Débora A. González

Calcium additional to that bound to the transport sites is required for full activation of the sarcoplasmic reticulum Ca-ATPase from skeletal muscle.

The sarcoplasmic reticulum Ca-ATPase is fully activated when approximately 1 microM [Ca2+] saturates the two transport sites; higher [Ca] inhibits the ATPase by competition of Ca-ATP with Mg-ATP as substrates. Here we describe a novel effect of EGTA and other chelators, raising the possibility of an additional activating effect of Ca in the sub- or low microM range. Sarcoplasmic reticulum membranes were isolated from rabbit skeletal muscles. The ATPase activity was measured after incubation at 37 degreesC in 3 mM ATP, 3 mM MgCl2, 50 mM MOPS-Tris (pH 7.2), 100 mM KCl, and variable CaCl2, EGTA and calcimycin. In the absence of added EGTA and Ca the ATPase activity is high due to contaminant Ca. The determination of the ATPase activity in the presence of increasing amounts of EGTA, without added Ca, yields a decreasing sigmoidal function. Ki ranged between 20 and 100 microM, depending on the enzyme concentration. Pi production is linear with time for several [EGTA] yielding suboptimal ATPase activities, which are inhibited by thapsigargin. These suboptimal Ca-ATPase activities are inhibited by preincubation of the enzyme in EGTA, at pH 7.2. This effect increases upon increasing EGTA concentration and preincubation time. The inhibitory effect of the previous exposure of the enzyme to EGTA is partially but significantly reverted by increasing [Ca2+] during incubations. Calcimycin and EDTA have similar effects as EGTA when added in preincubations. The effect of calcimycin is fully reverted by optimal [Ca2+] in incubations. The effects of EGTA, EDTA and calcimycin in preincubation are not additive. The results suggest that an additional calcium, lost during preincubations from a site with affinity near 1 microM, is necessary for full activation of the ATPase.

Manganese as a cosubstrate for the phosphorylation of the sarcoplasmic reticulum Ca-dependent adenosine triphosphatase with orthophosphate

The phosphorylation of the sarcoplasmic reticulum Ca-ATPase (EC 3.6.1.38) with P(i) was characterized using Mn as a Mg analogue. Steady state and transient fluorescence and radioisotopic techniques were used; the affinities of Mn and P(i) for the enzyme and the rate constants of the phosphorylation and dephosphorylation reactions were determined, under several conditions. The reactions were carried out at pH 5.5 to minimize the binding of contaminant Ca to the transport sites, thus avoiding the use of Ca chelators. The apparent affinity of Mn binding at low [Mn] is larger in the absence of P(i) (35 microM) than in the presence of saturating P(i) (70 microM). On the contrary, the apparent affinity of Mn for the formation of the phosphoenzyme increases, from 1.5 mM to 0.15 mM, upon increasing [P(i)] in the millimolar range. The apparent affinty of P(i) for the formation of the phosphoenzyme also increases, from 2.2 mM to 0.2 mM, upon increasing [Mn] in the millimolar range. The equilibrium of the phosphoenzyme with the noncovalent Mn.P(i). Enzyme complex favors the covalent species. The simulation of a reaction model including the random binding of 2 Mn and I P(i) per mol of ATPase and a noncovalent complex in equilibrium with the phosphoenzyme, using a set of equilibrium constants deduced from the results, agree with the experimental data.

Direct demonstration of an acid-labile phosphoenzyme in the cycle of the sarcoplasmic reticulum Ca2+-dependent adenosinetriphosphatase

The Ca2(+)-dependent adenosinetriphosphatase (Ca2(+)-ATPase) from the sarcoplasmic reticulum (SR) of rat skeletal muscles is phosphorylated by inorganic phosphate (Pi) in the absence of Ca2+. The reaction can be described by the following simplified scheme: [formula: see text] where E-P is a covalent, acid-stable and ADP-insensitive phosphoenzyme, and E.Pi is a noncovalent and acid-labile complex. The reaction is Mg2(+)-dependent. Membrane fragments deposited on Millipore filters were successively perfused with two solutions, at constant flow. The effluent samples were analyzed. The perfused solutions were Ca2+ free and always contained 40% dimethylsulfoxide (DMSO), plus other reactants. Following the successive perfusion of solutions without and with [32P]Pi, 32P binding is only detected in the presence of Mg2+, indicating the formation of the phosphoenzymes (E.Pi and E-P). Following perfusions of the phosphoenzymes with 5% trichloroacetic acid, 32P release indicates the amount of the acid-labile moiety (E.Pi). After phosphorylations, the filters were washed with acid and unlabeled Pi, and the remaining radioactivity was measured to evaluate the acid-stable phosphoenzyme (E-P). The acid-labile and acid-stable phosphoenzymes amounted, respectively, 0.72 +/- 0.12, and 1.48 +/- 0.10 nmol of Pi/mg of protein ( +/- S.E., n = 5), after phosphorylations with 20 microM Pi. The results indicate: (1) The method allowed the evaluation of the acid-labile intermediate of the SR Ca2(+)-ATPase cycle. Keq = k2/k-2), in the above scheme, approaches 2.0. (2) The substrate of the phosphorylation reaction, in the presence of DMSO, is likely to be the Mg.Pi complex, since Mg2+ is necessary for step 1 in the above scheme.

Demonstration of the simultaneous activation of Ca2+-independent and Ca2+-dependent ATPases from rat skeletal muscle microsomes.

The activation of the Ca2+-independent (basal) ATPase from rat skeletal muscle microsomes is demonstrated in the presence of enough Ca2+ to provide the simultaneous activation of the (Ca2+ + Mg2+)-ATPase. It was achieved taking advantage of the delayed inorganic phosphate (Pi) release due to the formation of a phosphoenzyme complex during the Ca2+-dependent enzymatic cycle, which is evidenced in fast experiments. The microsomes were immobilized on a filter and perfused at constant flow with an incubation medium which was briefly interrupted with a pulse of appropriate reactants to activate the ATPases, at 2 degrees C. Successive samples were collected after passing through the filter, at approx. 0.1 s intervals. The Pi effluent profile coincides with the pattern of the pulse when it activates only the Ca2+-independent ATPase, it appears delayed when the pulse activates only extra Pi production by the (Ca2+ + Mg2+)-ATPase, and it includes a rapid and a delayed component when both Ca2+-independent and Ca2+-dependent ATPases are activated simultaneously by the pulse.

Rat submandibular glands secrete nanovesicles with NTPDase and 5'-nucleotidase activities.

Extracellular nucleotides modulate a wide number of biological processes such as neurotransmission, platelet aggregation, muscle contraction, and epithelial secretion acting by the purinergic pathway. Nucleotidases as NTPDases and ecto-5′-nucleotidase are membrane-anchored proteins that regulate extracellular nucleotide concentrations. In a previous work, we have partially characterized an NTPDase-like activity expressed by rat submandibular gland microsomes, giving rise to the hypothesis that membrane NTPDases could be released into salivary ducts to regulate luminal nucleotide concentrations as was previously proposed for ovarian, prostatic, and pancreatic secretions. Present results show that rat submandibular glands incubated in vitro release membrane-associated NTPDase and ecto-5′-nucleotidase activities. Electron microscopy images show that released membranes presenting nucleotidase activity correspond to exosome-like vesicles which are also present at microsomal fraction. Both exosome release and nucleotidase activities are raised by adrenergic stimulation. Nucleotidase activities present the same kinetic characteristics than microsomal nucleotidase activity, corresponding mainly to the action of NTPDase2 and NTPDase3 isoforms as well as 5′-nucleotidase. This is consistent with Western blot analysis revealing the presence of these enzymes in the microsomal fraction.

Histamine stimulates secretion of extracellular vesicles with nucleotidase activity in rat submandibular gland

BACKGROUND Extracellular vesicles released by different cells have been isolated from diverse fluids including saliva. We previously reported that rat submandibular glands secrete nanovesicles that catalyze hydrolysis of ATP, ADP and AMP, which are actors of the purinergic signaling system along with adenosine. Extracellular nucleotides like ATP and adenosine are involved in the regulation of inflammatory processes and apoptosis. Histamine, a widely distributed biogenic amine, is involved in inflammatory response. OBJECTIVE To test if activation of histamine receptors in rat submandibular gland promotes changes in the release of vesicles with nucleotidase activity that could modulate purinergic signaling. METHODS Rat submandibular glands were incubated in the absence or presence of histamine and JNJ7777120, an antagonist for H4 receptors. Extracellular vesicles were isolated from incubation media by differential centrifugation. Vesicular nucleotidase activity was measured following Pi release by 3mM MgATP, MgADP or MgAMP. RESULTS Histamine increased the release of vesicles with nucleotidase activity in a concentration dependent manner. JNJ7777120 significantly reduced this effect. Vesicular nucleotidases obtained in the absence or presence of histamine promoted Pi production from ATP, ADP and AMP. CONCLUSION The results show a relationship between histamine and the regulation of purinergic signaling, which could be important in the modulation of inflammatory processes.

Mn as Cosubstrate for the Phosphorylation of the Sarcoplasmic Reticulum Ca‐ATPase by Pi

Phosphorylation of the sarcoplasmic reticulum Ca-ATPase with ATP occurs when the Ca transport sites of the enzyme are saturated, whereas phosphorylation with Pi occurs with the protonated form. Both reactions require Mg as cosubstrate, but it can be replaced by other divalent cations. The binding of Pi or ATP and Mg to the enzyme occurs randomly to form a noncovalent complex before the formation of the covalent phosphoenzyme. The stoichiometry of cations bound to the catalytic site remains an open question. In this study we used Mn as an Mg analog and combined fluorescence and radioactive techniques to study the phosphorylation of the Ca-ATPase by Pi. At pH 5.5, EDTA, Pi, or Mn alone has little or no effect on the fluorescence signal, whereas the addition of Ca alone or the simultaneous addition of Pi and Mn markedly increases it. These experiments indicate that the ATPase is Ca free at pH 5.5, which is a good condition in which to study the phosphorylation of the Ca-ATPase with Pi using Mn as cosubstrate in the absence of a chelating agent. The increase in the fluorescence signal is a sigmoidal function of log[Pi], or log[Mn], for several constants [Mn] and [Pi], respectively (data not shown). From these functions, the apparent affinities for Pi and Mn at various [Mn] and [Pi] were obtained (FIG. 1A and lB, respectively). 54Mn binding was measured in both the presence and the absence of Pi (FIG. 1C). The curves were drawn up to 1 mol of bound Mn per mol of ATPase (5-6 nmol/mg). The Scatchard plot supports the assumption that the first points indicate the binding of Mn to a first site with affinity 240 pM in the absence of Pi; the presence of Pi decreases the apparent affinity of the enzyme for this Mn.