Faculty Research 1980 - 1989

Title

Progesterone receptor binding to DNA: studies by sedimentation velocity methods.

Document Type

Article

Publication Date

1984

Keywords

Centrifugation-Density-Gradient, Chickens, DNA: me, DNA-Single-Stranded: me, Female, Kinetics, Macromolecular-Systems, Molecular-Weight, Oviducts: an, Progesterone: me, Receptors-Progesterone: me, SUPPORT-U-S-GOVT-P-H-S

JAX Source

J-Steroid-Biochem. 1984 Jan; 20(1):89-94.

Grant

HD07857

Abstract

Chicken oviduct progesterone receptor subunit A (Mr = 79000) was partially purified as a hormone-receptor complex labeled with [3H]progesterone. These complexes adsorb to DNA-cellulose; sucrose gradient ultracentrifugation showed a sedimentation coefficient of 3.6 S. Receptors were mixed with [32P]DNA fragments of various types derived from bacterial plasmid pBR322. DNA structures tested included linear double-stranded and single-stranded molecules as well as supercoiled intact plasmids. [3H]Receptors were mixed in various molar ratios with [32P]DNA (nominal sed. coeff. = 18 S) and the mixtures were analyzed by sedimentation velocity ultracentrifugation performed in 5-20% sucrose gradients. In the presence of excess DNA, all of the [3H]receptor sedimented with the DNA fraction. Titrations revealed an apparent Kdiss between 1 and 5 nM. Distribution of receptor counts over the slower-sedimenting edge of the DNA peaks allowed estimation of the half-life of these complexes, which was in excess of 35 min at 0 degree C. Double-label counting of [3H]receptor-[32P]DNA complexes revealed an increase in the sedimentation coefficient of linear double-stranded DNA as increasing receptor input was used. Analysis of this S-value shift showed evidence for cooperative receptor binding isotherms, causing dramatic compaction (or other topological constraints) on the DNA. A speculative schematic model of receptor A-DNA interaction is presented, consistent with the experimental results.

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