Lipocortin (Annexin) I heterotetramer binds to purine RNA and pyrimidine DNA.

Lipocortin I-like protein with a molecular weight of 94,000 Da as judged by Western analysis was found to bind to ssDNA rather than to dsDNA in a Ca(2+)-dependent manner. This protein was also bound to [(32)P]poly(rA) and [(32)P]poly(rG) as measured by EMSA. Poly(rG), poly(rA), poly(dC), and poly(dT) were competitive against binding of either [(32)P]poly(rA) or [(32)P]poly(rG), while poly(rC), poly(rU), and poly(dA) were less effective binding competitors. The binding of this protein to poly(rA) or poly(rG) was inhibited by immunoprecipitable anti-lipocortin I (calpactin II) and anti-S100 protein antibodies, but not by an anti-Ig antibody. Phospholipids such as phosphatidylserine and phosphatidylinositol enhanced the binding of lipocortin I to poly(rA). Taken together, our present observations suggest that the lipocortin I-S100 protein heterotetramer binds to either purine RNAs or pyrimidine ssDNAs in a Ca(2+)- and phospholipid-dependent manner.     

Monoclonal antibodies specific to a Ca2(+)-bound form of lipocortin I distinguish its Ca2(+)-dependent phospholipid-binding ability from its ability to inhibit phospholipase A2.

Lipocortin I, a Ca2(+)-and phospholipid-binding protein without EF-hand structures, has many biological effects in vitro. Its actual role in vivo, however is unknown. We obtained and characterized five monoclonal antibodies to lipocortin I. Two of these monoclonal antibodies (L2 and L4-MAbs) reacted with the Ca(+)-bound form of lipocortin I, but not with the Ca2(+)-free form, both in vivo and in vitro. Lipocortin I required greater than or equal to 10 microM-Ca2+ to bind the two antibodies, and this Ca2+ requirement was not affected by phosphatidylserine. L2-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I and inhibited its binding to Escherichia coli membranes and to phosphatidylserine in vitro. L4-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I, but did not affect its binding to E. coli membranes or to phosphatidylserine. These findings indicated that the inhibition of phospholipase A2 by lipocortin I was not simply due to removal or capping of the substrates in E. coli membranes. Furthermore, an immunofluorescence study using L2-MAb showed the actual existence of Ca2(+)-bound form of lipocortin I in vivo.     

DNA strand transfer catalyzed by the 5'-3' exonuclease domain of Escherichia coli DNA polymerase I.

A protein which promotes DNA strand transfer between linear double-stranded M13mp19 DNA and single-stranded viral M13mp19 DNA has been isolated from recA- E.coli. The protein is DNA polymerase I. Strand transfer activity residues in the small fragment encoding the 5'-3' exonuclease and can be detected using a recombinant protein comprising the first 324 amino acids encoded by polA. Either the recombinant 5'-3' exonuclease or intact DNA polymerase I can catalyze joint molecule formation, in reactions requiring only Mg2+ and homologous DNA substrates. Both kinds of reactions are unaffected by added ATP. Electron microscopy shows that the joint molecules formed in these reactions bear displaced single strands and therefore this reaction is not simply promoted by annealing of exonuclease-gapped molecules. The pairing reaction is also polar and displaces the 5'-end of the non-complementary strand, extending the heteroduplex joint in a 5'-3' direction relative to the displaced strand. Thus strand transfer occurs with the same polarity as nick translation. These results show that E.coli, like many eukaryotes, possesses a protein which can promote ATP-independent strand-transfer reactions and raises questions concerning the possible biological role of this function.     

Inhibition of Acanthamoeba myosin I heavy chain kinase by Ca(2+)-calmodulin.

The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosins I depends on phosphorylation of their single heavy chains by myosin I heavy chain kinase. Kinase activity is enhanced > 50-fold by autophosphorylation at multiple sites. The rate of kinase autophosphorylation is increased approximately 20-fold by acidic phospholipids independent of the presence of Ca2+ and diglycerides. We show in this paper that Ca(2+)-calmodulin inhibits phospholipid-stimulated autophosphorylation of myosin I heavy chain kinase and hence also inhibits the catalytic activity of unphosphorylated kinase in the presence of phospholipid. Ca(2+)-calmodulin does not inhibit kinase activity in the absence of phospholipid. Micromolar Ca(2+)-calmodulin also inhibits binding of myosin I heavy chain kinase to phospholipid vesicles and purified plasma membranes. Proteolytic removal of a 7-kDa NH2-terminal segment from the 97-kDa kinase prevents binding of both calmodulin and phospholipid; therefore, we propose that they bind to the same or overlapping sites. These data provide a mechanism by which Ca2+ could inhibit the actin-activated Mg(2+)-ATPase activity of the myosin I isozymes in vivo and thus regulate myosin I-dependent motile activities.     

Functional Approach to the Catalytic Site of the Sarcoplasmic Reticulum Ca2+-ATPase: Binding and Hydrolysis of ATP in the Absence of Ca2+

Isolated sarcoplasmic reticulum vesicles in the presence of Mg(2+) and absence of Ca(2+) retain significant ATP hydrolytic activity that can be attributed to the Ca(2+)-ATPase protein. At neutral pH and the presence of 5 mM Mg(2+), the dependence of the hydrolysis rate on a linear ATP concentration scale can be fitted by a single hyperbolic function. MgATP hydrolysis is inhibited by either free Mg(2+) or free ATP. The rate of ATP hydrolysis is not perturbed by vanadate, whereas the rate of p-nitrophenyl phosphate hydrolysis is not altered by a nonhydrolyzable ATP analog. ATP binding affinity at neutral pH and in a Ca(2+)-free medium is increased by Mg(2+) but decreased by vanadate when Mg(2+) is present. It is suggested that MgATP hydrolysis in the absence of Ca(2+) requires some optimal adjustment of the enzyme cytoplasmic domains. The Ca(2+)-independent activity is operative at basal levels of cytoplasmic Ca(2+) or when the Ca(2+) binding transition is impeded.     

Expression of annexins as a function of cellular growth state

Annexins are a structurally related family of Ca2+ binding proteins of undertermined biological function. Annexin I (also called lipocortin 1) is a substrate for the EGF-stimulated tyrosine kinase and is postulated to be involved in mitogenic signal transduction. To investigate further the involvement of lipocortin 1 in cell proliferation, we measured lipocortin 1 levels in normal diploid human foreskin fibroblasts (HFF) to determine whether its expression changed as a function of growth status. For comparison, the expression of annexin V (also called endonexin II) was measured in HFF cells. Endonexin II is a protein with similar Ca2+ and phospholipid binding properties as lipocortin 1, but it is not a substrate for tyrosine kinases. Quiescent HFF cell cultures were induced to proliferate by either subculture to lower cell density, EGF stimulation, or serum stimulation. In all three protocols, proliferating HFF cells contained three- to fourfold higher levels of lipocortin 1 and three- to fourfold lower levels of endonexin II than quiescent HFF cells. In contrast, the expression of annexin II (also called calpactin I) and annexin IV (also called endonexin I) remained relatively unchanged in growing and quiescent HFF cells. Lipocortin 1 synthesis rate was eightfold higher and its turnover rate was 1.5-fold slower in proliferating compared to quiescent HFF cells. Endonexin II synthesis rate remained constant but its turnover rate was 2.2-fold faster in proliferating compared to quiescent HFF cells. In a separate set of experiments, annexin expression levels were measured in cultures of rat PC-12 cells, a pheochromocytoma that ceases proliferation and undergoes reversible differentiation into nondividing neuronlike cells in response to nerve growth factor (NGF). After NGF treatment, PC-12 cells expressed fivefold higher levels of endonexin II and 32-fold higher levels of calpactin 1. Lipocortin 1 and endonexin I were not expressed in PC-12 cells. In summary, lipocortin 1 expression exhibited a positive correlation with cell proliferation in HFF cells. The increased expression of endonexin II in quiescent HFF cells and differentiating PC-12 cells implies that this protein may play a more prominent role in nondividing cells.     

Location of tryptophans in membrane-bound annexins

The annexins are a novel class of calcium-dependent membrane binding proteins with highly homologous sequences and similar binding characteristics. In order to better define structural parameters of the membrane-bound form, the localization of tryptophan residues in several of these proteins was studied by use of quenchers of their intrinsic fluorescence. Lipocortin I contains a single tryptophan located near its N-terminus, while the single tryptophan in lipocortin V is located in a repeated consensus sequence. Calcium-dependent binding to vesicles composed of 50% egg phosphatidylcholine and 50% bovine brain phosphatidylserine was accompanied by an increase in emission intensity resulting from a relief of internal quenching. The tryptophan fluorescence of bound lipocortin I was nearly unaffected by substituting the quencher 1-palmitoyl-2-(5-doxylstearoyl)-sn-glycero-3-phosphocholine (5-PC) for egg phosphatidylcholine, while that of the lipocortin V tryptophan was quenched significantly. With the quenching doxyl spin-label located deeper in the bilayer at the 12- and 16-positions of the acyl chain, the quenching was progressively weaker, suggesting an interfacial location for the tryptophan of lipocortin V. The same experiments with lipocortin I show almost no quenching in any case, suggesting that this tryptophan near the amino terminus is protected or oriented away from the membrane surface. Data on the bovine liver calelectrins are also presented showing that endonexin also has a tryptophan residue that interacts strongly with phospholipids.     

Voltage-dependent modulation of L-type calcium currents by intracellular magnesium in rat ventricular myocytes

Effects of changing cytosolic free Mg(2+) concentration on L-type Ca(2+) (I(Ca)) and Ba(2+) currents (I(Ba)) were investigated in rat ventricular myocytes voltage-clamped with pipettes containing 0.2 or 1.8mM [Mg(2+)] ([Mg(2+)](p)) buffered with 30mM citrate and 10mM ATP. Increasing [Mg(2+)](p) from 0.2 to 1.8mM reduced current amplitude and accelerated its decay under a variety of experimental conditions. To investigate the mechanism for these effects, steady-state and instantaneous current-voltage relationships were studied with two-pulse and tail current (I(T)) protocols, respectively. Increasing [Mg(2+)](p) shifted the V(M) for half inactivation by -20mV but dramatically decreased I(Ca) amplitude at all potentials tested, consistent with a change in gating kinetics that decreases channel availability. This conclusion was supported by analysis of I(T) amplitude, but these latter experiments also suggested that, in the millimolar concentration range, [Mg(2+)](p) might also inhibit permeation through open Ca(2+) channels at positive V(M).     

Tumor promoter-dependent phosphorylation of a Triton X-100 extractable form of lipocortin I in T51B rat liver cells

The phosphorylation of lipocortin (a substrate of EGF-receptor kinase, and a putative phospholipase A2 inhibitor) was examined in T51B cells. By using Western blot procedures and antisera specific to lipocortin I, we found that most immunoreactive lipocortin I was located in the cytosol (lipocortin(cvt] of cells extracted in Ca2+-free buffers These cells however had another pool of immunoreactive lipocortin I located in the particulate fraction that was Triton X-100 extractable (lipocortin(mem]. Increasing Ca2+ concentrations in the extraction buffer resulted in more lipocortin(mem) recovered. In vitro phosphorylation of endogenous proteins demonstrated that lipocortin I became phosphorylated in a Ca2+ and phosphatidylserine-dependent manner, suggesting an involvement of protein kinase C. Treatment of cells with 100 ng/ml 12-0-tetradecanoylphorbol-13-acetate (TPA) but not with 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD) resulted in the in vitro phosphorylation of lipocortin(mem) by protein kinase C. TPA also increased the phosphorylation of lipocortin(mem) in [32P]phosphate-labeled cells.     

Mg(2+) inhibits ATP-activated current mediated by rat P2X4 receptors expressed in Xenopus oocytes

To investigate the modulation of Mg(2+) on rat P2X4 receptors and its underlying mechanism, we transcribed cDNA coding for wild-type and mutant P2X4 receptors to cRNA in vitro, injected the cRNA to oocytes of Xenopus laevis using the microinjection technique and revealed the effect of Mg(2+) on ATP-activated currents (I(ATP)) mediated by P2X4 receptors using the two-electrode whole-cell voltage clamp technique. The effects of extracellular Mg(2+) on I(ATP) were as follows: (1) In oocytes expressing P2X4 receptors, Mg(2+) with concentration ranging from 0.5-10 mmol/L inhibited the amplitude of I(ATP) in a concentration-dependent and reversible manner, with a 50% inhibitory concentration value (IC(50)) of (1.24 ± 0.07) mmol/L for current activated by 100 μmol/L ATP. (2) Mg(2+) (1 mmol/L) shifted the dose-response curve for I(ATP) right-downward without changing the EC(50), but reduced the maximal current (E(max)) by (42.0 ± 2.1)%. (3) After being preincubated with Mg(2+) for 80 s, the inhibitory effect of the Mg(2+) on I(ATP) reached the maximum. (4) The inhibition of Mg(2+) on I(ATP) was independent of membrane potential from -120 mV to +60 mV. (5) Compared with the current activated by 100 μmol/L ATP in the wild-type P2X4 receptors, mutant P2X4 D280Q responded to the application of 100 μmol/L ATP with a smaller current. The peak current was only (4.12 ± 0.15)% of that seen in wild-type receptors. Mutant P2X4 D280E responded to ATP stimulation with a current similar to that observed in cells expressing wild-type receptors. (6) When Asp280 was removed from P2X4, the current amplitude of I(ATP) was increased almost one-fold, and Mg(2+) with concentration ranging from 0.5-10 mmol/L did not affect the I(ATP) significantly. The results suggest that Mg(2+) inhibits I(ATP) mediated by P2X4 receptors non-competitively, reversibly, concentration-dependently, time-dependently and voltage-independently. The inhibitory effect of Mg(2+) might be realized by acting on the site Asp280 of the P2X4 receptors.     

Enhancement of calcium sensitivity of lipocortin I in phospholipid binding induced by limited proteolysis and phosphorylation at the amino terminus as analyzed by phospholipid affinity column chromatography

A phospholipid column was prepared by coating siliconized porous glass beads with phospholipids. The analysis of the Ca2+ requirement of lipocortin I and its derivatives in the binding to phospholipids was carried out with this column. The Ca2+ concentration required for 50% binding to the phospholipid column at room temperature was about 30 microM for lipocortin I, while that was reduced to 15 microM when lipocortin I was phosphorylated by the epidermal growth factor receptor/kinase, and a further reduction in the Ca2+ requirement was observed with proteolytic cleavage at the N-terminal region. Cathepsin D and calpain I (low calcium-requiring form of calcium-activated neutral protease) rapidly cleaved human placental lipocortin I at Trp-12 and Lys-26, respectively. These N-terminal-truncated proteins required only 5 microM Ca2+ for 50% binding to the phospholipid column. This enhancement of Ca2+ sensitivity by limited proteolysis was also observed for porcine lung lipocortin I. Essentially the same results were obtained when the Ca2+ sensitivities of the modified lipocortins I were analyzed using dispersed phospholipid vesicles instead of the phospholipid affinity column. Equilibrium dialysis indicated that the release of the N-terminal region markedly increased the affinity of lipocortin I for Ca2+ in the presence of phosphatidylserine, without any appreciable change of the number of Ca2+-binding sites. Limited proteolysis by endogenous proteases such as calpain may be an important regulatory mechanism for the Ca2+ sensitivity of lipocortin I in phospholipid binding.     

Reductions in external divalent cations evoke novel voltage-gated currents in sensory neurons

It has long been recognized that divalent cations modulate cell excitability. Sensory nerve excitability is of critical importance to peripheral diseases associated with pain, sensory dysfunction and evoked reflexes. Thus we have studied the role these cations play on dissociated sensory nerve activity. Withdrawal of both Mg(2+) and Ca(2+) from external solutions activates over 90% of dissociated mouse sensory neurons. Imaging studies demonstrate a Na(+) influx that then causes depolarization-mediated activation of voltage-gated Ca(2+) channels (Ca(V)), which allows Ca(2+) influx upon divalent re-introduction. Inhibition of Ca(V) (ω-conotoxin, nifedipine) or Na(V) (tetrodotoxin, lidocaine) fails to reduce the Na(+) influx. The Ca(2+) influx is inhibited by Ca(V) inhibitors but not by TRPM7 inhibition (spermine) or store-operated channel inhibition (SKF96365). Withdrawal of either Mg(2+) or Ca(2+) alone fails to evoke cation influxes in vagal sensory neurons. In electrophysiological studies of dissociated mouse vagal sensory neurons, withdrawal of both Mg(2+) and Ca(2+) from external solutions evokes a large slowly-inactivating voltage-gated current (I(DF)) that cannot be accounted for by an increased negative surface potential. Withdrawal of Ca(2+) alone fails to evoke I(DF). Evidence suggests I(DF) is a non-selective cation current. The I(DF) is not reduced by inhibition of Na(V) (lidocaine, riluzole), Ca(V) (cilnidipine, nifedipine), K(V) (tetraethylammonium, 4-aminopyridine) or TRPM7 channels (spermine). In summary, sensory neurons express a novel voltage-gated cation channel that is inhibited by external Ca(2+) (IC(50)～0.5 μM) or Mg(2+) (IC(50)～3 μM). Activation of this putative channel evokes substantial cation fluxes in sensory neurons.     

Calcium-induced intracellular cross-linking of lipocortin I by tissue transglutaminase in A431 cells. Augmentation by membrane phospholipids.

Covalently cross-linked multimers of lipocortin I are shown to be present in human epidermoid carcinoma A431 cells treated with epidermal growth factor or the calcium ionophore A23187. This intracellular cross-linking of lipocortin I is suggested to be mediated by the action of tissue transglutaminase, a Ca2(+)-dependent protein cross-linking enzyme. Cross-linking of lipocortin I competes with proteolytic digestion of the protein, and pretreatment of the cells with inhibitors for calpain (Ca2(+)-dependent intracellular protease) markedly enhanced the cross-linking of lipocortin I. Cross-linked lipocortin I is shown to be present in the soluble fraction of A431 cells as well as in the particulate fraction; a 34-kDa fragment of lipocortin I was solubilized successfully by plasmin digestion of the latter fraction. Immunofluorescence microscopy using specific antilipocortin-I antibody showed that cross-linked lipocortin I forms an envelope-like structure, which is not extracted with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) or Triton X-100. In vitro incubation of purified lipocortin I with tissue transglutaminase resulted in the formation of covalently cross-linked lipocortin I dimer, tetramer, and so on. Amine incorporation and cross-linking studies using lipocortin I and its N-terminal truncated derivatives indicated that the cross-linking site is localized within the plasmin-susceptible N-terminal 29 amino acids of lipocortin I. The cross-linking of lipocortin I is shown to be accelerated more than 10 times by the addition of phosphatidylserine vesicles, on which lipocortin I molecules are most likely aligned in a conformation suitable for cross-linking. Collectively, these findings suggest that an increase of intracellular calcium concentration results in the attachment of lipocortin I onto the plasma membrane phospholipids through the C-terminal domain of the molecule where the membrane-bound lipocortin I is cross-linked by the action of tissue transglutaminase through the N-terminal domain.     

Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity. As seen by autoradiographic analysis of coomassie blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [γ-(32)P]ATP and Mg(2+) alone. Addition of Ca(2+) alone to the native PSD preparation had little or no effect on phosphorylation. However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 M(r). Similar results were also obtained in sonicated or freeze thawed native PSD preparations by addition of Ca(2+) alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions. The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulin-activatable phosphorylation, but not of the Mg(2+)-dependent activity, by the Ca(2+) chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca(2+). In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [γ-(32)P]ATP, Mg(2+) and Ca(2+), one could not induce a Ca(2+)-stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 M(r) PSD protein is the one that is phosphorylated and not the 51,000 M(r) component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 M(r) protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg(2+)-dependent kinase(s). Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins I(a) and I(b), the cyclic AMP-dependent protein kinase, which definitely does phosphorylate protein I(a) and I(b) and is present in the PSD, does not phosphorylate the 51,000 and 62,000 M(r) proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins.     

Corticosteroids increase lipocortin I in BAL fluid from normal individuals and patients with lung disease

Lipocortin I is a corticosteroid-inducible protein that has potent anti-inflammatory activity. To determine whether lipocortin I is present on the epithelial surface of the human lung, we used a specific polyclonal antibody by the technique of Western blotting to evaluate bronchoalveolar lavage (BAL) fluid of normal individuals and patients with idiopathic pulmonary fibrosis. Lipocortin I was a normal constituent of the epithelial surface of the normal lung and comprised 0.23 +/- 0.03% of BAL fluid proteins. Four separate immunoreactive species were detected, at 37, 36, 34, and 33 kDa, consistent with previously published results. Corticosteroids increased the amounts of lipocortin present in normal volunteers and in patients with idiopathic pulmonary fibrosis. These results demonstrate that lipocortin I is normally present in the human lung and further suggest that lipocortin I may be an important modulator of the anti-inflammatory effects of corticosteroids in the lung.     

Chemical modification of sarcoplasmic reticulum with methylbenzimidate. Stimulation of Ca2+ efflux.

Treatment of sarcoplasmic reticulum membranes with 12 mM-methylbenzimidate (MBI) for 5 min, in the presence of 5 mM-ATP at pH 8.5, resulted in a 2-3-fold stimulation of ATP hydrolysis and over 90% inhibition of Ca2+ accumulation. This phenomenon was strictly dependent upon the presence of nucleotides with the following order of effectiveness: adenosine 5'-[beta, gamma-imido]triphosphate greater than or equal to ATP greater than UTP greater than ADP greater than AMP. Divalent cations such as Ca2+, Mg2+ and Mn2+, when present during the MBI treatment, prevented both the stimulation of ATPase activity and the inhibition of Ca2+ accumulation. Modification with MBI had no effect on E-P formation from ATP, ADP-ATP exchange, Ca2+ binding or ATP-Pi exchange catalysed by the membranes. Membranes modified with MBI in the presence of ATP and then passively loaded with Ca2+ released about 80% of their Ca2+ content within 3 s. Control membranes released only 3% of their Ca2+ during the same time period. MBI modification inhibited Ca2+ accumulation by proteoliposomes reconstituted with the partially purified ATPase but not with the purified ATPase fraction. These results suggest that MBI in the presence of ATP stimulates Ca2+ release by modifying a protein factor(s) other than the (Ca2+ + Mg2+)-ATPase.     

Measurement of microsomal ATPase activities: a comparison between the inorganic phosphate-release assay and the NADH-coupled enzyme assay

The specific activity of the Mg2+-ATPase and the (Ca2+ + Mg2+)-ATPase has been measured in a microsomal fraction from pig antral smooth muscle with the phosphate-release assay and the NADH-coupled enzyme assay, and the release of inorganic phosphate as a function of time is compared with the concomitant production of ADP. Both assays are found to overestimate the true Mg2+-ATPase activity. The adenylate kinase inhibitor P1,P5-di(adenosine-5'-)pentaphosphate (Ap5A) reduces the specific activity of the Mg2+-ATPase measured in the NADH-coupled enzyme assay to about half of its original value; however, it does not affect the specific activity of the Mg2+-ATPase in the Pi-release assay. The considerable overestimation of the Mg2+-ATPase activity in the NADH-coupled enzyme assay results from a combined action of an ATP pyrophosphatase (ATP in equilibrium AMP + PPi) and adenylate kinase activity contaminating the microsomes. The adenylate kinase activity in the microsomes catalyses the conversion of AMP formed by the ATP pyrophosphatase together with ATP into two ADP's. Also the phosphate-release assay is prone to an overestimation artefact because an inorganic pyrophosphatase will degrade the pyrophosphate and thus lead to additional Pi-production. Measurements of AMP and NAD+ production by HPLC confirmed our proposed reaction scheme. The same (Ca2+ + Mg2+)-ATPase activity is found in both assays, because the (Ca2+ + Mg2+)-ATPase activity is calculated from the difference in ATPase activity in the presence and absence of Ca2+, so that as a consequence the interfering activities are automatically subtracted.     

Characterization of lipocortin I and an immunologically unrelated 33-kDa protein as epidermal growth factor receptor/kinase substrates and phospholipase A2 inhibitors

Reversible calcium-dependent association with a particulate fraction from human placenta was used as the first step in the purification of substrates for the epidermal growth factor-stimulated protein kinase. A protein with apparent Mr of 35,000 was purified to homogeneity, and the sequence was determined for approximately one-fourth of the protein. These residues could be aligned exactly with the previously published sequence of lipocortin I derived from the cDNA from a human lymphoma. Two other proteins that appear to be formed by proteolytic removal of 12 or 26 of the amino acids from the NH2 terminus of the protein also were isolated. Placental lipocortin I was phosphorylated in Tyr-21 in an epidermal growth factor-dependent manner by the kinase activity in a particulate fraction from A431 cells; half-maximal phosphorylation occurred at 50 nM lipocortin I. Lipocortin I phosphorylated on Tyr-21 was approximately 10-fold more sensitive to tryptic cleavage at Lys-26 than was the native protein. Placental lipocortin I and its two truncated forms were potent inhibitors of pancreatic phospholipase A2 activity. Another 33-kDa protein that was not related immunologically to lipocortin I or lipocortin II (calpactin I) also was purified from the EGTA extract of placenta. The unidentified protein inhibited phospholipase A2 but was not a substrate for the epidermal growth factor-stimulated kinase. The mechanism by which these proteins inhibit phospholipase A2 activity was investigated. Attempts to detect direct interaction between these proteins and the enzyme were unsuccessful. However, both the unidentified protein, lipocortin I, and 32P-labeled lipocortin I bound in a Ca2+-dependent manner to the [3H]oleic acid-labeled Escherichia coli membranes used as substrate in the phospholipase A2 assay. Heparin, which is known to block lipocortin I inhibition of phospholipase A2, also blocked binding of lipocortin I to E. coli membranes. The results of these and other experiments raise the possibility that placental lipocortin I inhibits phospholipase A2 activity in this assay by coating the phospholipid and thereby blocking interaction of enzyme and substrate.     

The different roles of metal ions and water molecules in the recognition and catalyzed hydrolysis of ATP by phenanthroline-containing polyamines

The phenanthroline bridging polyaza ligands L1, L2 and L3 can selectively and strongly bind nucleotides at physiological pH, and hence accelerate the hydrolysis rate of the bound ATP. It is interesting that a phosphoramidate intermediate at 2.88 ppm (should be added 5.63 ppm when compared with other models) was found in the hydrolysis process of L/ATP. By introduction of metal ions (critical Zn(2+) or hard Mg(2+), Ca(2+)) to the L/ATP system, recognition of the anionic substrates by the protonated ligands was greatly promoted. However, due to the different affinities of metal ions to the receptor and the substrate, ATP hydrolysis in Zn(2+)/L/ATP system and Mg(2+)(Ca(2+))/L/ATP system occurs through different mechanisms. By comparison with the M/ATP (M=Zn(2+), Mg(2+), Ca(2+)) system, the rates of ATP-hydrolysis in the Mg(2+)Ca(2+)/L/ATP system and the Zn(2+)/L/ATP system were enhanced and retarded, respectively. Moreover, the reasons contributing to large rate range of the L/ATP systems and M(2+)/L/ATP systems were given. The results show that metal ions vertically regulate the recognition and hydrolysis of ATP. On the other hand, water molecule participates in the hydrolysis reactions at different steps with different functions in the L/ATP systems and M(Zn(2+), Mg(2+), Ca(2+))L/ATP systems.