A group of hydrolytic enzymes, including phosphatases and nucleases, is selectively released from E. coli and certain other Gram-negative bacteria by a process designated as osmotic shock. This procedure involves exposure of the cells to ethylenediaminetetraacetate (EDTA) in 0.5 molar sucrose followed by a sudden osmotic transition to cold, dilute MgCl(2). Osmotic shock also results in an alteration of the permeability barrier of the bacterial cell and a depletion of the pool of acid-soluble nucleotides, but there is no loss of viability. On being restored to growth medium, the shocked cells recover after a lag period. Formation of spheroplasts by treatment with EDTA and lysozyme leads to selective release of the same group of enzymes. We believe that the selectively released enzymes are confined in a region between the bacterial cell wall and the cytoplasmic membrane. Histochemical studies indicate such a localization. Further, the enzyme activities are measurable with intact cells, even when the substrate is a nucleotide, to which whole cells are impermeable. Another piece of evidence concerns a mutant E. coli with a defective cell wall. In contrast to normal bacteria, this organism loses one of these enzymes into the medium in the course of growth. After osmotic shock, the bacteria show reduced uptake of sulfate,betagalactosides, galactose, and certain amino acids. Furthermore, the shock treatment causes the release of nondialyzable factors able to bind sulfate, galactose, and the same amino acids. A possible interpretation of these observations is the following: the binding proteins occupy sites near the bacterial surface, and they may be components of active transport systems responsible for the concentrative uptake of these nutrients.

Serum causes a 4-fold increase in "Rb+ (a K+ tracer) influx in quiescent 3T3 mouse fibroblast cells. It is one of the earliest changes caused by serum, being seen in 2 min and reaching a maximum in 10 min. Removaj of serum causes rapid reversal of this effect. Serum acts mainly by increasing the maximum velocity, Vmax, of entry. Ouabain inhibits entry of "Rb+ (82-90%) both in the presence and absence of serum, but does not alter exit. The rapid increase in cation influx is unaffected by cycloheximide and by changes in cyclic AMP and GMP. Low concentrations of insulin, epidermal growth factor, and prostaglandins (El and F2a) produced a smaller (80%) activation of 86Rb+ entry. Ouabain, at a level that inhibits cation influx, also prevents the onset of DNA synthesis following serum addition; this is a reversible effect dependent on the concentration of K+ in the medium. This suggests that cation pumping activity may be required for initiation of DNA synthesis. Cultured fibroblasts exist in two states of growth: one of active proliferation and one of reversible arrest in the GI phase of the cell cycle. Serum stimulates resting cells to reinitiate DNA synthesis and cell division (1). Changes in transport rates for Pi, nucleosides, and glucose (2-5) and in cyclic nucleotide levels (6-8) are, so far, the earliest events detected in this system. Whether or not K+ uptake is rapidly increased by serum is unknown and of importance because the asymmetric distribution of K+ and Na+ profoundly affects the regulation of transport of non-electrolytes (9), intracellular osmotic pressure (10), membrane potential (10), glycolytic enzymes (11), macromolecular synthesis (12-14), etc. Furthermore, lectin-stimulated lymphocytes (15) 0.1 mM RbCl) was added to the medium. After 10 min (unless otherwise indicated) at 370, 86Rb+ uptake was stopped by sucking off the medium and rapidly washing four times with ice-cold isotonic saline. The cells were extracted at 40 for 20 min with 1.5 ml of 5% trichloroacetic acid and 1 ml of extract was mixed with 10 ml of water to measure Cerenkov radiation. Control cultures were washed immediately after adding isotope. The radioactivity incorporated into cells was not significantly altered by washing at 260 or by using 10 mM Tris-HCI-200 mM choline C1, pH 7.4 in place of saline. To measure rate of efflux, cells were preloaded in the presence or absence of serum with 86Rb+ for 20 min, washed twice with prewarmed medium, and shifted to fresh medium without 86Rb+; at different times the 86Rb+ remaining in the cells was measured as in the uptake trials.DNA synthesis was estimated by exposing cells to [3H]thymidine for 24-26 hr and measuring incorporation into trichloroacetic-acid-insoluble material or by radioautography. For incorporation studies, levels of 1 0Ci/ml and 3 X 10-6 M were used; for autoradiography the values were 5 ,uCi/ml and 2 X 10-7 M. Saline-washed cells were assayed for protein (18). Epidermal growth factor and prostaglandins were the generous gift of Drs. S. Cohn, Vanderbilt, an...

The Mg2+-and Ca"-stimulated ATPase (EC 3.6.1.3; ATP phosphohydrolase) (bacterial coupling factor) was purified from two strains of E. coli by two different procedures: (a) method of Nelson, Kanner, and Gutnick [Proc. Nat. Acad. Sci. USA (1974) 71, 2720-27241 and (b) a modified procedure described in this paper. The ATPase purified from E. coli K12 (X) by the first procedure had 4 subunits (a, ,, -y, and e). It did not bind to a deficient membrane, nor did it reconstitute ATP-driven transhydrogenase activity. Our modified procedure (b) yielded 5 subunits (a, ,, -y, 6, and e). This ATPase could bind to a deficient membrane and reconstitute ATP-driven transhydrogenase. This finding suggests that the 5 subunit is required for the reaction with the membrane. The molecular weight of the 4-subunit ATPase was significantly lower than that of the 5-subunit ATPase, as judged by equilibrium centrifugation. The specific ATPase activities of both preparations were about the same. These two procedures were also applied to E. coli ML308-225.

Extracellular ATP in concentrations of 5-50 gLM displayed very little mitogenic activity by itself but it caused synergistic stimulation of [3H]thymidine incorporation in the presence of phorbol 12-tetradecanoate 13-acetate, epidermal growth factor, platelet-derived growth factor, insulin, adenosine, or 5'-(N-ethyl) These findings raise the possibility that exogenous ATP acts as a growth factor, and this was implied in two recent papers (23, 24), where ATP was compared to "more conventional mitogens." However, it is premature to call ATP a mitogen without more evidence than has been available. One must be careful to rule out the possibility that ATP is converted to adenosine by ubiquitous hydrolytic enzymes on the cell surface (ectoenzymes) (32). It was demonstrated some years ago in Rozengurt's laboratory (11-13) and by others (33) that adenosine is a potent mitogen for Swiss 3T3 cells, and this is also true for certain adenosine analogues such as NECA.In this paper, we show that exogenous ATP is a mitogen for 3T3, 3T6, A431, HFF (34), and DDT1-MF2 (35) cells. We confirm that adenosine is also mitogenic, but several lines of evidence show that ATP acts independently, in its own right, and not by being first converted to adenosine by ectoenzymes. MATERIALS AND METHODS Materials. [3H]Thymidine was purchased from Amersham.Culture media and fetal bovine serum (FBS) were from GIBCO. ATP, adenosine, epidermal growth factor (EGF), adenosine 5'-[8,y-imido]triphosphate (AdoPP[NH]P), and all other nucleotides were from Boehringer Mannheim. Bovine insulin, NECA, and phorbol 12-tetradecanoate 13-acetate (PTA) were from Sigma. Platelet-derived growth factor (PDGF) was obtained from Amgen Biologicals. All other chemicals were of the highest purity available.Cell Culture. Swiss 3T3 and 3T6 mouse fibroblasts, human A431 epidermoid carcinoma cells (14), and DDT1-MF2 cells (35), derived from a vas deferens tumor of the hamster, were maintained in Dulbecco's modified Eagle's medium (DMEM; GIBCO) containing 10 mM Hepes, 44 mM NaHCO3, penicillin (110 units/ml), streptomycin (100 ,ug/ml), and 0.5% (vol/vol) (3T6), 5% (A431, DDT1-MF2), or 10% (3T3) FBS.Cells were grown at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. Two days after seeding, the 3T3 cells were given fresh medium supplemented as before and then allowed to become confluent and quiescent for 5 days. The A431, DDT1-MF2, and 3T6 cultures were used 4, 5, or 7904The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.