Temperature dependence of cardiac sarcoplasmic reticulum Ca²âº-ATPase from rainbow trout Oncorhynchus mykiss.

In this work, the temperature dependence of the sarco-endoplasmic reticulum Ca(2+) -ATPase (SERCA2) activity from rainbow trout Oncorhynchus mykiss cardiac ventricles was measured and compared with the mammalian SERCA2 isoform. The rate of ATP-dependent Ca(2+) transport catalysed by O. mykiss vesicles was totally abolished by thapsigargin and the Ca(2+) ionophore A(23187) . At warm temperatures (25 and 30° C), the SERCA2 from O. mykiss ventricles displayed the same rate of Ca(2+) uptake. At 35° C, the activity of the O. mykiss enzyme decreased after 20 min of reaction time. The rate of Ca(2+) uptake catalysed by the mammalian SERCA2 was temperature dependent exhibiting its maximal activity at 35° C. In contrast to the rate of Ca(2+) uptake, the rate of ATP hydrolysis catalysed by O. mykiss SERCA2 was not significantly different at 25 and 35° C, but the rate of ATP hydrolysis catalysed by the rat Rattus norvegicus SERCA2 isoform at 35° C was two-fold higher than at 25° C. At low temperatures (5 to 20° C), the rate of Ca(2+) uptake from O. mykiss SR was less temperature dependent than the R. norvegicus isoform, being able to sustain a high activity even at 5° C. The mean ±s.e. Q(10) values calculated from 25 to 35° C for ATP hydrolysis were 1·112 ± 0·026 (n = 3) and 2·759 ± 0·240 (n = 5) for O. mykiss and R. norvegicus, respectively. Taken together, the results show that the O. mykiss SERCA2 was not temperature dependent over the 10 to 25° C temperature interval commonly experienced by the animal in vivo. The Q(10) value of SERCA2 was significantly lower in O. mykiss than R. norvegicus which may be key for cardiac function over the wide environmental temperatures experienced in this eurythermal fish.

Catalytic activity and heat production by the Ca(2+)-ATPase from sea cucumber (Ludwigothurea grisea) longitudinal smooth muscle: modulation by monovalent cations.

In muscle cells, excitation-contraction coupling involves the translocation of Ca(2+) between intracellular compartments and the cytosol. Heat derived from the hydrolysis of ATP by the sarcoplasmic reticulum Ca(2+)-ATPase of skeletal muscle plays an important role in the thermoregulation and energy balance of the cell. Although several Ca(2+)-ATPase isoforms have been described in vertebrates, little is known about Ca(2+) transport in invertebrates. In this report, a Ca(2+)-ATPase is identified in the microsomal fraction obtained from sea cucumber (Ludwigothurea grisea) smooth muscle. The activity of this enzyme is enhanced three- to fivefold by K(+) and Na(+). During Ca(2+) transport, the ATPase can synthesise ATP from ADP and inorganic phosphate (P(i)) using the energy derived from the Ca(2+) gradient formed across the microsomal membrane (ATP<->P(i) exchange). The apparent affinity of the enzyme for P(i) is increased by more than one order of magnitude by K(+). In the presence of K(+), the fraction of ATP synthesised during the exchange reaction by sea cucumber microsomes was found to be larger than that measured in microsomes derived from either rabbit or trout muscle. Like the isoforms found in skeletal muscle, the sea cucumber Ca(2+)-ATPase can convert osmotic energy into heat. The amount of heat produced after the hydrolysis of each ATP molecule increases two- to threefold when a Ca(2+) gradient is formed across the microsomal membrane.

Sarcoplasmic reticulum Ca(2+)-ATPase of sea cucumber smooth muscle: regulation by K(+) and ATP.

Although several Ca(2+)-ATPase isoforms have been described in vertebrates, little is known about Ca(2+)-transport in the muscle of invertebrates. In the microsomal fraction obtained from the sea cucumber (Ludwigothurea grisea) longitudinal body wall smooth muscle, we identified a Ca(2+)-transport ATPase that is able to transport Ca(2+) at the expense of ATP hydrolysis. This enzyme has a high affinity for both Ca(2+) and ATP, an optimum pH around 7.0, and - different from the vertebrate sarcoplasmic reticulum Ca(2+)-ATPases isoforms so far described - is activated 3- to 5-fold by K(+) but not by Li(+), at all temperatures, Ca(2+) and ATP concentrations tested. Calcium accumulation by the sea cucumber microsomes is inhibited by Mg/ATP concentrations >1 mM and the accumulated Ca(2+) is released to the medium when the ATP concentration is raised from 0.1 to 4.0 mM.

A sulfated polysaccharide from the sarcoplasmic reticulum of sea cucumber smooth muscle is an endogenous inhibitor of the Ca(2+)-ATPase.

Vesicles derived from the endoplasmic reticulum of sea cucumber smooth muscle retain a membrane bound Ca(2+)-ATPase that is able to transport Ca(2+) into the vesicles at the expense of ATP hydrolysis. In contrast with vesicles obtained from rabbit muscles, the activity of the Ca(2+)-dependent ATPase from sea cucumber is dependent on monovalent cations (K(+)>Na(+)>Li(+)). With the addition of highly sulfated polysaccharide to vesicle preparations from rabbit muscle, Ca(2+) uptake decreases sharply and becomes highly sensitive to monovalent cations, as observed with vesicles from sea cucumber muscle. These results led us to investigate the possible occurrence of a highly sulfated polysaccharide on vesicles from the endoplasmic reticulum of sea cucumber smooth muscle, acting as an "endogenous" Ca(2+)-ATPase inhibitor. In fact, vesicles derived from the invertebrate, but not from rabbit muscle, contain a highly sulfated polysaccharide. This compound inhibits Ca(2+) uptake in vesicles obtained from rabbit muscle and the inhibition is antagonized by monovalent cation. In addition, sea cucumber muscles contain high concentrations of another polysaccharide, which surrounds the muscle fibers, and was characterized as a fucosylated chondroitin sulfate. Possibly the occurrence of sulfated polysaccharides in the sea cucumber muscles is related with unique properties of the invertebrate body wall, which can rapidly and reversibly alter its mechanical properties, with change in length by more than 200%.

Modification of the pH dependence of animal and plant transport ATPases by sulfated polysaccharides.

The effects of heparin and dextran sulfate 8,000 on two isoforms of the sarco/endoplasmic reticulum Ca(2+)-ATPase of different animal tissues and on the corn root H(+)-ATPase were examined. In the absence of sulfated polysaccharides the pH profile's of the three transport ATPases were quite different, but after the addition of heparin or dextran sulfate 8,000 the pH profiles of the three enzymes became similar, all showed maximal activity at pH 7.0. Potassium and sodium antagonized the effects of sulfated polysaccharides on the three transport ATPases, but the antagonism was considerably reduced at acidic pH values.

Modulation of maize roots H(+)-ATPase by sulfated polysaccharides.

Vesicles derived from maize roots retain a membrane bound H(+)-ATPase that is able to pump H+ at the expense of ATP hydrolysis. In this work it is shown that heparin, fucose-branched chondroitin sulfate and dextran sulfate 8000 promote a shift of the H(+)-ATPase optimum pH from 6.0 to 7.0. This shift is a result of a dual effect of the sulfated polysaccharides, inhibition at pH 6.0 and activation at pH 7.0. At pH 6.0 dextran 8000 promotes an increase of the apparent K(m) for ATP from 0.28 to 0.95 mM and a decrease of the Vmax from 14.5 to 7.1 mumol Pi/mg x 30 min-1. At pH 7.0 dextran 8000 promotes an increase in Vmax from 6.7 to 11.7 mumol Pi/mg x 30 min-1. In the presence of lysophosphatidylcholine the inhibitory effect of the sulfated polysaccharides observed at pH 6.0 was not altered but the activation of pH 7.0 decreased. It was found that in the presence of sulfated polysaccharides the ATPase became highly sensitive to K+ and Na+. Both the inhibition at pH 6.0 and the activation promoted by the polysaccharide were antagonized by monovalent cations (K+ > Na+ > > Li+).