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Biomedical subjects

A Chrambach

Publications and source records attributed to A Chrambach.

At least 127 records · Page 7Linked to original sources
PubMed1988

Discontinuous buffer systems optimized for the agarose gel electrophoresis of subcellular particles.

Discontinuous buffer systems operative between pH 5.7 and 7.4, 0 degrees C, were generated, which are characterized by more rapidly displaced moving boundaries than applied previously. These allow one to resolve subcellular particles relatively rapidly and at relatively low agarose gel concentrations. A commercial mixture of DNA restriction fragments pre-stained with ethidium bromide was found to be a suitable tracking dye for these boundaries.

Buffers↗
PubMed1988

A motor-driven syringe-type gradient maker for forming immobilized pH gradient gels.

A motor driven gradient maker based on the commercial model (Jule Inc., Trumbull, CT) was designed for immobilized pH gradient gels to provide small volumes, rapid stirring and delivery, strict volume and temperature control and air exclusion. The device was constructed and by a convenient procedure yields highly reproducible gradients either in solution or on polyacrylamide gels.

Acrylic Resins↗
PubMed1988

Linear Ferguson plots of polystyrene sulfate size standards for the quantitative agarose gel electrophoresis of subcellular particles.

Accurately standardized commercial polystyrene sulfate particles in agarose gel electrophoresis yield linear Ferguson plots at pH 7.4 over a gel concentration range up to 0.9% agarose which do not exhibit any significant sigmoidal curve elements, using either a discontinuous buffer system or a continuous buffer. Ferguson plots of these standard-sized particles were evaluated using alternatively a linear or convex model, by means of a newly developed set of programs (to be used in conjunction with program M-LAB) which (i) is sufficiently user-friendly to allow for quantitative agarose gel electrophoresis of subcellular-sized spherical particles based on their convex Ferguson plots with the same operational simplicity previously available for linear Ferguson plots only; (ii) simultaneously and interactively analyzes the Ferguson plots of all particles under consideration on the basis of an extended Ogston model.

Electronic Data Processing↗
PubMed1988

Ferguson plots based on absolute mobilities in polyacrylamide gel electrophoresis: dependence of linearity of polymerization conditions and application to the determination of free mobility.

In contrast to Ferguson plots based on relative mobilities, Ferguson plots of proteins in polyacrylamide gel electrophoresis based on their absolute mobilities were found to be linear under unusual polymerization conditions which yield relatively wide gel fibers and a low total fiber length per unit weight, but not under previously and commonly used conditions. These linear Ferguson plots in gels of 1, 3 and 5% crosslinking intersect at a single gel concentration between 1 and 2% T (M-point). It is postulated that the measure of free mobility of the proteins is the M-point, and not the intercept of their Ferguson plots with the mobility axis as assumed previously. This postulate abolishes the well-known paradoxical interpretation of the increase with %C of the linearly extrapolated intercept of the Ferguson plot with the log(mobility) axis (designated Yo) in terms of free mobility. The postulate is also compatible with the interpretation of the points of intersection of the Ferguson plots of oligomeric series of proteins at finite gel concentrations (designated mu-points) as their common free mobilities.

Buffers↗
PubMed1988

Detection of turnip crinkle virus on agarose gel electropherograms at the nanogram load level.

The previous conditions for the physical characterization of turnip crinkle virus (TCV) by quantitative agarose gel electrophoresis [1, 2] were limiting the method to the microgram load level and were therefore insufficiently sensitive to satisfy the need in many areas of virology for detection of viruses containing single-stranded RNA at the nanogram level. The present report remedies that defect by presenting a technique compatible with the nanogram load level of such viruses. The technique is based on a reduction of gel thickness and on the use of silver staining.

Electrophoresis, Agar Gel↗
PubMed1988

The adsorption of large proteins in electrofocusing on immobilized pH gradients: I. Protein specificity and dependence on Immobiline and carrier ampholyte concentrations.

Phycoerythrin, ferritin, urease, beta-galactosidase and thyroglobulin, with molecular masses in excess of 200 kDa, adsorb and consequently fail to migrate to, and focus at, their pI positions in electrofocusing in immobilized pH gradients at a total Immobiline concentration of 20 mM while they do focus normally in pH gradients formed by carrier ampholytes. The addition of carrier ampholytes (pH range 3.5-9.5) at concentrations of 0.1 to 5% to the Immobiline-containing gels reduces adsorption (desorbs) some but not all of the 5 proteins at specific Immobiline concentrations. The adsorption is not due to water redistribution and consequent reduction in gel porosity; nor is it due to conductivity minima across the pH gradient. The hypothesis that the presence of oligomeric Immobiline contributed to the protein adsorption is the subject of the accompanying report.

Acrylamides↗
PubMed1988

The adsorption of large proteins in electrofocusing on immobilized pH gradients: II. Dependence on the oligomeric state of Immobiline.

Five proteins with molecular mass in excess of 200 kDa were found to adsorb onto gels during isoelectric focusing on immobilized pH gradients (IPGEF). To probe for the mechanism of that adsorption, the homogeneity of the six Immobiline preparations used to make IPGEF gels was tested. Five of these Immobiline preparations appear homogeneous in gel filtration of Sephadex G-10. The sixth Immobiline (pK 9.3) exhibits a minor component eluting ahead of the major peak and comprising less than 4% of the total Immobiline absorbing at 226 nm. The proportion of the minor component increases with column load. Major and minor components when isolated appear to equilibrate with one another. Judging by the results of mass spectrometry, all 6 preparations are free of small aggregates of less than 500-600 Da molecular mass. Ultrafiltration of the Immobiline preparations through a membrane with 500 Da nominal cutoff leads to partial desorption of only 3 of the 5 adsorbed proteins. CHAPS is ineffectual in desorbing the 5 proteins from the IPG gel made with ultrafiltered Immobilines. None of the 6 Immobiline preparations used precipitates ferritin. All large proteins that adsorb onto IPGEF gels in the pH range 4-9.5 also adsorb onto commercial IPGEF gels in the pH range 4-7.

Acrylamides↗
PubMed1988

Transformation of the glucocorticoid receptor in the cell-free cytosol of the neural retina of the chick embryo: changes in the size and charge of the receptor complex during transformation suggest a multistage process.

The physicochemical properties of the glucocorticoid receptors (GR), and the molecular changes induced during their transformation in the cell-free cytosol of the neural retina of the chick embryo, were investigated. The surface charge of the various size forms of the GR complex was determined on gel filtration and/or glycerol density gradient-isolated GR, by electrofocusing under nondenaturing conditions. The nontransformed molybdate-stabilized GR in hypotonic buffer (containing PMSF) appears as a 350 kilodalton (kDa) complex (Rs = 8.6 nm, S = 9.5), with an apparent pI value (pI') of 4.4 +/- 0.1. The GRs in heat or salt-activated cytosols appear as a 90 kDa hormone-receptor complex (Rs = 5.6 +/- 0.2, S = 3.9 +/- 0.1), which is resolved as a major peak with a pI' value of 6.2 +/- 0.1 and a minor peak with a pI' value of 5.4. The transformation of the 350 kDa oligomer to the 90 kDa monomer occurs in three stages. Two distinct dissociation steps were induced by 0.4 M KCl: (a) the dissociation of the 350 kDa complex to a 170 kDa complex (Rs = 7.8 +/- 0.2, S = 5.1 +/- 0.2), exhibiting a pI' value of 5.6 +/- 0.2, induced by salt and not inhibited by molybdate; and (b) the dissociation of the 170 kDa complex to the 102 kDa complex (Rs = 5.6 +/- 0.2, S = 4.4), also exhibiting a pI' value of 5.6 +/- 0.2, which is blocked by molybdate. The third step, the transition of the 102 kDa complex to the activated (nuclear-like), 90 kDa form, is dependent on cytosolic factors. It is induced in the isotonic milieu by physiological temperatures, and in the cold by exposing the crude cytosol to 0.4 M KCl. The nature of this cytosolic processing step is unknown. It occurs in the presence of PMSF, which presumably inhibits proteolytic GR degradation in the cytosol of the neural retina. Activated GR complexes tend to aggregate. Molybdate inhibits activation-induced GR-aggregation.

Animals↗
PubMed1987

Computer simulation of the variable agarose fiber dimensions on the basis of mobility data derived from gel electrophoresis and using the Ogston theory.

Agarose gel electrophoresis of viruses and proteins was evaluated for estimating fiber dynamics of agarose by computer simulation based on the extended Ogston theory. By introducing functions, in place of previously used constant parameters, into the equations derived from the Ogston theory it was demonstrated that the effective fiber properties are variable as a function of both gel concentration and the size of the particle passing through the gel. This variability accounts for the curvature of plots of log(mobility) vs gel concentration in agarose gel electrophoresis as well as for the apparent dichotomy between fiber properties obtained from the electrophoresis of either viruses or proteins. Specifically, computer simulation based on the electrophoretic mobility values of five viruses and seven proteins by use of an eight (gel-specific) parameter model yielded functions relating gel concentration and/or particle size with electrophoretic mobility of particles, the retardation coefficient, K'R, and effective fiber radius, length, and volume. The simulations give further insights into, as well as mathematical basis for, a number of previously made assumptions (such as variation of agarose fiber structure with gel concentration, variation of fiber volume with particle size, continuity of the K'R vs radius plot from 0 to 45 nm), thus demonstrating the continued usefulness of the Ogston model. The mathematical model provides the elements for an improved method for the determination of particle size, charge, and potentially shape by agarose gel electrophoresis, and can be regarded as the basis for future elaboration of a computer program for the routine determination of these parameters.

Computer Simulation↗
PubMed1986

Characterization of the filamentous hemagglutinin from Bordetella pertussis by gel electrophoresis.

A highly purified preparation of filamentous hemagglutinin (FHA) from Bordetella pertussis was analyzed for its protein composition by gel electrophoretic methods. In this preparation of FHA the following native species could be detected by polyacrylamide gel electrophoresis (PAGE) at pH 3.2: S1 and S2 (inactive subunits or fragments); two monomers, a major form designated Ia (144K), and a minor form Ib, differing only in net charge; and three oligomeric forms, designated II (213K), III (595K) and IV (1064K). Hemagglutinating activity was associated predominantly with component Ia. PAGE of FHA after derivatization with sodium dodecyl sulfate (SDS) showed there to be three major species, designated A, C and D. According to estimated molecular weight values, A, C and D are likely to correspond to S2, Ia and II respectively. Isolated components II, III and IV yield all three SDS-species upon derivatization with SDS. Both moving boundary electrophoresis and gel electrofocusing showed hemagglutinating FHA to be a basic protein. Its apparent pI is 8.1.

Bordetella pertussis↗
PubMed1985

Applicability of agarose gel electrophoresis to the physical characterization of clathrin-coated vesicles.

Agarose gel electrophoresis was found to be applicable to the physical characterization of clathrin-coated vesicles from bovine brain and rat liver. The vesicles from brain are smaller and, at pH 6.4, more highly charged than those from liver. Using a standard curve for spherical viruses, the mean radii of the brain vesicles averaged 40 +/- 3 nm for two preparations, and those of liver vesicles 53 +/- 5 nm. When the comparison between the two species is made within the same experiment, the size difference between rat liver-derived and bovine brain-derived clathrin-coated vesicles is significant at the 95% confidence level. The sizes are compatible with electron microscopy measurements. The mobilities (uncorrected for electroendosmosis), extrapolated to 0% agarose on the Ferguson plot, mu'0 (cm2/sec/V), were 0.76 +/- 0.04 for brain vesicles and 0.61 +/- 0.09 for liver vesicles. These are measures of the average surface net charge of the vesicles. The higher net charge of brain vesicles, compared to liver vesicles, is significant at the 95% confidence level whether the two species are compared intra- or interexperimentally. Charge differences between two different preparations from the same organ were significant at the 95% confidence level, while size differences were not.

Animals↗
PubMed1985

Electrophoretic characterization of active renin from human kidney and inactive renin from a human chorionic cell culture.

Enzymatically inactive human renin from chorionic cells in culture is significantly distinct in polyacrylamide gel electrophoresis (pH 8.17, 0 degree C) from active human kidney renin. The inactive renin is larger and more basic than the active renin; their molecular weights derived from gel electrophoretic retardation coefficients relate as 47.5/35.3 kDa, their valences (net protons/molecule) as 2.14/1.85. In gel electrofocusing conducted in a mixture of simple buffers, both inactive and active renins exhibit 2 components at the steady-state. The molecular size and basicity of inactive renin are consistent with the hypothesis that it may be a precursor (prorenin), although the possibility that it is an inhibitor complex cannot be ruled out.

Cells, Cultured↗
PubMed1985

Gel electrophoretic procedures potentially applicable to the isolation of human growth hormone from a transformed bacterial source at the gram-preparative scale.

A crude bacterial extract containing approximately 4 mg/ml protein, 25% of which was human growth hormone (hGH), was subjected to two alternative gel electrophoretic isolation procedures, designated I and II. Procedure I exploits the high electrophoretic net mobility (RM larger than 0.127) at pH 7.6, 0 degrees C, of the bacterial contaminants relative to hGH. This allows one to stack the contaminants at a protein load of 31.5 mg/cm2 of gel, using a "non-restrictive" gel concentration. Unstacked hGH is collected from the gel section between 0.3 and 0.6 of relative gel length and extracted electrophoretically as described previously. Alternatively, the unstacked hGH was concentrated on the gel by dispatching a second moving boundary behind the original stack ("re-stacking") and a gel section (relative gel length 0.45 to 0.6) between the two moving boundaries was excised and subjected to electrophoretic extraction. The yield of hGH ranged from 70 to 82%, and its purity (weight/Lowry) ranged from 86 to 115%. Procedure II exploits the high electrophoretic net mobility (RM larger than 0.064) at pH 10.5, 0 degrees C, of hGH relative to its bacterial contaminants at a gel concentration of 9 %T, 2 %CBis, at a protein load of 2.5 mg/cm2 of gel. The selectively stacked hGH is collected by preparative elution-PAGE, using an apparatus with 17.6 cm2 gel surface area. The yield of hGH was 90% and its purity ranged from 84-92%.

Cloning, Molecular↗
PubMed1984

Purification of the photoaffinity-labeled glucagon receptor by gel electrophoretic methods.

Rat liver plasma membrane glucagon receptor has been purified with a yield of 0.01% to an estimated homogeneity of 32-60%, using a 2-stage electrophoretic procedure. SDS-solubilized membrane proteins labeled by the photoaffinity-agent, Ne-4-azidophenylamidinoglucagon (APA-glucagon), were separated by polyacrylamide gel electrophoresis in SDS-containing buffers. Gel slices corresponding to the molecular weight of the receptor were excised, electrophoretically extracted and concentrated. The concentrate was subjected to isoelectric focusing on Sephadex to yield a purified product in which the photoaffinity-labeled receptor, with a molecular weight of 56K and a pI' of 5.9, is the sole major component.

Affinity Labels↗
PubMed1983

Heterogeneity of the glucocorticoid receptors: molecular transformations during activation, detected by electrofocusing.

Isoelectric focusing (IEF) of glucocorticoid receptor (GR) of the neural retina of the 14-day chick embryo was conducted under conditions that yielded quantitative recovery of binding activity. IEF of the cytosol, equilibrated with [3H]triamcinolone acetonide (TA) at 0-2 degrees C yielded three major TA-GR components with apparent isoelectric points (pI') of 5.4 +/- 0.3, 6.5 +/- 0.2, and 7.6 +/- 0.3, designated as I, II, and III, respectively. During temperature-induced activation (incubation at 30 degrees C for 60 min, in the presence of free [3H]TA and 0.15 M KCl), approximately 25% of the specifically bound TA was irreversibly lost. IEF reveals that this loss is accounted for by the complete loss of binding from I. During activation, II also decreases but correspondingly III increases, i.e., the sum of II and III remains unchanged. Only the bound TA of I is sensitive to the addition of KCl (a promoter of activation). This sensitivity of I is temperature dependent. Molybdate (an inhibitor of activation) protects the bound TA of I and suppresses the formation of III. These two effects of molybdate diminish simultaneously when the temperature is increased to 30 degrees C. III preferentially exhibits binding activity to nuclei. The data suggest that (i) the glucocorticoid-free cytosol contains two GRs, I and II, with possibly two different functions; (ii) activation involves the loss of bound TA from I and the transformation of II to III with increased pI; (iii) these two molecular events in GR activation are interdependent.

Animals↗
PubMed1982

Characterization of polynucleotide phosphorylase from Micrococcus luteus and isolation of the 13,000 base poly(A) product of the polymerization reaction.

A new purification procedure for polynucleotide phosphorylase from freeze-dried Micrococcus luteus cells gives approximately 20% yield of nearly homogeneous, primer-independent enzyme which is free of nucleic acid. The physicochemical properties of M. luteus polynucleotide phosphorylase are similar to those previously described for the enzyme from Escherichia coli in terms of Mr, subunit structure, and amino acid composition. The purified enzyme appears to be a trimer composed of three identical subunits (Mr 92,000), but it probably does not exist as such in the cell. Ferguson plot analyses of enzyme in cell extracts indicate that prior to purification the enzyme exists in oligomeric forms characterized by both higher charge and greater Mr. Changes in size and charge of oligomers which occur during purification are probably due to the dissociation of proteins and/or nucleic acids. Dissociation of the oligomers is achieved by dilution and electrophoresis, but reassociation does not occur after concentration. The poly(A) product of the initial polymerization stages migrates as a single band on both nondenaturing and urea-agarose gels. It is 13,000 +/- 2,000 nucleotides long, as measured by electron microscopy, and 8,000 nucleotides long by gel electrophoretic analysis. This poly(A) product remains bound to the enzyme after synthesis, yet can be easily obtained free of protein by proteinase K digestion.

Amino Acid Sequence↗