Positive Cooperative Interaction Research Articles

Purification and characterization of a Ca 2+- or Mg 2+-stimulated ATPase from plasma membrane enriched fractions of Dictyostelium discoideum

Evidence is presented for the presence of both diethylstilbestrol (DES)-sensitive and DES-insensitive Mg 2+-ATPase activities in plasma membrane enriched fractions of Dictyostelium discoideum. When removed from the membrane, the DES-sensitive activity is markedly less stable than the DES-insensitive activity, and the two activities display a number of quite distinct properties. The DES-sensitive enzyme has a decided preference for Mg 2+ over Ca 2+, displays saturation kinetics in response to ATP as substrate ( K m = 0.2 mM) and has a narrow pH optimum range. In contrast, the DES-insensitive activity is stimulated equally by Mg 2+ or Ca 2+, is not saturable by ATP within the mM concentration range and has a much broader pH optimum. The DES-insensitive activity has been purified extensively. The purified enzyme is inhibited by vanadate and fluoride, but is insensitive to N,N′-dicyclohexylcarbodiimide (DCCD), N-ethyl-maleimide and thimerosal. In the absence of divalent cations, the enzyme displays a sigmoidal activity curve in response to substrate concentration, which is abolished by addition of either Mg 2+ or Ca 2+, suggesting a binding site for a divalent cation and a positive cooperative interaction. The enzyme is capable of hydrolyzing other nucleotide triphosphates and ADP, but is without activity on AMP, p-nitrophenyl phosphate and pyrophosphate. The enzyme has an apparent molecular weight of approximately 64 000.

Cytoplasmic pH determines K+ conductance in fused renal epithelial cells.

The mineralocorticoid hormone aldosterone maintains acid-base balance and K+ homeostasis by regulating H+ and K+ secretory mechanisms in kidney epithelial cells. We have shown recently in the amphibian distal nephron that aldosterone activates a Na+/H+ exchange system in the luminal cell membrane, thus leading to transepithelial H+ secretion and cytoplasmic alkalinization. Since H+ secretory fluxes were paralleled by K+ secretion, it was postulated that the hormone-induced increase of intracellular pH activates the luminally located K+ channels. In "giant" cells fused from individual cells of the distal nephron, we measured simultaneously cytoplasmic pH and cell membrane K+ conductance during acidification of the cell cytoplasm. The experiments show that cell membrane K+ conductance is half-maximal at an intracellular pH of 7.42 and that a positive cooperative interaction exists between K+-channel proteins and H+ (Hill coefficient = 6.5). Moreover, the cellular K+ conductance is most sensitive to cytoplasmic pH in the range modified by aldosterone. This supports the hypothesis that intracellular H+ activity, regulated by the Na+/H+ exchanger, serves as the signal to couple aldosterone-induced K+ secretory flux to H+ secretion in renal tubules.

Mechanism of the estrogen receptor interaction with 4-hydroxytamoxifen.

The binding mechanism of the estrogen receptor with 4-[3H]hydroxytamoxifen was investigated. The equilibrium binding analysis with 4-[3H]hydroxytamoxifen indicated a positive cooperative interaction: the Scatchard plot was convex and the Hill coefficient was 1.4-1.5. This binding appears similar to the positively cooperative interaction of the estrogen receptor with [3H]estradiol. However, a competitive binding assay with a saturating concentration of [3H] estradiol and variable concentrations of 4-hydroxytamoxifen produced nonparallel displacement curves indicating that the binding mechanism of the receptor with these two ligands is different. The competitive binding assay with [3H]estradiol and 4-hydroxytamoxifen at constant molar ratios demonstrated that the receptor's affinity for estradiol was reduced and the receptor preferentially bound 4-hydroxytamoxifen. These data suggest that 4-hydroxytamoxifen interacts with the receptor differently than estradiol; it antagonizes the binding of estradiol when these two ligands are simultaneously present.

Characterization of the calf uterine progesterone receptor and its stabilization by nucleic acids.

The molecular and steroid hormone-binding properties of the calf uterine progesterone receptor and its interaction with nucleic acids were investigated. A positive cooperative binding interaction of [3H]progesterone with the receptor was evident from a nonlinear Scatchard plot and a Hill coefficient of 1.22 +/- 0.02. The range of progesterone receptor concentrations was 0.73-1.04 pmol/mg protein, approximately twice that of the estrogen receptor. Competitive binding assays revealed a high specificity for progesterone: R5020 greater than or equal to progesterone greater than deoxycorticosterone greater than 5 alpha-pregnane-3,20-dione much greater than 17 alpha-hydroxyprogesterone greater than or equal to 20 alpha-dihydroprogesterone greater than or equal to testosterone greater than or equal to estradiol greater than cortisol. Thus, a progesterone-specific receptor of high affinity and concentration is obtainable from calf uterus in large quantities without estrogen pretreatment. Thermal inactivation of the unoccupied progesterone receptor is inhibited by 10 mM sodium molybdate, whereas thermal inactivation of the ammonium sulfate-purified progesterone receptor is not. Thermal inactivation of the ammonium sulfate-purified receptor is inhibited by nucleic acids and polynucleotides; polyguanylate (poly G) is the most effective. DNA and poly G also effectively restore the progesterone-binding ability of the ammonium sulfate-purified receptor which had been lost due to heat inactivation. After incubation of the unoccupied receptor from 5-30 min at 25 C, the addition of poly G restored the receptor's [3H]progesterone-binding ability to control levels. These data suggest that the progesterone receptor's steroid-binding site is more readily inactivated by heat than is the DNA-binding site, and that nucleic acid binding induces a conformational change, which consequently restores the receptor's progesterone-binding site to functional activity.

The binding of thiamine pyrophosphate with transketolase in equilibrium conditions

The binding between thiamine pyrophosphate and transketolase, purified from baker's yeast, in equilibrium conditions has been studied. In the presence of Ca 2+, the enzyme molecule has been shown to possess two binding sites for the coenzyme, whose dissociation constants are 3.2 × 10 −8 and 2.5 × 10 −7M; besides, there are site(s) where the binding of the coenzyme is less firm. In the presence of Mg 2+, a positive cooperative interaction between the binding sites of thiamine pyrophosphate has been observed. Regardless of the cation used, the major part of the catalytic activity of the transketolase molecule manifests itself in the binding of one molecule of the coenzyme.