Isolation of amyloplasts from developing maize endosperm

Methods for the formation of protoplasts from developing maize endosperm and for the aqueous isolation of intact amyloplasts from such protoplasts are described. Protoplasts were obtained after incubating endosperm slices in a medium containing cellulase and pectolyase for 5 days at 4°C or 5 hours at 30°C. After purification in a Ficoll density gradient, the protoplasts were reptured by forcing the suspension through a Nitex mesh (20 micrometer) positioned at the lower end of a modified disposable syringe. The resulting filtrate was layered on a discontinuous Ficoll density gradient of 30, 15, and 10%. Each Ficoll solution contained 0.7 molar sucrose, 10 millimolar arginine, 10 millimolar dl-dithiothreitol, 50 millimolar 2-(N-morpholino)ethanesulfonic acid (pH 5.6), and 2 millimolar CaCl₂. After 3 hours in the cold, an amyloplast fraction 50 to 93% intact and free from cytoplasmic, mitochondrial, and glyoxysomal contamination was recovered in the 15% Ficoll layer. Amyloplast intactness was estimated by fluorescent microscopy and activity of certain amyloplast marker enzymes before and after rupture of the amyloplast membrane. Starch branching enzyme, ADPG-pyrophosphorylase, and nitrite reductase were used as amyloplast marker enzymes.     

Lysosomes can fuse with a late endosomal compartment in a cell-free system from rat liver

The passage of pulse doses of asialoglycoproteins through the endosomal compartments of rat liver hepatocytes was studied by subcellular fractionation and EM. The kinetics of disappearance of radiolabeled asialofetuin from light endosomes prepared on Ficoll gradients were the same as the kinetics of disappearance of asialoorosomucoid-horse radish peroxidase reaction products from intracellular membrane-bound structures in the blood sinusoidal regions of hepatocytes. The light endosomes were therefore identifiable as being derived from the peripheral early endosome compartment. In contrast, the labeling of dense endosomes from the middle of the Ficoll gradient correlated with EM showing large numbers of reaction product-containing structures in the nonsinusoidal parts of the hepatocyte. In cell-free, postmitochondrial supernatants, we have previously observed that dense endosomes, but not light endosomes, interact with lysosomes. Cell-free interaction between isolated dense endosomes and lysosomes has now been reconstituted and analyzed in three ways: by transfer of radiolabeled ligand from endosomal to lysosomal densities, by a fluorescence dequenching assay which can indicate membrane fusion, and by measurement of content mixing. Maximum transfer of radiolabel to lysosomal densities required ATP and GTP plus cytosolic components, including N-ethylmaleimide-sensitive factor(s). Dense endosomes incubated in the absence of added lysosomes did not mature into vesicles of lysosomal density. Content mixing, and hence fusion, between endosomes and lysosomes was maximal in the presence of cytosol and ATP and also showed inhibition by N-ethyl-maleimide. Thus, we have demonstrated that a fusion step is involved in the transfer of radiolabeled ligand from an isolated endosome fraction derived from the nonsinusoidal regions of the hepatocyte to preexisting lysosomes in a cell-free system.     

ISOLATION AND PARTIAL CHARACTERIZATION OF FRACTIONS ENRICHED IN SYNAPTOSOMES FROM CHICK BRAIN

–From a pool of hemispheres, optic lobes and cerebellum of chick 3 fractions containing synaptosomes have been prepared. They were obtained by subcellular fractionation of a homogenate and centrifugation of a crude mitochondrial suspension on a discontinuous Ficoll density gradient in iso-osmoticsucrose. The synaptosomal fractions were isolated from bands at the interface of 5–9, 9–12 and 12–16% Ficoll. The characterization of these fractions by marker enzymes, such as lactate dehydrogenase, acetyl-cholinesterase, monoamine oxidase, acid phosphatase and rotenone-sensitive and -insensitive NADH: cytochrome c reductase is reported. Electron microscopic analyses showed that the first fraction (AB) at the 5–9% Ficoll interface contained myelin and other membrane fragments as well as synaptosomes, the second fraction (C) at the 9–12% Ficoll interface contained mainly synaptosomes, and the third fraction (D) at the 12–16% Ficoll interface contained synaptosomes and free mitochondria. A fourth fraction (E) was obtained as a pellet, and was enriched in free mitochondria. There was fair agreement between the distribution pattern of the marker enzyme activities and the particles of the fractions seen by electron microscopy. The content of glycoprotein-bound N-acetylneuraminic acid and total phospholipid of these fractions has been determined. Relative to the mitochondrial fraction (E) the synaptosome fraction contained on basis of particulate protein, respectively, 2–3 times as much protein-bound N-acetylneuraminic acid and 10–20 per cent more total phospholipid.     

Isolation and characterization of the haemocytes of Calliphora vicina on density gradients of Ficoll

Discontinuous gradients of Ficoll have been used in an equilibrium density analysis of the haemocytes of Calliphora vicina. Using histochemical criteria, it was shown that the acid phosphatase-containing haemocytes decreased in mean density during larval life. Enzymatic analysis, and an analysis of the density distribution of labelled haemocytes at various times after an injection of [H³]-thymidine, provided evidence that a dense, replicating population of cells had been separated from a non-replicating acid phosphatase-containing population. The latter gained increasing amounts of the lysosomal enzymes acid phosphatase and protease as they aged.     

Macrophage heterogeneity in tumor resistance: Cytostatic and cytotoxic activity of corynebacterium parvum-activated and proteose peptone-elicited rat macrophages against Moloney sarcoma tumor cells

Peritoneal macrophages from proteose peptone and Corynebacterium parvum (CP)-treated Lewis and Brown Norway rats were separated into subpopulations by centrifugation on discontinuous gradients of Ficoll. Four macrophage subpopulations were prepared and tested for cytostatic and cytotoxic activity against syngeneic and allogeneic Moloney sarcoma tumor cells. Macrophages were cocultured with tumor cells for 48 hr, whereupon either the inhibition of [¹²⁵I]iododeoxyuridine uptake was measured (cytostasis) or the tumor monolayers were observed for cytotoxic effects. CP-Activated macrophages from heavy-density portions of the gradient (8–10% and 10%-pellet) were highly cytostatic and cytotoxic to both the syngeneic and allogeneic tumor cells while macrophages from the light-density portions (4–6 and 6–8% Ficoll bands) were not. Proteose peptone-stimulated macrophages from the heavy-density portions of the gradient were cytostatic but not cytotoxic to the tumor cells. The effector macrophages from the CP-activated pool were large, well-differentiated cells as determined by electron microscopic examinations and had enhanced phagocytic activity when contrasted with the noncytotoxic, less dense macrophages.     

Understanding enzyme behavior in a crowded scenario through modulation in activity,conformation and dynamics

Macromolecular crowding, inside the physiological interior, modulates the energy landscape of biological macromolecules in multiple ways. Amongst these, enzymes occupy a special place and hence understanding the function of the same in the crowded interior is of utmost importance. In this study, we have investigated the manner in which the multidomain enzyme, AK3L1 (PDB ID: 1ZD8), an isoform of adenylate kinase, has its features affected in presence of commonly used crowders (PEG 8, Dextran 40, Dextran 70, and Ficoll 70). Michaelis Menten plots reveal that the crowders in general enhance the activity of the enzyme, with the K_m and V_max values showing significant variations. Ficoll 70, induced the maximum activity for AK3L1 at 100 g/L, beyond which the activity reduced. Ensemble FRET studies were performed to provide insights into the relative domain (LID and CORE) displacements in presence of the crowders. Solvation studies reveal that the protein matrix surrounding the probe CPM (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin) gets restricted in presence of the crowders, with Ficoll 70 providing the maximum rigidity, the same being linked to the decrease in the activity of the enzyme. Through our multipronged approach, we have observed a distinct correlation between domain displacement, enzyme activity and associated dynamics. Thus, keeping in mind the complex nature of enzyme activity and the surrounding bath of dense soup that the biological entity remains immersed in, indeed more such approaches need to be undertaken to have a better grasp of the “enzymes in the crowd”.     

PREPARATION AND CHARACTERIZATION OF A PLASMA MEMBRANE FRACTION FROM ISOLATED FAT CELLS

A rapid method of preparing plasma membranes from isolated fat cells is described. After homogenization of the cells, various fractions were isolated by differential centrifugation and linear gradients. Ficoll gradients were preferred because total preparation time was under 3 hr. The density of the plasma membranes was 1.14 in sucrose. The plasma membrane fraction was virtually uncontaminated by nuclei but contained 10% of the mitochondrial succinic dehydrogenase activity and 25–30% of the RNA and reduced nicotinamide adenine dinucleotide cytochrome c reductase activity of the microsomal fraction. Part of the RNA and NADH-cytochrome c reductase activity was believed to be native to the plasma membrane or to the attached endoplasmic reticulum membranes demonstrated by electron microscopy. The adenyl cyclase activity of the plasma membrane fraction was five times that of Rodbell's "ghost" preparation and retained sensitivity to epinephrine. The plasma membrane ATPase activity was five times that of the homogenate and microsomal fractions. Electron microscopic evidence suggested contamination of the plasma membrane fraction by other subcellular components to be less than the biochemical data indicated.     

Separation of European flat oyster, Ostrea edulis, haemocytes by density gradient centrifugation and SDS-PAGE characterisation of separated haemocyte sub-populations

A two-step gradient centrifugation with Percoll and Ficoll successively as density medium was developed to separate European flat oyster, Ostrea edulis, haemocytes into three sub-populations representing granulocytes, large hyalinocytes and small hyalinocytes, respectively. After a Percoll gradient centrifugation, granulocytes and agranulocytes were separated and a pure fraction of granulocytes was obtained. The agranulocytes were further separated by centrifugation through a Ficoll gradient, and two haemocyte subpopulations representing large hyalinocytes and small hyalinocytes were obtained. No significant impact on the haemocyte viability was detected after separation with this two-step density gradient centrifugation. The three haemocyte sub-populations showed different protein patterns in SDS-PAGE.     

Induction of anthocyanin synthesis in relation to embryogenesis in a carrot suspension culture: Correlation of metabolic differentiation with morphological differentiation

A system in which anthocyanin synthesis could be induced under a defined condition, was established in a carrot suspension culture. A cell suspension culture of carrot ( Daucus carota L. cv. Kurodagosun) was subcultured for more than a year in a medium containing 5 × 10⁻⁷ M 2,4-dichlorophenoxyacetic acid (2,4-D). At every subculture the cultures were sieved through nylon screens and the cells and cell clusters collected in the size range of 31–81 μm were transferred to a fresh medium. When the cells were transferred to a medium without auxin, synthesis of anthocyanin was induced. Zeatin promoted anthocyanin synthesis in a medium lacking auxin, with maximum yields of anthocyanin obtained at 10⁻⁷ to 10⁻⁸ M zeatin, 2,4-D at higher concentrations than 10⁻⁷ M inhibited anthocyanin synthesis completely. The sieved cells were fractionated by Ficoll density gradient centrifugation. Somatic embryos were formed in the fraction of higher density (>14% of Ficoll) in a medium containing 10⁻⁷ M zeatin but lacking auxin, while synthesis of anthocyanin was hardly observed. On the other hand, cells in the fraction of lower density (<12% of Ficoll) synthesized anthocyanin in the same medium, but formed few embryos. Forty to fifty percent of the total cells in this lighter cell fraction synthesized anthocyanin at a maximum. The similarity between anthocyanin synthesis and embryogenesis was observed in the time course as well as in the effects of growth regulators. The correlation between metabolic and morphological differentiation is discussed.     

Analysis and purification of the blood-sinusoidal domain of rat liver plasma membrane

Methods are described for the analysis and purification of the blood-sinusoidal domains of rat liver plasma membranes using a combination of sucrose and Ficoll density gradient centrifugation. Use has been made of ¹²⁵I-labelled wheat-germ agglutinin and hormone-stimulated adenylate cyclase to identify the blood sinusoidal fraction, which may be resolved from Golgi and endoplasmic reticulum markers on Ficoll gradients.     

Protein phosphorylation associated with stimulation of rabbit gastric glands

Changes in protein phosphorylation associated with stimulation of acid secretion were investigated using isolated rabbit gastric glands labeled with 32P. The glands were stimulated by 100 microM histamine plus either 10 microM forskolin or 50 microM isobutylmethylxanthine, homogenized, and fractionated into a series of pellets: 40 X g, 5 min; 4000 X g, 10 min; 14,500 X g, 10 min; 48,200 X g, 90 min (microsomes); and supernatant. Stimulation induced a redistribution of H+/K+-transporting ATPase among the membrane fractions, i.e., a reduction in activity of the microsomal fraction, and a compensatory increase in the 4000 X g fraction. Further subfractionation of the 4000 X g pellet by Ficoll density gradient produced an 18% Ficoll layer, greatly enriched in the H+/K+-ATPase, and which is thought to be rich in apical membranes of parietal cells. SDS-polyacrylamide gel electrophoresis showed that the amount of 94 kDa peptide (the molecular size of the H+/K+-ATPase) was increased in the 18% Ficoll layer and decreased in the microsomal fraction by stimulation. Analysis of autoradiograms of the gels revealed that apparent changes in level of phosphorylation occurred in the 120, 94 and 80 kDa regions of the 18% Ficoll layer, and in the 94 kDa region of the microsomal fraction. The phosphorylation changes in the 94 kDa region may not reflect changes in specific activity of a single peptide but may be due to the heterogeneity of proteins in this region, which was demonstrated by selective heat treatment of the samples as well as two-dimensional electrophoresis. Phosphorylation of 120 kDa protein in the 18% Ficoll layer was clearly increased by stimulation, and this appeared to be associated with protein distribution changes as well as phosphorylation. The 80 kDa protein in the 18% Ficoll layer showed marked increased phosphorylation by stimulation, with little change in protein distribution. This 80 kDa protein was focused on two-dimensional gels as several sequential spots, with the most radioactive peptide focused toward the acidic side; thus, we propose isomeric forms of an 80 kDa protein with sequential phosphorylation sites. The phosphorylation changes observed in this study are considered to be important to the process of gastric acid secretion because they occurred in the putative apical membrane fractions in which biochemical and functional changes with stimulation have been demonstrated.     

Competitive Interactions of Ligands and Macromolecular Crowders with Maltose Binding Protein

Cellular signaling involves a cascade of recognition events occurring in a complex environment with high concentrations of proteins, polysaccharides, and other macromolecules. The influence of macromolecular crowders on protein binding affinity through hard-core repulsion is well studied, and possible contributions of protein-crowder soft attraction have been implicated recently. Here we present direct evidence for weak association of maltose binding protein (MBP) with a polysaccharide crowder Ficoll, and that this association effectively competes with the binding of the natural ligand, maltose. Titration data over wide ranges of maltose and Ficoll concentrations fit well with a three-state competitive binding model. Broadening of MBP ¹H­¹⁵N TROSY spectra by the addition of Ficoll indicates weak protein-crowder association, and subsequent recovery of sharp NMR peaks upon addition of maltose indicates that the interactions of the crowder and the ligand with MBP are competitive. We hypothesize that, in the Escherichia coli periplasm, the competitive interactions of polysaccharides and maltose with MBP could allow MBP to shuttle between the peptidoglycan attached to the outer membrane and the ATP-binding cassette transporter in the inner membrane.     

Isolation and Characterization of Vacuoles from the Ergot Fungus Claviceps purpurea

Protoplasts of Claviceps purpurea were prepared by treatment of mycelium with a lytic mixture of snail gut enzyme and cellulase from Trichoderma viride. Such protoplasts could be efficiently lysed by Triton X-100 treatment at high osmotic pressure without Ca²⁺ or Mg²⁺, allowing the release of intact vacuoles in high yields. Vacuoles obtained from cells grown in modified Vogel medium (vegetative-type cells not producing alkaloids) were isolated and purified by centrifugation from a 5% Ficoll 400 (wt/vol) phase into the interphase between two layers, one containing 0.25 M each of mannitol and sucrose, and one containing 0.5 M mannitol. Vacuoles derived from cells grown in a medium favoring ergot alkaloid synthesis (sclerotia-like cells) were isolated by gentle centrifugation of filtered protoplast lysates without addition of Ficoll 400. Biochemical analyses of the vacuole fraction isolated from either kind of cell revealed their function as compartments harboring several hydrolytic enzymes. However, the enrichment of free amino acids in vacuoles of sclerotia-like cells was less pronounced than that in vacuoles of vegetative-type cells, indicating a difference in metabolic compartmentation in the two types of cells.     

Isolation and Characterization of the Olfactory Epithelial Cells of the Catfish

A preparation enriched in olfactory receptor cells has beenobtained from the olfactory mucosa of the catfish (Ictaluruspunctatus). The tissue was treated successively with trypsin,DNase, trypsin inhibitor, EDTA in Ca^{+ +} , Mg^{+ +} free mediumaccording to a method derived from that of Cohen, et al.⁽¹⁾.After mechanical disruption of the isolated olfactory lamellae,the cells were isolated by centrifugation on a Ficoll gradient.Each type of cell was morphologically identified by comparingin situ and in vitro preparations by SEM. Small round cellswere collected on 10% Ficoll. The nature of these cells is notknown but part of them are certainly basal cells which havebeen shown⁽²⁾ to be the precursors of the constantly regeneratingolfactory neurons. Respiratory cells settled mainly on 20% Ficoll.A fraction containing 60% sustentacular cells was collectedon 33% Ficoll. Olfactory cells characterized by an axon, a dendriteand several cilia, were found on 37% Ficoll. This fraction alsocontains up to 40% sustentacular cells. A yield of 20% was measuredfor olfactory cell isolation. Vital staining and ability tosynthesize RNA indicate a viability of the final preparationof 70% to 80%. Further identification of the cells was performedby measuring the binding activity of a series of amino acidsto a preparation enriched in olfactory cells. A good correlationwas determined between the extent of the binding and the reportedelectrophysiological activities of these amino acids recordedin vivo. Although the final olfactory cell suspension is notpure, it constitutes the first step in the study of the olfactoryreceptor sites.     

Macromolecular crowding modulates α-synuclein amyloid fiber growth

The crowdedness of living cells, hundreds of milligrams per milliliter of macromolecules, may affect protein folding, function, and misfolding. Still, such processes are most often studied in dilute solutions in vitro. To assess consequences of the in vivo milieu, we here investigated the effects of macromolecular crowding on the amyloid fiber formation reaction of α-synuclein, the amyloidogenic protein in Parkinson’s disease. For this, we performed spectroscopic experiments probing individual steps of the reaction as a function of the macromolecular crowding agent Ficoll70, which is an inert sucrose-based polymer that provides excluded-volume effects. The experiments were performed at neutral pH at quiescent conditions to avoid artifacts due to shaking and glass beads (typical conditions for α-synuclein), using amyloid fiber seeds to initiate reactions. We find that both primary nucleation and fiber elongation steps during α-synuclein amyloid formation are accelerated by the presence of 140 and 280 mg/mL Ficoll70. Moreover, in the presence of Ficoll70 at neutral pH, secondary nucleation appears favored, resulting in faster overall α-synuclein amyloid formation. In contrast, sucrose, a small-molecule osmolyte and building block of Ficoll70, slowed down α-synuclein amyloid formation. The ability of cell environments to modulate reaction kinetics to a large extent, such as severalfold faster individual steps in α-synuclein amyloid formation, is an important consideration for biochemical reactions in living systems.     

Studies of oogenesis in Urechis caupo. I. Method for separating different size classes of oocytes

The immature oocytes of the echiuroid worm Urechis caupo develop while suspended as single cells in the coelomic fluid, free of any associated nurse or follicle cells. In any one female, size classes can be found ranging from 8 to 130 μm in diameter. A method is described for obtaining four to five relatively uniform size fractions by centrifuging the mixed oocyte population through a discontinuous Ficoll gradient and collecting the different size oocytes which accumulate at each step of the gradient. Phagocytosis of oocytes in vivo is also discussed.     

Plasmodium yoelii and Plasmodium berghei: Isolation of infected erythrocytes from blood by colloidal silica gradient centrifugation

Percoll (colloidal silica coated with polyvinylpyrrolidone) and Ficoll (MW 400,000) were used to separate erythrocytes infected with Plasmodium yoelii and Plasmodium berghei from uninfected red blood cells. Samples of blood collected from mice in different phases of malarial infection were overlaid on cushions of 55% Percoll, 20% Ficoll, or 28% Ficoll, respectively, centrifuged, and the interphase layers compared. The best yield of parasitized erythrocytes (PE) was achieved using Percoll when about 95% of the erythrocytes infected by the late developmental forms of the parasites (late trophozoites, schizonts, and gametocytes) were recovered from the gradient interphase, irrespective of the phase of the infection and the number of young erythrocytes in the sample. No alteration of antigenicity (assessed by immunofluorescence) or of osmotic fragility (over the range of 160–460 mOsm) could be detected in PE separated by Percoll or by Ficoll. In addition, parasites separated on Percoll gradients showed no significant ultrastructural changes and retained their normal infectivity to mice. Although both gradient media could be used for the separation of Plasmodium-infected erythrocytes, Percoll presented some advantages over Ficoll. Apart from the better reproducibility of the separation of high yields of very pure PE obtained with Percoll, its lower viscosity allowed easier handling, and lower centrifugal forces were needed to enable the cells to reach their isopycnic positions. Thus, Percoll fulfilled many of the criteria for an ideal density gradient medium. Parasitized erythrocytes were isolated by an easy, reproducible, and inexpensive procedure, and separated cells retained their normal structure, antigenicity, and infectivity.     

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.     

Toxoplasma gondii: large-scale purification by zonal density gradient centrifugation.

This study shows the feasibility of using density gradient centrifugation in the “A” zonal rotor for large-scale purification of Toxoplasma gondii tachyzoites from the host cells of mouse peritoneal exudate. Ficoll and Dextran 40 were used as gradients. Using Ficoll gradient, up to 70% of the toxoplasms were recovered by pooling purified fractions with the peak fractions giving recoveries around 38%. In the experiment using dextran gradients Toxoplasma recovery was of 41%, but the band of host cells was not so sharply formed.     

Macromolecular crowding modulates α-synuclein amyloid fiber growth

The crowdedness of living cells, hundreds of milligrams per milliliter of macromolecules, may affect protein folding, function, and misfolding. Still, such processes are most often studied in dilute solutions in vitro. To assess consequences of the in vivo milieu, we here investigated the effects of macromolecular crowding on the amyloid fiber formation reaction of α-synuclein, the amyloidogenic protein in Parkinson’s disease. For this, we performed spectroscopic experiments probing individual steps of the reaction as a function of the macromolecular crowding agent Ficoll70, which is an inert sucrose-based polymer that provides excluded-volume effects. The experiments were performed at neutral pH at quiescent conditions to avoid artifacts due to shaking and glass beads (typical conditions for α-synuclein), using amyloid fiber seeds to initiate reactions. We find that both primary nucleation and fiber elongation steps during α-synuclein amyloid formation are accelerated by the presence of 140 and 280 mg/mL Ficoll70. Moreover, in the presence of Ficoll70 at neutral pH, secondary nucleation appears favored, resulting in faster overall α-synuclein amyloid formation. In contrast, sucrose, a small-molecule osmolyte and building block of Ficoll70, slowed down α-synuclein amyloid formation. The ability of cell environments to modulate reaction kinetics to a large extent, such as severalfold faster individual steps in α-synuclein amyloid formation, is an important consideration for biochemical reactions in living systems.