Isolation of peroxisomes from rat liver using sucrose and Percoll gradients

Peroxisomes were isolated from the livers of both control and clofibrate-treated rats. Two procedures, one with a sucrose gradient, and a second with Percoll gradients, were utilized. The Percoll procedure allowed contamination of the isolated peroxisome fraction on protein basis, by lysosomes (8%), by mitochondria (5%) and by microsomes (2%). The peroxisome fraction isolated by the sucrose gradient showed no significant contamination with mitochondria, but the fraction contained 13% microsomes. In addition to established peroxisomal enzymes, the isolated peroxisomes also contained cytochrome b5, NADH-cytochrome c reductase and NADPH-isocitrate dehydrogenase. The peroxisomal membranes were also separated from the content, and they were found to have a relatively high phospholipid/protein ratio (0.55).     

Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic Percoll gradients

Techniques for the large-scale isolation of Cryptosporidium oocysts and sporozoites, obtained from the feces of experimentally infected Holstein calves, were developed employing discontinuous sucrose gradients and isopycnic Percoll gradients. The oocyst recovery method utilized 2 sequential discontinuous sucrose gradients followed by 1 Percoll gradient. Recovered oocysts were essentially free of debris and bacteria and represented 34% of the original oocyst suspension. Sporozoites were recovered from excystation mixtures on a single Percoll gradient. Sixty-three percent of the original sporozoites were recovered with 2.2% contamination by intact oocysts and virtually no oocyst walls.     

Localization of a proton-translocating ATPase on sucrose gradients

Ionophore-stimulated ATPase activity and ATP-dependent quinacrine quench were enriched in parallel when microsomal vesicles were prepared from corn (Crow Single Cross Hybrid WF9-Mo17) roots and collected on a cushion of 10% dextran. Activities were highest in the apical 1.5 centimeters of the roots. Vesicles collected on the dextran cushion also contained NADH cytochrome c reductase (enriched in the apical 0.5 cm of the root) and nucleoside diphosphatase (distributed throughout the first four cm). On continuous sucrose gradients, ATP-dependent proton transport and ionophore-stimulated ATPase activity coincided in a broad band extending from 1.08 to 1.15 grams per cubic centimeter with maximum activity at 1.10 to 1.12 grams per cubic centimeter. Large portions of the proton-translocating ATPase activity and ionophore-stimulated ATPase activity were clearly separable from mitochondrial membranes containing cytochrome c oxidase activity and azide-sensitive, pH 8.5 ATPase activity and from membranes bearing β-glucan synthetase I and II. The vesicles coincided with a minor portion of the NADH-cytochrome c reductase and nucleoside diphosphatase activities. It is suggested that the vesicles are of tonoplast origin.     

Isolation of transverse tubules by fractionation of sarcoplasmic reticulum preparations in ion-free sucrose density gradients

A new method for the preparation of transverse tubules (T-tubules) from rabbit skeletal muscles is reported. When crude sarcoplasmic reticulum (SR) preparations were centrifuged on sucrose density gradients containing buffering ions (buffered gradients) 70-80% of the material sedimented as a single heavy band in the region of 43% sucrose. When this fraction (or crude SR) was recentrifuged on sucrose gradients prepared free of buffer or other ions (ion-free gradients) the heavy band dissociated into three fractions of different densities. The lightest fraction sedimented at 28% sucrose and was identified as T-tubules on the basis of its nitrendipine and ouabain binding properties. The enzymatic properties, cholesterol contents, and protein compositions of the fractions obtained when SR is centrifuged on buffered and ion-free sucrose density gradients were measured. The T-tubules were enriched in cholesterol and in marker enzymes for surface membranes while the other fractions were shown to be terminal cisternae and longitudinal cisternae on the basis of their (Ca2+,Mg2+)-ATPase activities and characteristic protein profiles.     

Isolation of protein components from rat lung lamellar bodies

Lamellar bodies isolated from 10% (w/v) rat lung homogenates by discontinuous sucrose gradient centrifugation were shown to contain variable amounts of adhering proteins. These contaminating proteins could be removed by either Sepharose 4B gel filtration or precipitation of the crude preparation at pH 11.5. Both purification methods yielded membrane preparations with a phospholipid-to-protein ratio of 10.0 μmol/mg. Nearly complete separation of lamellar body phospholipid and protein could be achieved upon application of the purified membranes to DEAE-cellulose in the presence of 0.2% (v/v) Triton X-100. Phospholipid analyses showed that 83% of total lipid phosphorus was recovered in phosphatidylcholine. In phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine and phosphatidylinositol recoveries amounted to 4, 8, 2 and 2%, respectively. Molecular mass determinations of the isolated protein component of lamellar bodies by means of SDS polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue revealed the presence of three protein bands with molecular masses of 64, 33 and 31 kDa. Upon staining with silver a 16 kDa protein was also visible. Sephadex G-100 gel filtration showed only one protein peak corresponding to a molecular mass of 64 kDa when protein was assayed with Coomassie brilliant blue.     

A homemade device for linear sucrose gradients

We have developed a simple and inexpensive device to obtain linear sucrose gradients with commonly used laboratory materials—a syringe, a flask, a plastic tube, and a piece of Pongo (Play-Doh). Refractive index values measured on sucrose fractions collected using our system demonstrate both the linearity and reliability of the gradients obtained.     

Microvilli of the human term placenta. Isolation and subfractionation by centrifugation in sucrose density gradients.

Human placental microvilli were isolated and separated into two fractions by centrifugation in sucrose density gradients. Electron-microscopic morphology and morphometry, the distribution of enzymic activities and the results of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of proteins were used to assess the purity of the final preparations and to define their properties. The combined evidence strongly suggested that the preparations contained negligible material that was not plasma membrane. The two fractions of microvilli differed in buoyant density, protein composition, enzyme specific activities and microscopic appearance. Some of these differences were explained by the absence of internal structure in the microvilli of the lighter fraction.     

Zonal rate centrifugation of proteoglycans in sucrose gradients

A simple reverse-phase chromatographic system for separating deoxyribonucleoside monophosphates is described. Using isocratic elution at room temperature, clear separation of seven of the deoxyribonucleoside monophosphates that occur in either procaryotic or eucaryotic DNAs can be achieved in less than 2 h. Thus, this method allows a sensitive and rapid analysis of submicrogram quantities of ³²P-labeled deoxyribonucleoside monophosphates derived from DNA labeled in vivo with ³²P_i or from DNA labeled enzymatically in vitro at the 5′ or 3′ ends. The suitability of the method for studying methylation of mammalian DNAs is illustrated by presenting examples of its application to (a) quantitation of major and minor nucleotides in newly synthesized DNA, (b) determination of the specificity of in vitro methylation of DNA, and (c) quantitation of the extent to which specific restriction endonuclease sites are methylated in vivo.     

The heterogeneity of rat brain mitochondria isolated on continuous sucrose gradients

Abstract— The distribution of a series of enzymes in the post-nuclear supernatant of rat brain homogenates was investigated following continuous density-gradient centrifugation. The enzymes studied were acetyl coenzyme A synthetase, glutamic dehydrogenase, glutamine synthetase, glutaminase I, succinic dehydrogenase and monoamine oxidase. Each of these enzymes with the exception of glutamine synthetase appears predominantly in the mitochondrial region of the gradient. Although about 20 per cent of this enzyme is present in the crude mitochondrial pellet, on density gradient centrifugation no special association of glutamine synthetase with any of the mitochondrial fractions was observed. Each of the other enzymes studied was found to have a characteristic distribution in the gradient; this suggests that brain mitochondria may be heterogeneous both in buoyant density and in their enzyme content. Three principal fractions are described: (i) dense particles containing high concentrations of acetyl coenzyme A synthetase and glutamic dehydrogenase; (ii) a fraction comprising the bulk of the mitochondria with high levels of monoamine oxidase, succinic dehydrogenase and glutaminase I; and (iii) particles in the synaptic ending region of the gradient characterized by relatively high levels of monoamine oxidase and succinic dehydrogenase and containing only small amounts of the other enzymes studied. If the mitochondrial heterogeneity that is observed on centrifugation reflects the existence within brain cells of mitochondria with specialized function, a partial explanation may be available for multiple pools of tricarboxylic acid cycle intermediates which have been postulated from isotopie labelling experiments.     

Microfluorometric filter determination of DNA in sucrose density gradients

A simple filter method for the fluorometric estimation of DNA in alkaline and neutral sucrose gradient fractions with DABA·2HCl is discussed. Alpha-450 membrane filters of regenerated cellulose (Gelman Instrument Co., Ann Arbor, Michigan) were used. In the proposed method washing of the DNA precipitate as well as the reaction with DABA·2HCl were performed directly on the filters, thus avoiding repeated washing and centrifugations of DNA precipitates applied hitherto in analogous fluorometric techniques. A good coincidence of the results concerning localization of DNA sedimentation profiles determined by radioisotopic and fluorometric methods was obtained. The method is very convenient for DNA estimation in alkaline and neutral sucrose density gradient fractions obtained by ultracentrifugation of DNA of nonproliferating cells where DNA labeling is very difficult, and in the case of human lymphocytes even impossible without stimulation for blastic transformation. Its other advantages are a considerably simplified procedure and a higher precision with respect to other fluorometric methods for determination of DNA.     

Isolation and characterization of oat aleurone and starchy endosperm protein bodies

To compare oat (Avena sativa L. cv Froker) aleurone protein bodies with those of the starchy endosperm, methods were developed to isolate these tissues from mature seeds. Aleurone protoplasts were prepared by enzymic digestion and filtration of groat (caryopsis) slices, and starchy endosperm tissue was separated from the aleurone layer by squeezing slices of imbibed groats followed by filtration. Protein bodies were isolated from each tissue by sucrose density gradient centrifugation. Ultrastructure of the isolated protein bodies was not identical to that of the intact organelles, suggesting modification during isolation or fixation. Both aleurone and starchy endosperm protein bodies contained globulin and prolamin storage protein, but minor differences in the protein-banding pattern by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were evident. The amino acid compositions of the protein body fractions were similar and resembled that of oat globulin. The aleurone protein bodies contained phytic acid and protease activity, which were absent in starchy endosperm protein bodies.     

Isolation of metabolically distinct synaptosomes on Percoll gradients

Synaptosomes were prepared from whole rat brain by six different methods based on gradients of sucrose, Ficoll or Percoll. In these, the synthesis and calcium-specific release of amino acids were assessed by two different procedures. Preparations based on sucrose showed the least calcium-specific release, followed by Ficoll-derived synaptosomes. As previously described, Percoll gave two separate populations of synaptosomes, both very active in terms of release of aspartate, glutamate, and GABA. The data involving release and synthesis were not identical, but did agree in the following: in low-density synaptosomes, haloperidol blocked both the release and synthesis of glutamate, but was without effect in the heavier populatin. 2-chloroadenosine and 2-oxoglutarate affected both release and synthesis only in the high-density population. Dopamine blocked aspartate release and synthesis only in the high-density population. These results suggest that haloperidol interferes with glutamate release and synthesis via a mechanism which may not involve adenosine, serotonin, or dopamine.