적혈구막에서 Calcium ion 농도 변화에 따른 ATP의 phosphate incorporation에 관한 연구
It is generally accepted that calcium ion reduce membrane permeabilities to various substances and membrane excitability in excitable tissue (Gossweiler et al., 1954; Weidmann, 1955; Frankenhaeuser and Hodgkin, 1957). Ringer (1883) and Niedergerke (1963) demonstrated that Ca**++ plays an important role in maintaining
the contractility of the heart muscle. Also the requirements of Ca**++ for excitation-contraction coupling processes in broth cardiac and skeletal muscles was demonstrated (Heilbrumn and Wiercinski, 1947 ; Weber, and Herz, 1962; Ebashi and Lipman, 1962). Ca**++ movement through biological membranes was examined by Baker (1969) in the nerve cell, Niedergerke et al. (1963) and Lange and Frank (1972) in the cardiac muscle, Schatzman and Vincenzi (1969) in RBC membrane, Hasselbach (1964) and Ebashi and Lipman (1962) in sarcoplasmic reticulum of the skeletal muscle.
In 1965, Skou speculated that phosphate incorporation by ATP is an intermediate step in the sodium transport system of cell membranes. Ebashi and Lipman (1962) and Hasselbach (1964) reported that the sarcoplasmic reticulum of cardiac and skeletal
muscles play an essential role in the regulation of calcium ion concentrations in these muscle cells. These authors also reported that the fragments of the sarcoplasmic reticulum were capable of an exchange of terminal phosphate from ATP to ADP and speculated that a energy-rich prosphate bond was formed in the membrane
of sarcoplasmic reticulum and that the phosphorylated site could function as a carrier substance for the transfer of Ca**++ through the membranes. The terminal phosphate incorporation into the membrane has been demonstrated by many workers in sarcoplasmic reticulum of skeletal muscle (Yamamoto and Tonomura, 1967a, b; Martonosi, 1969; Makinose, 1969; Inesi et al., 1970), and of the cardiac muscle (Namm, 1972).
An active calcium transport hart been demonstrated in red blood cells and this process requires an energy supply to transfer calcium from the inside to the outside of the cell against an electrochemical gradient (Schatzman and Vincenzi, 1969). The present study is to test whether incorporation of the terminal phosphate of ATP into the RBC membrane and the effects of various ions on the formation of a phosphorylated intermediate.
(1) Preparation of RBC ghost membrane fragments: Fresh human blood was obtained from the blood bank and the membrane fragments were isolated by the method described by Dodge et al., (1963) and protein was measured by the method of Lowry et al., (1951). Membrane fragments (about 0.5mg protein) were spread over a cover
glass and dried in a desiccator containing silica gel in a cold room.
(2) Incubation and washing: Several cover glasses with the dried membrane fragments were introduced into a plexiglass rack which had perforated walls so that the incubation mixture or washing solution was freely accessible to the cover glasses. The rack with the cover glasses was immersed at room temperature for 10 minutes in a 2mM Tris solution (pH 7.0) and then incubated at 0℃ for 15 minutes in a solution mixture containing 150mM sucrose, 30mM Tris (pH 7.0), 1 mM MgCl^^2 , 1mM
ATP, and 0.1uc/ml (32)**P -ATP. After incubation, the rack was washed in a solution containing 1mM ATP, 1mM potassium phosphate, 2mM Tris (pH 7.0) and 5mM EGTA by dipping 6 times for 2 seconds each. Then the cover glasses were taken out and dried at room temperature. Each cover glass was introduced into a counting vial with a scintillation cocktail and the radioactivity remaining on the cover glass was counted by a Tri-Carb liquid scintillation counter (Model 3320, Packard Tri-Carb Instrument Co., Inc.).
(1) Time course of phosphate incorporation of ATP: Incubation was carried out for periods of 1.0, 5.0, 10, 20 and 40 minutes respectively. The amount of phosphate incorporation of ATP increased linearlly regardless whether calcium was present or
absent in the medium as the incubation time increased. The maximal capacity of phosphate incorporation of ATP was observed between 10 to 20 minutes.
On the basis of this experiment, incubation was carried out for a period of 15 minutes.
(2) Effect of Ca**++ concentration on phosphate incorporation of ATP: No significant difference was found in the calcium tested at concentrations between 10**-8 and 5×10**-4 moles, whereas at concentration of 10**-3 moles of calcium, (32)**P - incorporation from γ-(32)**P -ATP wag significantly increased and the maximum incorporation into membrane was observed at a Ca**++ concentration of 10**-2 moles.
(3) The phosphate incorporation into lipid extracted RBC membrane: The phosphate incorporation was decreased by about 10% in the lipid-extracted RBC membrane and the Ca**++ dependent phosphate incorporation was not observed.
(4) Effect of varying ATP concentrations on the phosphate incorporation: Amount of phosphate incorporation into RBC membrane increased as the ATP concentration increased and leveled off at 5 mM of ATP concentrations.
(5) Effect of Mg**++ on phosphate incorporation: At 0.1mM of Mg**++, the calcium-dependent phosphate incorpotation was highest but decreased at higher concentrations of Mg**++ .
(6) Effect of La**++ on phosphate incorporation: La**+++ at a concentration of 0.5mM increased the phosphate incorporation but 5mM of La**+++ in the medium decreased the phosphate incorporation of ATP. This result indicates that La**+++ alone increases the phosphate incorporations at low concentrations and decreases the incorporation at higher concentrations like Ca**++ , although Ca**++ and La**+++ were found to compete with each other at binding sites.