Sephacryl S-200 Gel Filtration Chromatography Research Articles

Immunologic release of neutrophil chemotactic activity from human lung tissue

The release of heat-stable neutrophil chemotactic activity (NCA) has been detected after challenge of isolated human lung tissue with anti-IgE. The major NCA released (NCA L) had similar physicochemical properties to the NCA detected in the circulation of asthmatic subjects after bronchial challenge with specific antigen (NCA AG). NCA L and NCA AG (1) had molecular weights of approximately 600,000 daltons as estimated by Sephacryl S-300 gel-filtration chromatography; (2) both eluted from DEAE-Sephacel (pH 7.8) between 0.1 and 0.2 molar NaCI; (3) had isoelectric points of between 6.5 and 6.8 as determined by chromatofocusing on Polybuffer Exchanger 94. In contrast to NCA AG, lung-derived neutrophil chemotactic activity appeared to be more heterogeneous after gel-filtration and anion-exchange chromatography. The release of NCA was complete by 15 min and there was no evidence of further release up to 12 hr. These observations indicate that high-molecular-weight NCA released from human lung tissue has similar properties to NCA AG and would support the view that NCA AG originates from lung tissue after antigen bronchial challenge in asthmatic subjects.

Unoccupied and in vitro and in vivo occupied 1,25-dihydroxyvitamin D3 intestinal receptors. Multiple biochemical forms and evidence for transformation.

The data presented herein indicate that the chick intestinal 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptor which is localized in the chromatin fraction of a low salt homogenate (Walters, M. R., Hunziker, W., and Norman, A. W. (1980) J. Biol. Chem. 255, 6799-6805) can exist in three distinct biochemical forms. The three 1,25(OH)2D3 receptor forms depended on the absence or presence of ligand and additionally whether the ligand was acquired in vitro (4 degrees C incubation for 3-4 h) or in vivo (13 nmol of 1,25(OH)2D3 administered intramuscularly 2 h prior to sacrifice). The receptor forms were distinguished by their relative KCl extractabilities from the target tissue chromatin preparation and from a reconstituted nontarget tissue (liver) chromatin preparation, as well as their relative elution from DNA-cellulose columns when applied as a mixture. In all cases the rank order "affinity" of the receptor for chromatin or DNA was: unoccupied 1,25(OH)2D3 receptors less than in vivo occupied receptors less than in vitro occupied receptors. These changes in DNA-binding "affinity" occurred without a major change in overall surface charge of the receptor molecules as evaluated by co-elution of all three receptor forms from DEAE-Sepharose columns. Similarly, these changes in DNA-binding characteristics were not accompanied by changes in the apparent molecular weights of these receptor species (91,900 +/- 3300; 99,700 +/- 9400; 93,100 +/- 5600, respectively) as assessed by Sephacryl S-200 gel filtration chromatography in the presence of the protease inhibitor phenylmethylsulfonyl fluoride, included throughout these experiments to protect from proteolytic damage. These results represent the first demonstration of biochemical heterogeneity in the 1,25(OH)2D3 receptor system and suggest the existence of a two-step transformation process for the 1,25(OH)2D3 receptor.