit of the inhibitory G-protein, associated in high relative amounts with canalicular plasma-membrane fractions, was partially lost in the last stage of purification; however, this subunit was retained by lateral plasma membranes. 4. Antibodies to the proteins of bile-canalicular vesicles were shown to localize to the hepatocyte surface in thin liver sections examined by immunofluorescent and immuno-gold electron microscopy. Two subsets of antigens were identified, one present on both sinusoidal and canalicular plasma-membrane domains and another, by using antisera pre-absorbed with sinusoidal plasma membranes, that was confined to the bile-canalicular domain.     

Purification of liver membranes highly enriched in phosphatidylinositol kinase.

Membranes highly enriched in phosphatidylinositol (PtdIns) kinase were purified from rat liver by sucrose density gradient centrifugation of a plasma membrane-depleted microsomal fraction. PtdIns kinase-containing membranes had a lower density than membranes containing Golgi and plasma membrane markers, both in sucrose and Nycodenz gradients, without being completely resolved from these other membranes. They also had a lower density than an endosomal marker. Furthermore, lectin affinity partitioning showed that PtdIns kinase did not reside in plasma membranes. PtdIns kinase in different membrane fractions was of type II and had similar kinetic properties. We suggest that the isolated membranes are the major site for phosphatidylinositol 4-phosphate formation in the liver cell, and that these membranes are part of the exocytic pathway. Thus, PtdIns kinase might be a convenient marker for the exocytic process.     

Phospholipid, cholesterol, polypeptide and glycoprotein composition of hepatic endosome subfractions.

The composition of hepatic endosome subfractions was compared directly with that of plasma membranes and Golgi-apparatus fractions. The neutral lipid and phospholipid composition of two endosome subfractions separated on Nycodenz gradients from a parent endosome fraction was similar to that of plasma membranes. The phospholipid/cholesterol ratios and the sphingomyelin contents were high, as in plasma membranes. However, the phosphatidylserine content was low. Endosomal subfractions contained a simpler polypeptide profile than did plasma membranes. However, a large number of glycoproteins were common to both fractions. Two endosome-specific glycoproteins, of Mr 59 000 and 38 000, were identified. Sialic acid was present at concentrations higher than in plasma membranes. The results indicate that endosomal membranes have a similar composition to plasma membranes that probably reflects their functional interaction during endocytosis and receptor recycling.     

Glutathione metabolism in yeast Saccharomyces cerevisiae. Evidence that gamma-glutamyltranspeptidase is a vacuolar enzyme

In a first experiment we have shown that S. cerevisiae beta-glutamyltranspeptidase is associated with a particulate fraction obtained by differential centrifugation. We have subsequently shown that this enzyme activity followed accurately the distribution of vacuolar markers. Liberation of vacuoles was carried out by mechanical disruption of spheroplast under isotonic conditions and the vacuoles were purified by centrifugation of Ficoll gradients. Yeast beta-glutamyltranspeptidase could be implicated in the exchanges of amino acids between the cytoplasm and the vacuolar sap.