Contact-mediated changes in ATPase activity at the surface of primary cultured hepatoma cells

Summary Hepatoma cells grown in monolayer culture display certain alterations in their Mg-ATPase activity present on the cell surface as a function of time during a exponential growth. Levels of enzyme estimated biochemically and expressed as activity per cell increase as the cell population density increases. Histochemical investigation shows that Mg-ATPase activity is located intensively on the surface of cell contact and the activity is not encountered on the cell surface facing the free space. No enzyme activity is detected histochemically on the cell surface of sparse culture. Deposits of acidic polysaccharide are also seen on the surface of cell contact.     

Membrane phosphatase activities in isolated and cultured adult rat hepatocytes

Membrane bound phosphohydrolysing enzymes, such as Na-K-ATPase, Mg-ATPase, ALPase and G-6-Pase were assayed in intact liver, in freshly isolated cells and in cultured hepatocytes to evaluate the effects of the isolation procedure and culture on these enzyme activities. Na-K-ATPase and Mg-ATPase are significantly reduced following cell dispersion while ALPase and G-6-Pase are nearly unaffected. During culture, Na-K-ATPase is restored to the "in vivo" level within the first two days, but rapidly declines in the following days. The Na dependent, energy requiring AIB uptake shows a similar pattern; Mg-ATPase is practically unmodified. A significant increase in ALPase activity and a net decrease of G-6-Pase activity, as a function of the culture time has been observed.     

Different effects of inhibitors on two mutants of Escherichia coli K12 affected in the Fo portion of the adenosine triphosphatase complex.

The effects of the inhibitors dicyclohexyl-carbodiimide (DCCD), bathophenanthroline and tertiary octylcatechol, on some enzyme activities in membranes from strains of Escherichia coli carrying mutations in the uncB or uncC genes have been studied. Membranes prepared from uncC mutants retain a normal DCCD-sensitive Mg2+-stimulated adenosine triphosphatase (Mg-ATPase) activity whereas in uncB mutants this enzyme activity is insensitive to DCCD. The membrane-bound Mg-ATPase activity from the uncC mutant strain, as compared with that from the normal strain, is only partially sensitive to the inhibitors bathophenanthroline or tertiary-octylcatechol. Both of these inhibitors stimulate the membrane-bound Mg-ATPase from uncB mutant strains. A DCCD-insensitive Mg-ATPase activity is found in the cytoplasmic fraction following cell disruption of either the uncB or the uncC mutants. The lipophilic chelators bathophenanthroline and tertiary-octylcatechol stimulate the activity of the 'soluble' Mg-ATPase in the uncB mutant but partially inhibit the activity in the uncC mutant. The NADH oxidase activities in membranes from both mutant and normal strains are strongly inhibited by tertiary-octylcatechol and bathophenanthroline but not by DCCD.     

Activation of Mg-ATPase (spectrin-dependent ATPase) by Ca2+.

The dependence of saponin-stimulated Mg-ATPase activity in the erythrocyte membrane on Ca2+ concentration was studied. In the membrane of freshly sampled human erythrocytes we found for this enzyme and Ca2+ an apparent dissociation constant of 0.611 mumol/l (SE +/- 0.106 mumol/l) and Hill coefficient of 0.93 (SE +/- 0.05). The enzyme is in most probability identical with Ca,Mg-ATPase of high affinity to Ca2+ described also as spectrin-dependent Ca,Mg-ATPase.     

Some properties of hog thyroidal membrane-bound adenosine tri-phosphatase: proteolytic activation of Mg-dependent activity.

The ATPase preparations from the hog thyroid was preincubated with various amounts of trypsin. The activity of Mg-ATPase was consistently elevated. On the contrary, the Na, K-ATPase activity decreased with increasing amounts of trypsin. The effects were similar to those which were observed in the enzyme preparations treated with basis polyamino acids as previously reported. This phenomenon seemed to be specific in the preparations from the thyroid. The Mg-dependent activity was increased after pretreatment with trypsin or poly-L-lysine (PLL) when CTP, ITP and UTP were used as substrate. Thus the substrate specificity of Mg-ATPase was low. The enzyme-kinetics using ATP as substrate showed that the increase in activity was due to an increase in Vmax and not to a change in Km. The activity of Mg-ATPase was increased even after 30 min of preincubation with trypsin, while the Na, K-ATPase activity was almost diminished. These results suggest that the activity of Mg-ATPase in the preparation from the thyroid is specifically changed by the modification of the molecular environment of the enzyme with trypsin or basic polyamino acids.     

Calmodulin-dependent regulation of Ca,Mg-ATPase activity in plasma membranes of the swine myometrium

Highly purified plasma membrane (PM) preparations of pig myometrium were found to contain 0.91 +/- 0.22 microgram calmodulin per mg of PM protein. Treatment of membranes with 1 mM EGTA in the presence of 0.2 M NaCl causes the diminution of the calmodulin content down to 3% of the original level. The activity of Ca, Mg-ATPase is thereby decreased by 40%. Exogenous calmodulin restores the enzyme activity up to 1.94 +/- +/- 0.30 mumol Pi/mg protein/hour. The maximal activation of Ca, Mg-ATPase is observed with 10(-7) M calmodulin. Calmodulin increases the total ATPase activity of myometrium PM without affecting the Mg-ATPase activity. Trifluoroperazine (20 microM) diminishes the activating effect of exogenous calmodulin on Ca, Mg-ATPase. Calmodulin stimulates Ca, Mg-ATPase at low concentrations of Ca2+(10(-8)-10(-6) M) by decreasing Km for Ca2+ from 0.4.10(-6) M to 2.10(-8) M as well as by increasing Vmax--from 0,8 to 1.42 mumol Pl/mg protein/hour. It is supposed that the activating effect of calmodulin on Ca, Mg-ATPase is based on electrostatic interactions of Ca2+-free calmodulin with the enzyme.     

Specific in vitro activation of Ca,Mg-ATPase by vitamin D-dependent rat renal calcium binding protein (calbindin D28K)

The vitamin D-dependent, calcium-binding protein from rat kidney, calbindin D28k (renal CaBP) specifically stimulates Ca,Mg-ATPase activity of human erythrocyte plasma membranes in a dose-dependent, calcium-sensitive manner. This stimulation was about two-fold compared to a three-fold stimulation by calmodulin. The effect was specific since other calcium-binding proteins and low molecular weight proteins did not stimulate Ca,Mg-ATPase activity. Renal CaBP did not stimulate cyclic nucleotide phosphodiesterase at concentrations greater than those which stimulated Ca,Mg-ATPase activity. This is the first report of a specific in vitro effect of renal CaBP on an enzyme system.     

Tributyltin inhibits the oligomycin-sensitive Mg-ATPase activity in Mytilus galloprovincialis digestive gland mitochondria

Tributyltin (TBT), widely employed in the past in antifouling paints, is one of the most toxic organic pollutants. Although recently banned, it still threatens coastal water ecosystems and accumulates in filter-feeding molluscs. TBT is known to act as a membrane-active toxicant; however data on mussels are scanty and exposure effects on mitochondrial ATPase activities remain hitherto unexplored. TBT effects on the mitochondrial Mg-ATPase activities in the digestive gland of Mytilus galloprovincialis were investigated both in vitro and in TBT-exposed mussels. Both an oligomycin-sensitive Mg-ATPase (OS Mg-ATPase) (70% of total Mg-ATPase activity) and an oligomycin-insensitive ATPase (OI Mg-ATPase) (30%) were found. The OS-Mg-ATPase was as much as 70% in vitro inhibited by 0.7 μM (203 μg/L) TBT, while higher concentrations promoted a partial inhibition release up to 5.0 μM TBT; higher than 10.0 μM TBT concentrations yielded nearly complete enzyme inhibition. Concentrations higher than 1 μM TBT enhanced the OI Mg-ATPase. Mussels exposed to 0.5 and 1.0 μg/L TBT in aquaria showed a 30% depressed OS Mg-ATPase activity, irrespective of TBT dose and exposure time (24 and 120 h). The OI Mg-ATPase activity was apparently refractory to TBT exposure and halved both in control and TBT-exposed mussels after 120 h exposure.     

cAMP-dependent regulation of the active transport of Ca2+ in plasma membranes of the swine myometrium

Plasma membranes of pig myometrium show the ability for endogenous phosphorylation (160 +/- 45 pmol 32P/mg.min); the initial rate of this process increases 2.5-fold in the presence of 10(-6) cAMP. Micromolar concentrations of cAMP activate the ATP-dependent transport of Ca2+ in myometrium plasma membranes; cAMP at concentrations of 10(-9)-10(-4) M has no effect on Ca,Mg-ATPase. Myometrium plasma membranes possess the Mg2+-dependent phosphatase activity. Dephosphorylation of membranes is accompanied by a decrease (by 25-50%) of the Ca,Mg-ATPase activity and Ca2+ uptake, respectively. The exogenous catalytic subunit of cAMP-dependent protein kinase increases the activity of Ca,Mg-ATPase in native and dephosphorylated membranes. Tolbutamide diminishes the activity of Ca,Mg-ATPase in native membranes by 25% without causing any appreciable influence on the enzyme activity in dephosphorylated membranes. Taking into account the similarity of dependence of Ca2+ uptake on Ca2+ concentration in native and cAMP-phosphorylated vesicles, it can be assumed that the cAMP-dependent phosphorylation affects the enzyme turnover number but not its affinity for Ca2+. The dephosphorylation-induced inhibition of Ca,Mg-ATPase activity and accumulation of Ca2+ are reversible processes.     

Comparison of the rat microsomal Mg-ATPase of various tissues

The microsomal Mg-ATPase from various rat tissues was compared. After fractionating the microsomal vesicles by sucrose gradient centrifugation, the highest specific activity of the Mg-ATPase was found in the low-density vesicles which contained plasma membrane. A large fraction (25-90%) of the microsomal Ca-independent Mg-ATPase found in each tissue had the following properties: (1) the Km for ATP was 0.2 mM; (2) the rate of ATP hydrolysis by the Mg-ATPase was nonlinear due to an ATP-stimulated inactivation of the enzyme; (3) wheat germ agglutinin, concanavalin A, glutaraldehyde, and antiserum prevented inactivation induced by ATP or AdoPP[NH]P; (4) detergents at relatively low detergent:protein ratios increased the rate of inactivation with little change in the initial rate of ATP hydrolysis; (5) the Mg-ATPase was inactivated by irradiation in the presence of 8-azido ATP. (6) in addition to ATP, the Mg-ATPase was able to hydrolyze CTP, GTP, UTP, ITP, and GTP but was unable to hydrolyze any of the 10 nonnucleotide phosphocompounds which were tested; (7) the bivalent cation requirement of the Mg-ATPase could be provided by Mg2+, Ca2+, Mn2+, Zn2+, or Co2+ but the enzyme was inactive in the presence of Cu2+, Sr2+, Ba2+, or Be2+; (8) the Mg-ATPase activity was not altered by ionophores or inhibitors of the Na,K-ATPase, the Ca,Mg-ATPase or the mitochondrial F1ATPase. These data suggest that a major portion of the microsomal, basal Mg-ATPase activity is due to one unique enzyme found in most if not all tissues.     

Effect of cell contact on regionalization of mouse embryos

Cytochemical demonstrations of 5'-nucleotidase and alkaline phosphatase reveal the activity of these enzymes on regions of cell apposition from the late four-cell stage onward. These enzyme activities also appear on regions of artificial cell contact between aggregated embryos having more than four cells. Cytochemistry of single two-cell embryos does not reveal 5'-nucleotidase nor alkaline phosphatase activity, however, these enzyme activities appear at both the artificial and natural contacts in chimaeras of two two-cell embryos. We interpret these results as meaning: (1) that cell contact causes the regionalization of 5'-nucleotidase and alkaline phosphatase activity on the cell surface, (2) that these enzyme activities can be induced or enhanced by contact between two two-cell embryos, (3) that a signal is transmitted from the artificial to the natural contact.     

Mg-ATPase and Torpedo Cholinergic Synaptic Vesicles

The reported presence of Mg-ATPase activity in cholinergic synaptic vesicles from the electric organ of Torpedo marmorata was reinvestigated in view of possible contamination of vesicles by other subcellular fractions. After dilution in concentrated sucrose, the vesicular fraction isolated on a sedimentation sucrose gradient was purified further on a flotation density gradient. It appears that this treatment allows separation of the vesicles according to their content. The two vesicular content markers, acetylcholine and ATP, are recovered as sharp coincident peaks at a density close to 0.48 M sucrose. Empty vesicles are identified in denser regions by the protein pattern on gel electrophoresis which is identical to the pattern obtained for filled vesicles. Refractionation of vesicles depleted of their acetylcholine content by valinomycin leads to an extreme picture, with a massive shift of the vesicles toward denser regions. We have then shown that a ouabain-insensitive Mg-ATPase is indeed associated with the vesicle membrane, but the activity is fully apparent only when vesicles are permeabilized either as the result of the fractionation procedure or after detergent treatment. The relative insensitivity of the Mg-ATPase associated with the synaptic vesicles to oligomycin, N,N'-dicyclohexylcarbodiimide, and azide indicates that this enzyme differs from the classic F1F0 mitochondrial enzyme. The most striking finding is the sensitivity to vanadate of the vesicular Mg-ATPase, which suggests the involvement of a phosphorylated intermediate. On the basis of both the difference in inhibitor sensitivity between untreated and detergent-treated vesicles and of the pronase experiments, the possibility that the enzyme has an inward orientation is discussed.     

Evidence for the presence of H+-ATPase in the membrane of brain synaptic vesicles in the rat

Mg-ATPase of rat brain synaptic vesicles (SV) is considerably (by 85%) inhibited by dicyclohexyl carbodiimide (200 microM), a blocker of proton pumps, whereas orthovanadate (100 microM) does not produce any influence on the enzyme. Oligomycin (5 micrograms/ml) does not alter Mg-ATPase activity of the SV, whereas N-ethylmaleimide (300 microM) reduces it to a moderate degree, namely by 35%. This indicates that Mg-ATPase of the SV differs from mitochondrial ATPase. The protonophore p-trichloromethoxycarbonyl cyanide phenylhydrazone (20 microM) and bicarbonate anions (20 mM) stimulate slightly (by 12 to 25%) Mg-ATPase of the SV. Bicarbonate (20 mM) raises 1.8-2.1-fold Mg-ATPase activity of the mitochondria isolated from rat brain. It is assumed that the membrane of brain SV contains proton ATPase (H+-ATPase) differing from mitochondrial H+-ATPase in some of the properties.     

Density-dependent regulation of cell surface gamma-glutamyl transpeptidase in cultured glial cells.

A decline in cell surface gamma-glutamyl transpeptidase specific activity was previously observed to be concomitant with C6 glial cell proliferation. To elucidate the underlying factor(s) mediating gamma-glutamyl transpeptidase down-regulation, the effects of C6 cell density and culture conditions on cell surface transpeptidase activity levels were investigated. After 24 h of culture, the transpeptidase specific activities were inversely related to the initial plating densities. The lower-density cultures showed an induction within 24 h of plating. As the cultures proliferated, the specific transpeptidase activities declined to a common low level at post-confluency. The gamma-glutamyl transpeptidase down-regulation was unrelated to cell growth rate and was most pronounced during logarithmic proliferation. Induction and down-regulation of gamma-glutamyl transpeptidase activity at low cell densities were not a result of trypsinization. Supplementation of low-density cultures with conditioned medium, use of matrix-coated wells, or periodic replacement of growth media to prevent conditioning had minor effects on the decline of cell surface activity. Kinetic analysis showed that the Michaelis constants and the reaction mechanism were unaltered by cell density, indicating that down-regulation was not due to allosteric factors or an alteration in enzyme character. A reduction in the maximal velocity of cell surface transpeptidation at higher cell densities suggested that gamma-glutamyl transpeptidase down-regulation is related to the concentration of enzyme at the cell surface. Immunocytochemical localization of gamma-glutamyl transpeptidase demonstrated that gamma-glutamyl transpeptidase antigen levels decrease as C6 cell density increases. These results led us to propose that cell-cell contact stimulates the disappearance of gamma-glutamyl transpeptidase from the surface of cultured C6 glial cells.     

Ultrastructural cytochemical studies of plasma membrane phosphatase activities during the HeLa S3 cell cycle.

Alkaline phosphatase (AP), 5'-nucleotidase (5'N), Mg2+-activated ATPase (Mg-ATPase) and Ca2+-activated ATPase (Ca-ATPase) were studied in sychronized HeLa S3 cells with cytochemical methods and electron microscopy. It was found that AP activity, as determined by the deposition of lead phosphate reaction product (r.p.) was most active in mitotic (M), early and middle G1 cells, less active in late G1 and almost undetectable in S phase cells. Most AP enzyme activity was found to be associated with undulations (mainly microvilli) of the plasma membrane. Fluctuations and the redistribution of 5'N were also observed; the reaction for 5'N was positive in all phases of the cell cycle studied, it was strongest in M cells and in the majority of middle G1 cells. Mg-ATPase activity was present in the plasma membranes of cells throughout the cell cycle, but did not show noticeable fluctuations in activity and distribution. Ca-ATPase activity appeared in plasma membranes and in limited areas of cell nuclei but was evident only in S phase cells. The results of the present study confirm and extend previous biochemical observations and indicate that changes in membrane phosphate activities are associated with enzyme activity redistributions within the plasma membrane during the HeLa S3 cell cycle.     

Activity and thermal stability of the enzymes of energy support in the common frog

The activity and heat resistance of succinate dehydrogenase (SDG), Na, K-ATPase and Mg-ATPase of the grass frog (39 specimens) have been determined. No correlation was found between individual levels of heat resistance both of either enzyme examined and of the same enzyme but taken from different tissues (SDG of liver and muscles). The average level of heat resistance of SDG in liver is significantly higher than that in m. gastrocnemius. A statistically significant correlation was observed between individual levels of enzyme activity in the internal organs (SDG of liver, Na, K-ATPase and Mg-ATPase of kidney). The activity of SDG of muscles does not correlate with that of either partner.     

Differential Response of Adenylate Cyclase and ATPase Activities After Chronic Ethanol Exposure of PC12 Cells

The direct effects of chronic ethanol administration on adenylate cyclase, Na,K-ATPase, and Mg-ATPase activities in a cell containing neuronal characteristics were investigated using PC12 pheochromocytoma cells. Exposure of PC12 cells to 0, 75, and 150 mM ethanol for 4 days caused a dose-dependent increase in the stimulation of adenylate cyclase by in vitro ethanol without altering activation of the enzyme by GTP, NaF, MnCl2, or 2-chloroadenosine. Conversely, a 4-day treatment with 150 mM ethanol increased Na,K-ATPase and Mg-ATPase activities without altering the inhibitory effects of in vitro ethanol. The increase in Na,K-ATPase activity was associated with an increase in Vmax without any change in the Km for KCl. Chronic ethanol exposure also increased the amount of [3H]ouabain specifically bound to PC12 cell membranes. Except for the increase in Mg-ATPase activity, the above results were also observed when chronic ethanol treatment was carried out in the presence of pyrazole. Although ethanol slowed PC12 cell growth, observed changes were not due to an ethanol-induced reduction in cellular density. A 4-day exposure of a nonneuronal cell line (Madin Darby canine kidney cell) to 150 mM ethanol did not alter adenylate cyclase or ATPase activities. The present study indicates that the direct effects of chronic ethanol exposure of a neuronal-like cell involve an increase in the density of sodium pumps per cell and an enhanced sensitivity of adenylate cyclase to activation by ethanol.     

Characterization of Mg-ATPase and (Na+ + K+)-stimulated Mg-ATPase in smooth muscular cells of the sheep's common carotid artery.

The Mg-ATPase and (Na+ + K+)-stimulated Mg-ATPase in the mitochondrial and microsomal fraction of smooth muscular cells of the sheep's common carotid artery have been characterized in more detail. Optimal enzyme activities were found for all ATPases to be at pH 7.5-8.0 and 45 degrees C-50 degrees C. The energies of activation were found to be at 5-9 kcal/mole for both ATPases. Two-thirds of the (Na+ + K+)-stimulated Mg-ATPase were found to be ouabain-sensitive and thus attributed to the coupled (Na, K)-transport system. The pI 50 values of ouabain for microsomal and mitochondrial fractions are 6.3 and 6.0, respectively. The highest activity of (Na+ + K+)-stimulated Mg-ATPase is at 5-10 mM K+ and more than 50 mM Na+. One-third of the (Na+ + K+)-stimulated Mg-ATPase activity was found to be due to a stimulation of Mg-ATPase by Na+ alone, which is not inhibited by ouabain. The relationship of this activity to the ouabain-sensitive part of the (Na+ + K+)-stimulated Mg-ATPase and to Na+-transport is discussed. For the Mg-ATPases apparent KM(ATP) values were determined to be 1.4 and 1.0 mM, resp., and for the (Na+ + K+)-stimulated Mg-ATPases 0.15 and 0.14 mM, resp.     

Dependence of alkaline phosphatase activity in human cultured fibroblasts on the proliferative status of the culture

The alkaline phosphatase activity was studied in embryonic lung human fibroblasts grown in vitro. The total enzyme activity per flask was constant during the log-phase of culture growth to increase significantly after confluence. The increase in the activity did not depend on the rate of culture growth, and was seen to start after the mean density equal to (37.5 +/- 5.9) X 10(3) cells/cm2 flask surface. The stimulation of cell growth may presumably slow down the synthesis of alkaline phosphatase, which resumes after the contact inhibition of DNA replication.     

Post-mortem changes in cytochemical localization and enzymological measurement of marker enzymes of the mitochondria, SDH and Mg-ATPase, of porcine muscle stored at 4 degrees C, -18 degrees C, or -80 degrees C

As a series of studies on postmortem changes in the fine structure of porcine muscle, activity of two mitochondrial marker enzymes, succinate dehydrogenase (SDH) and magnesium dependent adenosine triphosphatase (Mg-ATPase), was measured and localized in cardiac, red and white muscles stored at 4 degrees C, -18 degrees C or -80 degrees C. The postmortem loss of SDH activity was most remarkable in cardiac muscle. The variation of SDH activity was proportional to the amount of absolute activity. The postmortem change of Mg-ATPase was more variable than SFH, though the activity was well preserved up to 15 weeks in all three types of porcine muscle stored at -80 degrees C. The loss of Mg-ATPase was most remarkable in red muscle stored at -18 degrees C or -80 degrees C. Cytochemical localization of SDH was between the outer and the inner mitochondrial membranes while that of Mg-ATPase was on the inner surface or matrix side of the inner membrane. Those localization was not altered by the difference in temperature and the duration of storage.