Sephadex LH-20 Gel Chromatography Research Articles

Regional distribution and characterization of kinin in the CNS of the rat.

The distribution of kinin in the CNS of the rat, which was extracted with n-butanol from an acidified homogenate, was determined using a bradykinin (BK) radioimmunoassay system. The immunoreactive kinin was widely distributed throughout the brain. The highest content was found in the pituitary gland (4,135 fmol BK Eq/g), followed by the medulla oblongata (912 fmol/g), cerebellum (549 fmol/g), and cortex (512 fmol/g). The kinin in the posterior pituitary was concentrated 4.5 times as much as in the anterior lobe. Serial dilution of brain extracts produced binding curves parallel to the standard radioimmunoassay curve. The purified brain kinin comigrated with authentic BK during CM-cellulose chromatography and Sephadex LH-20 gel chromatography. Its molecular weight was estimated to be 1,127 +/- 45 by gel filtration, which coincides well with that of BK. Chymotrypsin degraded the extracted kinin and authentic BK, but trypsin did not. These data demonstrate that a peptide indistinguishable from BK exists in the rat brain. Furthermore, pituitary kinin was separated into BK (87%), Lys-BK (10%), and Met-Lys-BK (3%), using reverse phase HPLC.

In vitro study of frog ( Rana ridibunda pallas) interrenal function by use of a simplified perifusion system : V. Influence of adrenocorticotropin upon progesterone production

The dynamics of progesterone production by perifused frog adrenal explants has been investigated under pituitary stimulation. Specific progesterone antibodies have been raised in rabbits and a radioimmunoassay has been developed. The sensitivity of the assay and the low cross-reactivity of the antiserum with the major corticosteroids produced by the interrenal tissue, mainly corticosterone (0.7%) and aldosterone (0.002%), made it possible to measure progesterone concentrations in 150-μl samples of effluent perifusate, without prior extraction of purification. The validity of the method was confirmed on Sephadex LH-20 gel chromatography. A direct effect of temperature variations on progesterone output was demonstrated in the temperature range 5–30°, which strongly suggested that external temperature controls progesterone synthesis by the interrenal gland in vivo. In the presence of distal lobe homogenates a linear log-dose increase in progesterone output was observed. The use of ACTH 1–24 as a reference standard made it possible to compare the biological activity of crude distal lobe homogenates from frog pituitary and synthetic ACTH. The effect of neurointermediate lobe extracts on progesterone production was also studied. At a concentration as low as 0.006 neurointermediate lobe eq/ml, a 2.1-fold increase in progesterone output was observed suggesting a very high concentration of ACTH-like material in frog pars intermedia. As far as is known the present data provide the first unequivocal evidence that, in frog, intact interrenal cells produce significant amounts of progesterone in vitro. Our results demonstrate also that synthetic ACTH and pituitary extracts stimulate progesterone output and that the dynamics of the steroidogenic response to various stimulants was virtually the same with progesterone, corticosterone, or aldosterone.

Analytical and biological analyses of test materials from the synthetic fuel technologies III. Use of sephadex LH-20 gel chromatography technique for the bioassay of crude synthetic fuels

To determine the health effects associated with newly emerging energy technologies, we have subjected a group of synthetic fuels to mutagenicity evaluation, using the Ames Salmonella assay. Coupling of chemical fractionation to the mutagenicity assays was necessary. Fractions obtained by use of Sephadex LH-20 gel chromatography on crude-coal-derived oils and shale oil were tested for mutagenicity with strain TA98 (with Aroclor S9 mix). Mutagenicity results obtained with synthetic fuels were compared with those from a mixture of naturalpetroleum crude oils. Merits of the Sephadex LH-20 separation technique and precautions in interpreting experimental results are discussed.