Selective release of enzymes from bacteria.

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.

The phosphorylation of calmodulin and calmodulin fragments by kinase fractions from bovine brain.

The phosphorylation of intact calmodulin and of fragments obtained by trypsin digestion was studied, using a protein kinase partially purified from bovine brain. Brain extracts were made in the presence of the detergent CHAPS (3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate). The protein kinase catalyzed the incorporation of nearly 1 mol of 32P from [gamma-32P]ATP into calmodulin fragment 1-106. Incorporation was exclusively into serine 101. With fragment 78-148, the extent of phosphorylation was somewhat less and 32P appeared mainly in threonine residues. Fragment 1-90 was also a fairly good substrate, but the phosphorylation of intact calmodulin never exceeded 0.01 mol per mol. Little or no phosphorylation was seen with parvalbumin, the brain Ca2+-binding protein (CBP-18) and intestinal calcium-binding protein. The protein kinase had no requirement for cAMP or phospholipids. High levels of Mg2+ (60-70 mM) stimulated phosphorylation of the fragments 20-fold. Millimolar concentrations of Ca2+ were inhibitory. It is suggested that the calmodulin fragments were in a conformation more favorable for phosphorylation than intact soluble calmodulin.

The effect of osmotic shock on release of bacterial proteins and on active transport.

Osmotic shock is a procedure in which Gram-negative bacteria are treated as follows. First they are suspended in 0.5 M sucrose containing ethylenediaminetetraacetate. After removal of the sucrose by centrifugation, the pellet of cells is rapidly dispersed in cold, very dilute, MgCl(2). This causes the selective release of a group of hydrolytic enzymes. In addition, there is selective release of certain binding proteins. So far, binding proteins for D-galactose, L-leucine, and inorganic sulfate have been discovered and purified. The binding proteins form a reversible complex with the substrate but catalyze no chemical change, and no enzymatic activities have been detected. Various lines of evidence suggest that the binding proteins may play a role in active transport: (a) osmotic shock causes a large drop in transport activity associated with the release of binding protein; (b) transport-negative mutants have been found which lack the corresponding binding protein; (c) the affinity constants for binding and transport are similar; and (d) repression of active transport of leucine was accompanied by loss of binding protein. The binding proteins and hydrolytic enzymes released by shock appear to be located in the cell envelope. Glucose 6-phosphate acts as an inducer for its own transport system when supplied exogenously, but not when generated endogenously from glucose.

A comparative study of permeabilization induced by extracellular ATP and by Sendai virus.

Both Sendai virus and extracellular ATP induce membrane changes in 3T6 cells which allow passage of phosphorylated metabolites and normally impermeant aqueous solutes. The two processes share many features in common, including their kinetics and the effects of temperature, Ca2+, and various metabolic inhibitors. Furthermore, in each case permeabilization is preceded by net changes in intracellular cations. However, there are significant differences in that only ATP-dependent permeabilization is influenced by changes in the ionic strength of the medium, by inhibition of the Na+, K+, Cl- cotransporter and by preincubation of 3T6 cells with dithiothreitol.

The effect of oncomodulin on cAMP phosphodiesterase activity.

Oncomodulin was purified from Morris rat hepatoma according to the procedure of Durkin, J.P., Brewer, L.M. and MacManus, J.P. (1983) Cancer Res. 43, 5390-5394. The preparation, in general, had the properties and amino acid composition of the material which they described. However, we were unable to confirm the reported stimulation of cyclic nucleotide phosphodiesterase under conditions where calmodulin gave the usual stimulation.

Control of glycolysis and the pentose phosphate shunt in transformed 3T3 cultures rendered permeable by ATP.

Exogenous ATP has been shown earlier to activate a permeability change in transformed 3T3 cultures leading to massive efflux of the acid-soluble pools. This leads to reduction of the basal rate of glycolysis to a very low level so that glycolysis becomes almost totally dependent on the addition to the medium of glucose, inorganic phosphate and ADP in order to restore the rate to that of untreated cells. No such depression of glycolysis is observed in untreated transformed cells or in ATP-treated normal 3T3 cells. In such permeabilized cultures, phosphorylated intermediates such as glucose-6-phosphate and fructose-1,6-diphosphate can serve as effective substrates for lactic acid formation. ATP treatment of cultured cells also allows molecules as big as NADP to enter the cells and participate in the pentose phosphate shunt pathway. This ability to temporarily and differentially render transformed cells permeable allows a review of several aspects of cellular metabolism and biosynthesis in the intact cell where the cellular organization is maintained. Furthermore, it deserves serious consideration as a means to achieve differential cytotoxicity of transformed cells by chemotherapeutic agents which, on their own, are indiscriminate in their action.

Serum rapidly stimulates ouabain-sensitive 86-RB+ influx in quiescent 3T3 cells.

Serum causes a 4-fold increase in 86Rb+ (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. Removal of serum causes rapid reversal of this effect. Serum acts mainly by increasing the maximum velocity, Vmax, of entry. Ouabain inhibits entry of 86Rb+ (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 (E1 and F2alpha) 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 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.

Methods for rapidly altering the permeability of mammalian cells.

Various agents alter mammalian cells so that they rapidly become nonspecifically permeable to substances that ordinarily do not penetrate intact cells. Thus, toluene renders liver cells permeable to nucleotides and macromolecules. Tween 80 and Tween 60 act on similar fashion, and the effect is reversible. Dextran sulfate reversibly alters the permeability of Ehrlich ascites tumor cells, which offers a tool for studying the control of macromolecular syntheses and other processes. Brief exposure to external ATP alters the permeability of certain transformed mouse cells but not of untransformed cells. The effect of ATP is rapidly reversible.

Evidence for a role of G protein beta gamma subunits in the enhancement of cAMP accumulation and DNA synthesis by adenosine in human cells.

The expression of both A1- and A2a-adenosine receptors occurs in human foreskin and lung fibroblasts (Ahmed et al., 1995, Biochem. Biophys. Res. Commun. 208:871-878). Studies with highly specific A1- and A2a-adenosine receptor agonists provide indirect evidence that binding of adenosine activates Gs and Gi, after which Gs alpha interacts with beta gamma subunits released from Gi. The interaction of Gs alpha with beta gamma augments cyclic adenosine monophosphate (cAMP) accumulation, more than does Gs alpha alone. In the present study, we have provided direct evidence for a role of the beta gamma complex in the augmentation of cAMP accumulation by using a recombinant His6 fusion protein containing the carboxyl third of beta ARK1. This portion of beta ARK1 contains G beta gamma binding sequences and acts as a specific beta gamma scavenger (Koch et al., 1994, Proc. Natl. Acad. Sci. USA 91:12706-12710). In permeabilized fibroblasts, the His6 fusion protein inhibited the augmentation of cAMP accumulation resulting from adenosine binding to both A1 and A2a receptors. In addition, the specific G beta gamma scavenger inhibited the further rise in cellular cAMP levels caused by pretreating cells with pertussis toxin before incubation with adenosine. Finally, we observed that specific A1-adenosine receptor agonists augmented the cAMP accumulation stimulated by A2a-receptor agonists, and this cAMP augmentation was also suppressed by the G beta gamma scavenger. Similar results were obtained when the cells were treated with extracellular ATP and lysophosphatidic acid (LPA) to stimulate Gs and release G beta gamma subunits, respectively.

Evidence for a GTP-dependent increase in membrane permeability for calcium in NG108-15 microsomes.

The effect of GTP on Ca2+ uptake and release was studied in a microsomal fraction isolated from neuroblastoma x glioma hybrid NG108-15 cells. GTP did not alter the ATP-dependent initial uptake of Ca2+ but markedly enhanced the efflux of Ca2+ from microsomes. GTP-dependent Ca2+ release requires the presence of millimolar concentration of Mg2+. The effect of GTP was not mimicked by other nucleotides and was competitively blocked by the thiophosphate analogue of GTP, GTP gamma S but not by the non-hydrolyzable nucleotide GMP-PNP. Addition of an inhibiting concentration of GTP gamma S after completion of GTP-induced calcium release did not result in a re-uptake of Ca2+, showing the irreversibility of the releasing effect of GTP. Our data are consistent with the hypothesis of Ca2+-dependent GTP-induced opening of a channel responsible for vectorial transport of Ca2+ ions from one intracellular compartment to another. A model is proposed suggesting that the GTP-binding protein is a GTP-specific diacylglycerol kinase.

Stimulation of aged human lung fibroblasts by extracellular ATP via suppression of arachidonate metabolism.

The mitogenic effect of extracellular ATP on IMR-90 human fibroblasts subjected to in vitro aging was studied. ATP stimulated DNA synthesis and cell proliferation in young cells as much as epidermal growth factor (EGF) or insulin, while it stimulated aged cells to a much greater extent than seen for any other growth factor tested. When combined with EGF or insulin, ATP restored the greatly reduced mitogenic responsiveness of aged cells nearly to the level noted for young cells. Addition of prostaglandin E2 or other agents that elevate cAMP levels resulted in inhibition of DNA synthesis stimulated by EGF or insulin. Furthermore, the basal release of arachidonic acid and prostaglandin E2 and the endogenous levels of cAMP rose during aging and became much greater than in young cells. All three of these changes were suppressed by extracellular ATP. ATP-dependent suppression of cAMP accumulation was pertussis toxin-sensitive. Protein kinase C down-regulation inhibited arachidonate metabolism and enhanced DNA synthesis stimulated by ATP. These studies suggest that ATP exerts its mitogenic effect, especially on aged IMR-90 cells, at least partially by suppression of arachidonate metabolism.

Extracellular ATP and ADP stimulate proliferation of porcine aortic smooth muscle cells.

The mitogenic effect of extracellular ATP on porcine aortic smooth muscle cells (SMC) was examined. Stimulation of [3H]thymidine incorporation by ATP was dose-dependent; the maximal effect was obtained at 100 microM. ATP acted synergistically with insulin, IGF-1, EGF, PDGF, and various other mitogens. Incorporation of [3H]thymidine was correlated with the fraction of [3H]thymidine-labeled nuclei and changes in cell counts. The stimulation of proliferation was also determined by measurement of cellular DNA using bisbenzamide and by following the increase of mitochondrial dehydrogenase protein. The effect of ATP was not due to hydrolysis to adenosine, which shows synergism with ATP. ATP acted as a competence factor. The mitogenic effect of ATP, but not adenosine, was further increased by lysophosphatidate, phosphatidic acid, or norepinephrine. The inhibitor of adenosine deaminase, EHNA, stimulated the effect of adenosine but not ATP. The adenosine receptor antagonist theophylline depressed adenosine-induced mitogenesis. ADP and the non-hydrolyzable analogue adenosine 5'-[beta, gamma-imido]triphosphate (AMP-PNP) were equally mitogenic. Thus extracellular ATP stimulated mitogenesis of SMC via P2Y purinoceptors. The mechanism of ATP acting as a mitogen in SMC was further explored. Extracellular ATP stimulated the release of [3H]arachidonic acid (AA) and prostaglandin E2 (PGE2) into the medium, and enhanced cAMP accumulation in a dose-dependent fashion similar to ATP-induced [3H]thymidine incorporation. Inhibitors of the arachidonic acid metabolism pathway, quinacrine and indomethacin, partially inhibited the mitogenic effect of ATP but not of adenosine. Pertussis toxin inhibited ATP-stimulated DNA synthesis, AA release, PGE2 formation, and cAMP accumulation. Down-regulation of protein kinase C (PKC) by long-term exposure to phorbol dibutyrate (PDBu) partially prevented stimulation of DNA synthesis and activation of the AA pathway by ATP. The PKC inhibitor, staurosporine, antagonized mitogenesis stimulated by ATP. No synergistic effect was found when PDBu and ATP were added together. Therefore, a dual mechanism, including both arachidonic acid metabolism and PKC, is involved in ATP-mediated mitogenesis in SMC. In addition, ATP acted synergistically with angiotensin II, phospholipase C, serotonin, or carbachol to stimulate DNA synthesis. Finally, the possible physiological significance of ATP as a mitogen in SMC was further studied. The effect of endothelin and heparin, which are released from endothelial cells, on ATP-dependent mitogenesis was investigated. Extracellular ATP acted synergistically with endothelin to stimulate a greater extent of [3H]thymidine incorporation than was seen with PDGF plus endothelin. Heparin, believed to have a regulatory role, partially inhibited the stimulation of DNA synthesis caused both by ATP and PDGF.(ABSTRACT TRUNCATED AT 400 WORDS)

Extracellular ATP induces the release of calcium from intracellular stores without the activation of protein kinase C in Swiss 3T6 mouse fibroblasts.

Exposure of Swiss 3T6 mouse fibroblasts to extracellular ATP stimulated the formation of inositol phosphates and mobilized intracellular calcium. The mobilization of intracellular calcium was verified by imaging of fura-2 fluorescence in individual cells and by monitoring the efflux of 45Ca2+ from preloaded cells. However, we found no activation of protein kinase C as measured by phosphorylation of an 80-kDa acidic protein and by transmodulation of the receptor for epidermal growth factor. A careful examination of the kinetics of the phosphorylation reaction (from 30 sec to 10 min) revealed no activation of protein kinase C by extracellular ATP at any time. The lack of activation of protein kinase C was demonstrated even when a concentration of ATP 10-fold higher than that required to give a strong Ca2+ signal was used. Extracellular ATP did not inhibit protein kinase C activation by fetal bovine serum, platelet-derived growth factor, or phorbol esters. The effects of ATP were also produced by UTP but not by ADP, AMP, or adenosine. These findings demonstrate that it is possible to induce the mobilization of intracellular calcium by an inositol phosphate-mediated pathway without the activation of protein kinase C.

Control of membrane permeability by external and internal ATP in 3T6 cells grown in serum-free medium.

Cultures of 3T6 cells were plated in serum-free medium and grown in the presence of insulin (1 microgram/ml) and epidermal growth factor (0.5 ng/ml). External ATP (250 microM) applied to such cultures caused a rapid efflux of acid-soluble pools labeled with [3H]uridine, 2-deoxy[3H]glucose, or 86Rb+ and allowed the entry of p-nitrophenylphosphate. This increase in passive membrane permeability depended on ATP concentration, pH, and time of ATP contact with the cells, and it was not produced by GTP, UTP, or Pi. In the presence of compounds that decrease intracellular ATP, low concentrations of external ATP (40 microM) caused a massive synergistic stimulation of efflux. The efflux of acid-soluble pools was stopped (sealing) by bringing the cultures of 3T6 cells to neutral pH in the presence of Ca2+ and Mg2+. Exposure of 3T6 cells grown in serum-free medium to [gamma-32P]ATP under the conditions of permeabilization led to the selective labeling of a membrane protein with a molecular weight of 44,000 as revealed by NaDodSO4 polyacrylamide gel electrophoresis and autoradiography. The results show that the control of membrane permeability by ATP is completely independent of serum-deprived proteins. Furthermore, the protein band (Mr, 44 x 10(3)) that shows selective labeling by [32P]ATP during permeabilization is not an adsorbed serum component.

Partial purification of active delta and epsilon subunits of the membrane ATPase from escherichia coli.

We have partially purified active delta and epsilon subunits of the E. coli membrane-bound Mg2+-ATPase (ECF1). Treating purified ECF1 with 50% pyridine precipitates the major subunits (alpha, beta, and gamma) of the enzyme, but the two minor subunits (delta and epsilon), which are present in relatively small amounts, remain in solution. The delta and epsilon subunits were then resolved from one another by anion exchange chromatography. The partially purified epsilon strongly inhibits the hydrolytic activity of ECF1. The epsilon fraction inhibits both the highly purified five-subunit ATPase and the enzyme deficient in the delta subunit. The latter result indicates that the delta subunit is not required for inhibition by epsilon. By contrast, two-subunit enzyme, consisting chiefly of the alpha and beta subunits, was insensitive to the ATPase inhibitor, suggesting that the gamma subunit may be required for inhibition by epsilon. The partially purified delta subunit restored the capacity of ATPase deficient in delta to recombine with ATPase-depleted membranes and to reconstitute ATP-dependent transhydrogenase. Previously we reported (Biochem, Biophys. Res. Commun. 62:764 [1975]) that a fraction containing both the delta and epsilon subunits of ECF1 restored the capacity of ATPase missing delta to recombine with depleted membranes and to function as a coupling factor in oxidative phosphorylation and for the energized transhydrogenase. These reconstitution experiments using isolated subunits provide rather substantial evidence that the delta subunit is essential for attaching the ATPase to the membrane and that the epsilon subunit has a regulatory function as an inhibitor of the ATPase activity of ECF1.

Biochemical and cytochemical evidence for the polar concentration of periplasmic enzymes in a "minicell" strain of Escherichia coli.

A number of "surface" enzymes of Escherichia coli (i.e., among those selectively released by osmotic shock) all displayed higher specific activities in extracts of minicells than in extracts of typical rod forms; these enzymes included alkaline phosphatase, cyclic phosphodiesterase, acid hexose monophosphatase, 5'-nucleotidase, and ribonuclease I. In addition, alkaline phosphatase, cyclic phosphodiesterase, and acid hexose monophosphatase were cytochemically localized to regions of minicell periplasm that resembled reactive polar enlargements of the periplasm in rod forms. In contrast, a number of "internal" cytoplasmic enzymes (inorganic pyrophosphatase, beta-galactosidase, glutamine synthetase, polynucleotide phosphorylase, and ribonuclease II) showed elevated or similar specific activities in extracts of rod forms versus extracts of minicells. A specific heat-labile inhibitor for 5'-nucleotidase, known to occur in the cytoplasm, also showed no enrichment in minicells. These findings indicate that the "surface" enzymes are segregated in vivo into the terminal minicell buds, possibly because these enzymes are concentrated in the polar enlargements of the periplasm in typical rod forms.

Purification and properties of reconstitutively active and inactive adenosinetriphosphatase from Escherichia coli.

The Mg(2+)- and Ca(2+)-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, 2720-2724] and (b) a modified procedure described in this paper. The ATPase purified from E. coli K12 (lambda) by the first procedure had 4 subunits (alpha, beta, gamma, and epsilon). It did not bind to a deficient membrane, nor did it reconstitute ATP-driven transhydrogenase activity. Our modified procedure (b) yielded 5 subunits (alpha, beta, gamma, delta, and epsilon). This ATPase could bind to a deficient membrane and reconstitute ATP-driven transhydrogenase. This finding suggests that the delta 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 is a mitogen for 3T3, 3T6, and A431 cells and acts synergistically with other growth factors.

Extracellular ATP in concentrations of 5-50 microM 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)carboxamidoadenosine. Cultures of Swiss 3T3, Swiss 3T6, A431, DDT1-MF2, and HFF cells were used. The percent of cell nuclei labeled with [3H]thymidine and cell number were also increased. ADP was equally mitogenic, while UTP and ITP were much less active. The effect of ATP was not due to hydrolysis by ectoenzymes to form adenosine, a known growth factor. Thus, the nonhydrolyzable analogue adenosine 5'-[beta, gamma-imido]triphosphate was mitogenic. In addition, it was found that ATP showed synergism in 3T6 and 3T3 cells when present for only the first hour of an incorporation assay, during which time no significant hydrolysis occurred. Furthermore, prolonged preincubation of cells with ATP reduced the mitogenic response to ATP but not to adenosine; preincubation with adenosine or N6-(R-phenylisopropyl)adenosine had the reverse effect. Finally, the effect of adenosine, but not of ATP, was inhibited by aminophylline. We conclude that extracellular ATP is a mitogen that interacts with P2 purinoceptors on the plasma membrane.