Selective affinity chromatography of DNA polymerases with associated 3' to 5' exonuclease activities

The use of 5'-AMP as a ligand for the affinity chromatography of DNA polymerases with intrinsic 3' to 5' exonuclease activities was investigated. The basis for this is that 5'-AMP would be expected to act as a ligand for the associated 3' to 5' exonuclease. The requirements for binding of Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus DNA polymerase delta, all of which have associated 3' to 5' exonuclease activities, to several commercially available 5'-AMP supports with different linkages of 5'-AMP to either agarose or cellulose were examined. The DNA polymerases which possessed 3' to 5' exonuclease activities were bound to agarose types in which the 5'-phosphoryl group and the 3'-hydroxyl group of the AMP were unsubstituted. Bound enzyme could be eluted by either an increase in ionic strength or competitive binding of nucleoside 5'-monophosphates. Magnesium was found to reinforce the binding of the enzyme to these affinity supports. DNA polymerase alpha, which does not have an associated 3' to 5' exonuclease activity, did not bind to any of these columns. These differences can be used to advantage for the purification of DNA polymerases that have associated 3' to 5' exonuclease activities, as well as a means for establishing the association of 3' to 5' exonuclease activities with DNA polymerases.     

A spectroscopic study of metal ion and ligand binding to β-lactamase II

β-Lactamase II has two metal-binding sites. The electronic spectra of Cd(II)- and Co(II)-substituted β-lactamase II have been investigated. It is suggested that a thiol ligand is involved in metal binding at the first site. The stoichiometric dissociation constants for Co(II) binding to β-lactamase II were estimated to be 0.13 and 2.66 mM (pH 6.0, 4°C, 1 M NaCl) by equilibrium dialysis. Competition between Zn(II) and Co(II) for the first metal binding site suggests a value of 0.7 μM (pH 6.0, 30°C, 1 M NaCl) for the dissociation constant o Zn(II).The electronic spectra of the Co(II) enzyme lead to the suggestion that the coordination geometries around the metal ions in the first and second sites are related to those of a distorted tetrahedron and octahedron, respectively.     

Rat and human embryo and post-natal sera contain a potent endogenous competitor of estrogen-rat alpha-fetoprotein interactions

A highly active inhibitor of the binding of estrone and estradiol-17β to rat alpha-fetoprotein is demonstrated for the first time in embryo, immature and adult rat sera as well as in fetal and adult human sera. The competitive character and the narrow specificity of this inhibition effect is shown. The major compound responsible for this activity is isolated by successive column Sephadex LH₂₀ and thin layer chromatography : it is characterized as a nonpolar, nonphenolic, dialysable and thermostable substance, unreactive towards anti-estrone and anti-estradiol-17β anti-bodies. The possible biological role of an endogenous non-estrogen ligand of rodent fetoproteins is discussed.     

Improved synthesis of 16alpha-hydroxylated androgens: intermediates of estriol formation in pregnancy.

16alpha-Hydroxyandrostenedione (16alpha-hydroxyandrost-4-ene-3,17-dione), 16alpha-hydroxytestosterone (16alpha,17beta-dihydroxyandrost-4-en-3-one) and 16alpha-hydroxydehydroepiandrosterone 3-sulfate (3beta, 16alpha-dihydroxyandrost-5-en-17-one 3-monosulfate) were synthesized by a new chemical approach with much improved yield. 16alpha-Bromoandrostendione was converted to the hydrazone of 16alpha-hydroxyandrostenedione which gave 16alpha-hydroxyandrostenedione on acid hydrolysis in total 63% yield. Oxidation of 16alpha-hydroxydehydroepiandrosterone with Jones' reagent also selectively afforded 16alpha-hydroxyandrostenedione. 16alpha-Hydroxytestosterone was observed by selective reduction of 16alpha-hydroxyandrostenedione with sodium borohydride. Reaction of 16alpha-hydroxydehydroepiandrosterone with chlorosulfonic acid in pyridine selectively gave the 3-monosulfate. The structure of the sulfate was deduced from its solvolysis to the starting material, and its acetylation and subsequent solvolysis to 16alpha-hydroxydehydroepiandrosterone 16-acetate. All procedures are suitable for large scale synthesis without the use of microorganisms.     

Enriched populations of rat retinal ganglion cells: Studies using a cell-type specific surface marker

Cells dissociated from adult and neonatal rat retinas were separated by density gradient centrifugation. Previous work had shown that rat retinal cells labelled by an immunofluorescence assay for the Thy-1 antigen were chiefly or exclusively ganglion cells, and so the proportion of Thy-1 positive cells in the density gradient fractions was used as an index of the enrichment of ganglion cells. The proportion of Thy-1 positive neonatal cells was increased from about 0.4% in the initial dissociate to about 8% in the most enriched fraction of a Percoll step gradient. Amongst adult cells the initial 0.7% Thy-1 positive cells were increased to roughly 2% in the best fraction of a metrizamide step gradient.

The presence of relatively large numbers of Thy-1 positive cells in other fractions suggested that it would be difficult to further increase the proportion of rat ganglion cells by methods based on their sedimentation properties. These results demonstrate the importance of cell-type specific markers in attempts to purify cells from the central nervous system.     

Stabilization of rat liver lysosomes by new anti-inflammatory steroids in vitro

Steroid acid esters, synthesized by modifying the 17-ketol side chain of prednisolone, were tested for their in vitro ability to stabilize heavy mitochondrial lysosomes prepared from rat liver. Membrane stabilization was determined by assessing capability of steroids to decrease extrusion of the marker enzymes (acid phosphatase, beta-glucuronidase and aryl sulfatase) from lysosomes incubated in hypo-osmotic sucrose-Tris acetate buffer. Results indicated that prednisolone (1) significantly inhibited the lysosomal release of acid phosphatase as did the new anti-inflammatory steroid, methyl 20-dihydroprednisolonate. Methyl prednisolonate exhibited weak membrane stabilization capacities and 20-dihydroprednisolonic acid, a metabolic product of methyl 20-dihydroprednisolonate, showed virtually no membrane stabilization.     

A respiration-dependent primary sodium extrusion system functioning at alkaline pH in the marine bacterium Vibrioalginolyticus

The membrane potential generated at pH 8.5 by K⁺-depleted and Na⁺-loaded $\text{Vibrio}\text{alginolyticus}$ is not collapsed by proton conductors which, instead, induce the accumulation of protons in equilibrium with the membrane potential. The generation of such a membrane potential and the accumulation of protons are specific to Na⁺-loaded cells at alkaline pH and are dependent on respiration. Extrusion of Na⁺ at pH 8.5 occurs in the presence of proton conductors unless respiration is inhibited while it is abolished by proton conductors at acidic pH. The uptake of α-aminoisobutyric acid, which is driven by the Na⁺-electrochemical gradient, is observed even in the presence of proton conductors at pH 8.5 but not at acidic pH. We conclude that a respiration-dependent primary electrogenic Na⁺ extrusion system is functioning at alkaline pH to generate the proton conductor-insensitive membrane potential and Na⁺ chemical gradient.