The study deals with the interrelationship of the phosphate-transferring activities of the calciumtransporting sarcoplasmic reticulum membrane vesicles : the phosphate exchange between nucleoside triphosphate (NTP) and nucleoside diphosphate (NDP) (NTP-NDP exchange), the calcium-dependent NTPase, and the phosphorylation of NDP by inorganic phosphate in the presence of NTP (NTP-Pi exchange). Different nucleotides were used as phosphate donors and acceptors. It is demonstrated for the phosphate transfer from ITP to GDP that the NTP-NDP exchange exhibits ping-pong kinetics with Mg-ITP and unliganded GDP as substrates. The apparent affinities of the enzyme for the nucleoside diphosphate and triphosphate species are deduced according to this mechanism. The enzyme's affinity for the nucleoside triphosphates and diphosphates depends on its functional state being considerably lower under conditions of NTP-NDP exchange than during NTP splitting or NTP synthesis. ATP and GTP are split with the same low rates when calcium-activated NTPase is inhibited by high internal calcium concentrations after calcium transport has reached steady state. The rates of the NTP-NDP exchange reactions, however, differ by a factor of about 10 being E 3 pmol . mg-' . min-' for ATP-ADP and only E 0.3 pmol . mg-' . min-' (22°C) for GTP-GDP. When the sarcoplasmic reticulum vesicles are made calcium-permeable, the calcium transport ATPase is turned on and the rates of GTP and ATP splitting increase about tenfold. Yet, while the rate of ATP-ADP exchange is little reduced, the rate of GTP-GDP exchange drops by approximately 50 %. The persisting exchange activity of calcium-permeable vesicles demonstrates that high internal calcium concentrations are not required for the transfer of the protein-bound phosphoryl group to NDP during NTP-NDP exchange.

The reversible inhibition of the sarcoplasmic-reticulum calcium-transport enzyme by pressure at room temperature is accompanied by a significant enhancement of the accessibility of the enzyme to tryptic cleavage dependent on the presence of calcium. The calcium-transport enzyme activity was monitored with dinitrophenyl phosphate as substrate. Pressure in the range 0.1 -100.0 MPa affects trypsin cleavage of the control substrate N-a-benzoyl-~-arginine-4-nitroanilide hydrochloride little in the presence and absence of calcium. In contrast, application of 100.0 MPa to the calcium-transport enzyme at room temperature accelerates subsequent tryptic cleavage at the T2 but not at the T I cleavage site [C. J. Brandl et al. (1986) Cell 44, 597 -6071. Pressure application during tryptic digestion likewise solely affects cleavage at T2 which proceeds slowly in the absence but rapidly in the presence of calcium. At atmospheric pressure in the absence of calcium and at high pressure in the absence and presence of calcium new cleavage sites are exposed giving rise to new subfragments B1 -in addition to the established peptides Al and A2. Under pressure and in the presence of calcium, Al and A2 rapidly disappear indicating the presence of calcium-binding sites in these peptides. In contrast, the B1 -peptides which are most likely derivates of the B fragment accumulate in the presence and absence of calcium. In contrast to tryptic cleavage at atmospheric pressure, tryptic cleavage of the A as well as of the B fragment tends to completion under pressure. In parallel to the disappearance of the A and B fragments calcium-dependent substrate hydrolysis vanishes. Computation of activation volumes for pressure-induced reversible enzyme inhibition and for tryptic cleavage furnished closely related volumes of opposite signs of 20 -40 ml/mol and 80 -100 ml/mol in the ranges 0.1 -40.0 MPa and 40.0 ~ 100.0 MPa, respectively. Thus pressure produces reversible changes in the calcium-transport enzyme which activates and modifies tryptic-cleavage patterns at the T2 site of the A segment and at sites in its subfragments in the presence of calcium, i.e. if the enzyme resides in its El state. In contrast tryptic cleavage of the B fragment is accelerated by pressure independently of the presence of calcium.The effect of pressure on the activity of various enzymes, including transport enzymes has been studied with the intention of gaining information concerning activity-related changes in structure and/or hydration [l, 21. The effect of pressure on enzymatic activity furnishes volume changes of the functioning enzyme complex. De Smedt et al. al. [5] have recently applied high pressure at low temperature to the sarcoplasmic-reticulum calcium-transport enzyme and observed an irreversible inactivation connected with a considerable acceleration of tryptic cleavage of the enzyme and the appearance of new peptides when the pressurized preparation was digested with trypsin after pressure release. For this irreversible alteration of the enzyme, activatio...

The effect which hydrostatic pressure exerts on the binding of vanadate to the calcium-transport enzyme was determined. The recent unavailability of radioactive vanadate prevented direct measurements of vanadate binding.The vanadate-free enzyme fraction was instead monitored by phosphorylating it with ATP according to Medda and Hasselbach [Medda, P. & Hasselbach, W. (1983) Eur. J . Biochem. 137, 7 -141. Vanadate binding is reduced with rising pressure at first markedly and subsequently, above 30 MPa, relatively little. The biphasic pressurebinding relationship was analysed by applying a biexponential fitting procedure to the experimental data. The biphasicity of the pressure-binding relationship indicates that the description of vanadate binding requires at least a two-step reaction sequence. The volume increments which predominate at lower pressure values, range from 200 -400 ml . rno1-l depending on the composition of the reaction medium containing 5 pM and 20 pM vanadate and no or 15% (by vol.) MezSO. The binding volumes deduced for the higher pressure range amount to 20-40 ml . mol-l. Vanadate binding is reduced in the presence of 30 pM calcium, and simultaneously both binding volumes are diminished by 100 ml . mol-' and 20 ml . mol-' for the low and high pressure values, respectively, as one can expect for mutual interactions between the two ligands of the transport enzyme.In recent studies we tried to monitor structural changes of the sarcoplasmic reticulum calcium pump connected with its transport cycle [l -31. We determined reaction volume changes by measuring the effect of pressure on the calciumdependent hyrolysis of two ATP substrate analogues, 4-nitrophenyl phosphate and dinitrophenyl phosphate. The intention of these studies was to find out if the transition between the well-characterized reaction intermediates could be related to the kinetically derived volume changes. Two characteristic reaction volumes were obtained. We assigned a small volume change of approximately + 20 ml . mol-' to the slow transition between the phosphate-free intermediates Ez and El and a large one of 80-100 ml . rno1-l to the transition of EIPCa to E2P which is connected to ion translocation.Since the resulting volume changes were obtained from the effect of pressure on the reaction rates of the forward running pump, only activation volumes could be derived. Their values cannot be directly related to net volume changes connected with the transition between corresponding reaction intermediates.To determine real volume changes unambiguosly it is necessary to measure the effect of pressure on equilibria between the intermediates under consideration. As a reaction equilibrium which can most simply be established we considered to study phosphorylation of the calcium-free protein Ez which results in the reaction intermediate EzP having a low calcium affinity. In the past, formation and decay of this intermediate could quite easily be studied due to its acid Correspondence to W. Hasselbach, Max-Planck-lnstitut fur Medizinische Fo...