Calcium pump phosphoenzyme from young and old human red cells.

Area of Science:

• Cell Biology
• Gerontology
• Biochemistry

Background:

• Intracellular calcium (Ca2+) increases in aging human erythrocytes (red blood cells).
• Aged erythrocytes exhibit diminished active Ca2+ extrusion capacity.
• This defect is hypothesized to stem from calcium pump proteolysis via calpain activation.

Purpose of the Study:

• To investigate whether reduced Ca2+ pumping activity in aged erythrocytes is caused by proteolysis of the Ca2+ pump.
• To compare Ca2+ pump phosphorylation in young versus old human erythrocytes.

Main Methods:

• Percoll-separated young and old human erythrocytes were used.
• Ca2+ pump phosphorylation was studied using [gamma-(32)P]ATP.
• Phosphoenzyme levels were assessed after short and prolonged phosphorylation periods.
• Acidic electrophoresis and Western blotting with a specific monoclonal antibody (5F10) were employed.

Main Results:

• After 30s of Ca2+ phosphorylation, young erythrocyte membranes produced 50% of the phosphoenzyme compared to old cells.
• In the presence of Ca2+ and La3+, phosphoenzyme levels were similar in both young and old cells.
• Prolonged phosphorylation (50-90s) resulted in nearly identical equilibrium phosphoenzyme levels in both preparations.
• Both young and old erythrocyte preparations showed a single Ca2+-dependent radioactive band (~150 kDa) after acidic electrophoresis and Western blotting.

Conclusions:

• The molecular mass and number of active Ca2+ pump units are unaltered during erythrocyte aging.
• The reduced Ca2+ pumping activity in aged erythrocytes does not result from pump proteolysis.
• Calpain activation is unlikely to be the cause of impaired Ca2+ extrusion in aging red blood cells.