Tryptic Fragmentation of the Calcium Transport System in the Sarcoplasmic Reticulum

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...

Section: Efyect Of Pressure On Tryptic Cleavage At a Low Trypsin/ca2'supporting

confidence: 93%

“…This change is due to the combined effects of trypsin and pressure because trypsin digestion alone does not affect enzymatic activity under these conditions [8].…”

Section: Efyect Of Pressure On Tryptic Cleavage At a Low Trypsin/ca2'mentioning

confidence: 99%

Effect of pressure and calcium on the reversible inhibition of the sarcoplasmic‐reticulum calcium‐transport enzyme and on its tryptic cleavage pattern

Ronzani

Hasselbach

Stephan

1990

“…In this context it is interesting to note that ATPase was found to split in subunits with molecular weights from 50000 to 60000 after mild treatment with trypsin [5,6]. Thus the possibility exists that the cooperativity is associated with the interaction of four, not necessarily equal, subunits, two of them constituting the well known ATPase unit, 100000 in molecular weight.…”

Section: Discussionmentioning

confidence: 99%

“…70"d of the protein content is due to a calcium and magnesium dependent ATPase with a molecular weight close to 100000; tryptic fragments between 50000 and 60000 have been observed [5,6].…”

mentioning

confidence: 99%

The Binding of Calcium and Magnesium to Sarcoplasmic Reticulum Vesicles as Studied by Manganese Electron Paramagnetic Resonance

Kalbitzer

Stehlik

Hasselbach

1978

The binding pattern of the biologically relevant ions calcium and magnesium has been investigated via the binding of the ion-analogue manganese. The binding parameters of manganese are obtained conveniently by standard electron paramagnetic resonance techniques, the binding of calcium and magnesium is inferred from competition experiments.The quantitative analysis was carried out with a computer program which was able to treat the competition of three kinds of ions for three classes of independent and one class of cooperative binding sites.It was possible to correlate specific binding classes with biological functions of the sarcoplasmic reticulum membrane. The magnesium and manganese specific binding class of medium affinity is related to the catalytic function of these ions in the ATP-splitting. For the first time a manganese and calcium specific clash of cooperative binding sites has been observed and it can be related to the inhibition of the calcium transport at high concentrations of manganese or calcium.From the binding results a model can be developed which allows a full description of the role of the divalent ions and their specific binding sites during calcium transport.

“…Gel electrophoresis of sarcoplasmic reticulum phosphorylated from [32P]~rthophosphate, dissolved in phenol/acetic acid/water ( l / l / l ) was carried out on 7 % polyacrylamide equilibrated with phenol/ acetic acid/water [7,28]. Both electrode chambers contained 10 % acetic acid.…”

Section: Polyacrylamide Gel Electrophoresismentioning

confidence: 99%

Phosphorylation of the Calcium‐Transport Adenosine Triphosphatase of Cardiac Sarcoplasmic Reticulum by Orthophosphate

Winkler¹,

Suko²

1977

Membranes of sacroplasmic reticulum prepared from dog hearts are phosphorylated by [32P]-orthophosphate in the presence of a calcium load and magnesium. The [32P]phosphate incorporation into sarcoplasmic reticulum follows Michaelis-Menten kinetics with an apparent Michaelis Constant of 0.5 mM for orthophosphate (pH 7.0). The phosphorylation is pH-dependent, with an optimum pH of 6.0-6.2 (maximum phosphoprotein steady state level 0.6-0.8 nmoljmg protein).The phosphorylation of sarcoplasmic reticulum by [32P]orthophosphate is strongly inhibited by calcium in the medium. Half-maximum inhibition occurs at an ionized calcium concentration of about 0.8 pM. The phosphoprotein steady-state level is reduced by 85 -90 % by phospholipase-A treatment or solubilization of calcium-preloaded sarcoplasmic reticulum with Triton X-100.The phosphoprotein of sarcoplasmic reticulum formed from [32P]orthophosphate is dephosphorylated by ADP with resultant in ATP synthesis. Phosphoprotein formation and ATP synthesis by sarcoplasmic reticulum are unaltered by azide, dinitrophenol, dicyclohexyl carbodiimide and oligomycin.The phosphoprotein is acid-stable. The trichloroacetic-acid-denaturated 32P-labelled membrane complex is dephosphorylated by hydroxylamine, indicating that the phosphorylated protein is an acylphosphate.Polyacrylamide gel electrophoresis (performed with phenol/acetic acidjwater) of sarcoplasmic reticulum phosphorylated by [32P]orthophosphate demonstrates that the phosphate incorporation occurs into a protein with a molecular weight of about 100000, as in the case of phosphoprotein formation from ATP.The data suggest that the phosphoprotein of the calcium-dependent ATPase formed from inorganic phosphate represents a high-energy intermediate of the reverse reaction of the calcium pump of cardiac sarcoplasmic reticulum.ATP-driven calcium uptake by cardiac sarcoplasmic reticulum [l -31 occurs via the formation of a phosphorylated intermediate of the calciumdependent ATPase [4-71, similar to the process described for skeletal muscle sarcoplasmic reticulum [8 -111. However, there are several quantitative differences between the calcium pumps of cardiac and skeletal muscle sarcoplasmic reticulum insofar as the rates of calcium uptake, calcium-dependent ATP splitting and ATP-ADP phosphate exchange, as well as the phosphoprotein steady-state level formed from ATP are lower in cardiac sarcoplasmic reticulum [l -71 as compared with skeletal muscle sarcoplasmic reticulum [12 -161. These observations do not appear to be fully explained by the lower yield of ATPase protein present in cardiac sarcoplasmic reticulum fractions [7].Moreover, ATP synthesis from ADP and inorganic phosphate by sarcoplasmic reticulum [17 -201, which proceeds via the phosphorylation of the calciumdependent ATPase by inorganic phosphate [17-191, has been demonstrated to occur in sarcoplasmic reticulum of skeletal muscle, but not in cardiac sarcoplasmic reticulum.Phosphorylation of the calcium-dependent ATPase of dog hearts by inorganic phosphate was investi...