Mobility measurements by photometric analysis of zone electrophoresis in a sucrose gradient column

A density gradient zone electrophoresis apparatus has been designed for measuring electrophoretic mobilities on small amounts (about 10 μg) of viruses or other ultraviolet-absorbing materials. Dialysis membranes separate the gradient column from electrode chambers and eliminate the need to maintain hydrostatic equilibrium between the buffer chambers. The virus zone was located by pumping the gradient column through an ultraviolet flow cell, a procedure which did not disturb the virus zone. The apparatus was tested by measuring the mobilities of tobacco mosaic and brome mosaic viruses in different buffers, in sucrose, glucose, and glycerol gradients, at 0° and 15°, and during ascending and descending electrophoresis.     

Velocity sedimentation of organelles at low centrifugal force in an isokinetic gradient.

Mast-cell granules and polystyrene microspheres (0.600 and 1.011 micrometer in diameter) were sedimented in a previously described [Pretlow (1971) Anal. Biochem. 41, 248--255] isokinetic gradient in a low-speed centrifuge. For the analytical velocity sedimentation of organelles, this gradient offers several advantages over gradients that are commonly used for the sedimentation of organelles: (a) the density gradient (0.0008 g.ml-1.cm-1) is small, and the effective densities of organelles will change relatively little during sedimentation; (b) the densities at all points in the gradient (1.017--1.027 g/ml) are less than those in gradients commonly used for the sedimentation of organelles, the effective densities of sedimenting organelles are consequently relatively large, and the effect of density as a determinant of velocity of sedimentation is less limiting than in conventional gradients; (c) the small slope of the gradient is associated with a relatively slow increase in the viscosity encountered by the sedimenting organelle; (d) the iso-osmotic gradient is not significantly affected by the gradient medium (Ficoll), and the osmolarity can be adjusted to the desired value by the selection of an appropriate salt solution as the solvent for the Ficoll; (e) the gradient will be isokinetic for particles of densities similar to most organelles. An ultracentrifuge is not required for work with this gradient.     

High resolution KSCN/CsSCN equilibrium gradients effectively separate a population of density labeled proteins from unlabeled proteins

The persistence of proteins in a number of biological systems has been analyzed by density labeling techniques; however, the utility of this approach has been severely hampered by poor resolution between density-labeled and unlabeled proteins on equilibrium gradients. A high resolution equilibrium salt gradient composed of KSCN/CsSCN has been developed to effectively separate density-labeled proteins (13C-15N-2H-substituted) from unlabeled proteins. The resolution of this system is approximately twofold greater than that previously achieved with cesium formate/guanidine hydrochloride equilibrium gradients which have been used in many recent protein density labeling studies. In order to examine the extent of cross-contamination between density-labeled and unlabeled proteins in a KSCN/CsSCN gradient system, density-labeled chick epidermal proteins were mixed with unlabeled Drosophila larval proteins and then separated on these equilibrium gradients. From individual gradient fractions proteins were recovered and fractionated on a sodium dodecyl sulfate-polyacrylamide gel, demonstrating the virtually complete separation between the two populations. The general utility of this system for protein stability studies is also demonstrated.     

Construction of a thermotaxis chamber providing spatial or temporal thermal gradients monitored by an infrared video camera system.

A thermotaxis chamber was constructed to quantitatively study thermotaxis in eukaryotic amoeboid cells. The apparatus provided either spatial or temporal temperature gradients in an observation chamber set in an inverted microscope. With an infrared video camera system, spatial thermal gradients were monitored directly and the temperature at the actual location of the cells could be estimated accurately. This enabled a precise determination of the strength of thermal stimuli. With this apparatus, we were able to simultaneously measure temperature and observe cellular behavior directly. This feature permits quantitative studies on stimulus-response relationships. The utility of the apparatus was demonstrated by thermotaxis assay under a spatial thermal gradient in polymorphonuclear leukocytes. Since this apparatus can also provide temporal thermal gradients, it may have several applications in studies of temperature-dependent phenomena in cell biology.     

Rapid, multisample isoelectric focusing in sucrose density gradients using conventional polyacrylamide electrophoresis equpiment: a two-peak transient in the approach-to-equilibrium.

Using a semiporous plug of agar gel to support a sucrose density gradient column without restricting electrical conductivity, Massey and Deal [J. Biol. Chem.248, 56 (1973)] were able to use a conventional polyacrylamide gel electrophoresis apparatus to carry out single tube isoelectric focusing experiments in density gradients in only 2 hr using minute amounts (50 μg) of sample and very little ampholyte (0.18 ml); no cooling apparatus was required. In this work we report that 1) polyacrylamide provides a superior gel plug and 2) that ten isoelectric focusing tubes can easily be run simultaneously in a conventional polyacrylamide gel electrophoresis apparatus. In addition, the isoelectric points of eight proteins, with pI values ranging from 5.1 to 8.8 have been determined and the kinetics of the approach-to-isoelectric-focusing-equilibrium have been analyzed. Of special interest is the discovery that in the initial stages of focusing, in these sucrose density gradients, a major peak is formed at each end of the column; these two peaks migrate toward each other and finally coalesce into a single peak. Similar, although less pronounced, effects were previously observed by Catsimpoolas and Wang [Anal. Biochem.39, 141 (1971)] in focusing experiments in polyacrylamide gels. With all other conditions constant, the time required to reach equilibrium is 1) less in broad range (e.g., 3–10) pH gradients than it is in narrow range (e.g., 5–8) pH gradients and 2) generally greater with higher molecular weight substances than with lower molecular weight substances. Explanations are given for all of these kinetic phenomena.     

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.     

Tumor cell haptotaxis on covalently immobilized linear and exponential gradients of a cell adhesion peptide

The movement of cells up an adhesive substratum gradient has been proposed as a mechanism for directing cell migration during development and metastasis. Critical evaluation of this hypothesis (haptotaxis) benefits from the use of quantifiable, stable substratum gradients of biologically relevant adhesion molecules. We report covalent derivatization of polyacrylamide surfaces with quantifiable gradients of a nonapeptide containing the adhesive Arg-Gly-Asp sequence. Cell migration was studied by seeding derivatized surfaces evenly with B16F10 murine melanoma cells. Within 8 hr, cells on gradients redistributed markedly; higher cell densities were found at gel positions having higher immobilized peptide densities. In contrast, cells seeded on control gels with uniform concentrations of adhesive peptide did not redistribute. Redistribution occurred on gradients in both serum-free and serum-containing media. Experiments with uniform density peptide-derivatized gels demonstrated that redistribution on gradients was not due to preferential initial cell attachment or preferential growth on the higher density of immobilized peptide, but must have been due to cell translocation. Cells on exponential gradients of immobilized peptide migrated to a position on the gel surface corresponding to the highest immobilized peptide density, while cells on linear gradients of the same peptide migrated to a position of intermediate peptide density. These data suggest that the B16F10 cells respond to proportional changes in immobilized peptide density rather than to absolute changes, implying a sensing mechanism which utilizes adaptation. These results demonstrate that (1) a gradient of a small adhesive peptide is sufficient to generate redistribution of cell populations and (2) controlled quantifiable substratum gradients can be produced and used to probe the underlying cellular mechanisms of this behavior.     

Localization of organelle proteins by isotope tagging (LOPIT)

We describe a proteomics method for determining the subcellular localization of membrane proteins. Organelles are partially separated using centrifugation through self-generating density gradients. Proteins from each organelle co-fractionate and therefore exhibit similar distributions in the gradient. Protein distributions can be determined through a series of pair-wise comparisons of gradient fractions, using cleavable ICAT to enable relative quantitation of protein levels by MS. The localization of novel proteins is determined using multivariate data analysis techniques to match their distributions to those of proteins that are known to reside in specific organelles. Using this approach, we have simultaneously demonstrated the localization of membrane proteins in both the endoplasmic reticulum and the Golgi apparatus in Arabidopsis. Localization of organelle proteins by isotope tagging is a new tool for high-throughput protein localization, which is applicable to a wide range of research areas such as the study of organelle function and protein trafficking.     

Automated kinetic analysis of pyruvate kinase

An apparatus for the automatic determination of enzyme kinetics of pyruvate kinase is described. A continuous plit of velocity versus substrate concentration is obtained using quantities of enzyme and substrates comparable to manual determinations. The automated procedure offers a number of advantages over manual methods including elimination of repetitive pipetting, simpler reaction temperature regulation, reduced analysis time, and possible on-line computer analysis. The apparatus utilizes a commercially available column uv flow monitor to measure NADH/NAD changes in the coupled lactic dehydrogenase reaction at 340 nm in a continuous flow system. The optical density changes are directly related to the velocity of the enzyme-catalyzed reaction. A linear substrate gradient is generated from a density gradient maker to provide the required relationship between velocity and substrate concentration. The system is calibrated by forming a gradient from a hemoglobin solution of known concentration. The procedure has been evaluated by determination of the kinetic parameters of three of the isozymes of pyruvate kinase. Values obtained by the continuous flow method are in close agreement with those obtained by individual point determination in a recording spectrophotometer.     

Scanning isoelectric focusing in small density-gradient columns: I. Use of a Standard Spectrophotometer Cuvette for Focusing. Chemical Modification of Proteins by Migrating Reactive Ions

By the method of isoelectric focusing in a sucrose density gradient, small protein samples (less than 100 μg) have been separated and analyzed within 2 hr, using as electrolysis column a commercial standard quartz spectrophotometer cuvette, equipped with platinum electrodes and placed in an optical scanning device. Preparation of the cuvette prior to an isoelectric focusing experiment required about half an hour with no external apparatus, as the density gradient was created spontaneously in the cell by free interdiffusion of sucrose solutions. The cuvette temperature could be controlled by circulating water. The optical detection device permitted repeated scanning of the cuvette during the electrolysis process, thus providing information about the events occurring to a protein during focusing or prolonged electrolysis. By scanning with wavelength at the positions where the proteins have focused, their absorption spectra were obtained. the isoelectric points of separated proteins were estimated by fractionation of the cell contents and subsequent pH measurements on the fractions.The present paper also describes how individual Ampholine components, or groups of components, in their focused states gave rise to easily detected refractive-index gradients within the cell. The optical scanning device has been built in such a way that interference of these gradients with absorption measurements was abolished.Application of the technique to the isoelectric separation of commercial sperm whale myoglobin is reported. Ferrous or ferric forms of the focused myoglobin components were obtained by migration of reducing or oxidizing agents through the zones.     

Synthesis of Arabinose-Containing Cell Wall Precursors in Suspension-Cultured Tobacco Cells I. Intracellular Site of Synthesis and Transport

The distribution of arabinose-containing macromolecules in suspension-culturedtobacco cells was examined using sucrose density gradients.Exogenously applied ¹⁴Carabinose was scarcely converted intoother sugars, and concentrated in the Golgi-rich fraction (1.15g/cm³) and then secreted to the cell wall. ¹⁴C-Arabinose wasalso incorporated in a lower sucrose density fraction (1.11g/cm³), which contains small vesicles presumably originatedfrom the Golgi apparatus. The arabinose-containing macromoleculesin this fraction was more easily solubilized in water than thosein the Golgi-rich fraction. Alkaline hydrolysis of the macromoleculesindicated that cell-wall glycoprotein is a major component ofthe macromolecules and that the degree of glycosylation is slightlygreater in the lower density fractions than in the Golgi-richfraction. Based on these results, a scheme is suggested in whichthe glycoproteins and polysaccharides are glycosylated in theGolgi apparatus and secreted to the cell wall via secretionvesicles in the low density fraction. The possibility of ¹⁴C-arabinose-containingmacromolecules, in the early phase of synthesis, being a markerof the plant Golgi apparatus is also proposed. (Received September 21, 1980; Accepted January 27, 1981)     

Density-gradient sedimentation in silica sols. Anomalous shifts in the banding densities of polystyrene "latex" beads.

When polystyrene "latex" beads are centrifuged to equilibrium in gradients of Nalcoag 1030, Nalcoag 1034A and Ludox 130M, which are commercial formulations of colloidal silica, the beads at densities that may be markedly lower or higher than the bulk density. Addition to the gradient of small amounts of certain polymers restores the banding densities towards the expected value. These findings closely mimic previously observed "density shifts" of biological particles. A possible model for this anomalous behavior is discussed.     

Preparative immobiline isoelectric focusing of plasma apolipoproteins on vertical slab gels

An effective preparative isoelectric focusing method has been developed using the LKB Immobiline system in a vertical slab gel apparatus. Advantages of this procedure are ease of sample application, excellent resolution, and the direct visualization of focused bands. Narrow pH gradients have been used to separate apolipoprotein E3 isoforms (pH gradient 4.9-5.9) and to resolve the apolipoprotein C mixture (pH gradient 4.0-5.0). Recoveries ranged from 40 to 70%. The method should be valuable for protein and isoform purification.     

An apparatus for density gradient forming and nonpuncturing fractionation

A technique is described which allows gradient formation, sample layering, and nonpuncturing gradient fractionation in one apparatus. The demonstrated device can be adapted to centrifuge tubes of different sizes and volumes and connected to almost any device which is used for gradient formation or analysis with flow cuvettes and/or fraction collectors. The operation technique can be standardized. The construction gives reproducible gradients with sharp starting bands of the samples and provides good resolution after centrifugation.     

Fundamental limits to position determination by concentration gradients

Position determination in biological systems is often achieved through protein concentration gradients. Measuring the local concentration of such a protein with a spatially varying distribution allows the measurement of position within the system. For these systems to work effectively, position determination must be robust to noise. Here, we calculate fundamental limits to the precision of position determination by concentration gradients due to unavoidable biochemical noise perturbing the gradients. We focus on gradient proteins with first-order reaction kinetics. Systems of this type have been experimentally characterised in both developmental and cell biology settings. For a single gradient we show that, through time-averaging, great precision potentially can be achieved even with very low protein copy numbers. As a second example, we investigate the ability of a system with oppositely directed gradients to find its centre. With this mechanism, positional precision close to the centre improves more slowly with increasing averaging time, and so longer averaging times or higher copy numbers are required for high precision. For both single and double gradients, we demonstrate the existence of optimal length scales for the gradients for which precision is maximized, as well as analyze how precision depends on the size of the concentration-measuring apparatus. These results provide fundamental constraints on the positional precision supplied by concentration gradients in various contexts, including both in developmental biology and also within a single cell.     

Simulation of density gradients of astral microtubules at cell surface in cytokinesis of sea urchin eggs

Astral microtubules extend close to the cell surface just before cytokinesis in sea urchin eggs. At this time, a small region with a constant area is considered around a point on the egg surface. To calculate the number of microtubules that reach the surface region, i.e. the microtubule density at the point, a simple mathematical model was set up. The density was estimated at many surface points in multipolar and distorted eggs by using the model. A contour map was drawn to investigate the density gradients. The gradient patterns were compared with the distributions of contractile-ring microfilaments. The simulated cases were: (1) an unusual distribution of contractile-ring microfilaments in an egg that had polyasters and was compressed by a coverslip; (2) formation of contractile-ring microfilaments at the equatorial region in compressed eggs with a centrally-located mitotic apparatus; (3) normal furrowing in the plane of the spindle midpoint in eggs inserted into a glass loop or confined in a capillary; (4) failure of furrow formation in spherical eggs treated with ethyl urethane and revival of furrowing by pushing the equatorial surface close to the spindle. These simulations proposed the hypothesis that contractile-ring microfilaments form at surface regions where the microtubule density has a local minimum, not a local maximum. In addition, it was suggested that the probability of the formation of the contractile-ring microfilaments is dependent on how abruptly the density gradient changes at the local-minimum point. These results support the idea that the gradient pattern of the microtubule density determines whether and where contractile-ring microfilaments appear.     

Purification of very high density lipoproteins by differential density gradient ultracentrifugation

Differential density gradient ultracentrifugation procedures, utilizing a vertical rotor, were developed for the preparative purification of very high density lipoproteins (VHDL, density greater than 1.21 g/ml). The VHDLs of several insect species were purified as follows. An initial density gradient ultracentrifugation step removed lipoproteins of lower density from the VHDL-fraction, which partially separated from the nonlipoproteins present in the infranatant. A complete separation was achieved by a second centrifugation step employing a modified gradient system. The use of a vertical rotor and specially designed discontinuous gradients allows a relatively fast, efficient, and economical isolation of the class of very high density lipoproteins. Similar gradient systems should be useful for the detection and purification of VHDLs from other sources.     

Preconstruction of density gradients in cells of the analytical ultracentrifuge

The principles and techniques of zonal centrifugation are now well established (1), but despite the advantage of requiring smaller amounts of material than conventional experiments, this procedure has not been widely applied to the analytical ultracentrifuge. In general, the method has been limited to experiments where separations are based on differences in density. Usually, this involves the generation in situ of gradients, and has been widely used in the analysis of nucleic acids (3,4); recently, the scope of this technique was enlarged by a method for fractionating the zones produced by this type of separation (5). However, Rosenbloom and Schumaker (2) showed that a preformed gradient can be constructed in the analytical cell prior to running, and this stabilised the sedimentation of a boundary or a zone of nucleic acid. Although the use of a preformed density gradient (containing initially a uniform distribution of the macromolecule(s)) could reduce the time to reach isopycnic equilibrium it is not a prerequisite for the experiment, however, a preformed gradient is essential when measuring the velocity of a zone, as in Cohen, Giraud, and Messiah (8). This communication describes a simple technique for generating density gradients in the analytical cell prior to running and with the minimum of disturbance.     

A model for sedimentation in inhomogeneous media. I. Dynamic density gradients from sedimenting co-solutes

Macromolecular sedimentation in inhomogeneous media is of great practical importance. Dynamic density gradients have a long tradition in analytical ultracentrifugation, and are frequently used in preparative ultracentrifugation. In this paper, a new theoretical model for sedimentation in inhomogeneous media is presented, based on finite element solutions of the Lamm equation with spatial and temporal variation of the local solvent density and viscosity. It is applied to macromolecular sedimentation in the presence of a dynamic density gradient formed by the sedimentation of a co-solute at high concentration. It is implemented in the software SEDFIT for the analysis of experimental macromolecular concentration distributions. The model agrees well with the measured sedimentation profiles of a protein in a dynamic cesium chloride gradient, and may provide a measure for the effects of hydration or preferential solvation parameters. General features of protein sedimentation in dynamic density gradients are described.     

Ion transport and the vibrating probe.

The theory of ion transport in the vicinity of a vibrating probe is developed. It is shown that the convection loops produced by the probe will not affect the electrical current density, assuming that the action of the probe does not affect the sources of the current in the biological system. However, the convection loops will significantly alter the ion concentration gradients in the unstirred layer near a tissue or cell surface. The concentration gradients within each convection loop will be reduced, while the concentration gradients between the loops and outside of the loops will be increased relative to the gradients existing without the probe. As a consequence, the electrical potential gradients can be changed relative to the potential gradients existing in the absence of the convection caused by the probe. If the mobility of the ion species carrying the electrical current is greater than the average ion mobility in the medium, then a decrease in ion concentration gradient will be accompanied by an increase in electrical potential gradient, while an increase in concentration gradient will be accompanied by a decrease or even a reversal of electrical potential gradient. Thus, the electrical potential gradient measured by the probe will depend on the concentration gradient in the vicinity of the probe, which will depend in turn on the spatial relation of the convection loops to the probe. An example of the effect of the convection loops on ion concentration and electrical potential is obtained from the theory via a numerical computer calculation. Experimental tests of this theory are discussed.