Isopycnic Sucrose Gradient Centrifugation Research Articles

Particulate isocitrate lyase and malate synthase in Caenorhabditis elegans

Biochemical evidence is presented suggesting the particulate nature of some of the glyoxylate cycle enzymes in the free-living nematode Caenorhabditis elegans. A crude homogenate of freshly grown nematodes was prepared by gentle grinding. Isopycnic sucrose gradient centrifugation of the supernatant fraction obtained by low-speed centrifugation yielded four protein bands. The glyoxylate cycle enzymes, isocitrate lyase and malate synthase, appeared in the lowermost band at a density of 1.25 g/cm 3, while the mitochondrial enzymes, fumarase and NADH oxidase, equilibrated at a density of 1.18 g/cm 3. The glyoxylate cycle and the mitochondrial enzymes were released differentially from the particulate fraction either by sonic treatment or by treatment with 0.1% Triton X-100. The specific activities of isocitrate lyase and malate synthase in the supernatant fraction obtained after a sonic treatment of the particulate fraction were always higher than those observed in the parent fraction.

The isolation of plasma membrane from protoplasts of soybean suspension cultures.

A procedure for the isolation of plasma membranes from protoplasts of suspension-cultured soybean is described. Protoplasts were prepared by enzymic digestion of the cell wall and the plasma membrane was labelled with radioactive diazotized sulphanilic acid. The membrane systems from broken protoplasts were separated by continuous isopycnic sucrose gradient centrifugation. Radioactivity was localized in a band possessing a buoyant density of 1-14 g ml-1. The activities of NADPH- and NADH-cytochrome c reductase, fumarase, Mg2+-ATPase, IDPase and acid phosphodiesterase in the various regions of the density gradient were determined. A plasma membrane fraction was selected which was relatively uncontaminated with membranes derived from endoplasmic reticulum, tonoplasts and mitochondria. The results indicated that Mg2+-ATPase and possibly acid phosphodiesterase were associated with the plasma membrane.

The isolation and characterisation of particulate sn-glycerol-3-phosphate oxidase from Trypanosoma brucei

1. 1. A method for the isolation of sn-glycerol-3-phosphate oxidase from T. brucei is described. After pre-treatment with saponin, bloodstream trypanosomes can be easily disrupted by gentle homogenisation. The particulate glycerophosphate oxidase can be purified 10-fold by differential centrifugation, sedimentation occurring at 24,000 g. Further purification to 24-fold has been achieved by isopycnic sucrose gradient centrifugation. The oxidase forms a symmetrical peak with a modal density of 1.17 g/ml. sn-Glycerol-3-phosphate: (acceptor) oxidoreductase (E.G. 1.1.99.5) distributes identically with the glycerophosphate oxidase in subcellular fractions and in sucrose gradients and is therefore considered to be the dehydrogenase component of the holo-enzyme. 2. 2. FAD has been identified as a coenzyme for the glycerophosphate oxidase. Flavin, copper and iron are present in partially purified preparations in a ratio of 1:4:8. Iron liganded to acid-labile sulphide is not present. EPR studies revealed an iron signal at g = 4.1 together with a cuproprotein with a signal at g = 2.05. The latter is released from the oxidase by freezing and thawing and can not be considered a component of the oxidase. The copper remaining in the preparations after freezing and thawing can not be detected by EPR.

The Stichosome and Its Secretion Granules in the Mature Muscle Larva of Trichinella spiralis

The stichosome of the mature muscle larva of Trichinella spiralis consists of a single row of 45 to 55 stichocytes. Each stichocyte is about 25 mum in diameter and possesses a single nucleus. A duct leads from each stichocyte to the lumen of the esophagus. The stichocyte cytoplasm contains mitochondria, structures resembling Golgi-complexes, rough endoplasmic reticulum, and usually 1 of 2 types of secretory granules. The alpha-granule measures about 800 mn in diameter, contains a prominent inclusion, and has a granular matrix. The beta-granule is about 600 mn in diameter and is homogeneous in appearance. Both granule types are surrounded by a single membrane. Ten to thirteen stichocytes containing alpha-granules are confined to the posterior portion of the stichosome. After isopycnic centrifugation in sucrose gradient of large granule fractions obtained from cell-free homogenates, the alpha- and beta-granules show characteristic distribution patterns as revealed by the morphology of the fractions. The median equilibrium density of the alpha-granules is 1.245, while that of the beta-granules is 1.230. There is a correlation between the distribution of the granules and of antigens reacting with hyperimmune antitrichinella seruma. At least 4 unique antigens can be attributed to each of the granule types. Fractions enriched in mitochondria do not contain specific antigens. Antigens from both types of secretory granules cross react totally with those present in the excretion-secretion products of living muscle larvae. Cytoimmunochemical data show that antigens are distributed in a patchy fashion throughout the stichocyte cytoplasm. This finding is consistent with the distribution of the secretory granules in the intact stichocyte.

Respiratory syncytial virus proteins

Respiratory syncytial (RS) virus grown in BS-C-1 cells was concentrated from the fluid of infected cultures by precipitation with polyethylene glycol (PEG) and banded by isopycnic centrifugation in sucrose or metrizamide density gradients. At least six virus-specified polypeptide bands, one of which was heterogeneous, could be resolved by continuous SDS-polyacrylamide gel electrophoresis (PAGE) and an additional band by discontinuous SDS-PAGE. The three predominant viral polypeptides were a glycopolypeptide of 48 × 10 3 (VGP48), a nucleocapsid polypeptide of 41 × 10 3 (VP41), and a polypeptide of 27 × 10 3 molecular weight (VP27). Three minor viral polypeptides have been assigned the molecular weight of 38 × 10 3 (VP38), 32 × 10 3 (VP32) and 25 × 10 3 (VP25). A minor glycopolypeptide of molecular weight 42 × 10 3 (VGP42) may exist also. Partial purification was accompanied by the loss of high molecular weight glycopolypeptides; however, one high molecular polypeptide (P2) remained consistently associated with the presumptive polypeptides and may represent an eighth virus-specified polypeptide. VP27 can be obtained in relatively pure form by sedimentation of detergent-treated RS virus in a metrizamide gradient containing detergent.

Subcellular localization of glyoxylate cycle enzymes in Ascaris suum larvae.

Evidence is presented on the particulate nature of glyoxylate cycle enzymes in metazoa with the use of 15-day old larvae of the nematode Ascaris suum. Homogenization procedures were developed to disrupt the resistant nematode cuticle. Malate synthase and isocitrate lyase, key enzymes of the glyoxylate cycle, consistently sedimented with mitochondrial enzymes in differential pellets while catalase, a major peroxisomal enzyme, was always soluble. Isopycnic sucrose gradient centrifugation of the differential pellet yielded two protein peaks: one at 1.18 g/cm3 (characteristic for mitochondria), and another at 1.23 g/cm3 (common for glyoxysomes and peroxisomes). Electron microscopy of these fractions revealed that the lighter peak consisted primarily of mitochondria, while the heavier band contained proteinaceous bodies termed "dense granules" morphologically resembling microbodies. SIgnificantly, both malate synthase and isocitrate lyase cosedimented with the mitochondrial marker enzymes in the lighter peak (1.18 g/cm3) and not with the dense granules. Further purification of mitochondria, accomplished by separating dense granules with a step gradient before isopycnic centrifugation, substantiated the evidence that microbodies (glyoxysomes) do not occur in these nematode larvae. Rough-surfaced membranes were alternatively considered as the subcellular site, but the evidence tends to favor localization of the glyoxylate bypass enzymes in the mitochondria.

The purification and characterization of plasma membranes and the subcellular distribution of adenylate cyclase in mouse parotid gland

1. 1. Plasma membranes have been purified 17-fold from mouse parotid gland homogenates prepared in hypertonic sucrose media using differential centrifugation. The method is fast and simple. The membranes were characterised by electron microscopy, enzyme composition and chemical composition. Further purification was achieved by isopycnic centrifugation in discontinuous sucrose gradients. 2. 2. The purified membranes contain an adenylate cyclase activity which is stimulated by isoproterenol and fluoride. Only 50% of the total adenylate cyclase activity sedimented in the plasma membrane fraction. The rest of the activity resided in the crude nuclear and mitochondrial pellets. However, this adenylate cyclase activity was not associated with these organelles but with membrane fragments in the pellets. Purified nuclei did not contain adenylate cyclase activity. 3. 3. Adenylate cyclase activity was also localised by electron microscopic cytochemistry. Besides being found at the plasma membrane, large amounts of adenylate cyclase were found in a small proportion of the vesicles within the acinar cells, which appeared to be secondary lysosomes. 4. 4. Adenylate cyclase activities, under standard assay conditions, are proportional to the time of incubation and the concentration of enzyme. The enzyme requires both Mg 2+ and Ca 2+ for activity. Isoproterenol increases activity 2-fold and this increase is abolished by β-adrenergic blocking agents.

Isolation and characterization of two outer membrane preparations from Escherichia coli

A method was developed for releasing specifically a part of outer membrane during spheroplast formation. A highly purified outer membrane (outer membrane I) was obtained from the spheroplast medium by isopycnic sucrose gradient centrifugation. The remaining outer membrane (outer membrane II) and cytoplasmic membrane was also isolated from the spheroplasts by the isopycnic centrifugation. Two outer membrane preparations were different from the cytoplasmic membrane in protein composition, enzyme localization, phospholipid composition, lipopolysaccharide content and electron micrographs. Although outer membranes I and II were almost the same in various respects, they seemed to be different from each other under electron microscope and in cardiolipin content. It is suggested that the outer membrane I and the outer membrane II, at least a part of the outer membrane II, are integrated in a different fashion in the outer-most layer of Escherichia coli cell surface.

Electron Microscopy of a Tumor Virus RNA

The structure of Friend leukemia virus (FLV) has been studied by electron microscopy. The virus was harvested from the plasma of infected mice and purified by techniques including differential velocity and isopycnic sucrose gradient centrifugation. Viral nucleic acid was isolated using GuHCl followed by precipitation with ethanol and resuspension in 1 mM dithiothreitol. In initial experiments, the RNA solution was diluted 1:10 into either H2O or 4-8 M urea. Prior to spreading, 1 M ammonium acetate and cytochrome c were added to a final concentration of 0.4 M and 0.01% respectively. Fifty μ1 of this mixture was spread using a glass slide inclined in a ten cm2 trough filled with 15 mM ammonium acetate. Samples were picked up on collodion filmed grids, stained with uranyl acetate and rotary shadowed with Pt:Pd. This method is very similar to the conditions used to study the RNA of avian myeloblastosis virus, murine leukemia virus, and mammary tumor virus (MTV).

Artifactual subcellular distribution of dopamine β-hydroxylase: Effects of dilution and non-specific protein on the dopamine β-hydroxylase activity of membranes of adrenomedullary and sympathetic nerve vesicles

Dopamine β-hydroxylase (EC 1.14.2.1.) activity showed a bimodal distribution pattern on isopycnic sucrose gradient centrifugation analysis of a crude preparation of membranes of bovine adrenomedullary vesicles. The two peaks of DBH activity corresponded to the peak of catecholamines (CA) and to the peak of the mitochrondrial “marker enzyme” cytochrome c oxidase. The high DBH activity of the latter peak was found to be due to stimulation by mitochondrial contamination. The specific activity of dopamine β-hydroxylase in membranes of highly purified bovine adrenomedullary vesicles was shown to be reduced by approx. 85 per cent upon dilution. This effect of dilution was abolished, and the activity enhanced, by the presence of a mitochondrial fraction. This enhancing effect is probably due partly to nonspecific stimulation of the DBH activity by mitochondrial protein and partly to catalase present in the mitochondrial preparation. A similar effect has been observed with sympathetic nerve vesicles. The implications of this effect for other density gradient studies are discussed.

Membranes of glyoxysomes from castor-bean endosperm. Enzymes bound to purified-membrane preparations.

A procedure was developed for the isolation of membranes of glyoxysomes from the endosperm of germinating castor beans (Ricinus communis L. var. zanzibariensis). These membranes were prepared by osmotic‐shock treatment of isolated glyoxysomes. In some cases this was followed by mild sonication. They were isolated by centrifugation on to a 57% (w/w) sucrose layer and purified by isopycnic sucrose gradient centrifugation.The membrane preparations were characterized by electronmicroscopy and assayed for enzyme activities. They consisted mainly of ghost‐type vesicles and were found to contain a major part of fatty acid β‐oxidation, malate synthase, citrate synthase and antimycin‐A‐insensitive NADH‐cytochrome c reductase activities of the whole glyoxysomes. Malate dehydrogenase may also be bound to the membrane to a significant extent. A tentative scheme is postulated describing the structural organization of fatty acid β‐oxidation and of the glyoxylate cycle in glyoxysomes.

Rapid method for isolation of large quantities of outer membrane from Escherichia coli K-12 and its application to the study of envelope mutants.

A rapid method for the isolation of large quantities of bacterial outer membrane is described. This cell envelope component was removed from plasmolyzed cells of Escherichia coli K-12 by lysozyme-ethylenediaminetetra-acetic acid treatment, aggregated by lowering the pH to 5.0, and recovered by centrifugation. Aggregates of membrane fragments were clearly identified in an electron microscope. A criterion of homogeneity of the preparation was obtained by isopycnic sucrose gradient centrifugation. A single band appeared at a density of 1.24 g/cc. The cytoplasmic membrane marker, succinate dehydrogenase activity, was 40 times lower in the outer membrane preparation than in complete cell envelope preparations. A rich activity was, however, found for the outer membrane marker, phospholipase A. The compositions of outer membranes from a transductant pair were compared. One transductant was a chain-forming, antibiotic-supersensitive envA strain, whereas the other contained the envA(+) allele. The envA strain showed a slightly modified protein pattern and a lower relative content of phosphatidylglycerol.

Incorporation of labeled precursors into the RNA and proteins of lactic dehydrogenase virus.

Lactic dehydrogenase virus was grown in primary mouse embryo cells and labeled with (3)H-uridine and (3)H-amino acids. Concentrated and purified virus was banded by isopycnic centrifugation in sucrose gradients, and infectivity and radioactivity were found to correspond at a density of 1.17 g/cm(3). The extracted viral RNA was resolved by electrophoresis in polyacrylamide-agarose mixed gels, and the mol wt was estimated to be 6.0 x 10(6).

The importance of strict temperature control during gradient centrifugation of osmotically active cell organelles

A temporary drop in temperature below 0°C in the rotor tube during isopycnic sucrose gradient centrifugation is not usually detectable with the available temperature gauges, but such transient sub-zero temperatures will cause mitochondria to rupture and to redistribute throughout the gradient. Swing-out titanium rotors are particularly prone to such temperature variations as titanium in relation to aluminium is a poor heat conductor.

Use of the Macrophage Migration Inhibition Test to Monitor Fractionation of Soluble Antigens of Chemically Induced Sarcomas of Inbred Guinea Pigs

Abstract Soluble tumor antigen was prepared from two transplanted sarcomas of strain 13 guinea pigs, induced with 3-methylcholanthrene or 9,10-dimethyl-1,2-benzanthracene, by extraction with isotonic saline followed by ultracentrifugation. Further fractionation by ammonium sulfate precipitation, gel filtration on Sephadex G-150 or Biogel A-5m, and DEAE Sephadex chromatography was performed. Antigenic activity was assayed in vivo by skin tests and in vitro by the macrophage migration inhibition method. Antigenic activity was not restricted to a single species of molecules as defined by size or charge. The major part of the activity was associated with two fractions, one excluded by Sephadex G-150, and the other included in Sephadex G-150. The behavior of the antigen preparations in isopycnic sucrose gradient centrifugation indicated that they were truly soluble and not membrane associated. The active fractions were nontoxic for the skin and for peritoneal cells and stable under the conditions of the macrophage migration test. Their antigenic activity was specific for each of the two tumors. In terms of the amount of antigen required to give a positive reaction, the migration inhibition assay was more sensitive than the skin tests.

The characterization of rat kidney plasma membranes prepared by zonal centrifugation.

1. Plasma membranes were isolated from a 10000g-min pellet prepared from a renal cortical homogenate in 20mm-NaHCO(3) by isopycnic centrifugation in a linear sucrose gradient in an ;A'-type zonal rotor. 2. The preparation was characterized by electron microscopy, and alkaline phosphatase, 5'-nucleotidase, l-leucine beta-naphthylamidase and l-leucine p-nitroanilidase activities were found to be selectively associated with the renal plasma membrane. 3. The preparation had a high degree of purity, as indicated by the presence of low activities of marker enzymes associated with subcellular organelles. A preliminary chemical analysis indicated that the chemical composition resembled that of plasma membranes of other tissues. 4. Plasma membranes were also prepared from tubular fragments and their enzyme contents were found to be similar to those of plasma membranes prepared from cortical homogenates. 5. l-Leucine beta-naphthylamidase, l-leucine p-nitroanilidase and 5'-nucleotidase were not enriched to the same extent as alkaline phosphatase in the preparation of plasma membranes from tubular fragments. A possible explanation for this finding is discussed.

Isolation and characterization of ribonucleoprotein substructures from Rous sarcoma virus

A combination of velocity and isopycnic density centrifugation in sucrose gradients has been employed to isolate ribonucleoprotein substructures (cores) from Rous sarcoma virus. The cores band at a density of 1.27–1.29 g/cc in sucrose gradients, compared with the density of 1.16–1.18 g/cc for intact virus. The cores contain the viral RNA and one of the structural proteins of the virion. This core-associated protein has an estimated molecular weight of 11,000 daltons. In addition, there are up to nine minor proteins in core preparations. Some of these represent trace amounts of major virion proteins; however, there are some proteins which differ in electrophoretic mobility from any of the major virion proteins. The cores contain no detectable phospholipid and are noninfectious in a focus-forming assay.

Resolution of proteins from subfractions of nerve endings

An ultracentrifugation procedure is described for the isolation of ostomically sensitive nerve endings (synaptosomes) using an isotonic Ficoll-sucrose discontinuous gradient. These nerve endings wee lysed and their individual components isolated by isopycnic centrifugation in discontinuous sucrose gradients. With this procedures, highly purified fractions of nerve ending soluble material, synaptic vesicles, synaptic plasma membranes and nerve ending mitochondria were prepared. Polyacrylamide gel electrophoresis of the proteins from these subractions revealed that the soluble component had 19 detectable bands, the synaptic vesicles had 22 bands, the synaptic plasma membrane had 20 bands, and the mitochondria had 22 bands. The protein gel profile of the nerve ending mitochondria resembled the profile of the mitochondria obtained from the cell bodies of neurones and glia. Comparison of the profiles of the soluble proteins of the cell bodies (neuronal plus glial) and the nerve endings revealed one qualitative and 3 quantitative differences between the two. The synaptic vesicles had a complex gel pattern which did not resemble the pattern of any of the other components in the nerve endings. It was possible to obtain an insoluble fraction containing, in addition to large quantities of electron dense amorphous material, structures resembling synaptic complexes by treating the synaptic plasme membranes with Triton X-100. This insoluble fraction had a protein gel pattern consisting of 13 bands; 5 of these bands were not found in the protein profile of the soluble material.

Membrane-associated replication complex in arbovirus infection.

Cytoplasmic extracts of chicken embryo fibroblast cells infected with Semliki Forest virus were subjected to isopycnic centrifugation in discontinuous sucrose gradients. Seven distinct bands were usually formed. The four upper bands contained predominantly smooth membranes and the lowest band was enriched in rough endoplasmic reticulum. One fraction (fraction 5), banding at a density of 1.16 g/cm(3), was found to be heavily enriched in pulse-labeled ribonucleic acid (RNA), viral RNA polymerase, and viral RNA forms associated with RNA replication. Thus, fraction 5 evidently contained a membrane-associated viral replication complex of a type previously defined in picornavirus infections. Fraction 5 was also consistently enriched with unique membranous structures previously observed in intact cells as type 1 cytopathic vacuoles (CPV-1). When the CPV-1 in fraction 5 were isolated from cells briefly incubated with (3)H-uridine and (3)H-adenosine prior to cell disruption, a large proportion was found to be labeled by high-resolution autoradiography. Thus, ultrastructural, biochemical, and biological evidence were all consistent with the interpretation that the CPV-1 membranes represent a significant element of the viral replication complex.

Mechanism of Assembly of the Outer Membrane of Salmonella typhimurium: SITE OF SYNTHESIS OF LIPOPOLYSACCHARIDE

The site of synthesis of lipopolysaccharide has been investigated in cytoplasmic and outer membrane fractions isolated by isopycnic sucrose gradient centrifugation of the total membrane fraction from lysozyme-EDTA spheroplasts. Evidence that synthesis of O-antigen occurs exclusively in cytoplasmic membrane was obtained by pulse-chase experiments in vivo and by assay of biosynthetic enzymes in isolated membrane fractions. O-Antigen chains pulse-labeled in vivo with [14C]mannose appeared initially in cytoplasmic membrane, but were rapidly transferred to outer membrane during a subsequent chase with nonradioactive mannose. In accord, the specific activities of enzymes of O-antigen synthesis in isolated cytoplasmic membrane fractions were 15- to 30-fold greater than in outer membrane. The cytoplasmic membrane fractions were also enriched for glycosyltransferase activities involved in biosynthesis of the core region of lipopolysaccharide. However, unequivocal localization of core transferase enzymes in the isolated membranes was not possible since these activities were also found with soluble fraction, and secondary binding of soluble enzyme to both cytoplasmic and outer membrane was shown to occur during the isolation procedure. Evidence that the cytoplasmic membrane is the site of synthesis of the core region was derived from pulse-chase experiments in a mutant in which [14C]galactose is incorporated exclusively into the core portion of the polysaccharide. Over 90% of the [14C]galactose incorporated into lipopolysaccharide during a 1-min pulse was recovered in the cytoplasmic membrane fraction. The mechanism of translocation of lipopolysaccharide to the outer membrane is unknown. Evidence suggesting that the process is not readily reversible was obtained in experiments in mutants with conditional defects in lipopolysaccharide synthesis. Incomplete lipopolysaccharides incorporated into outer membrane during growth under nonpermissive conditions could not be completed following subsequent shift to permissive growth conditions.