Kinetics and mechanism of dissociation of cooperatively bound T4 gene 32 protein-single-stranded nucleic acid complexes. 2. Changes in mechanism as a function of sodium chloride concentration and other solution variables

The dissociation kinetics of bacteriophage T4 coded gene 32 protein-single-stranded nucleic acid complexes have been examined as a function of monovalent salt concentration, temperature, and pH in order to investigate the details of the dissociation of cooperatively bound protein. Fluorescence stopped-flow techniques were used, and irreversible dissociation was induced by a combination of [NaCl] jumps and mixing with excess nucleic acid competitor. This made it possible to directly investigate the irreversible dissociation process over a wide range of NaCl concentrations [e.g., from 50 mM to 0.60 M for the gene 32 protein-poly(A) complex], in the absence of reassociation. Over the entire salt range, the only dissociable species observed is the singly contiguously bound gene 32 protein which dissociates from the ends of protein clusters. However, the [NaCl] dependence of the dissociation rate constant suggests that two competing pathways exist for dissociation of cooperatively bound gene 32 protein from the ends of protein clusters. At high monovalent salt concentrations, dissociation is dominated by a single-step process, with log ke/log [NaCl] = 6.5 +/- 0.5; i.e., the dissociation rate constant increases with increasing NaCl concentration due to the uptake of approximately six monovalent ions upon dissociation. This indicates that singly contiguous protein dissociates directly into solution. However, at much lower [NaCl] the data suggest that gene 32 protein, when bound at the end of a protein cluster, dissociates by first sliding off the end to form a noncooperatively bound intermediate which subsequently dissociates. A quantitative model which incorporates the sliding pathway [Berg, O. G., Winter, R. B., & von Hippel, P. H. (1981) Biochemistry 20, 6929-6948] in the dissociation mechanism fits the data reasonably well and suggests that noncooperatively bound monomers of gene 32 protein may be capable of one-dimensional translocation along single-stranded nucleic acids as suggested by independent kinetic data on the association reaction [Lohman, T. M., & Kowalczykowski, S. C. (1981) J. Mol. Biol. 152, 67-109]. It is also observed that both the absolute dissociation rate constant for T4 gene 32 protein and its salt dependence are sensitive to the average molecular weight and polydispersity of the nucleic acid sample used. This is a general phenomenon exhibited by proteins that bind to nucleic acids in a highly cooperative manner.     

Terminal degeneration in the mediodorsal cerebral cortex of the lizard Agama agama: Light and electron microscopy

The degeneration of axon terminals in the small-celled part of the mediodorsal cortex (sMDC) of the lizard Agama agama has been studied after lesions in the dorsal cortex at various survival periods. The Fink-Heimer stain was used to map and demonstrate terminal degeneration with the light and electron microscope. Electron microscopy was used to identify and describe degenerating boutons ultrastructurally. One sham-operated and three unoperated animals served as controls. Between 6 and 21 days postsurgically, degenerating terminals can be seen through 80% of the superficial plexiform layer, the zone adjacent to the cellular layer remaining free of degeneration. Swelling of dendrites in the outer part of the superficial plexiform layer and increased numbers of vacuolar invaginations, both present at short (24 hr–6 days; peak at 48–54 hr) survival periods, can be regarded as reaction to the surgical trauma. Degeneration of axon terminals takes three forms, all of the electron-dense type: gray boutons, degenerating bouton-dendritic spine complexes surrounded or engulfed by glia, and degeneration debris inside glial processes. Several forms of terminal degeneration occur concomitantly at any short (3–12 days) survival time. At longer survival times (15–21 days) only debris is present. From 6 days on, considerable numbers of degenerating structures are present, but the majority of degenerating boutons and debris are associated with reactive glia rather than with dendrites. From these observations it is concluded that in this lizard application of the combined degeneration-Golgi-EM technique would probably lead to little success. Electron microscopy of Fink-Heimer-stained sections suggests that degenerating bouton-dendritic spine complexes and degeneration debris accumulate silver particles, whereas gray boutons do not.     

Kinetics and mechanism of dissociation of cooperatively bound T4 gene 32 protein-single-stranded nucleic acid complexes. 1. Irreversible dissociation induced by sodium chloride concentration jumps

The dissociation kinetics of cooperatively bound bacteriophage T4 gene 32 protein from a variety of single-stranded homopolynucleotides has been investigated by stopped-flow techniques. Irreversible dissociation of the complexes was induced by rapidly increasing the salt concentration and monitoring the increase in tryptophan fluorescence upon dissociation of the gene 32 protein. The dependence of the apparent dissociation rate constant on initial fractional saturation of the nucleic acid lattice as well as the observation of zero-order kinetics when the lattice is initially fully saturated with protein indicates that dissociation occurs only from the ends of protein clusters and not from doubly contiguous molecules. The data for the entire time course are quantitatively fit by a kinetics model specifying irreversible dissociation of only singly contiguously bound protein [Lohman, T.M. (1983) Biopolymers 22, 1697-1713]. This model is used to extract molecular rate constants for the dissociation of isolated, singly contiguously and doubly contiguously bound protein. It is also shown that the polynucleotide specificity observed for the cooperative binding constant, K omega, and the cooperativity itself are intrinsic properties of the dissociation rate of the various complexes.