AMP deaminase in Dictycostelium discoideum: Increase in activity following nutrient deprivation induced by starvation or hadacidin

Summary AMP deaminase, the activity that catalyzes the deamination of AMP to form IMP and NH₃ has been measured in Dictyostelium discoideum. A new procedure to assay the activity of this enzyme was developed using formycin 5-monophosphate, a fluorescent analog of AMP as the substrate, and ionpaired reverse phase HPLC to separate the reactants and products. Quantitation of the formycin containing compounds was accomplished at 290 nm. At this wavelength adenosine containing compounds were not detected and activity could be monitored in the presence of its activator ATP. The AMP deaminase activity in vegetative cells was 7.4 nmols/min/mg proteins while the activity in cells measured at 2 and 6 hrs after starvation-induced growth-arrest was 376 nmols/min/mg protein... a 51-fold increase. When vegetative cells were treated with hadacidin, a drug that restricts de novo AMP synthesis and pinocytosis, the activity of the AMP deaminase was 511 nmols/min/mg protein... a 70-fold increase compared to that in untreated vegetative cells. Smaller increases were noted following the inhibition of growth with the drugs cerulenin and vinblastine, as well as after the inhibition of de novo GMP synthesis with the drug mycophenolic acid or the partial inhibition of de novo AMP synthesis with analogs of hadacidin, N-hydroxyglycine and N-formylglycine. In addition, when the activity of two other enzymes involved in purine metabolism, namely adenosine kinase and hypoxanthine-guanine phosphoribosyl transferase, was measured in vegetative cells, and the activity of both compared to that measured in starvation and hadacidin induced growth-arrested cells, showed no significant changes. These data suggest that the changes in the activity of the AMP deaminase are in response to nutrient deprivation and further, that as a consequence of the increase in AMP deaminase activity, ammonia will be produced and an increase in pH should follow. The production of ammonia and its effect on development implicates the AMP deaminase in the early differentiation of this organism.     

Histone H1: Ultracentrifugation studies of the effects of ionic strength and denaturants on the solution conformation

The conformation of histone H1 has been examined under native and denaturing conditions in the absence of DNA or chromatin. Sedimentation coefficients were determined for Histone H1 in 0.1 m KCl and in 6 m guanidine hydrochloride solutions at pH 7.4. The influence of ionic strength on the conformation of histone H1 has been determined by measurement of the sedimentation coefficient in tetramethylammonium chloride solutions of up to 2.5 m and extrapolated to infinite ionic strength. Results from these experiments suggest that the native conformation of histone H1 is very asymmetric in shape. The molecule is best described as a prolate ellipsoid with axes of 312 Å (2a) and 16 Å (2b) in low ionic strength media and also as a prolate ellipsoid with axes of 202 Å (2a) and 20 Å (2b) at high ionic strength or when associated with polyanions, e.g., DNA. Denaturation of histone H1 by guanidine hydrochloride was found to be completely reversible. In 6 m guanidine hydrochloride, the H1 molecule collapses to a sphere but the original extended conformation of the protein is readily restored on dialysis. This suggests rigid conformational requirements for the H1 molecule as incorporated into chromatin. The shape and dimensions for the H1 molecule at high ionic strength are not sufficiently conclusive to locate H1 in the chromatin structure. It is proposed, however, that viable models for chromatin architecture must be consistent with the histone H1 solution dimensions obtained here.     

Respiratory chain of the alkalophilic bacterium Bacillus firmus RAB and its non-alkalophilic mutant derivative

The membrane-bound respiratory chain components of alkalophilic Bacillus firmus RAB were studied by difference spectroscopy and oxidation-reduction potentiometric titrations. Cytochromes with the following midpoint potentials were identified at pH 9.0: a-type cytochromes, +110 and +210 mV; b-type cytochromes, +20, -120, -280, and -400 mV; and cytochrome c, +60 mV. Only the higher-potential cytochrome a showed an upward shift in midpoint potential when titrated at pH 7.0. Parallel studies of a non-alkalophilic mutant derivative of B. firmus RAB, strain RABN, revealed the presence of only one species each of a-, b-, and c-type cytochromes which exhibited midpoint potentials of +110, -150, and +160 mV, respectively, at pH 7.0. Membranes of both strains were found to contain menaquinone. At pH 9.0, NADH caused the reduction of essentially all of the cytochromes that were seen in fully reduced preparations of wild-type B. firmus RAB membranes. By contrast, at pH 7.0, NADH failed to appreciably reduce the b-type cytochromes. These findings may relate to our recent proposal that an inadequacy in energy transduction (production of a proton motive force) by the alkalophilic respiratory chain at pH 7.0 is what precludes the growth of B. firmus RAB at a neutral pH.     

Neem (Azadirachta indica) cultivated in Haiti

So that valuable products from the neem tree (Azadirachta indica) will be readily available in the future, countries in the neotropics should emulate Haiti and grow the species where it thrives even in eroded, semiarid, depauperate areas suitable for few other trees.     

A new variety of coreopsis Californica (Compositae) Endemic to Arizona

Coreopsis californica var. newberryi, a new variety, differing from typical material ofC. californica in achene morphology, is proposed. Achene spotting in the two varieties exhibits maternal inheritance.     

Calcium-dependent activation and deactivation of rod outer segment phosphodiesterase is calmodulin-independent

ATP-dependent activation and deactivation of retinal rod outer segment phosphodiesterase is affected by calcium [Kawamura, S. and Bownds, M. D., J. Gen. Physiol. 77:571-591(1981)]. Our data demonstrate that although calmodulin has been found in rod outer segments [Liu, Y. P. and Schwartz, H., Biochim. Biophys. Acta 526:186-193(1978); Kohnken, R. E. et al, J. Biol. Chem. 256:12517-12522(1981)], this protein is not involved in calcium-dependent phosphodiesterase activation at light levels at which calcium clearly affects this enzyme's activity. Furthermore, calmodulin does not mediate the calcium-dependent deactivation of phosphodiesterase.     

Purification and characterization of the membrane (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B

The membrane (Na⁺ + Mg²⁺)-ATPase of Acholeplasma laidlawii B has been solubilized with a Brij-58/sodium deoxycholate mixture and purified by a combination of gel filtration and ion-exchange chromatography. The purified, partially delipidated ATPase has a specific activity of 195 μmol P_i/mg protein per h, which could be enhanced by 25% upon the addition of exogenous phospholipids. The kinetic properties of the purified enzyme are similar to those of the native membrane-bound enzyme, suggesting that it has not been substantially altered during the purification procedure. The enzyme is an assembly of five polypeptide species and its kinetic properties suggest that it is dissimilar to other known ATPases.