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J Steensgaard

Publications and source records attributed to J Steensgaard.

12 recordsLinked to original sources
PubMed1992

GTP-binding proteins in luminal and basolateral membranes from pars convoluta and pars recta of rabbit kidney proximal tubule.

The GTP-binding proteins on luminal and basolateral membrane vesicles from outer cortex (pars convoluta) and outer medulla (pars recta) of rabbit proximal tubule have been examined. The membrane vesicles were highly purified, as ascertained by electron microscopy, by measurements of marker enzymes, and by investigating segmental-specific transport systems. The [35S]GTP gamma S binding to vesicles, and to sodium cholate-extracted proteins from vesicles, indicated that the total content of GTP-binding proteins were equally distributed on pars convoluta, pars recta luminal and basolateral membranes. The membranes were ADP-ribosylated with [32P]NAD+ in the presence of pertussis toxin and cholera toxin. Gel electrophoresis revealed, for all preparations, the presence of cholera toxin [32P]ADP-ribosylated 42 and 45 kDa G alpha s proteins, and pertussis toxin [32P]ADP-ribosylated 41 kDa G alpha i1, 40 kDa G alpha i2 and 41 kDa G alpha i3 proteins. The 2D electrophoresis indicated that Go's were not present in luminal nor in basolateral membranes of pars convoluta or pars recta of rabbit proximal tubule.

Animals↗
PubMed1979

A new gradient former and a simplified procedure for zonal centrifugation of immune complexes.

A new gradient former for production of optimized sucrose gradients for B-XIV zonal rotors is described. Optimized gradients are gradients with an initial shelf to provide capacity and a shape that ensures isokinetic sedimentation conditions. By exhaustive computer calculations it has been found that optimized gradients can be produced by a very simple two-compartment gradient former. The design is given. Moreover, graphs for choice of gradient strength and run duration for preparative and analytical runs of samples containing soluble immune complexes are calculated. The new gradient former allows the use of a very simple procedure for zonal centrifugation.

Antigen-Antibody Complex↗
PubMed1979

On the composition of IgG anti-IgG immune complexes.

The formation of different immune complexes of IgG was followed in a system comprising human IgG as antigen and rabbit IgG directed against the Fc portion of human IgG as antibody. Soluble IgG complexes were analyzed by analytical zonal centrifugation. In antigen excess, 16S complexes predominated. 16S complexes are oligomers of IgG, mainly trimers and tetramers. In decreasing antigen excess larger and larger complexes were formed. It was, however, found consistently that oligomers were always formed in the largest amounts. The largest complexes detectable by this method consisted of about twenty IgG molecules. The solubility of different complexes in polyethylene glycol was also studied. Low concentrations of polyethylene glycol preferentially precipitate large complexes. Four and six per cent polyethylene glycol precipitated all types of IgG complexes although not completely. Polyethylene glycol was seemingly not bound directly to soluble immune complexes, but caued otherwise soluble complexes to precipitate by an indirect mechanism.

Animals↗
PubMed1979

A theoretical approach to precipitin reactions: insight from computer simulation.

The theoretical consequences of different hypotheses of the mechanism of precipitin reactions have been evaluated by means of computer simulation. It has been found that the formation of compositionally different complexes in different antigen/antibody mixtures provides a valid explanation of the zoning phenomenon, but this concept fails to explain the absence of free antigen and of antigen in soluble complexes at the point of maximum percipitation. It is found that the following hypothesis provides an improved qualitative and quantitative explanation of percipitin reactions. In the first stage of the total reaction a series of compositionally different complexes is formed. As the second stage of the total reaction two kinds of processes are proposed. Inherently insoluble complexes precipitate causing the remaining soluble complexes to participate in mutual rearrangements to re-establish a new state of equilibrium in the supernatant. The inherently insoluble complexes, moreover, create a hydrophobic phase, distinct from the supernatant and cause the remaining otherwise soluble complexes to distribute themselves between the two phases according to a partition coefficient. A mathematical apparatus to study the consequences of this hypothesis is presented, and it is demonstrated that the features of precipitin curves can be explained nearly completely this way.

Antigen-Antibody Complex↗
PubMed1979

Measurements of precipitin reactions by difference turbidimetry: a new method.

A new method for the measurement of precipitin and flocculation reactions between antibodies and antigens has been developed. The technique, called difference turbidimetry, involves the use of tandem cuvettes providing the opportunity of using separated and unmixed antigen and antibody as blank solutions for spectrophotometric readings in the ultraviolet wavelength range. By use of this technique genuine difference turbidity spectra have been recorded for the reaction between human serum albumin and rabbit-anti-human serum albumin IgG. It was found that difference turbidimetry at low wavelengths (e.g. 280 nm) allows the construction of precipitin curves with a very clearly expressed zoning phenomenon at a sensitivity which in terms of antigen and antibody concentrations is more than twice the sensitivity of conventional procedures. It is of special interest that the zone of equivalence differs when the same reaction between an antigen and its antibody is measured by difference turbidimetry, by absorbance of washed and redissolved precipitate, and by amount of precipitated antigen.

Antigen-Antibody Reactions↗
PubMed1979

Fc-mediated immune precipitation. II. Analysis of precipitating immune complexes by rate-zonal ultracentrifugation.

The formation of immune complexes was studied by analytical rate-zonal ultracentrifugation using isomolar solutions of rabbit anti-human serum albumin IgG and of the corresponding F(ab')2 fragments. The F(ab')2 fragments retained full ability to react with the antigenic determinants and to form genuine antigen-antibody complexes. Thus, the difference between the IgG and the F(ab')2 systems was supposed solely to reflect the lack of the Fc portion. The complexes formed with F(ab')2 fragments in the zone of low and moderate antigen excess were found to be distinctively mor soluble than those formed with intact IgG. The data indicated that there were two kinds of precipitating immune complexes, namely antibody-rich and antigen-rich complexes. In the antibody-excess zone and the first part of the equivalence zone the immune complexes precipitated due to their antibody richness. Antigen-rich complexes formed in the zone of low antigen excess precipitate only in the presence of antibody-rich insoluble complexes. It is believed that this type of precipitation was due to an Fc-Fc interaction. This new function of the Fc portion of IgG was designated Fc-mediated immune precipitation.

Animals↗
PubMed1977

The properties of immune complex-forming systems. A new theoretical approach.

A new mathematical model for antigen-antibody interactions has been developed. The new model is based on the assumption that the formation of complexes between a bivalent antibody and a multivalent antigen is determined thermodynamically by the concentrations and valences of antigen as well as antibody, together with one association constant which is common to all mutual interactions. Formulae have been derived for calculation of the distributions of compositionally different antigen-antibody complexes either from knowledge of equilibrium concentrations of free antigen and antibody, or from knowledge of total amounts of antigen and antibody in the system. A computer program for these calculation is described. The model is found to yield precise predictions of the formation of soluble immune complexes, as studied by zonal centrifugation. It is found through use of the model that 'complex formation' as such differs in binding characteristics from adsorption, especially for high concentrations of antigens and antibodies. 'Complex formation' implies that association constants estimated through a Sips plot method will vary with antibody concentration, and that certain curvatures of the lines in a Sips plot reflect inherent properties of complex-forming systems.

Antigen-Antibody Complex↗
PubMed1975

The effects of overloading in density-gradient centrifugation.

The effects of overloading of the sample zone in density gradient centrifugation have been studied by use of a three-component shelf-lavered sample in which the total protein concentration was increased by addition of different amounts of albumin. It is found that overloading of the gradient gives rise to particle movements which are not predictable from the Svedberg equation. The two typical effects of overloading are dislocation of the zone mass centres and changes in the zone shapes. It is found that the magnitude of the calculated sedimentation coefficients increases nearly linearly with increasing sample load. The changes in zone shapes are found to depend on the specific load and two different patterns may be distinguished. The zone of the sample component which causes the overloading is defined as primarily overloaded and the others as secondarily overloaded. In primarily overloaded zones the original Gaussian shape is lost, while in secondarily overloaded zones the Gaussian zone shape is maintained, although a zone broadening is seen. Extreme high loads are found to be able to divide single zones. As a whole these experiments show that evidence for a non-overloaded set of experimental conditions must be provided, when density gradient centrifugation is used for determination of sedimentation coefficients. For preparative gradient centrifugations the power of resolution will decrease with increasing sample load. A simple method to detect overloading in density gradient centrifugations is described.

Animals↗
PubMed1975

Properties and immunochemical reactivities of carboxy-modified human serum albumin.

Seven carboxy modified and four amino modified derivatives of human serum albumin have been prepared and studied by optical rotatory dispersion measurements, gel filtration, immunoelectrophoresis, immunodiffusion and by use of an ammonium sulphate technique. It is found that carboxy groups are of major importance for the maintenance of the structure and function of human serum albumin, and that carboxy modification has a much more profound effect than amino modification has to the same relative extent.

Ammonium Sulfate↗
PubMed1975

Computer simulation of immunochemical interactions.

A computer model for simulation of the interactions between a macromolecular antigen and its corresponding IgG has been developed. The model takes all possible immune complexes into account, and it calculates the most probable immune complex distribution patterns on the basis of basic thermodynamic principles from the valences and initial concentrations of antigen and antibody, respectively, together with an association constant assumed to be common to all mutual interactions. In antigen excess small antigen-rich complexes are predicted. At or near equivalence a rich variety of relatively small complexes is predicted, while in antibody excess complexes of the type AgAbn are found to be the most probable. By further assuming that the precipitate consists of antibody excess complexes, a precipitin curve can be calculated. The agreement between calculated results and experimentally obtained data is found to be good. It is of special interest that this theory implies that the outcome of immunochemical interactions depend equally well on the concentrations of antigen and of antibody.

Animals↗