Eigil Bojesen

Specificity and thermodynamic properties of the corticosteroid binding to a receptor of rat thymocytes in vitro

Abstract Thymocytes isolated from recently adrenalectomized rats and incubated in a medium with low concentrations of [1,2-3H2]corticosterone (0.01 to 10 nM) were found to accumulate the hormone at 4, 17 and 37°. The rate of uptake and washout were greatly temperature-dependent and the degree of accumulation increased as the temperature decreased. On the basis of saturation kinetics, a receptor mechanism has been postulated, and the number of receptor sites per cell has been calculated as approximately 2400 with a dissociation constant of the corticosterone receptor complex of 1.25 nM at 4°, 4.5 nM at 17° nM at 37°. A thermodynamic analysis of the data supports the assumption of a single receptor site. The hypthetical receptor does not undergo qualitative changes in the temperature interval studied (4 to 37°). The temperature effects of kinetics and states of equilibrium can consistently be understood on the basis thermodynamic functions of a single process. The specificity of the receptor was investigated by the addition of other steroids to the incubation medium and determination of the displacement of [1,2-3H2]-corticosterone from the receptor sites. Among the series investigated only steroids with a 3-keto-4-ene group and an α-ketol side-chain were powerful competitors. The 11β-hydroxy group of corticosterone was also found to be of importance since 11-dehydrocorticosterone was only a weak competitor. In general, however, no parallelism could be observed between the affinities and the hormonal activities in vitro of different steroids. This observation suggests therefore that the steroid receptor complex itself is involved in the next step of events, eventually causing the known hormonal effects.

Evaluation of the chemical parameters of four systems of radioimmunological assay

An iterative indirect model fitting (IIMF) procedure has been designed to evaluate antibody binding capacities and ligand affinities of fairly simple RIA systems. The information is provided by one or more series of standard equilibrium assays, the mass of tracer ligand, the residual fraction of free ligand which contaminates measured bound fractions, and the chemical purity of the tracer. The procedure uses theoretical models which describe systems with one or two antibodies with identical or different affinities towards the hot and the cold ligands. The tracer may be the purified radioactive ligand or a mixture of the hot and the cold ligands with the cold species as the major component. The theoretical models corresponding to two antibody systems are simplified and only strictly valid within limited ranges of ligand concentrations. The chemical parameters of both antibodies may in suitable cases be estimated by two runs of assays with different doses of antiserum and tracer. The IIMF procedure involves the computation of the regression line of a dose function for which the dependent variable is the model derived bound fraction over the measured bound fraction multiplied by the dose. Two tallies are combined of which the statistics of means of replicates being on the regression line has a higher priority than the minimal deviation of the slope of the regression line from 1. The procedure is applied to three RIA systems using carbon adsorption of the free ligand: prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha) and angiotensin 1 (Ang. 1), and to one using antibody precipitation-arginine vasopressin (AVP). The consistency of the results was tested by repeated runs of standards or by varying the concentrations of the reactants and the temperature.

Prostaglandine E1 high affinity binding sites of rat thymocytes. Specificity and blockade by non-steroidal antiinflammatory drugs and localization in a plasma membrane enriched fraction

Great specificity is demonstrated for the prostaglandin E1 high affinity binding sites of rat thymocytes. Whereas prostaglandin E2 has the same affinity as prostaglandin E1, 13 other prostaglandin derivatives and antagonists are bound with 2-1000 times smaller affinities. 50% inhibition of the high affinity binding of prostaglandin E1 to rat thymocytes is demonstrated for three non-steroidal antiinflammatory drugs, indomethacin (3.6. 10(-5) M), salicylic acid (2.9. 10(-3) M) and acetylsalicylic acid (2.10(-2) M). The low affinity binding of prostaglandin E1 is enhanced by the same concentration of indomethacin, however, to a lesser degree and more variable than the inhibition of the high affinity binding of prostaglandin E1. Like intact cells a 50-fold purified plasma membrane fraction, isolated from a homogenate of rat thymocytes, shows reversible high affinity binding of prostaglandin E1 as well as irreversible binding of unidentified tritiated compounds. The binding data are compatible with a localization in the plasma membrane of high affinity sites for reversible binding with a considerably higher dissociation constant than that found for whole cells. Their identity remains to be demonstrated.

An isotope derivative method for the analysis of prostaglandins E1 and E2, in serum using 2-amino-[35S]thiazole as the reagent

Abstract An isotope derivative method for analysis of the prostaglandins E 1 and E 2 is described. The method is based upon the addition of 3 H-labelled internal standard and the formation of 2- [ 35 S]thiazolyl-N-prostaglandin amide , by means of a mixed carbonic anhydride technique. The derivatives are obtained in greater than 50% yield and purified in a four-step procedure using thin-layer chromatography and two secondary transformations of the derivatives as the main tools. The average yields of the 3 H-labelled standards are 6.5 and 5.3% for prostaglandins E 1 and E 2 , respectively. The sensitivity of the method is about 2 ·10 -13 M and is a little better for prostaglandin E 1 than for E 2 . Although systematic errors have been removed by the technique described, two sources of random errors remain, counting errors and errors due to isotope fractionation on thin-layer chromatography. The method has been applied to rat sera from mixed arterial and venous blood. Two pools of such sera, one from normally fed and another from fasting rats, had indistinguishable prostaglandin E 1 levels (90 pg/ml), whereas prostaglandin E 2 levels of the two pools were markedly different, viz . 265 and 430 pg/ml, respectively.

In vitro and in vivo synthesis of long-chain fatty acids from [1-14C]acetate in the renal papillae of rats

1. The relationship between the rate of [1-14C] acetate incorporation into the fatty acids of renal papillary lipids and the acetate concentration in the medium has been measured. 2. [1-14C] acetate was incorporated mainly into fatty acids of phospholipids and triacylglycerols. Only a few per cent of the radioactivity was found in the free fatty acid fraction. 3. The major part of the [1-14C] acetate was found to be incorporated by a chain elongation of prevalent fatty acids. The major component of the poly-unsaturated fatty acids in triacylglycerols and the major product of fatty acid synthesis from [1-14C] acetate in vitro was demonstrated by mass spectrometry to be docosa-7,10,13,16-tetraenoic acid. 4. The radioactivity of docosa-7,10,13,16-tetraenoic acid accounted for 40% of total radioactivity in triacylglycerol fatty acids (lipid droplet fraction) and 20% of total radioactivity in membrane phospholipid fatty acids.

Sterol biosynthesis in vitro of rat renal inner medulla

Abstract In vitro studies of lipid production of rat renal papillae with [2-14C]acetate or [2-14C]mevalonate have shown that sterologenesis is an important part of the total lipogenesis in this tissue. However, only a very small amount of labelled cholesterol could be detected in the sterol fraction. The lipid extract of tissue slices was directly subjected to thin-layer chromatography or reversed-phase column chromatography. The purified sterols were analyzed by radio gas-liquid chromatography on three different stationary phases, argentation thin-layer chromatography and digitonin thin-layer chromatography. By these techniques it was demonstrated that nearly all the sterol radioactivity could be accounted for by 4,4,14α-trimethyl-5α-cholesta-8,24-dien-3β-ol (lanosterol) and three sterols, which were slightly more hydrophilic than cholesterol. Two of these sterols had the same chromatographic mobilities as cholesta-5,24-dien-3β-ol (desmosterol) and 5α-cholest-7-en-3β-ol (lathosterol), respectively. The third compound, which accounted for the largest fraction of the radioactivity in the sterol fraction, could be tentatively identified as 5α-cholesta-7,24-dien-3β-ol.

Exchange efflux of [3H]palmitate from human red cell ghosts to bovine serum albumin in buffer. Effects of medium volume and concentration of bovine serum albumin

[3H]Palmitate, PA, exchange efflux kinetics is recorded from human erythrocyte ghosts to buffer with bovine serum albumin, BSA, at 0 degrees C. The effects have been investigated of three medium/ghost volume ratios: 36, 80 and 500, of six BSA concentrations, [BSA]: 0.01, 0.02, 0.05, 0.2, 1 and 2% (1.5, 3.0, 7.5, 30, 150 and 300 microM) and of various v, molar ratios of palmitate to BSA, between 0.15 and 0.94. Data are analyzed in terms of a virtually closed three-compartment model. In theory, the tracer efflux is biexponential and the rate coefficients differ at least 20 fold [1]. The efflux rate at 2% BSA is monoexponential beyond our resolution time of about 1 s, but nearly biexponential at or below 0.2% BSA with a well-defined smallest-rate coefficient beta. beta depends strongly on [BSA] but is remarkably v independent. The medium/ghost volume ratio has no effect on beta when [BSA] > or = 0.2%, although beta measured at 2% BSA is almost 2-fold higher than at 0.2%. This suggests the presence of an unstirred layer, USL. According to our model, the observations are understood quantitatively on basis of our previously published dissociation rate constants of the PA-BSA complex, as well as PA equilibrium bindings to ghost membranes (Bojesen, I.N. and Bojesen, E. (1991) Biochim. Biophys. Acta 1069, 297-307). Essentially, beta is theoretically a function of two terms, one comprising the membrane transport parameters and the other the medium-dependent variables. Most important is the clearance with respect to monomer concentration adjacent to the membrane. The clearance is calculated on basis of quasi-stationary diffusion in USL. The data are compatible with a planar USL of 6 microns depth and with the same area as a ghost but not with a spherical USL.

Reversible Inhibition of LCAT by Penicillins. Kinetic and Mechanistic Investigations

A simple and rapid method is described for labelling serum lipoproteins with 3H cholesterol. According to the tracer data as well as cholesterol analyses, the velocity of ester formation in rat sera and beta-lipoprotein-depleted human sera decreased during incubations as a first order reaction. This suggests a level of chemical activity for cholesterol far below the Km of the enzyme. The reversible inhibition by three different penicillins showed a dependency of the inhibitor concentration which supports this notion. 50 per cent inhibition of the rat enzyme was obtained with 0.1 mM dicloxacillin, 0.3 mM benzylpenicillin, and 1.4 mM methicillin (water phase concentrations). The human enzyme was about 10-fold less sensitive. The mechanism of penicillin inhibition, investigated by stripping-recombination experiments with the 20-fold purified rat enzyme chromatographed on penicillin-loaded Sephadex G-25 columns, was found to be a dissociation of the enzyme into two components, an inactive apoenzyme and a coenzyme with a molecular weight below 1000.