Yves Dupont

Fluorescence studies of the sarcoplasmic reticulum calcium pump

Abstract Intrinsic Tryptophan fluorescence has been used to reveal conformational changes of the Sarcoplasmic Reticulum Calcium pump. It is shown that upon binding of calcium ions the fluorescence is enhanced. The effect being reversed after removal of dependence calcium ions by EGTA. The calcium concentration dependence of this fluorescence change and the effect of inhibitors is compared with the activation of calcium dependent ATPase. We conclude that calcium ions induces a conformational change of the calcium pump and that this change is responsible for the activation of the ATPase activity.

Transient non-native secondary structures during the refolding of α-lactalbumin detected by infrared spectroscopy.

Stopped-flow Fourier-transform infrared spectroscopy (SF-FTIR) was used to identify native as well as non-native secondary structures during the refolding of the calcium-binding protein α-lactalbumin. Infrared absorbance spectra were recorded in real time after a pH jump induced refolding of the protein. In the presence of calcium, the refolding is fast with concerted appearance of secondary structures; in its absence, folding is much slower and intricate, with transient formation and disappearance of non-native β-sheet. The possibility of detecting native as well as non-native structures at the same time is especially valuable in providing insight into the complexity of the refolding process of a protein.

Low-temperature studies of the sarcoplasmic reticulum calcium pump: Mechanism of calcium binding

The mechanism of the sarcoplasmic reticulum Ca2+-ATPase was investigated at low temperatures (0 to -12 degrees C). Transient states of the enzyme were studied by two complementary techniques: intrinsic protein fluorescence and rapid filtration on Millipore filters. Intrinsic fluorescence was used to distinguish conformational states of the protein and to evaluate the rate of conversion between these states. Filtrations were used to measure the evolution of the active sites during the transition; the time resolution was 2-5 s. At sub-zero temperatures this time is shorter than the lifetime of most of the enzymatic states which have been detected. In this paper the mechanism of Ca2+ binding to the protein is investigated in the absence of nucleotides. Two basic experiments are described; (1) Kinetics of calcium binding and dissociation over a wide range of calcium concentration. (2) Kinetics of calcium exchange (45Ca2+ in equilibrium 40Ca2+) at constant concentration. The results obtained in the first series of experiments are consistent with a sequential binding to two interacting Ca2+ binding sites. Calcium ions have very fast access to a site with low apparent affinity (Kd approximately 25 microM). Occupation of this site induces a slow conformational change which increased its apparent affinity and reveals a second site of high apparent affinity. At equilibrium the two sites are not equivalent in terms of rate of exchange. Two different rates were detected k fast greater than 0.2 s-1, k slow approximately 0.015 s-1 at -10 degrees C. Removal of Ca2+ from the fast exchanging site by addition of EGTA accelerates the rate of release of the slow exchanging one. A model is proposed with two interacting Ca2+-binding sites. A set of parameters has been obtained which produces correctly the Ca2+-binding curve and the fluorescence level at equilibrium as well as the rate constants of the calcium-induced fluorescence changes over a very wide range of Ca2+ concentrations (0.02 to 150 microM). The non-equivalence of the two classes of site and the meaning of the initial low-affinity binding are discussed.

Evaluation of H2O activity in the free or phosphorylated catalytic site of Ca2+-ATPase.

The sarcoplasmic reticulum Ca2+‐ATPase catalyses a reversible calcium transport coupled to phosphate transfer between ATP and water. It has been proposed [Biochemistry (1980) 19, 4252–4261] that the reactivity of the acyl‐phosphate bond is dependent on the water activity within the catalytic site. We have tested this hypothesis and found that the polarity in the free catalytic site is lower than that of water, a further and large decrease is observed when the enzyme is phosphorylated by Pi. Phosphorylation by ATP indicates that this polarity change is specifically associated with the formation of the ADP‐insensitive phosphoenzyme

A rapid filtration technique for membrane fragments or immobilized enzymes measurements of substrate binding or ion fluxes with a few millisecond time resolution

The construction and use of a filtration system with milliseconds time resolution is described here. This apparatus allows measurements of substrate binding to immobilized enzyme or ion fluxes through membrane vesicles to be performed over a very large time scale, from 10 ms to seconds. The main advantage of this system compared to the widely used quench-flow technique is that it does not require the use of an inhibitor. Following adsorption of the enzyme in an adequately chosen filter, the reaction is allowed to proceed within the filter during a forced filtration of a buffer containing the reactive substrate (or of a washing solution in the case of efflux measurements). The design allows the duration of filtration and buffer flux to be finely and reproducibly controlled. This paper illustrates the use of this rapid-filtration system for time-resolved measurements of calcium binding and transport by sarcoplasmic reticulum Ca-ATPase and of the initial phase of ADP transport by the ADP/ATP carrier of intact mitochondria.

Vanadate inhibition of the Ca2+‐dependent conformational change of the sarcoplasmic reticulum Ca2+‐ATPase

Vanadate has been shown to be a potent inhibitor of many phosphohydrolases which include a covalent phosphoenzyme intermediate in their enzymatic cycle [ 11. The similar structure of pentavalent vanadium and phosphorus was suggested to be responsible for this inhibitory effect [2]. Extensive work has been done on the inhibitory effect of vanadate on the (Na’,K+)-ATPase [3-S]. Binding to the phosphorylation site is facilitated by K+ and Mg2+ and locks the enzyme in the Ez state [5-61. Vanadate inhibition of Ca2+-ATPase activity of sarcoplasmic reticulum has been reported in [7,8], the apparent affinity for the inhibitor being however much lower than for the __ (Na’,K+)ATPase. These results prompted us to investigate the effect of vanadate on the major conformational change of the Ca2+-ATPase which is induced by Ca2+ binding to the high affinity sites. This change can be followed by intrinsic fluorescence measurements [9]. The main conclusions of this report are: (i) Vanadate binds with high affinity (Kd = 0.2 PM) to the calcium free state E of the Ca’+-ATPase; (ii) The binding is a slow process which requires Mg2+ and is competitively antagonized by orthophosphate; (iii) Binding of vanadate results in stabilizing the calcium free conformation of the Ca2+-ATPase.

Expression of the sarcoplasmic reticulum Ca2+-ATPase in yeast

We describe here an easy system for the production of mg amounts of the rabbit Ca2+‐ATPase SERCA 1a in the yeast S. cerevisiae. The protein is present in several membranes, including the plasma membrane of the yeast, in a native conformation. It can be purified by immunoprecipitation and can be phosphorylated from ATP in a Ca2+‐dependent manner. Using a temperature‐sensitive secretion mutant strain, the fully active protein can also be obtained in secretory vesicles.

Titration of the nucleotide binding sites of Sarcoplasmic Reticulum Ca2+-ATPase with 2′, 3′-0-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate and 5′-diphosphate

Abstract Titration of nucleotide binding sites of the Sarcoplasmic Reticulum ATPase were performed with the ribose-modified nucleotides: 2′, 3′-0-(2,4,6-trinitrophenyl) adenosine-5′-triphosphate and diphosphate. Binding studies were carried out by three independent methods: fluorescence, difference absorbance spectra and direct binding measurements with (γ-32P) TNP-ATP. These three measurements indicate that TNP-ATP and TNP-ADP bind with high affinity to the Ca2+-ATPase protein. The number of TNP nucleotide binding sites ranged from 6.0 to 7.0 nmoles/mg of protein therefore indicating that the Ca2+-ATPase probably possesses one nucleotide binding site per polypeptide chain of molecular mass 110,000.

Abnormal rapid Ca2+ release from sarcoplasmic reticulum of malignant hyperthermia susceptible pigs

Using the rapid filtration technique to investigate Ca2+ movements across the sarcoplasmic reticulum (SR) membrane, we compare the initial phases of Ca2+ release and Ca2+ uptake in malignant hyperthermia susceptible (MHS) and normal (N) pig SR vesicles. Ca2+ release is measured from passively loaded SR vesicles. MHS SR vesicles present a 2-fold increase in the initial rate of calcium release induced by 0.3 microM Ca2+ (20.1 +/- 2.1 vs. 6.3 +/- 2.6 nmol mg-1 s-1). Maximal Ca2+ release is obtained with 3 microM Ca2+. At this optimal concentration, rate of Ca2+ efflux in absence of ATP is 55 and 25 nmol mg-1 s-1 for MHS and N SR, respectively. Ca(2+)-induced Ca2+ release is inhibited by Mg2+ in a dose-dependent manner for both MHS and N pig SR vesicles (K1/2 = 0.2 mM). Caffeine (5 mM) and halothane (0.01% v/v) increase the Ca2+ sensitivity of Ca(2+)-induced Ca2+ release. ATP (5 mM) strongly enhances the rate of Ca2+ efflux (to about 20-40-fold in both MHS and N pig SR vesicles). Furthermore, both types of vesicles do not differ in their high-affinity site for ryanodine (Kd = 12 nM and Bmax = 6 pmol/mg), lipid content, ATPase activity and initial rate of Ca2+ uptake (0.948 +/- 0.034 vs. 0.835 +/- 0.130 mumol mg-1 min-1 for MHS and N SR, respectively). Our results show that MH syndrome is associated to a higher rate of Ca2+ release in the earliest phase of the calcium efflux.

Mechanism of the sarcoplasmic reticulum calcium pump. Fluorometric study of the phosphorylated intermediates.

Abstract After removal of calcium ions bound to the high affinity sites the sarcoplasmic reticulum calcium pump can be phosphorylated by inorganic phosphate. The intrinsic fluorescence of the protein is used to follow conformational changes of the pump and an intensity change can be observed upon addition of phosphate. This effect is activated by internal calcium ( K 1 2 = 10 mM ) and inhibited by external calcium ( K 1 2 = 0.4 μM ) and the apparent affinity for phosphate is high (0.2 mM). We conclude that the change observed is linked to the formation of the gradient-dependent phosphorylated intermediate. It is compared with previous results concerning the enzymatic cycle of the pump.