Expansion of the cellular content of ribonucleoside triphosphates induces cell shrinkage and KCl loss in Ehrlich ascites tumor cells and in Chinese hamster ovary cells.

The conversion to corresponding triphosphate derivatives of various ribonucleosides has been studied in Ehrlich ascites tumor cells and in Chinese hamster ovary cells under conditions that are optimal for cellular uptake of orthophosphate. The initial cellular uptake of orthophosphate is followed by a cellular loss of Cl- which might be consistent with a H2PO4-/Cl- exchange mechanism. Subsequent addition of ribonucleosides to the medium leads to cellular accumulation of the corresponding triphosphate and to a concomitant loss of KCl and to sustained cell volume reduction. The latter two events are quite unspecific with regard to the nucleobase moiety of the ribonucleoside triphosphate accumulated (adenine, guanine and purine being almost equally effective) and they depend in a rather simple way on the increase of the cellular content of these compounds. The KCl loss seems to depend on opening of the separate K+ and Cl- channels. The pharmacological profile of the putative ion channels could not be identified in spite of experiments with conventional blockers. In the case of purine riboside the accumulation of the corresponding triphosphate and concomitant loss of KCl and cell water may be followed by a regain of cell volume due to a continued purine riboside triphosphate accumulation, which apparently depends on the uptake of orthophosphate by cotransport with Na+ and which for osmotic reasons is accompanied by the uptake of water and hence volume increase. The possibility that the nucleoside triphosphate induced opening of a putative Cl- channel may be due to a direct effect of triphosphate on a channel protein is discussed.

Net uptake of orthophosphate in Ehrlich ascites tumor cells in the presence of purine riboside may be rate limiting for the expansion of the pool of ribonucleotides.

Incubation with adenosine or with structural analogs thereof may in several cell types under some conditions result in the cellular accumulation of abundant amounts of the corresponding triphosphates. In the present work we have found that incubation of cells at high concentrations of orthophosphate (Pi) results in increased intracellular levels thereof, although they become not as high as the extracellular concentration. In the presence of purine riboside (nebularine, Pr) and high concentration of Pi the intracellular Pi is, however, kept at a low steady-state level, probably because it immediately upon uptake is being trapped primarily as the triphosphate of purine riboside. The latter compound accumulates at a constant rate for at least 1 h. The rate of accumulation of the sum of phosphate residues present in Pi, adenosine phosphates and purine riboside phosphates appears to be proportional to the extracellular concentration of Pi and to be highly dependent on pH (6.5 and 7.0 being optimal and 7.9 nonpermissible) but it is unaffected by substitution of Na+ by choline.

Different relationships between cellular adenosine or 3'-deoxyadenosine phosphorylation and cellular adenine ribonucleotide catabolism may be obtained.

Treatment of BALB/c-3T3 mouse fibroblasts with 3'-deoxyadenosine led to a rapid accumulation of 3'-deoxyadenosine phosphates and the kinetics of this process has been determined. Concomitant with accumulation of these compounds, the adenine ribonucleotide pool was reduced. The kinetics of the two processes suggested that they were tightly coupled. The inhibitory effect of relatively high concentrations of coformycin indicated that IMP was an intermediate in the catabolic pathway. Similar experiments with Ehrlich ascites tumor cells were performed in Ringer-Hepes solution at pH 6.5 or 7.5 and with varying concentrations of orthophosphate. The experiments were performed with cells where ATP was [3H]-labeled. This allowed the determination of the catabolism of adenine ribonucleotides to labeled nucleosides under conditions where added adenosine was phosphorylated. The results showed that at low phosphate concentration (5.8 mM) at pH 6.5 adenosine may be phosphorylated at a rate that was completely balanced to the concomitant catabolism of adenine ribonucleotides; that is, there was apparently a tight kinetic coupling between anabolism of adenosine and catabolism of adenine ribonucleotides. With 3'-deoxyadenosine a corresponding effect was obtained although the apparent coupling between phosphorylation of 3'-deoxyadenosine and catabolism of adenine ribonucleotides was not complete. When experiments were performed at the same pH but at high concentration of phosphate (45 mM) there was in contrast no coupling between the two processes; that is, ATP was present in constant amounts while 3'-deoxyadenosine phosphates accumulated at a high rate. In experiments with adenosine under these conditions there was still some although a relatively limited degree of apparent coupling between phosphorylation of adenosine and catabolism of adenine ribonucleotides. In both lines of cells used and with both adenosine and 3'-deoxyadenosine, the main products of the catabolism of adenine ribonucleotides were inosine and hypoxanthine. With 3'-deoxyadenosine there was in addition (about 20%) formation of xanthosine, suggesting that IMP dehydrogenase had also been activated. These results lead to the suggestion that adenosine (or 3'-deoxyadenosine) may be phosphorylated in two ways. 1) Phosphorylation may depend on an adenosine kinase unrelated to catabolism of adenine ribonucleotides. 2) Phosphorylation may be tightly coupled to catabolism of adenine ribonucleotides. A nucleoside phosphotransferase may catalyze the transfer of a phosphoryl group from IMP to adenosine (or 3'-deoxyadenosine) to form AMP (or 3'-dAMP) and inosine, a process that may be tightly coupled to an AMP deaminase reaction. The IMP formed in the latter reaction may not be released but transferred to the phosphotransferase.(ABSTRACT TRUNCATED AT 400 WORDS)

The rate of catabolism of dATP to deoxyadenosine during the growth of different cell lines in vitro.

A method has been developed for the determination of the rate of formation of deoxyadenosine from dATP in cultured cell lines. The lowest rate was found in the T-cell-derived Molt cell line while it was about 70-fold higher in Balb c/3T3 mouse fibroblasts. In the B-cell-derived Raji cells and in the murine sarcoma cell line SEWA it had intermediary values. It is concluded that in some cell types like the 3T3-cells the catabolism of dATP to deoxyadenosine may have a significant regulatory effect on the cellular content of dATP.

Adenosine induction of rapid catabolism of adenine ribonucleotides and independent elevation of the ATP content in quiescent mouse fibroblasts.

The influence of adenosine on the ribonucleotide metabolism in quiescent BALB/c 3T3 cells was studied. The cellular adenine ribonucleotides were labelled by pretreating the cells with [2-3H]-adenine. After addition of adenosine to the cell cultures, the amount and radioactivity of the cellular purine ribonucleotides and the radioactivity of the purine compounds in the medium were determined. It appeared that adenosine gave rise both to rapid catabolism of adenine ribonucleotides with inosine 5'-monophosphate (IMP) as an intermediate and to expansion of the cellular adenosine 5'-triphosphate (ATP) pool. The maximal rates and the apparent activation constants for the two processes have been determined. Experiments with varying concentrations of coformycin (an inhibitor of adenosine 5'-monophosphate [AMP] deaminase and adenosine deaminase) and of 5'-amino-5'-deoxyadenosine (an inhibitor of adenosine kinase), respectively, showed that each compound may almost completely inhibit the adenosine-induced catabolism. This effect can be obtained under conditions where there was little or no effect by the two inhibitors on the rate of expansion of the cellular ATP pool. These results may best be explained by assuming that the process of expansion of the ATP pool is independent of the induced catabolism of adenine ribonucleotides, even though both processes seem to depend on the phosphorylation of adenosine to AMP. The total increase in the pool size of ATP and of guanosine 5'-triphosphate (GTP), both caused by adenosine, seems not to have regulatory effect on adenine ribonucleotide catabolism.

Diagnosis of storage pool deficiency by determination of diadenosine 5', 5'''-P1,P4-tetraphosphate in whole blood.

Platelets from cat and cattle with Chediak-Higashi disease were found completely devoid of Ap4A as measured by high performance liquid chromatography. Using a very sensitive firefly biolumnescence method 6% of the normal content of Ap4A was, however, found in platelets from sick animals. A content of Ap A of 1.90 +/- 0.11 X 10 M (means +/- SEM, n = 10) was found in whole normal human blood as measured by firefly bioluminescense method in trichloroacetic acid extracts of the blood samples. This concentration corresponds to the contribution from the platelets, thus the contribution of Ap4A from erythrocytes and the "buffy-coat" is negligible. Using the same method an Ap4A contents in platelets of 0.063 and 0.021 nmol/mg of protein compared to the normal content of 0.42 nmol/mg of protein (1) was found in two patients with severe myeloproliferative disorder calculated in this way on basis of platelet counts and on the assumption that 10(11) platelets contain 189 mg of protein (2). Comparison of these figures with parallel HPLC analyses on acid extracts of platelets isolated from the same patient were in agreement. The storage pool deficiency of adenine nucleotides in this disease found by others on basis of release experiments (3) can thus be diagnosed by rapid and simple measurements of Ap4A in whole blood using the advantage of Ap4A being a specific components stored in dense granules.

On the mechanism of action of free purine bases on DNA synthesis in serum-starved L-cells treated with platelet extract.

It has previously been found that there is a synergistic effect of free purine bases and low concentrations of dialyzed platelet extract on net synthesis of DNA in serum-starved fibroblast-like mouse L-cells. Experiments with a mutant line of L-cells that was deficient in hypoxanthine phosphoribosyl transferase (EC 2.4.2.8) indicated that purine bases had a stimulatory effect only if they were incorporated into cellular ribonucleotides. In the present paper it was shown that platelet extract induced the incorporation of hypoxanthine or adenine into both ATP and DNA. The induced net synthesis of DNA appears to take place in the nuclei and it requires that platelet extract is present in the medium only initially while free purine bases have to be present only later in the period of the experiment when DNA is being synthesized. The induction of both incorporation of free purine bases into DNA and ATP and of net DNA synthesis is dependent on heat-labile components in platelet extract. The extract cannot be substituted for by platelet derived growth factor.

Platelet-derived growth factor stimulates chemotaxis and nucleic acid synthesis in the protozoan Tetrahymena.

Platelet-derived growth factor (PDGF) is in concentrations of a few nanograms per ml a very active chemoattractant for the free-living ciliated protozoan Tetrahymena; at the same time it induces a rapid increase in incorporation of radioactive nucleic acid precursors into RNA and DNA. We find it remarkable that this lower eukaryote responds to platelet-derived growth factor in very much the same way as fibroblastic cells.

Both hypoxanthine and adenosine stimulate DNA synthesis independently in serum-starved L cells treated with platelet protein.

The effect of human platelet factors and purine derivatives on DNA synthesis has been investigated in mouse fibroblast-like L cells whose growth was arrested by serum starvation. When such cells were exposed to diluted platelet extract (e.g., 35 micrograms of protein per ml), a stimulatory effect on net DNA synthesis was observed. This effect was almost abolished by dialysis of the extract. The stimulation was, however, recovered by supplementing the diluted and dialyzed extract with hypoxanthine or adenosine. Similar phenomena were observed in pulse-labeling experiments performed with [3H]thymidine. In this case, however, there was a marginal stimulatory effect of adenosine or hypoxanthine alone. When the cells were treated with saturating concentrations of pure platelet-derived growth factor (PDGF), a stimulatory effect on pulse labeling was again obtained by the simultaneous presence of hypoxanthine or adenosine. In serum-starved cells of a mutant line of L cells deficient in hypoxanthine phosphoribosyltransferase, there was, however, no stimulatory effect on pulse labeling by hypoxanthine when it was added alone or together with either PDGF or diluted dialyzed platelet extract. It is suggested that the stimulation of DNA synthesis by the purine derivatives in the presence of a certain type of platelet proteins, probably involving PDGF, may be explained by their function as precursors for a purine ribonucleotide pool that is specifically related to DNA synthesis. Treatment of serum-starved L cells with high concentrations of dialyzed platelet extract (e.g., 240 micrograms of protein per ml) showed that platelets contain an additional type of factor that may substitute for the requirement of adenosine or hypoxanthine for DNA synthesis to take place. It is suggested that the effect of this type of factor may be to activate the catabolic activity of the purine salvage pathway.

Abundant amounts of diadenosine 5',5"'-P1,P4-tetraphosphate are present and releasable, but metabolically inactive, in human platelets.

Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) may be formed in the back reaction of the amino acid-activation reaction [Zamecnik, Stephenson, Janeway & Randerath (1966) Biochem. Biophys. Res. Commun. 24, 91-98]. On the basis of a number of observations of the properties of Ap4A it has been suggested that it may have a signal function for the initiation of DNA replication in eukaryotic cells] Grummt (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 371-375]. In the present paper human platelets have been shown to contain relatively large amounts of Ap4A. The compound is apparently metabolic inactive in platelets, but it is almost quantitatively released when platelets are activated to aggregate by treatment with thrombin. The results are discussed in connection with the known growth-stimulating activity of platelets.

Differential effect of N-carboxymethylisatoylation on the DNA polymerase activity, the 5' leads to 3'-exonuclease activity and the 3' leads to 5'-exonuclease activity of DNA polymerase I of Escherichia coli.

Treatment with native DNA polymerase I of Escherichia coli with the acylating agent N-carboxymethylisatoic acid anhydride (NCMIA) results under specific conditions in a rapid loss of polymerase activity, an increase in 5' leads to 3'-exonuclease activity and in unchanged 3' leads to 5'-exonuclease activity. When a nucleoside triphosphate and Mg2+ was present the polymerase activity was completely protected against the effect of NCMIA. Treatment with higher concentration of the acylating agent under these conditions led to a loss of 3' leads to 5'-exonuclease activity without any appreciable loss of polymerase activity. Treatment with NCMIA of the two catalytically active fragments of the enzyme led to very similar results. In this case both the polymerase activity and the 3' leads to 5'-exonuclease activity deteriorated more rapidly on treatment with the acylating reagent. The increase in 5' leads to 3'-exonuclease activity as a result of modification of the native enzyme appeared to be due to a change in the optimum conditions with regard to concentration of the assay buffer used. These changes are very similar to those seen when the polymerase is cleaved by limited proteolysis. From the results obtained it is concluded that NCMIA reacts primarily with a site at or near the triphosphate-Mg2+ complex binding site, leading to an almost complete loss of polymerase activity. The acylating reagent reacts also with another group on the native enzyme resulting in a modification of the 5' leads to 3'-exonuclease activity, and at high concentrations with a group leading to a slow loss of 3' leads to 5'-exonuclease activity.

Iolation and charcterization of a DNA-binding non-histone protein from Tetrahymena pyriformis.

Three proteins (A, B and C) that bind specifically to single-stranded DNA have been isolated from the eukaryotic organism Tetrahymena pyriformis. Their molecular weights are 47 000, 41 000 and 32 000. The amino acid composition of the A protein indicates that it is a non-histone protein and sucrose gradient centrifugation shows that it binds to bacteriophage M13 DNA and to oligo (dT)100 in a cooperative manner. The exonucleolytic degradation of oligo (dT)100 is prevented when it is bound to the A protein. The effect of A protein on the exonucleolytic reaction confirms the cooperative manner of binding of A protein binding to oligo (dT)100 and shows that this process may be prevented by high ionic strength. The A protein seems to be without enzymatic activity.

Involvement of Escherichia coli DNA polymerase-I-associated 5' in equilibrium 3' exonuclease in excision-repair of UV-damaged DNA.

From comparative studies between Escherichia coli PolA107 cells (lacking 5' in equilibrium 3' exonucleoytic activity associated with DNA polymerase I) and the isogenic wild-type strain, and between the purified DNA polymerase I preparations isolated from these strains, it can be concluded that the 5' in equilibrium 5' exonuclease is involved in excision of pyrimidine dimers in E. coli. Evidence is presented that the polA107 mutation is located on that part of the DNA polymerase I gene coding for the small fragment on which 5' in equilibrium 3' exonucleolytic activity is found.

Selective elimination of the exonuclease activity of the deoxyribonucleic acid polymerase from Escherichia coli B by limited proteolysis.

Purification of DNA polymerase from E. coli B has in two cases each time led to the isolation of two separate polymerase activities, enzyme A and enzyme B. Enzyme A was in contrast to enzyme B almost completely devoid of exonuclease activity. Each of the two enzymes yielded a single symmetrical activity peak in gel filtration chromatograms. From the elution volumes the molecular weights were estimated to be about 70,000 for enzyme A and about 150,000 for enzyme B. Treatment of enzyme B with subtilisin led to an increase of about 30 per cent of the polymerase activity while the exonuclease activity almost completely disappeared. The product of the subtilisin treatment (enzyme C) gave rise to a single symmetrical polymerase activity peak in a gel filtration chromatogram. The elution volume was identical to that obtained with enzyme A. It is concluded that enzyme A and enzyme C are formed by limited proteolysis of enzyme B.