The use of beta, gamma-methyleneadenosine 5'-triphosphate to determine ATP competition in a scintillation proximity kinase assay.

A novel method for characterizing the kinetics of protein kinase inhibitors is described. This method uses glycogen synthase kinase beta as the model protein kinase and looks at the shift in IC50 of inhibitors using the nonhydrolyzable ATP analog, beta, gamma-methyleneadenosine 5'-triphosphate, also known as AMP-PCP. Due to its inability to be hydrolyzed, AMP-PCP is being used to characterize known glycogen synthase kinase inhibitors by determining the shift in IC50 at concentrations above its calculated Ki of 490 microM. The assay format for the detection of inhibition is a scintillation proximity assay which is robust and reproducible at very low levels of [gamma-33P]ATP. The use of AMP-PCP coupled with the use of the scintillation proximity assay allows this characterization of inhibition without increasing [gamma-33P]ATP and without significantly diluting the overall assay signal. We have used this method in kinetic analyses to demonstrate that we can detect a significant shift in IC50 with the known ATP competitive inhibitors, staurosporine, Ro 31-8220, and olomoucine. The IC50 for glycogen synthase peptide and lithium chloride, which has been reported to be uncompetitive, remains unchanged.     

Evidence for a calcium-sensitive factor which alters the alkaline pH sensitivity of sarcoplasmic reticulum calcium transport

Oxalase-supported, ATP-dependent Ca2+ uptake by cardiac and skeletal muscle sarcoplasmic reticulum (SR) exhibits a pH profile with the maximal rate of Ca2+ uptake at pH 6.6-6.8 and marked inhibition (90-95%) at pH 7.4-7.6, a point at which Ca2+-dependent ATPase activity is optimal. These observations are noted when the SR is first preincubated in media containing no added Ca2+. This alkaline pH inhibition is not caused by an irreversible perturbation since the Ca2+ uptake rate is fully restored by changing the alkaline pH preincubation medium to pH 6.8. When SR is preincubated with added Ca2+, Ca2+ uptake at alkaline pH (7.4-7.6) is only inhibited by 10-30%. Ca2+ uptake at pH 6.8 is the same regardless of preincubation conditions. A depressed oxalate permeability is not a factor in the observed alkaline pH inhibition of Ca2+ uptake. At alkaline pH, the relationship between the preincubation Ca2+ concentration and the rate of Ca2+ uptake is hyperbolic; the half-maximal free Ca2+ concentration for stabilization of Ca2+ uptake is 8-15 microM with a Vmax equal to the velocity at the optimal pH. The Hill coefficient is 1.0, implying a single class of Ca2+-requiring sites for stabilization at alkaline pH. In contrast to its effect on Ca2+ uptake, the presence of Ca2+ during preincubation does not alter the pH sensitivity of Ca2+-dependent ATPase activity. Thus, the presence of Ca2+ during preincubation may stabilize a state of the CaATPase, conducive to the coupling of net Ca2+ translocation to Ca2+-dependent ATPase activity, which is ordinarily opposed by alkaline pH. The data suggest a single class of Ca2+-requiring sites which favors this coupled state.     

Distribution of Interdivisional Times In Proliferating and Differentiating Friend Murine Erythroleukaemia Cells

Abstract. The interdivisional times of Friend murine erythroleukaemia cells which are growing continuously, or during terminal erythroid differentiation after exposure to dimethyl sulphoxide (DMSO), were determined by time lapse video photography. the median interdivisional times were found to increase from 11.75 hr before exposure to DMSO, to 24.0 hr at 72 hr after exposure. This increase in median interdivisional time was accompanied by an increase in heterogeneity of interdivisional times (% CV = 8-5 → 40.8), by an increase in the similarity of sister interdivisional times (r_yy= 0.622 → 0.925), and by a decrease in the fraction of cells observed to divide ( F = 1. 0 → 0.807). Cells exposed to DMSO for 72 hr can be induced to divide at least once with nearly normal interdivisional times, if they are resuspended at a tenfold higher cell concentration. Computer simulations of cell cycle regulation, based on the opposing reactions model of Murphy, generate interdivisional time distributions which resemble the experimental data better than the single transition probability model of Smith and Martin.     

Ribosomes from a relC mutant strain of Escherichia coli show altered activity with bacterial release factor-1

Ribosomes from a relC mutant of Escherichia coli, JF505, are altered in the large subunit protein L11. This protein has abnormal mobility on gel electrophoresis. The ribosomes have a lowered specific activity for release factor-1 which is intermediate between that found for ribosomes containing normal L11 and that for L11 lacking ribosomes. JF505 ribosomes are as sensitive to inactivation of in vitro termination by thiostrepton as normal ribosomes when the antibiotic is added in dimethylsulphoxide but less sensitive when it is added in ethanol.     

Anion effects on in vitro sarcoplasmic reticulum function. The relationship between anions and calcium flux

Isolated sarcoplasmic reticulum vesicles exhibited different functional characteristics in the presence of zwitterionic as compared to anionic buffers. In the absence of oxalate, dicarboxylic anions (e.g. maleate, succinate) in a dose-dependent manner enhanced ATP-supported Ca2+ accumulation, the ensuing spontaneous Ca2+ release, and Ca2+-dependent ATPase activity compared to zwitterionic buffers (e.g. piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (Hepes). This was not attributed to ionic strength and osmotic effects. The additional anion-dependent Ca2+ accumulation was linked to augmented Ca2+-dependent ATPase activity, and both could be induced by the addition of anion at any time during Ca2+ accumulation as long as ATP was present. Since the initial Ca2+ accumulation rates and acyl phosphoenzyme formation were the same between the two buffer classes, and the presence of either oxalate (a Ca2+-precipitating anion) or A23187 (a Ca2+ ionophore) abolished differences in Ca2+-dependent ATPase activity between the two buffer classes, it is likely that conditions favoring high intravesicular Ca2+ concentration allow the expression of the observed effect of the anions. Initial spontaneous Ca2+ release in the presence of maleate was not caused by ATP depletion, and it was virtually absent in Pipes buffer. The rate of spontaneous release was also stimulated in a dose-dependent manner by the dicarboxylic anions, with the time of release being related to the time of anion addition and not ATP addition. A later, more rapid release phase in either maleate or Pipes buffer corresponded to ATP depletion, and could be duplicated at any time in the Ca2+ accumulation/release cycle by the addition of an ATP trap. With an ATP-regenerating system present or with very high ATP concentrations, the maximal peak Ca2+ accumulation in Pipes buffer could approach that in maleate buffer. The data suggest that dicarboxylic anions stimulate the filling of a Ca2+ compartment from which spontaneous Ca2+ release occurs.