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

Abstract

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.

Characteristics of the Sarcoplasmic Reticulum Ca2+-dependent ATPase from Masticatory Muscles

“…1A). The addition of 1 mmol/L EGTA reduced the enzymatic activity due to contaminant calcium, as previously reported (Alonso et al, 1998), indicating the calcium-dependence of the ATPase. The ATPase activity measured in the presence of 0.1 mmol/L EGTA was inhibited by thapsigargin (Fig.…”

Involvement of EP1 and EP2 receptors in the regulation of the Na,K-ATPase by prostaglandins in MDCK cells

“…Previous observations that PGE 1 and PGE 2 cause an increase in intracellular Ca 2+ and cAMP in MDCK cells are consistent with the involvement of EP1 and EP2 receptors in mediating the effects of these two prostaglandins in MDCK cells [10,16,19,30,[39][40][41][42]. The increase in intracellular Ca 2+ that occurs in PGE 1 and PGE 2 treated MDCK cells may cause a number of Ca 2+ mediated pathways to be activated (in addition to PKC), which may all contribute to the PGE 1 stimulation.…”

“…6D shows that the PGE 1 stimulation (5.4 ± 0.3-fold) was reduced to 3.3 ± 0.3-fold in the presence of 0.1 μM thapsigargin. Thapsigargin releases sequestered Ca 2+ by inhibiting endoplasmic reticulum Ca 2+ ATPases, without affecting PKC [39][40][41].…”

PfMDR1 Transport Rates Assessed in Intact Isolated Plasmodium falciparum Digestive Vacuoles Reflect Functional Drug Resistance Relationship with pfmdr1 Mutations

“…For this purpose, the chelating agent ethylene glycolbis(β-aminoethyl ether)-N,N,N ,N -tetraacetic acid (EGTA) was implemented for free Ca 2+ control. As the ATPase of PfMDR1 is at risk of being inhibited by EGTA concentrations higher than 0.07 mM [25], Ca 2+ containing buffers were prepared with 0.01 mM EGTA, solely in plastic containers. Because the stability of free Ca 2+ concentrations in this buffer is limited, strict incubation and preparation times must be followed.…”