Purification Of Organelles Research Articles

Immunoaffinity purification of subcellular particles and organelles.

The application of immunoaffinity techniques to subcellular fractionation is reviewed and the basic principles underlying the various methods that have been successfully employed, identified. The requirement for organelle-specific antigens and high-avidity antibodies is discussed, as is the widespread use of indirect immunoadsorbents. Approaches for the optimization of immunoaffinity-based subcellular fractionation are suggested.

Preparative agarose gel electrophoresis for the purification of small organelles and particles

A novel preparative method of quantitative flatbed agarose gel electrophoresis has been used to separate a number of small subcellular structures, such as ribosomes, coated vesicles, smooth vesicles, and ferritin. The technique utilizes continuous elution of a second, electrophoretically “downstream”, well in the agarose gel. The elution occurs concurrently with the electrophoresis, so essentially no additional time is required for the recovery of the structures. The technique is nondestructive, relatively simple and inexpensive, and can be used by modifying any nonsubmerged horizontal agarose gel system. The preparative separation of small organelles and subcellula structures according to their charge allows the purification of small structures previously difficult to isolate by conventional techniques. Two novel structures purified by this technique are described: a short intermediate filament-like species consisting of a single polypeptide of M r 142,000, and an ovoid species (70 × 35 nm) whose protein composition is dominated by a polypeptide of M r 104,000.

Shift of equilibrium density induced by 3,3'-diaminobenzidine cytochemistry: a new procedure for the analysis and purification of peroxidase-containing organelles.

Galactosylated BSA (galBSA) and its conjugate to horseradish peroxidase (galBSA-HRP) enter the galactose-specific pathway of hepatocytes. 10 min after intravenous injection, structures containing either ligand sediment mostly between 33,000 and 3 X 10(6) g X min (LP fraction) and have an equilibrium density of 1.11-1.13 g/ml in sucrose gradients (Quintart, J., P. J. Courtoy, J. N. Limet, and P. Baudhuin, 1983, Eur. J. Biochem., 131:105-112). Such low density fractions, prepared from rats given galBSA-HRP, were incubated for 30 min at 25 degrees C in 5.5 mM 3,3'-diaminobenzidine (DAB) and 11 mM H2O2 in buffered sucrose. Upon equilibration in a second sucrose gradient, the galBSA-HRP distribution shifted towards higher (approximately 1.19 g/ml) density, but the bulk of protein remained at low density. In the absence of H2O2, galBSA-HRP distribution was also found at low density. As observed by electron microscopy, particles equilibrating at higher density after DAB cytochemistry were largely made of vesicles or tubules filled with DAB reaction product. The density shift of galBSA-HRP-containing organelles after incubation with DAB and H2O2 is attributed to the trapping of HRP-oxidized DAB inside the host organelles. If the low density fractions isolated from a rat injected with [3H]galBSA-HRP were mixed in vitro with similar fractions from another rat given [14C]galBSA, the 3H distribution shifted after DAB cytochemistry, but the 14C distribution was essentially unaffected. By contrast, if both derivatives were injected simultaneously, a concomitant density shift was observed. In conclusion, the DAB-induced density shift was specific to ligand-HRP-containing organelles. The potentials of the method include the purification of HRP-containing particles and the study of their association to ligands, fluid-phase tracers, or marker enzymes.

Suitability of silica sol gradients for purification of cell organelles

Homogeneous populations of different chloroplast classes (organelles with morphologically intact, swollen and broken external envelopes) have been separated by combinations of differential and silica sol density gradient centrifugation. Organelles with intact envelopes purified in silica (Ludox) gradients are rather stable against osmotic rupture. The ultrastructural analysis of isolated organelles revealed colloid precipitates predominantly at the organelle membranes. Evidence is presented that Mg 2+ and strong cationic membrane groups participate in the colloid/membrane interaction. Physiological activities of silica gradient-purified unbroken chloroplasts were compared with those of organelles isolated in silica sol-free media (chloroplastic NAD-dependent malic dehydrogenase, Mg 2+-independent; NAD-dependent phosphoglycerate kinase/triosephosphate dehydrogenase, Mg 2+-dependent; light-dependent phosphoglycerate and CO 2 reduction; incorporation of deoxyribonucleotide triphosphates in an acid-insoluble, DNase-sensitive form). Altered or no activities were recovered with silica-treated plastids. Care should therefore be taken if silica gradient-purified organelles are used for investigations of physiological processes. However, due to the high resolving power of the gradient and the isotonicity of the isopycnic steps the method is recommended and has successfully been applied for the isolation of pure high mol wt substances from cell organelles, e.g. for higher plant chloroplast and nuclear DNA.

Lung Concentric Laminar Organelle: HYDROLASE ACTIVITY AND COMPOSITIONAL ANALYSIS

An analytical study was made of the concentric lamellar organelles (CLO) isolated from rabbit lung. These subcellular structures, which are normally located in the type II cells of mammalian lungs, were purified by flotation using a discontinuous sucrose density gradient. Electron microscopy of the isolated CLO revealed many intact structures about 1 µm in diameter with densely packed trilaminar membranes having, also, a granular core and an outer limiting membrane. The CLO contained a preponderance of polar lipids, mostly as phosphatidylcholine and phosphatidylethanolamine, with trace amounts of neutral lipid. A phospholipid to protein ratio of approximately 12:1, was repeatedly obtained and served as a criterion for organelle purification. Acrylamide gel electrophoresis of the detergent-solubilized CLO revealed a prominent staining band migrating with albumin. These structures had numerous hydrolases similar to those observed in lysosomes. High specific activities were observed for such enzymes as acid phosphatase, aryl sulfatase and β-N-acetylglucosaminidase. Multiple forms of the latter enzyme were observed in zonal electrophoresis studies. Purification of the CLO resulted in a 20- to 40-fold increase in specific activity over homogenates for most of the hydrolases. Cathepsin D, acid DNase, acid RNase and neuraminidase, other lysosome-associated hydrolases, were not detected in the CLO fraction. It is suggested from these and other studies that CLO are secreted to the acellular lining of lung where they contribute surfactant phospholipids, and possibly, functional hydrolases. The lung CLO also appear to have many properties in common with those subcellular structures associated with certain human genetic lipidoses.

A method for the isolation and purification of normal rat liver and hepatoma nuclear “ghosts” by zonal centrifugation

A method is presented for the isolation of nuclear membranes in the form of large fragments and intact nuclear “ghosts” from normal rat liver and an aminoazo dye induced rat hepatoma. The preparation involves overnight incubation at 4°C of the nuclear pellet suspension from hypotonic homogenates, followed by rate-dependent fractionation on a sucrose gradient in an A-XII Zonal rotor. The fraction sedimenting between mitochondria and nuclei was observed using phase contrast microscopy to be rich in large membrane fragments and nuclear “ghosts”. Isopycnic centrifugation of this fraction on sucrose gradients, in conventional swinging-bucket and zonal rotors resulted in a purification of the nuclear membranes. No significant difference in the density of normal rat liver and hepatoma nuclear membranes was detected. The purity and homogeneity of the nuclear membranes was confirmed by electron microscopy using both thin sectioning and negative contrast staining. Both electron microscopic techniques revealed the presence of pore complexes on the nuclear membranes, there being a marked absence of membranes not displaying these morphological features in the purified preparation. An assessment of the degree of contamination of the membranes with chromatin was obtained by thin sectioning and by measurement of DNA: protein ratios in purified preparations of both nuclear membranes and intact nuclei. The method developed for the preparation of nuclear membranes is an extension of those devised for the isolation of plasma membranes under hypotonic conditions, and the preparative procedures are discussed in relation to previous studies upon both plasma membranes and nuclear membranes. The method is thought to be significant as an addition to the use of rate and isopycnic zonal centrifugation for the purification of cellular organelles.