Eliezer Rapaport

Ap4A and Ap5A prefer folded unstacked conformations at ph 4-5, in sharp contrast with Ap2A and Ap3A

Acid-soluble nucleotides have been implicated as metabolic signals in regulation and coordination of ~trace~ular functions. Their metabolic lability, which allows their pool sizes to fluctuate rapidly in response to changes in extracellular conditions, enables this class of compounds to act in intracellular mediation of environmental changes [l]* Diadenosine S’,!?“-PI, PQ-tetraphosphate (Ap+A) has recently been suggested, based on its high metabolic lability and its pool sizes which were found directly related to the prolife~ati~ activity of mammalian cells, to act as a positive growth signal [2]. This nucleotide is readily formed in a cell free protein synthesizing system as a product of the back reaction of the amino acid activation step [3]. Its intracellular pool, however, is small and fluctuates widely in response to extracellmar conditions which affect growth f2]. Addition of Ap& to permeabilized Gr-arrested baby hamster kidney cells yielded initiation of DNA replication in the resting cells 141. A recent study has shown highly specific binding of Ap,& to mammalian DNA polymerase a: which was suggested to account for the ability of Apa to trigger DNA replication [S]. The conformation of Ap& in solution is of primary importance in determining its mode of interaction with proteins or DNA. Proton magnetic resonance studies of adenine nucleotides of the type A(5’~~(5’)A @=2--S) suggest preferred stable intramolec~arly stacked conformations for all of these compounds at physiological temperatures and pD 7. Ap& and ApsA alone, at pD 4-5, assume a unique ‘folded’ unstacked conformation as their preferred confo~~tion. Space Blling models indicate that a chain of 4 or 5 phosphate residues is ideal for this conformation.

Charcoal adsorption assay for measurement of colchicine binding and tubulin content of crude tissue extracts

Abstract A rapid, reproducible, and quantitative colchicine-binding assay for tubulin content of crude tissue extracts is described and applied to the high speed supernatant fraction of rat liver homogenates. Utilizing Scatchard plots, the colchicine-tubulin association constant is found to be in general agreement with values reported for purified tubulin from other sources.

ELEVATED NUCLEAR ATP POOLS AND ATP/ADP RATIOS MEDIATE ADENOSINE TOXICITY IN FIBROBLASTS

We have recently suggested that the nuclear compartment pools of ATP and nuclear ATP/ADP ratios act as regulators of DNA replication in S phase 3T6 (mouse fibroblast) cells. A decrease in nuclear ATP/ADP ratios has been observed upon entry of 3T6 cells into the S phase of their cycle. High ATP/ADP ratios were shown to be inhibitory to DNA replication in isolated S phase 3T6 nuclei. The decrease in nuclear ATP/ADP ratios upon entry into S phase is probably produced by DNA-dependent ATPases which have been associated with increases in the proliferative activities of a variety of mammalian cells. This report indicates that inhibition of DNA synthesis observed in log phase 3T6 cells incubated in the presence of adenosine, is a result of an increase in nuclear ATP pools and ATP/ADP ratios. Adenine or inosine which yield increases in total cellular ATP pools and ATP/ADP ratios similar to those promoted by adenosine, do not produce similar increases in the nuclear compartment and consequently do not inhibit DNA synthesis in log phase 3T6 cells. An inverse relation is demonstrated between the incorporation of adenosine (at physiological levels) and the proliferative activity of cells. The data reported here suggest that adenosine incorporation, which was shown to yield compartmentalized nuclear ATP pools, may be used as an intracellular growth regulatory mechanism. This mechanism would be mediated by the nuclear compartments ATP pools and ATP/ADP ratios.