Dixie J. Goss

Kinetic studies of the rates and mechanism of assembly of the protein synthesis initiation complex

Rate constants for a number of the assembly reactions involved in forming Escherichia coli ribosome initiation complexes have been measured. These reactions were monitored in a stopped-flow device in which Rayleigh scattering and fluorescence anisotropy were followed as a function of time. Fluorescence was induced by laser excitation modulated at 50 kHz. Aminoacyl-tRNA, initiation factor 3 (IF3), and 70S ribosomes were labeled with fluorescent probes. The light-scattering and fluorescence data show that the antiassociation model for IF3 function cannot be correct. IF3 can be considered to act as an effector in an allosteric model for ribosome function. Fluorescence anisotropy stopped-flow experiments provided rate constants for the binding of IF3 to both 30S subunits and to the intact 70S ribosome. Aminoacyl-tRNAs and nucleotide triplets appear to bind rapidly to 70S ribosomes and then a slow first-order conformational change occurs.

Rapid micro-isolation of mammalian oxymyoglobin for biophysical studies

A rapid, single-step procedure is described for the isolation of mammalian oxymyoglobin for physical studies. The procedure employs a high-capacity, reusable, Hg-resin through which the myoglobin is filtered. The mammalian myoglobins for which sequences have been reported lack reactive cysteine. The column binds hemoglobin and other sulfhydryl-containing proteins, and retards other proteins. The procedure requires less than twenty minutes for the isolation of oxymyoglobin from homogenized muscle tissue. Virtually no metmyoglobin is formed. The yields are 2-3 times greater than by other methods. The procedure can easily be scaled down for isolation of myoglobin from a few milligrams of muscle. Identical rate constants for oxygen association, dissociation, and CO association were measured by laser photolysis for beef heart myoglobin prepared by this procedure and that of Yamazaki et al. (Yamazaki, I., Yokota, K. and Shikama, K. (1964) J. Biol. Chem. 239, 4151). Gel electrophoresis indicates that the preparation is at least 95% pure.

The kinetics of ligand binding for diverse mammalian myoglobins and the effects of substitutions outside the heme cavity.

1. We report rate constants for oxygen dissociation and for oxygen, carbon monoxide, azide, and cyanide binding to whale, horse, dog, beef and human myoglobins. 2. For azide binding, rate constants can vary by at least a factor of two for substitutions outside the heme cavity. Azide binding may be affected by a substitution at residue 66 in the E helix, a site suggested by Case & Karplus (1979) J. molec. Biol. 132, 343-368, to be on a reactive path to the heme. 3. The oxygen and CO data show that substitutions outside the heme cavity can affect rate constants by at least a factor of 1.5. 4. The oxygen equilibrium constant was correlated with the metabolite rate of the corresponding species, in accord with the Wyman (1966) J. biol. Chem. 241, 115-121, model for facilitated diffusion of oxygen.