The isolation and characterization of specific 3-methylcholanthrene-binding proteins from rat liver cytosol

The major proteins to which 3-methylcholanthrene specifically binds have been purified over 480-fold with a 45% yield compared to a rat liver 100,000g supernate. The procedure involved a batch ion-exchange technique together with hydrophobic gel filtration and chromatofocusing chromatography. The multiple, specific 3-methylcholanthrene-binding proteins obtained from this protocol had apparent isoelectric points of pH 6.3, 6.0, 5.7, and 5.5 on elution from a chromatofocusing column. They all shared a common sedimentation coefficient as determined by sucrose gradient analysis of 4.4 S. Gel filtration on Sephadex G-75 gave a common Stokes radius of 27 A. An analysis of these chromatofocusing peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed those which eluted at pH 6.3 and 6.0 to contain two major protein bands of Mr 32,000 and 34,000, together with several contaminating proteins. In contrast, the peaks from chromatofocusing which eluted at pH 5.7 and 5.5 contained three major proteins of Mr 40,000, 25,000, and 14,000. The specific binding capability of these chromatofocusing peaks was found to be unstable to temperatures of -30 degrees C and below. Competition studies showed that these proteins were not steroid receptors, and that only polycyclic aromatic hydrocarbons which could induce cytochrome P-450c were able to displace 3-methylcholanthrene from the binding site. A marked preference was noted for polycyclic aromatic hydrocarbons with four to five benzene rings arranged in a nonlinear fashion, suggesting the stereochemical requirements of the protein binding site. The stability of the noncovalent interaction between the proteins and 3-methylcholanthrene was in the range of pH 7 to 9.     

Conservation of Glycerolated Cultures of Ochromonas danica and O. malhamensis at −10 C

SYNOPSIS. Ochromonas danica in a complex natural growth medium dies at 6–10 C in 4 days; O. malhamensis in ∼2 days. O. danica grown in the medium supplemented with 4.0% glycerol survived at −10±2 C for 35 days, and with 8% glycerol 29 days. O. malhamensis lasted only to 5 days in these media supplemented with 4% glycerol. Ethylene glycol and dimethylsulfoxide were too toxic to be effective. Difficulties in freeze-preservation of certain other phagocytic cells, notably blood granulocytes having comparatively simple flexuous outer membranes, add interest to use of O. danica and O. malhamensis as test organisms for preservation methods, especially in the convenient, inexpensive -10 to -20 C range. Biphasic media with an overlay of distilled water serve for conservation at room temperature. Problems of mutational erosion of these photosynthetic phagotrophs are discussed.