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Isolation and partial characterization of magnesium ion- and calcium ion-dependent adenosine triphosphatase activity
Abstract:
Microsomal fraction was prepared by ultracentrifugation of homogenates of cortical tissue from bovine brains. The preparation displayed ATPase (adenosine triphosphatase) activity in the presence of Mg(2+) (6.4mumol of P(i)/h per mg of protein) and Ca(2+) (3.4mumol of P(i)/h per mg of protein). Kinetic analysis of the activation of the enzyme preparation by Ca(2+) resulted in the demonstration of two apparent K(m) values for Ca(2+) (6.0x10(-8)m and 1.2x10(-6)m). Treatment of the microsomal membranes with Triton X-100 resulted in solubilization of the ATPase, though with some loss of activity. The solubilized microsomal proteins were incorporated into liposomes. By incubation of the liposomes in media containing (45)Ca(2+) an ATP-dependent uptake of Ca(2+) was demonstrated. The solubilized preparation was subjected to preparative isoelectric focusing in granulated gel beds. Two distinct peaks of Mg(2+)- and Ca(2+)-dependent ATPase activity were observed at pH4.8 (peak 4.8) and at pH6.3 (peak 6.3). The material isolated in peaks 4.8 and 6.3 was focused in polyacrylamide gel with pH gradients. The material corresponding to peak 4.8 consisted of a single protein, whereas peak 6.3 contained one major and at least one minor protein. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis confirmed these results and indicated that the major component of peak 4.8 and the protein of peak 6.3 both had a molecular weight of 105000. The material in peaks 4.8 and 6.3 was assayed for ATPase activity in the presence of various concentrations of Ca(2+). Kinetic analysis of the results for peak 4.8 demonstrated an apparent K(m) value for Ca(2+) of 4.1x10(-8)m. The enzyme isolated at pH6.3 had an apparent K(m) value of 3.8x10(-6)m. However, when the material from peak 4.8 was incubated in the presence of 1mm-Mg(2+) the ATPase could not be activated by Ca(2+).
Insights
Researchers isolated two distinct adenosine triphosphatase (ATPase) enzymes from bovine brain microsomes. One enzyme, activated by calcium, showed specific calcium affinity, while the other was magnesium-dependent, highlighting distinct roles in cellular calcium regulation.
Area of Science:
- Biochemistry
- Neuroscience
- Molecular Biology
Background:
- Microsomal fractions from bovine brain cortical tissue were prepared using ultracentrifugation.
- The preparation exhibited adenosine triphosphatase (ATPase) activity in the presence of magnesium (Mg2+) and calcium (Ca2+).
Purpose of the Study:
- To characterize and isolate Ca2+-dependent and Mg2+-dependent ATPase activities from bovine brain microsomes.
- To investigate the kinetic properties and molecular characteristics of the isolated ATPase enzymes.
Main Methods:
- Enzyme activity assays for ATPase in the presence of Mg2+ and Ca2+.
- Solubilization of microsomal membranes using Triton X-100 and incorporation into liposomes.
- Separation of ATPase activities using preparative isoelectric focusing and polyacrylamide gel electrophoresis.
- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for molecular weight determination.
- Kinetic analysis to determine apparent Km values for Ca2+.
Main Results:
- Two distinct peaks of ATPase activity were identified at pH 4.8 and pH 6.3 after isoelectric focusing.
- Peak 4.8 contained a single protein, while peak 6.3 contained one major and at least one minor protein, both with a molecular weight of 105,000 Da.
- Kinetic analysis revealed different apparent Km values for Ca2+ for the enzymes isolated at pH 4.8 (4.1x10(-8) M) and pH 6.3 (3.8x10(-6) M).
- The enzyme from peak 4.8 could not be activated by Ca2+ in the presence of Mg2+.
Conclusions:
- Bovine brain microsomes contain at least two distinct Mg2+- and Ca2+-dependent ATPase enzymes.
- The enzyme isolated at pH 4.8 exhibits high affinity for Ca2+ and its activity is inhibited by Mg2+.
- These findings suggest distinct roles for these ATPases in cellular calcium handling and signaling pathways within the brain.