[Röentgen diagnosis of the esophagus, stomach and intestinal tract in adults. New methods and contrast media].
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Adult↗
Biomedical subjects
G Fleischmann
Publications and source records attributed to G Fleischmann.
Photoaffinity labeling of steroid binding proteins with unmodified ligands.
Photoactivation of the alpha,beta-unsaturated ketones of natural and synthetic steroid molecules by light of lambda greater than or equal to 330 nm allows their covalent attachment to steroid-binding proteins. The general validity of this method is demonstrated with two steroid hormone receptors and the steroid-binding protein uteroglobin. Progesterone can be covalently attached to the partially purified progesterone receptor and to uteroglobin, and comigrates with the binding proteins upon electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Similarly the synthetic glucocorticoid triamcinolone acetonide can be covalently bound to the partially purified glucocorticoid of rat liver. This method allows the identification of steroid hormone receptors after electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Labeling with radioactive steroids is specific since it can be prevented by the addition of an excess of non-radioactive ligand. Digestion of the labeled binding proteins with trypsin or chymotrypsin yields a defined pattern of radioactive peptides, demonstrating that covalent attachment takes place at specific binding sites.
Affinity Labels↗
Activation of the progesterone receptor of rabbit uterus.
The influence of several parameters on the kinetics of activation of the progesterone receptor in the cytosol of rabbit uterus is described. The estimation of the proportion of activated receptor is based on the differential affinity of the activated and non-activated forms of the receptor for phosphocellulose. Under appropriate conditions binding to phosphocellulose can be used as a test of activation and gives results similar to those obtained with DNA--cellulose, or isolated cell nuclei. The kinetics of receptor activation is temperature-dependent and compatible with a first-order reaction at all temperatures tested. The thermodynamic activation energy of this reaction is 67.8 kcal mol-1. The progesterone receptor can be activated to various extents by increased ionic strength or by dilution of the cytosol with buffers of low ionic strength, and in all cases the activation follows apparent first order kinetics. At a concentration of 0.4 M NaCl, 70--80% of the receptor can be converted into the activated form. The activated and non-activated forms of the receptor appear to be in equilibrium. Salt-activated and heat-activated receptor can be transformed to a non-activated form by decreasing either the salt concentration, or the temperature of incubation. The rate of dissociation of the steroid from the activated form of the receptor is indistinguishable from that observed with the non-activated form, but the activated receptor is more thermolabile. Upon centrifugation on sucrose gradients there are no major differences in the sedimentation behaviour of the two forms of the receptor.
Animals↗
Effect of phospholipases and lysophosphatides on partially purified steroid hormone receptors.
Treatment with phospholipase A2 of crude or partially purified preparations of the glucocorticoid receptor of rat liver results in an inactivation of the receptor, which cannot be attributed to contaminating proteases. Similar enzymatic treatment of the progesterone receptor of rabbit uterus does not affect its steroid-binding activity. At various stages during purification the preparations of glucocorticoid receptor contain 10 to 50-fold higher concentrations of lipid phosphate than the corresponding preparations of progesterone receptor, suggesting that the effect of phospholipase A2 on the hepatic receptor could be mediated by lysophosphatides produced during hydrolysis of endogeneous phospholipids. In fact, mixing experiments show that in the presence of the glucocorticoid receptor, phospholipase A2 also inactivated the progesterone receptor. Both partially purified receptors are inactivated by similar concentrations of added lysophosphatides but are not affected by incubation with phospholipase C, which does not produce ionic detergents. In addition, the effects of phospholipase A2 and of added lysophosphatides can be overcome by an excess of bovine serum albumin, indicating that free lysophosphatides are involved in receptor inactivation, possibly due to their strong detergent properties.
Animals↗