Plasma Membrane ATPase Activity following Reversible and Irreversible Freezing Injury.

Plasma membrane ATPase has been proposed as a site of functional alteration during early stages of freezing injury. To test this, plasma membrane was purified from Solanum leaflets by a single step partitioning of microsomes in a dextran-polyethylene glycol two phase system. Addition of lysolecithin in the ATPase assay produced up to 10-fold increase in ATPase activity. ATPase activity was specific for ATP with a K(m) around 0.4 millimolar. Presence of the ATPase enzyme was identified by immunoblotting with oat ATPase antibodies. Using the phase partitioning method, plasma membrane was isolated from Solanum commersonii leaflets which had four different degrees of freezing damage, namely, slight (reversible), partial (partially reversible), substantial and total (irreversible). With slight (reversible) damage the plasma membrane ATPase specific activity increased 1.5- to 2-fold and its K(m) was decreased by about 3-fold, whereas the specific activity of cytochrome c reductase and cytochrome c oxidase in the microsomes were not different from the control. However, with substantial (lethal, irreversible) damage, there was a loss of membrane protein, decrease in plasma membrane ATPase specific activity and decrease in K(m), while cytochrome c oxidase and cytochrome c reductase were unaffected. These results support the hypothesis that plasma membrane ATPase is altered by slight freeze-thaw stress.

Binding of 5 alpha-dihydroprogesterone and other progestins to female rat anterior pituitary nuclear extracts.

The specific binding of 5 alpha-dihydroprogesterone (5 alpha-DHP), progesterone and R5020 to anterior pituitary nuclear extracts was studied using ovariectomized rats treated with estradiol benzoate and progesterone. The binding equilibrium association constant for 5 alpha-dihydroprogesterone with different preparations of nuclear extract ranged from 4.0 +/- 0.54 microM-1 to 59 +/- 10 microM-1. The association constants for progesterone and R5020 were 0.39 +/- 0.81 nM-1 and 1.5 +/- 0.15 nM-1, respectively. The binding of 5 alpha-DHP was specific in that it could be competed only by R5020, progesterone and 5 alpha-DHP and not by other progesterone metabolites and other hormonal steroids tested. With [3H]-progesterone and [3H]R5020 as ligands the most efficient competitors also were R5020, progesterone and 5 alpha-DHP. Estrogen priming of ovariectomized rats consistently and significantly increased the number of binding sites for all three progestins and subsequent progesterone treatment enabled their detection at higher levels in the nuclei.

Antibodies directed against guanylic acid and guanosine are nucleolus specific probes.

Purified antibodies against guanylic acid and guanosine binding to RNA at guanosine residues were used to probe human lymphocyte preparations by indirect immunofluorescence. Neither antibody gave any banding pattern with metaphase chromosomes but both showed binding to specific sites in the interphase nuclei. Evidence presented indicates that these sites are guanosine residues on rDNA transcripts at the nucleolar organizer regions.

Antibodies to guanylic acid: fractionation and specificities.

Guanylic-acid-specific antibodies were elicited in rabbits, using as immunogen pG linked through 5'-phosphate to thyroglobulin. Specificity and affinity of antibodies to nucleotides, nucleosides, DNA, and RNA were studied by their binding to radioactive ligands and competition experiments. Guanylic-acid-specific antibodies do not bind to deoxyguanylic acid and have an average association constant of 10(7) M-1 at 4 degrees C. Binding of the antibodies to 3H-RNA is G-specific. The antibodies do not bind to 32P-ssDNA or 32P-dsDNA. The pG-specific antibodies could be separated into different fractions by affinity chromatography. These fractions, though specific to pG, differ in their cross-reactivities to nucleosides and nucleotides.

Antibodies to guanosine. Fractionation and specificities.

Bovine serum albumin conjugates of guanosine prepared by the periodate method was used as immunogen to elicit guanosine antibodies in rabbits. The specificities of the antibodies were studied by the inhibition of their binding to [3H]Gox-red, [32P]DNA and [3H]RNA by related non-radioactive compounds. A population of antibodies is specific to Gox-red with an average association constant of around 10(7) M-1 at 4 degrees C. There are a population of antibodies which bind to [32P]ssDNA and [3H]RNA specifically at guanosine residues. RNA binding antibodies were separated into two populations by affinity chromatography.