Preformed frozen sucrose gradients--a new laboratory aid

A useful method for preparing large numbers of virtually identical gradients of sucrose has been described. This technique involves storage of preformed gradients at ?60°C for future use after a 90 min thaw at room temperature. The method is applicable to salt-containing sucrose gradients with certain limitations, noted above.     

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 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.     

Effect of sucrose gradient composition on resolution of RNA species

Resolution of RNA samples on sucrose gradients was strikingly dependent upon the sucrose concentration in the gradients. Linear 10–70% gradients separated peaks which were barely discernible as shoulders in 10–20% gradients. The resolution obtained in steep sucrose gradients was comparable to that observed upon electrophoresis in acrylamide/agarose gels.     

Detecting immunocomplex formation in sucrose gradients by enzyme immunoassay: application in determining epitope accessibility on ribosomes.

A sensitive method using enzyme immunoassay and sucrose gradient to analyze immunocomplexes of biological particles has been developed. The sensitivity and application of this method were demonstrated by that the in situ accessibility of ribosomal protein epitopes could be easily determined. We used sucrose gradients to separate the ribosome-bound and the free antibodies and traced the antibodies in the gradients by an enzyme-linked immunosorbent assay. Epitopes exposed in situ are bound by specific antibodies, which in turn are detected in sucrose gradients migrating with ribosomes. This method of detecting antibody migration is more sensitive than the conventional means of using A260nm to monitor the antibody-mediated dimerization of ribosomes. Furthermore, an epitope defined by a biotin-labeled monoclonal antibody can be analyzed in the presence of other unlabeled antibodies. Thus, the relationship of different accessible epitopes in situ can be readily examined. Versatility and sensitivity of this method should make it useful in analyzing a variety of immunocomplex systems.     

Identification and purification of collagen-synthesizing polysomes with anti-collagen antibodies.

Antibodies against embryonic chick bone collagen were prepared in rabbits and were purified by affinity and ion exchange chromatography until collagen-specific and RNase-free. 125I-anti-collagen antibodies were used to locate the collagen-synthesizing polysomes of 8-day chick embryo wings and legs on sucrose gradients by measuring the polysome associated radioactivity. The 125I-anti-collagen antibodies bound predominantly to polysomes in the heavy region of sucrose gradients. These binding sites could only be saturated with homologous anti-collagen antibodies. Further evidence for the specificity of this reaction was provided by a correlation of the amount of anti-collagen antibodies bound in the heavy regions of sucrose gradients with the amount of collagen being synthesized by a particular tissue. The validity of this immunochemical method was confirmed by localizing collagen-synthesizing polysomes by an independent method which utilizes their ability to incorporate [3H]proline into collagen peptides in a cell-free system. The collagen-synthesizing polysomes are found in a single, rather broad peak in these gradients. The results of shortening the centrifugation time indicate that larger species of collagen-synthesizing polysomes are not present in these tissues. Partial purification of the collagen-synthesizing polysomes may be achieved by specifically sedimenting them after treatment with anti-collagen antibodies followed by goat anti-rabbit antibodies.     

A semiautomated system for the production and analysis of sucrose density gradients

A semiautomated system permitting considerable accuracy, speed and reproducibility in the making and fractionation of sucrose density gradients is described. The system consists of a modified Beckman gradient forming device which makes six gradients simultaneously and delivers them into six 12.5 ml polyallomer centrifuge tubes in such a manner that new material is continuously added to the meniscus of the gradient. The gradients are fractionated three at a time and up to 100 fractions per gradient can be collected automatically directly into scintillation vials with a choice of drop counting or time mode with rinse and automatic addition of scintillation fluid to each vial. The system can process up to six gradients per hour but centrifugation time is usually the limiting factor. With neutral sucrose gradients, sharp, reproducible, monodisperse peaks containing up to 100% of the gradient radioactivity are usually obtained but a smaller monodisperse peak containing as little as 3.5% of the gradient radioactivity can be detected under conditions where some pairs of molecules might tangle or dimerize. The resolution and reproducibility of this system when used with neutral sucrose gradients is at least the equal if not superior to that commonly claimed for alkaline sucrose gradients.     

DETERMINATION OF GUSTATORY SUCROSE THRESHOLD IN WATER BY USE OF A LINEAR SUCROSE GRADIENT

A novel experimental method was developed which allows the determination of the threshold concentration of sucrose by use of a linear sucrose gradient in water. With this method a continuous tasting of the test-liquid is possible. A panel of 15 persons experienced in taste-testing was used. Three gradients of different steepness were applied: 0 to 1.5% (w/w) sucrose in 2 min (I), 3 min (II) and 4 min (III). The results of the new method were compared with those of the standard method (DIN). With gradients I and II we found values which were significantly higher than those of the standard method (I: 0.49% (w/w); II: 0.46% (w/w); DIN: 0.31% (w/w)), whereas with gradient III the same threshold value was found as with the DIN-Method (III: 0.32% (w/w)).     

Isolation of Cryptosporidium sp. oocysts from human fecal samples]

Experiments using two sequential discontinuous sucrose gradients, performed with 12%, 18%, 21% and 24% solutions, for the isolation of Cryptosporidium sp. oocysts from human fecal samples were undertaken. The sucrose gradients were centrifuged at 4000 rpm during 20 min. at 10 degrees C or room temperature. After that, 3 bands were observed. Oocysts were recovered mainly from the second band (75.5% after the first gradient, and 44.4% in the next). The two sequential discontinuous sucrose gradients would permit an efficient isolation of Cryptosporidium sp. oocysts from human fecal samples.     

beta-Glucosidase Activity in Corn Roots: Problems in Subcellular Fractionation

Preliminary results from differential centrifugation experiments, washing treatments, and enrichment in linear sucrose gradients at a density of 1.09 grams per cubic centimeter all indicated that β-glucosidase activity in corn root homogenates was associated with a membrane such as tonoplast. A subsequent sucrose density gradient centrifugation time course showed that the β-glucosidase was actually a soluble enzyme which moved into the gradients. The problem of soluble enzymes contaminating light density membranes in sucrose gradients and the question of centrifugation time necessary for membrane vesicles to reach isopycnic conditions are addressed.     

Filtration-fluorescence determination of DNA sedimentation profiles

A method for determination of DNA sedimentation profiles in density gradients of sucrose or salts is proposed. The method consists in isolation and purification of DNA from the fractions by molecular filtration and a subsequent determination of DNA content by the fluorescence of the DNA-ethidium bromide complex.     

Density Gradient Study of Victorin-Binding Proteins in Oat (Avena sativa) Cells

Victorin-binding proteins (VBPs) in oat (Avena sativa) cells were identified using native victorin and anti-victorin polyclonal antibodies. Homogenates of oat tissues were fractionated in continuous or discontinuous sucrose density gradients or with an aqueous two-phase method, and covalent binding sites of victorin were detected by western blotting. In a 20 to 45% (w/w) sucrose continuous density gradient, the 100-kD VBP was located in fractions of 37 to 44% sucrose, with a peak at 39% sucrose. Based on marker enzyme assays, plasma membranes peaked at 39 to 41% sucrose, mitochondria peaked at 41%, but Golgi and endoplasmic reticulum were in lower density fractions, peaking at 28 to 29% and 22 to 24% sucrose, respectively. The 100-kD VBP was not found in plasma membranes purified by the aqueous two-phase method or in mitochondria purified by discontinuous density gradient centrifugation. Victorin binding to 65- and 45-kD proteins was detected in all fractions in the continuous sucrose density gradients. The 65- and 45-kD proteins were both detected in purified plasma membranes, but only the 65-kD protein was detected in purified mitochondria. The subcellular location of VBPs was the same in sensitive and resistant oat cells.     

Generation of density gradients. Approximations to equivolumetric gradients for zonal rotors

The use of a “density gradient generating function” allows the concentration profile of a density gradient to be written explicitly in terms of the required distribution of sedimentation coefficients in place of the previous implicit formulations. This function, which is easily implemented in a computer program, permits calculation of density gradients for a number of applications. This approach is applied to computation of a variety of equivolumetric gradients of sucrose for zonal rotors and yields a formula for the calibration of such gradients. An accurate approximation has been found which allows the generation of virtually all equivolumetric gradients of sucrose for a given rotor using a single program for the gradient generator employed; the adjustment for different particle densities and for different concentrations at the top of the gradient is made by varying only the initial and final concentrations of sucrose used.     

Rate of DNA chain elongation in ultraviolet light-irradiated mammalian cells as estimated by a bromodeoxyuridine photolysis method.

Using a new method, we show that, when the DNA chain elongation rate is measured for short time periods, ultraviolet light does not decrease this rate. The method is based on the photolysis of bromouracil-containing DNA by 313 nm light and alkaline sucrose gradients.     

Inducing activity of fractionated microsomal material from theXenopus laevis gastrula stage

Summary The postmitochondrial supernatant fromXenopus gastrulae has been fractionated on sucrose gradients. Part of the microsomal material was treated with EDTA, which dissociates most of the polysomal and monosomal material into ribosomal subunits. In addition, a series of pooled fractions from the EDTA treated gradients has been applied to discontinuous gradients in more concentrated sucrose to separate membranous material from the remaining microsomal components.Pooled fractions from all gradients have been tested for inductive activity on amphibian gastrula ectoderm. The spinocaudal (trunk and tail) inducing activity was to some extent eneriched in the membrane fractions.     

Tissue-culture fractionation. Zonal centrifugation of Lettrée cells homogenized by glycerol-induced lysis: subfractionation of membranes in sucrose gradients.

1. Lettrée cells were grown intraperitoneally in MF-1 mice. 2. Cells that were loaded with glycerol were swollen in 0.1 M-sucrose and disrupted by Dounce homogenization. 3. Early-passage Lettrée cells were more easily disrupted than late-passage cells by this method, and the former produced larger fragments of plasma membrane. 4. The membranes were fractionated initially in sucrose gradients (on the basis of sedimentation rate) in a BXIV zonal rotor. 5. Fractions from this gradient were further resolved in isopycnic sucrose gradients. 6. Plasma-membrane and endoplasmic-reticulum fractions were recovered in good yield and high purity.     

Generating sucrose gradients in three minutes by tilted tube rotation

A technique which permits rapid preparation of sucrose gradients with highly reproducible profiles is described. Tubes are filled with equal volumes of light and heavy sucrose solutions, sealed, and rotated for 3 min tilted 80 degrees from vertical. Linear 5-20% and 5-30% gradients and nonlinear but useful 5-45% gradients are obtained.     

ISOLATION AND PROPERTIES OF SECRETORY GRANULES FROM RAT ISLETS OF LANGERHANS : I. Isolation of a Secretory Granule Fraction

A method has been devised for the isolation of a secretory granule fraction from isolated rat islets of Langerhans. The islets were homogenized in buffered sucrose, and the homogenate was separated into nuclear, mitochondrial, secretory granule, and microsomal fractions by differential centrifugation. The secretory granule fraction was purified by differential centrifugation in discontinuous sucrose density gradients. A greater degree of purification could be achieved by the use of two successive gradients of this type, although the final yield was greatly reduced. Biochemical and morphological characterization of the fractions was obtained; the secretory granule fraction contained both insulin and glucagon. The limiting membranes of the granules remained intact and the general appearance of the granules was similar to that seen within the whole islet cells.     

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.     

Induction of DNA single-strand breaks in barley by sodium azide applied at pH 3.

Sodium azide (1 to 50 mM), adjusted to pH 3 and applied for 2 h to presoaked barley seeds, induced a dose-dependent frequency of single-strand breaks in DNA of non-germinating embryos. This was demonstrated by sedimentation analyses of isolated DNA samples in alkaline sucrose gradients and in neutral sucrose gradients with 80% formamide. The doses applied also inhibited dose dependently the root length, seed germination and partially the seedling height. Only the sub-lethal doses (10 and 12.5 mM) induced a low frequency of chromatid breaks and translocations in the root tip metaphases. The sedimentation rate (in alkaline sucrose gradients) of calf thymus DNA treated with sodium azide at pH 3, was similar to that of the control DNA treated with buffer (pH 3) alone.