Acetyl Phosphate as Substrate for Ca2+ Uptake in Skeletal Muscle Microsomes

In skeletal muscle microsomes, Ca2+ uptake activated by acetyl phosphate is inhibited by alkali ions. The inhibitory activity of these ions depends on the acetyl-P concentration in the assay medium. For 0.2 mu acetyl-P the pattern of inhibition is I3 > Na+ > K+ = Rbf = Csf and for 2.0 mu acetyl-P it is Naf > Li+ 2 K+ = Rb+ = Cs+. In assay media containing 7.1 pnd Ca2+ and either 20 mM KCl, 120 mM KCl, or 120 mM NaCl, the plot of the initial rate of Cazf uptake against acetyl-P concentration yields hyperbolic saturation curves. In an assay medium containing 120 mu LiCl, this saturation curve has a sigmoidal shape. In the presence of 2 mM acetyl-P, the initial rate of Ca2f uptake measured as a function of the Ca2f concentration in the assay medium displays a saturation curve with a sigmoidal shape. However, if ATP is used as substrate, a hyperbolic saturation curve is obtained. For 2 mM acetyl-P, the inhibitory activity of the alkali ions depends on the Ca2f concentration in the assay medium. For Ca2+ concentrations ranging from 1 to 2 PM, the pattern of inhibition is Lif >_ Na+ > Kf, and for CaZ+ concentration of 7.1 PM, the pattern of inhibition is Na+ > Li+ 2. K+.


SUMMARY
In skeletal muscle microsomes, Ca2+ uptake activated by acetyl phosphate is inhibited by alkali ions. The inhibitory activity of these ions depends on the acetyl-P concentration in the assay medium.
In assay media containing 7.1 pnd Ca2+ and either 20 mM KCl, 120 mM KCl, or 120 mM NaCl, the plot of the initial rate of Cazf uptake against acetyl-P concentration yields hyperbolic saturation curves.
In an assay medium containing 120 mu LiCl, this saturation curve has a sigmoidal shape. In the presence of 2 mM acetyl-P, the initial rate of Ca2f uptake measured as a function of the Ca2f concentration in the assay medium displays a saturation curve with a sigmoidal shape.
However, if ATP is used as substrate, a hyperbolic saturation curve is obtained. For 2 mM acetyl-P, the inhibitory activity of the alkali ions depends on the Ca2f concentration in the assay medium. For Ca2+ concentrations ranging from 1 to 2 PM, the pattern of inhibition is Lif >_ Na+ > Kf, and for CaZ+ concentration of 7.1 PM, the pattern of inhibition is Na+ > Li+ 2. K+.
The relaxation of skeletal muscle is related to the ATP-dependent Ca2+ uptake of sarcoplasmic reticulum. This process has been studied in vitro by means of sarcotubular fragments isolated from the microsomal fraction of skeletal muscle homogenates (1,2).
It has been shown in previous reports that acetyl phosphate can be used as substrate for Cazf uptake by skeletal muscle microsomes (3)(4)(5)(6).
The Ca*f uptake supported by acetyl-P was shown to be inhibited by Li+, Na+, and K+. This inhibition was not observed when 4 mru ATP was used as substrate (4). In this paper, the effect of alkali ions on the acetyl-P-supported Ca2+ uptake was further studied.
The data shows that, when * Visiting scientist at the Max-Plan&-Institut. Permanent address, Instituto de Biofisica, Universidade Federal do Rio de Janeiro, Av. Pasteur 458, Rio de Janiero-Gb, Brasil.
acetyl-P is used as substrate, the kinetics of Ca2f transport by skeletal muscle microsomes resembles that of allosteric enzymes.

Preparation
of &licrosomesThese were prepared by the method of Hasselbach and Makinosel which is the standard procedure of the laboratory.
All operations were performed at 4". The hind leg and back muscles of a rabbit were ground in a meat grinder.
Approximately 500 g of the minced muscle were mixed with 1,500 ml of a cold solution containing 100 mM KCl, 2 mM EDTA, 2.5 KH2P04, and 2.5 mM K2HPOI. The mixture was homogenized in a Waring Blendor for 2 min. The myofibrils were sedimented by centrifugation at 6,500 X g for 15 min. The supernatant was centrifuged at 10,000 x g for 15 min to remove mitochondria.
The supernatant was centrifuged in a Spinco model L preparative ultracentrifuge at 44,000 x g for 1 hour.
The pellet was suspended in 160 ml of a solution containing 1 M sucrose and 50 mM KCl.
The material was dispersed in a Potter-Elvehjem tissue grinder and centrifuged at 4,500 x g for 15 min. To the supernatant were added 120 ml of KCl, 2 M; 8 ml of ATP, 0.1 M; 8 ml of MgC&, 0.1 n%; and 104 ml of water.
The mixture was centrifuged at 80,000 x g for 90 min. The pellet was washed twice with 200 ml of KCI, 0.1 M. In each washing, the material was dispersed in a Potter-Elvehjem tissue grinder and centrifuged at 80,000 x g for 1 hour.
The pellet was then resuspended in 25 ml of KCl, 0.1 M, and stored at 5". This preparation proved to be active for at least 1 week. Protein was estimated by the micro-Kjeldahl method assuming the nitrogen content of the protein to be 160/;. Standard Assay--Unless otherwise stated, the incubation medium consisted of 10 mM Tris-maleate buffer, pH 7.0, 5 mM MgCls, 0.10 mM 45CaC12, 0.15 InM EGTA,2 4 mM potassium oxalate, and the specified concentrations of acetyl-P and either NaCl, KCl, or LiCl.
For these CaC12 and EGTA concentrations, the free calcium ion concentration calculated was 6.5 x 10m6 M. The reaction was started by the addition of microsomes, 0.15 mg of protein per ml. After 2 min of incubation at 37", the reaction was stopped by removal of particles with Millipore filters.
In all experiments, controls were performed both (a) Xeasurement of Ca2+ Uptake-45CaC12 was determined with a liquid scintillation counter.
The percentage of Ca2f bound to microsomes was calculated from the radioactivity of the microsome-free medium and that of the microsome-containing sample. In the range of microsomal protein concentration used, the amount of Ca2f bound to microsomes in the absence of acetyl-P was less than 0.025 pmole per mg of protein, independent of the concentration of monovalent cations in the assay medium.
Apparent Binding Constant of EGTA for Calcium---The solubility method described by Murphy and Hasselbach (7) was used to determine the extent of calcium-EGTA complex formation in the presence of alkali ions. The difference in soluble calcium concentration with and without the addition of EGTA to a suspension of a sparingly soluble calcium salt was measured. 2. Inhibition of Ca2f uptake by alkali ions. Incubation medium and experimental conditions were as described under "Methods." In each experiment, the percentage of inhibition was calculated relative to a control containing 15 mM KC1 in the assay medium. The values of each column represent the average f S.E. of 8 experiments.
45Ca-Labeled oxalate monohydrate crystals with a high specific activity were prepared according to McComas and Rieman (8).
To 75 ml of 0.134 M ammonium osalate, 48 ml of 0.25 M 4"CaC12 were slowly added with vigorous stirring. Then, 7.4 ml of 0.1 M HCl were added. The precipitate was digested at 100" for 24 hours and washed several times with cold deionized water. Solubility measurements were made by adding an excess of the crystals to a mixture containing 10 rn~ Tris-malea.te buffer, pH 7.0, 5 mM MgC12, potassium oxalate ranging from 5 to 15 mM, and different concentrations of either KU, NaCl, or LiCl. Parallel experiments were performed with the same mixture plus EGTA ranging from 0.1 to 0.2 mM. After 2 to 3 hours of incubation at 37", aliquots of these mixtures were filtered through Millipore filters with average pore diameter of 0.1 ~111, and the 4jCa content was determined with a scintillation counter.
Equilibrium between calcium oxalate and its sxturated solution was attained in about 1 hour. The concentration of the free calcium ions was calculated by means of the solubility product of calcium oxalate measured in EGTA-free solutions.
The calcium-EGTA concentration was given as the difference between the soluble calcium concentration obtained with and without EGTA. The free EGTA in the mixture was determined as the difference between the total EGTA added and the calcium-EGTA measured. The apparent dissociation constant of the complex calcium-EGTA was calculated according to the relation &iss-(calcium free) (EGTA free) /(calcium-EGTA).
All the other chemicals were analytical grade.

Ca2+
Uptake as Function of Acetyl-P Concentration-In these experiments the initial velocity of Ca2+ uptake was measured. When one of the activating divalent cations or the substrate was varied, the other components were added at optimal concentrations.
This experimental procedure was used to study the inhibition of Ca2+ uptake by monovalent cations. Fig. 1 shows Ca*+ uptake as a function of acetyl-P concentration in the assay medium.
In the presence of a low concentration of monovalent cations (control in Fig. l), a hyperbolic saturation curve was observed. The apparent K, measured in five different microsomal preparations was found to range from 2.0 X 10m4 to 2.5 x 10W4 nI. When the monovalent cation concentration of the assay medium was increased to 120 nlM with either KC1 or NaCl, the uptake of Cazf was inhibited (Fig. 1). Na+ was a more effective inhibitor than K+ at any of the acetyl-P concentrations tested.
In both cases a hyperbolic relationship between Ca2+ uptake and acetyl-P concentration was obtained.
On the other hand, this relationship changed to a sigmoid curve when 120 mM LiCl was added to the control medium.
Thus, the relative inhibition of Ca2+ uptake by alkali ions varies with the acetyl-P concentration in the assay medium. Fig. 2 shows that, at an acetyl-P concentration of 0.2 111~, their relative inhibitory activity was Li+ > Na+ > K+. At 2.0 m acetyl-P it changed to Na+ > Li+ >_ K+. Fig. 3 shows the progressive inhibition induced by increasing concentrations of alkali ions at acetyl-P concentrations of 0.2 and 2.0 mM. RbCl and CsCl also inhibit microsomal Cazf uptake. It was found in four preparations tested, that the inhibitory activity of Rb+ and Cs+ was essentially the same as that of Kf both at 0.2 and 2.0 mM acetyl-P.
In control experiments, the Ca2+ uptake was measured in 0.2 mM acetyl-P both with 10 m&r KC1 and with 10 mM KC1 plus 240 mM sucrose. It was observed that the amount of CaZf taken up was essentially the same, thus excluding the possibility of an osmotic influence on Ca2f uptake.

Eflect of Monovalent Cations on Dissociation Constant of Calcium-EGTA
Complex-In order to study the Ca2f uptake as a function of the Ca2+ concentration in the assay medium, a CaC12 plus EGTA system was used. It has been shown that the apparent dissociation constant of the complex calcium-EGTA varies with the composition of the assay medium (9-12).
To determine whether the different concentrations of monovalent cations used modify this dissociation constant, the solubility method described under "Methods" was used. In 15 different determinations with 120 mM of either KC1 or NaCl, the value found for Kdiss was 3.95 X lop6 M f S.E. 0.24. This value is in the range reported by Ebashi and Ogawa at pH 6.8 and is about 20 times higher than the value calculated from Schwarzenbach's data for pH 7.0 (9-13). Table I shows that this constant was not significantly altered when the KC1 concentration of the medium was raised from 15 to 120 mM or when NaCl and LiCl were substituted for KC1 in the same concentration range. In addition, no significant differences were found when: (a) the free calcium concentration of the medium was modified by increasing the oxalate concentration from 4 to 16 IYIM; (b) the EGTA concentration was raised from 0.1 to 0.2 mM; or (c) 2 mM acetyl-P or 2 InM phosphoenolpyruvate plus 10V5 M ATP were added to the medium.
Thus, in the following experiments, the value of Kdjss = 3.95 X 10m6 M was used to calculate the free calcium concentrations for given CaCl2 and EGTA concentrations.
Cay+ Uptake as Function of Ca2+ Concentration- Fig.  4 shows Cn2+ uptake in media containing 2 mM acetyl-P, a low concentration of KU, and different concentrations of ionic calcium. Sipmoidal saturation curves were observed in the several mi- This implies that the apparent K, for Ca2+ was progressively modified by increasing concentrations of this cation (14). Kinetics such as this have been deemed characteristic of allosteric enzymes. In a separate paper (15), it has been shown that a hyperbolic saturation curve is obtained when ATP was used as substrate in concentrations of 10m5 M or higher. Fig. 4 shows this experiment, repeated as a control, with the same microsomal preparation used for the test in the presence of acetyl-P.
Marked differences between the saturation curves obtained with ATP and acetylP can be observed. Fig. 5 shows the inhibitory effect of alkali ions. In the presence of 120 mM KC1 or 120 mM NaCl, sigmoidal curves were obtained with a shape similar to that observed with a control medium containing a low concentration of KCl. However, when 120 mM LiCl was added to the control assay medium, a sigmoidal curve of different shape was observed.
At lower Ca2f concentrations, Li+ was a strong inhibitor, at least as strong as Na+. Its activity decreased when the Ca2+ concentration was raised, and at 3.8 pM Cazf the activity of Li+ was similar to that of K+. Addition of excess Mg2+ inhibited Ca2+ uptake. The pattern of this inhibition was similar for the two concentrations of acetyl-P used. DISCUSSION The data presented show that when acetyl-P was used as substrate the Ca*+ transport system of skeletal muscle microsomes displayed kinetics similar to that described for allosteric enzymes (14).
Different kinetics data toward Ca2f were observed depending on the choice of substrate.
When acetyl-P was used, the saturation curve toward Ca2f showed a sigmoidal shape indicating a cooperative effect, where at least two calcium ions interact with the carrier system. The binding of one ion in some manner facilitates the binding of the next, i.e. Ca2f was acting both as substrate and as activator (14,16). On the other hand, an ATP concentration of 1OV M elicited first order kinetics toward Ca2f. These data suggest that the binding of ATP to the microsomal membrane promotes a conforrnational change in the Ca* carrier system which results in a modification of its affinity for Ca2+.
In a separate paper (15), data have been presented showing that the alkali ions do not damage the microxomal membrane nor do they interfere with the precipitation of calcium oxalate in the interior of the vesicles. Thus, the described inhibition of Ca* transport is related to a specific effect of these alkali ions at the site of the Ca2+ pump.
The data of Figs. 1 and 2 show that the inhibitory effect of the alkali ions decreases when the acetyl-P concentration of the assay medium was raised. Yamamoto and Tonomura (17), measuring the Ca2+-dependent ATPase activity of sarcoplasmic reticulum, have shown that the Ca2+ affinity of the vesicles was enhanced by raising the ATP concentrations in the assay medium. It is possible that as in the case with ATP, increasing concentrations of acetyl-P also increased the affinity of the Ca 2+ transport system for calcium ions and that the observed inhibition was the result of different degrees of competition between Ca2f and the alkali ions. In a  (15), it has been shown that the alkali ions inhibit the Ca2f transport supported by ATP concentrations of 1 to 10 PM in a manner similar to that described in this paper for acetyl-P preparations.
Finally, it should be emphasized that there is additional evidence that the Ca2+ transport system exhibits characteristics similar to those described for allosteric enzymes (17,18). Weber, by means of the heavy fraction of the sarcoplasmic reticulum, has shown that the effect of caffeine on Ca2f transport varies with the ATP concentration in the assay medium (18).