Purification of chick nuclear thyroid-hormone-receptor protein.

A crude nuclear thyroid-hormone-receptor protein preparation from chick liver (an ammonium sulfate fractionation of high-ionic-strength-solubilized chromatin proteins) binds both triiodothyronine and thyroxine with high affinity. This crude preparation has characteristics similar to preparations from a variety of animal tissues, reported by several different laboratories, and is used for the further purification of the receptor protein. For this purification an affinity chromatography medium, 4-[N-(3,5,3'-triiodothyronine)-2-amino-3-hydroxypropoxy]-butylpropoxy -Sepharose ether, is used to take advantage of the observation that hydroxymercuribenzoic acid causes a reversible dissociation of the complex between triiodothyronine and the receptor protein. The hydroxymercuribenzoate treatment greatly increases this rate of dissociation at low temperatures compared with other methods, such as free triiodothyronine competition or an increase in ionic strength or pH. This procedure results a in purified fraction (1000-10000-fold with respect to binding triiodothyronine), which has a molecular mass of approximately 65 kDa and which retains a high degree of the original thyroid-hormone-binding activity.

Immobilization of reticulocyte elongation factor EF-2.

Conditions are described whereby the ADP-ribosylation (from NAD+) of reticulocyte elongation factor EF-2, catalyzed by diphtheria toxin, is essentially complete and whereby the reverse of this process may be carried out with recovery of 60--70% of the original EF-2 activity. Both reactions proceed well at room temperature. The reverse reaction is much slower than the ADP-ribosylation process and requires high nicotinamide concentrations. For the reverse reaction to occur at a significant rate it is necessary to lower the pH to 6.5 (from the 7.5 used for the forward reaction). NAD+ covalently linked to agarose may replace NAD+ in the diphtheria toxin reaction. The characteristics of this reaction are similar to those of the reaction employing free NAD+ except that the velocity is reduced and the concentration of NAD+ moieties greatly increased. NAD+ immobilized on agarose through the C-8 of the adenine ring is a superior substrate compared with NAD+ linked to agarose via its periodate-oxidized ribose moieties. Preliminary experiments indicate that reversal of this latter reaction with recovery of biological activity may be possible.

Reaction of N-(3-pyrene)maleimide with thiol groups of reticulocyte ribosomes.

The reaction of N-(3-pyrene)maleimide with thiol groups of rabbit reticulocyte ribosomes offers a possible fluorescent probe for studying ribosomal structure and conformation. At relatively low concentrations of N-(3-pyrene)maleimide a group of 30-40 readily reactive sulfhydryl residues is derivatized. The major ribosomal proteins containing these thiol groups are identified as S2 + S3, S5, S7, S8, S29, S31, S32, L1, L5, L6, L10 + L14, L15, L18 + L19, and L36. Ribosomal activity, as measured by the nonenzymic binding of phenylalanyl-tRNA and polyphenylalanine synthesis, is inhibited by this degree of reaction with N-(3-pyrene)maleimide. The inhibition is relieved by the prior binding of polyuridylic acid to the ribosomes while the extent of derivatization by N-(3-pyrene)-maleimide is diminished only slightly. The average relative polarization of the fluorescence of the ribosomal bound N-(3-pyrene)maleimide changes significantly with the degree of derivatization of ribosomal thiol groups or with the binding of polyuridylic acid, indicating the value of such a fluorescent thiol-derivatizing agent as a probe of ribosomal structure.