FT-IR study of pyridoxamine 5' phosphate

Aqueous solutions of pyridoxamine 5' phosphate (PMP) at several pH conditions have been studied using FT-IR spectroscopy using the attenuated total reflection (ATR) technique. In spite of the strong intense OH stretching and bending bands of water, most of the vibrational structure of solute can be observed from 900 to 1500 cm(-1). With increasing pH, very intense changes in the spectra have been observed due to concentration changes of the hydrogen bonded species. Spectra of the different ionic species have been calculated from the mathematical fitting of experimental absorption spectra as a function of pH. Spectra are characterized by the presence of broad band-like structures in the 2400-3500 cm(-1) region, with extended continua that indicate very large proton polarizability of hydrogen bonds. Contributions of the phosphate group to the total absorption have been analyzed by comparison with pyridoxamine spectra.     

Analogs of adenosine 3',5'-cyclic phosphate. II. Synthesis and enzymatic activity of derivatives of 1,N6-ethenoadenosine 3',5'-cyclic phosphate

The reactions of chloroacetaldehyde with adenosine 3′,5′-cyclic phosphate, and with several analogs modified at C₈ of the purine ring or C₅, of the sugar, lead to the corresponding 1,N⁶-etheno derivatives^d. Similar reactions using other 2-bromoaldehydes or phenacyl bromide give 1,N⁶-ethenonucleotides substituted at the α- or β-positions of the etheno bridge respectively. The ability of these compounds to activate the protein kinases from rabbit muscle and calf brain has been evaluated over a wide range of concentrations. While no derivative proved to be more active than adenosine 3′,5′-cyclic phosphate itself using the enzyme from rabbit muscle, a wide spectrum of activities was found using that from calf brain.     

Cyclic cuanosine 3'5' phosphate, guanylate cyclase and cyclic guanosine phosphodiesterase in the eye lens

While cGMP levels of rat lenses are in the range of those of other tissues, in calf lenses their values are much lower. Guanylate cyclase activities are rather high in proliferating epithelial cells of the lens and decrease strongly with cell differentiation and aging. cGMP phosphodiesterase activities are also reduced with aging in lens epithelial cells. A slight increase seems present in differentiated cortical fibers.     

The 3' 5' exonucleases

Over the past few years, several new 3' 5' exonucleases have been identified. In vitro studies of these enzymes have uncovered much about their potential functions in vivo, and certain organisms with a defect in 3' 5' exonucleases have an increased susceptibility to cancer, especially under conditions of stress. Here, we look at not only the newly discovered enzymes, but also at the roles of other 3' 5' exonucleases in the quality control of DNA synthesis, where they act as proofreading exonucleases for DNA polymerases during DNA replication, repair and recombination.     

Parallel helical domains in DNA branched junctions containing 5',5' and 3',3' linkages

The Holliday junction is a central intermediate in genetic recombination. It contains four strands of DNA that are paired into four double helical arms that flank a branch point. In the presence of Mg2+, the four arms are known to stack in pairs forming two helical domains whose orientations are antiparallel but twisted by about 60 degrees. The basis for the antiparallel orientation of the domains could be either junction structure or the effect of electrostatic repulsion between domains. To discriminate between these two possibilities, we have constructed and characterized an analogue, called a bowtie junction, in which one strand contains a 3',3' linkage at the branch point, the strand opposite it contains a 5',5' linkage, and the other two strands contain conventional 3',5' linkages. Electrostatic effects are expected to lead to an antiparallel structure in this system. We have characterized the molecule in comparison with a conventional immobile branched junction by Ferguson analysis and by observing its thermal transition profile; the two molecules behave virtually identically in these assays. Hydroxyl radical autofootprinting has been used to establish that the unusual linkages occur at the branch point and that the arms stack to form the same domains as the conventional junction. Cooper-Hagerman gel mobility analyses have been used to determine the relative orientations of the helical domains. Remarkably, we find them to be closer to parallel than to antiparallel, suggesting that the preferred structure of the branch point dominates over electrostatic repulsion. We have controlled for the number of available bonds in the branch point, for gel concentration, and for the role of divalent cations. This finding suggests that control of branch point structure alone can lead to parallel domains, which are generally consistent with recombination models derived from genetic data.     

Investigation of the adenosine 3', 5'-cyclic phosphate binding site of pig brain histone kinase with the aid of some analogues of adenosine 3', 5'-cyclic phosphate.

2'-O-Chloroacetyl cyclic AMP, 2'-O-acrylyl cyclic AMP and N-6, 2'-O-diacrylyl cyclic AMP were synthesized by the reaction of cyclic AMP with chloroacetic and acrylic anhydrides, respectively. Selective O-deacylation of N-6, 2'-O-diacrylyl cyclic AMP yielded N-6 -monoacrylyl cyclic AMP. In the reaction of gamma-mercaptobutyric acid with 8-bromo cyclic AMP, 8-(gamma-carboxypropylthio) cyclic AMP was obtained. The compounds synthesized and other cyclic AMP analogues (8-bromo cyclic AMP and adenosine 3', 5'-cyclic sulphate) were tested for ability to interact with the highly purified pig brain histone kinase. All compounds under study were found to be activators of the enzyme. The highest activating potency was manifested by 8-bromo cyclic AMP and 8-(gamma-carboxypropylthio) cyclic AMP; adenosine 3', 5'-cyclic sulphate was the least potent in this respect. All compounds were shown to inhibit binding of cyclic [-3-H]AMP to histone kinase. The inhibition was competitive with respect to cyclic AMP in all cases. All compounds, except for 2'-O-chloroacetyl cyclic AMP may indicate the formation of a covalent bond between this analogue and the enzyme. These findings suggest that an active site of the regulatory subunit of the histone kinase contains at least three specific areas responsible for cyclic AMP binding.     

Activity of tubercidin-, toyocomycin-, and sangivamycin-3',5'-cyclic phosphates and related compounds with some enzymes of adenosine-3',5'-cyclic phosphate metabolism

Rat liver nuclei contain at least two DNA polymerases that can be separated by extracting the nuclei with 5% Brij 58. The loosely-bound activity increases little or not at all after partial hepatectomy and is insensitive to cytosine arabinoside triphosphate (araCTP). The tightly-bound enzyme activity rises along with DNA replication and is inhibited by araCTP.