Tohru Kanazawa

Occurrence and Characteristics of a Rapid Exchange of Phosphate Oxygens Catalyzed by Sarcoplasmic Reticulum Vesicles

Abstract Sarcoplasmic reticulum vesicles prepared from rabbit skeletal muscle catalyze a rapid Pi ⇄ HOH exchange in the presence of Mg2+ and absence of ATP and Ca2+. The capacity for oxygen exchange is about 14 times the potential capacity for ATP cleavage in the presence of Ca2+. No detectable exchange is found without added MgCl2. The exchange is unaffected by oligomycin, 2, 4-dinitrophenol, or ouabain, but strongly inhibited by low concentrations of Ca2+ in the medium. The Ca2+ ion concentration giving a half-maximum inhibition is 2.0 µm in the presence of 5 mm MgCl2. A Hill plot of the Ca2+ inhibition gives a straight line with a Hill coefficient of 1.8. The Ca2+ inhibition is competitively overcome by additional Mg2+. The Pi ⇄ HOH exchange is almost completely inhibited by the detergent Triton X-100 at low concentrations in which the ATPase activity is not disturbed. Sarcoplasmic reticulum vesicles are phosphorylated by Pi in the presence of Mg2+ and absence of Ca2+ under conditions similar to those for the Pi ⇄ HOH exchange. The phosphorylation requires Mg2+ and is strongly inhibited by low concentrations of Ca2+. The response of the phosphorylation to Ca2+ is quite similar to that of the Pi ⇄ HOH exchange; the Ca2+ ion concentration giving a half-maximum inhibition is 2.0 µm in the presence of 5 mm MgCl2, and the Hill coefficient is about 2.0. The various properties of the exchange give strong support to the probability that it results from reversal of steps in the over-all process associated with Ca2+ transport driven by ATP cleavage.

Slow transition of phosphoenzyme from ADP-sensitive to ADP-insensitive forms in solubilized Ca2+, Mg2+-ATPase of sarcoplasmic reticulum: evidence for retarded dissociation of Ca2+ from the phosphoenzyme.

Abstract Solubilized Ca2+, Mg2+-ATPase of sarcoplasmic reticulum was phosphorylated with ATP without added MgCl2. The phosphoenzyme formed was ADP-sensitive. Ca2+ in the medium was chelated after phosphorylation. This induced a slow transition of the phosphoenzyme from ADP-sensitive to ADP-insensitive forms. The ADP-sensitivity was restored by subsequent addition of CaCl2. These results showed that the transition was caused by dissociation of Ca2+ bound to the phosphoenzyme. Further observations indicated that, when Ca2+ in the medium was chelated, Ca2+ bound to the phosphoenzyme was dissociated much more slowly than Ca2+ bound to the dephosphoenzyme. This suggests a possible formation of the occluded form of the Ca2+-binding site in the phosphoenzyme.

Quasi-irreversible inactivation of the sarcoplasmic reticulum Ca2+-ATPase by simultaneous tight binding of magnesium and fluoride to the catalytic site

The sarcoplasmic reticulum Ca(2+)-ATPase was inactivated quasi-irreversibly by the treatment with KF in the presence of Mg2+ and absence of Ca2+. This inactivation was Mg(2+)-dependent, and prevented by high-affinity Ca2+ binding. The enzyme was completely protected by ATP against the inactivation with an affinity consistent with that of the catalytic site for ATP. The affinity for Mg2+ in this inactivation was in agreement with that for Mg2+ in phosphorylation of the enzyme with Pi. Mg.ATP did not bind to the inactivated enzyme, whereas metal-free ATP did bind to it with a high affinity. These findings suggest that the Mg2+ binding sub-site in the catalytic site of the inactivated enzyme is occupied by tightly-bound Mg2+. The enzyme was completely protected by Pi against the inactivation with an affinity consistent with that of the catalytic site for Pi. The inactivated enzyme showed neither phosphorylation with Pi nor high-affinity vanadate binding. These findings suggest that the phosphorylation site of the inactivated enzyme is occupied by tightly-bound F-. The contents of tightly-bound Mg2+ and F- in the inactivated enzyme were determined after unbound Mg2+ and F- were removed by gel filtration. 2.3 mol of Mg2+ and 3.7 mol of F- per mol of phosphorylation sites were tightly bound to the enzyme. The tight binding of these ligands depended on the presence of each other, and was completely prevented by high-affinity Ca2+ binding. Linear relationships were found between the contents of the tightly-bound ligands and the extent of the enzyme inactivation. The tightly-bound Mg2+ and F- were entirely released by low-affinity Ca2+ binding, and correspondingly the ATPase activity was restored. It is concluded that the observed enzyme inactivation is caused by simultaneous tight binding of Mg2+ and F- to the catalytic site.

Mutations of Arg198 in sarcoplasmic reticulum Ca2+‐ATPase cause inhibition of hydrolysis of the phosphoenzyme intermediate formed from inorganic phosphate

Arg198 of sarcoplasmic reticulum Ca2+‐ATPase was substituted with lysine, glutamine, glutamic acid, alanine, and isoleucine by site‐directed mutagenesis. Kinetic analysis was performed with microsomal membranes isolated from COS‐1 cells which were transfected with the mutated cDNAs. The rate of dephosphorylation of the ADP‐insensitive phosphoenzyme was determined by first phosphorylating the Ca2+‐ATPase with 32Pi and then diluting the sample with non‐radioactive Pi. This rate was reduced substantially in the mutant R198Q, more strongly in the mutants R198A and R198I, and most strongly in the mutant R198E, but to a much lesser extent in R198K. The reduction in the rate of dephosphorylation was consistent with the observed decrease in the turnover rate of the Ca2+‐ATPase accompanied by the steady‐state accumulation of the ADP‐insensitive phosphoenzyme formed from ATP. These results indicate that the positive charge and high hydrophilicity of Arg198 are critical for rapid hydrolysis of the ADP‐insensitive phosphoenzyme.

Deletions or Specific Substitutions of a Few Residues in the NH2-terminal Region (Ala3 to Thr9) of Sarcoplasmic Reticulum Ca2+-ATPase Cause Inactivation and Rapid Degradation of the Enzyme Expressed in COS-1 Cells

Amino acid residues in the NH2-terminal region (Glu2 – Ala14) of adult fast twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) were deleted or substituted, and the mutants were expressed in COS-1 cells. Deletion of any single residue in the Ala3–Ser6 region or deletion of two or more consecutive residues in the Ala3–Thr9 region caused strongly reduced expression. Substitution mutants A4K, A4D, and H5K also showed very low expression levels. Deletion of any single residue in the Ala3–Ser6 region caused only a small decrease in the specific Ca2+ transport rate/mg of SERCA1a protein. In contrast, other mutants showing low expression levels had greatly reduced specific Ca2+ transport rates. In vitroexpression experiments indicated that translation, transcription, and integration into the microsomal membranes were not impaired in the mutants that showed very low expression levels in COS-1 cells. Pulse-chase experiments using [35S]methionine/cysteine labeling of transfected COS-1 cells demonstrated that degradation of the mutants showing low expression levels was substantially faster than that of the wild type. Lactacystin, a specific inhibitor of proteasome, inhibited the degradation accelerated by single-residue deletion of Ala3. These results suggest that the NH2-terminal region (Ala3 –Thr9) of SERCA1a is sensitive to the endoplasmic reticulum-mediated quality control and is thus critical for either correct folding of the SERCA1a protein or stabilization of the correctly folded SERCA1a protein or both.

Stoichiometry of Phosphorylation to Fluorescein 5-Isothiocyanate Binding in the Ca2+-ATPase of Sarcoplasmic Reticulum Vesicles

In an attempt to establish the stoichiometry of phosphorylation in the Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles, phosphorylation by ATP (or Pi) or labeling by fluorescein 5-isothiocyanate (FITC) was performed with the SR vesicles under the conditions in which almost all the phosphorylation sites or FITC binding sites are phosphorylated or labeled. The resulting vesicles were solubilized in lithium dodecyl sulfate and then the Ca2+-ATPase was purified by size exclusion high performance liquid chromatography. Peptide mapping and sequencing of the tryptic digest of the purified enzyme showed that Lys-515 of the Ca2+-ATPase was exclusively labeled with FITC, in agreement with the previously reported findings. The content of the phosphoenzyme from ATP (4.57 nmol/mg of Ca2+-ATPase protein) or from Pi (4.94 nmol/mg of Ca2+-ATPase protein) in the purified enzyme was approximately half the content of the FITC binding site (8.17-8.25 nmol/mg of Ca2+-ATPase protein) and also half the content of the Ca2+-ATPase molecule (9.06 nmol/mg of Ca2+-ATPase protein) calculated from its molecular mass (110,331 Da). These results show that there is one specific FITC binding site per molecule of the Ca2+-ATPase (in agreement with the previously reported findings) and that the stoichiometry of phosphorylation to FITC binding is approximately 0.5:1.0. All the above findings lead to the conclusion that only half of the Ca2+-ATPase molecules present in the SR vesicles can be phosphorylated. FITC binding completely inhibited the ATP-induced phosphorylation before the binding reached its maximum level. This finding indicates that FITC preferentially binds to a part of the Ca2+-ATPase molecules and that this binding is primarily responsible for the inhibition of phosphorylation, suggesting an intermolecular ATPase-ATPase interaction.

Modification of arginine-198 in sarcoplasmic reticulum Ca2+-ATPase by 1,2-cyclohexanedione causes inhibition of formation of the phosphoenzyme intermediate from inorganic phosphate.

Sarcoplasmic reticulum vesicles were modified with 1,2-cyclohexanedione (CHD), a specific arginine-modifying reagent, in sodium borate (pH 8.0 or 8.8). Phosphoenzyme formation from Pi in the Ca2+-ATPase (reversal of hydrolysis of the phosphoenzyme intermediate) was almost completely inhibited by the modification with CHD. Tight binding of F− and Mg2+ and high affinity binding of vanadate in the presence of Mg2+, either of which produces a transition state analog for phosphoenzyme formation from the magnesium-enzyme-phosphate complex, were also markedly inhibited. In contrast, phosphoenzyme formation from acetyl phosphate in the forward reaction was unaffected. The enzyme was appreciably protected by tight binding of F− and Mg2+ or by high affinity binding of vanadate in the presence of Mg2+, but not by the presence of 20 mm MgCl2 alone or 150 mm Pi alone, against the CHD-induced inhibition of phosphoenzyme formation from Pi. Peptide mapping of the tryptic digests, detection of peptides containing CHD-modified arginyl residues with Girard’s reagent T, sequencing, and mass spectrometry showed that Arg-198 was a single major residue protected by tight binding of F− and Mg2+ against the modification with CHD. These results indicate that modification of Arg-198 with CHD is responsible for at least a part (the portion reduced by the transition state analogs) of the CHD-induced inhibition of phosphoenzyme formation from Pi and suggest that Arg-198 is located in or close to the catalytic site in the transition state for phosphoenzyme formation from the magnesium-enzyme-phosphate complex.

Modification of Histidine 5 in Sarcoplasmic Reticulum Ca2+-ATPase by Diethyl Pyrocarbonate Causes Strong Inhibition of Formation of the Phosphoenzyme Intermediate from Inorganic Phosphate

Sarcoplasmic reticulum vesicles were modified with diethyl pyrocarbonate (DEPC), a histidine-modifying reagent. Phosphoenzyme formation from Pi in the Ca2+-ATPase (reversal of hydrolysis of the phosphoenzyme intermediate) was almost completely inhibited by this modification. Tight binding of F− and Mg2+ and high affinity binding of vanadate in the presence of Mg2+, both of which produce transition state analogs for phosphoenzyme formation from the magnesium-enzyme-phosphate complex, were also inhibited. Formation of the phosphoenzyme from acetyl phosphate in the forward reaction was only weakly inhibited, but hydrolysis of the phosphoenzyme was strongly inhibited. The enzyme was protected by tight binding of F−and Mg2+ or by high affinity binding of vanadate in the presence of Mg2+ against the DEPC-induced inhibition of phosphoenzyme formation from Pi. The enzyme was also protected by tight binding of F− and Mg2+against the DEPC-induced inhibition of phosphoenzyme hydrolysis. Peptide mapping of the tryptic digests, detection of peptides containing DEPC-modified histidine by UV absorption at 240 nm, amino acid analysis, sequencing, and mass spectrometry showed that His-5 was a single major residue protected by the above transition state analogs against the modification with DEPC. These results indicate that modification of His-5 with DEPC is responsible for the DEPC-induced inhibition of phosphoenzyme formation from Pi and of phosphoenzyme hydrolysis and suggest that His-5 is located in or very close to the catalytic site in the transition state for phosphoenzyme formation from the magnesium-enzyme-phosphate complex and is likely involved in the catalytic process of this reaction step.

Characterization of the phosphoenzyme that is involved in the Ca2+-Ca2+ exchange catalyzed by the Ca2+-ATPase of sarcoplasmic reticulum vesicles

The rate of Ca2+ efflux was determined with 45Ca2+ -loaded sarcoplasmic reticulum vesicles (mainly with the light fraction of vesicles) at pH 6.5 and 0 degrees C. The efflux depended on external Ca2+, Mg2+, ATP and ADP, but it was not activated by AMP. The results indicate that the efflux is derived from Ca2+ -Ca2+ exchange mediated by the phosphoenzyme (EP) of membrane-bound Ca2+ -ATPase. EP was formed with Ca2+ -loaded vesicles (light fraction) under similar conditions without added ADP. The subsequent addition of EGTA and ADP induced triphasic EP dephosphorylation. Three species of EP (EP1, EP2, and EP3) were distinguished on the basis of this dephosphorylation kinetics, EP1, EP2, and EP3, corresponding to the first, second, and third phases of the dephosphorylation. Dephosphorylation of EP1 and EP2 resulted in stoichiometric ATP formation, while dephosphorylation of EP3 led to stoichiometric Pi liberation. The rate of Ca2+ efflux was compatible with that of EP2 dephosphorylation, whereas it was much lower than the rate of EP1 dephosphorylation and much higher than the rate of EP3 dephosphorylation. The intravesicular Ca2+ concentration dependence of the rate of EP2 dephosphorylation agreed with that of the rate of Ca2+ efflux. The results suggest that isomerization between EP1 and EP2 is the rate-limiting process in the Ca2+ -Ca2+ exchange and that EP3 is not involved in this exchange.

Identification of arginyl residues located at the ATP binding site of sarcoplasmic reticulum Ca2+-ATPase. Modification with 1,2-cyclohexanedione.

Sarcoplasmic reticulum vesicles were treated with 1,2-cyclohexanedione (CHD) in sodium borate (pH 8.0). The Ca2+-ATPase activity was completely inhibited. Inhibition of Mg·ATP and Mg·ADP binding to the high affinity ATP binding site as well as inhibition of phosphorylation with ATP occurred simultaneously with the inhibition of the Ca2+-ATPase activity. Phosphorylation with acetyl phosphate was not inhibited. The Ca2+-ATPase was strongly protected by Mg·ATP, Mg·ADP, and Mg·AMP against this inhibition. Binding of acetyl phosphate or Pi to the enzyme gave no protection. Phosphorylation with acetyl phosphate also had no protective effect. Peptide mapping of the tryptic digests, detection of peptides containing CHD-modified arginyl residues with Girards reagent T, and sequencing revealed that Arg-489, Arg-505, and Arg-678 were modified with CHD. Arg-489 and Arg-678 were almost completely protected by Mg·ATP against this modification, but partially protected by prelabeling with fluorescein 5-isothiocyanate, which occupies the adenosine binding region in the ATP binding site. In contrast, Arg-505 was slightly protected by Mg·ATP and almost completely protected by prelabeling with fluorescein 5-isothiocyanate. Taken together, these findings suggest that Arg-489 and Arg-678 are located in or near the region occupied by the triphosphate moiety of ATP, either or both of these residues being in or close to the region occupied by the α-phosphoryl group in the high affinity ATP binding site and involved in the CHD-induced inhibition of this enzyme and that Arg-505 is very close to (but slightly out of) the adenosine binding region in the ATP binding site. The acetyl phosphatase activity and phosphorylation with Pi were also inhibited by the CHD treatment, but the inhibitions were considerably slower than those described above. This suggests that the arginyl residues involved in these inhibitions are distinct from that involved in the inhibition of the Ca2+-ATPase activity.