Alkalinization stimulates the purified plasma-membrane Ca2+ pump by increasing its Ca2+ affinity.

Area of Science:

• Biochemistry
• Cell Physiology
• Membrane Transport

Background:

• Plasma-membrane Ca2+-ATPase (PMCA) activity is influenced by its surrounding charges.
• Negatively charged phospholipids activate PMCA, while polycations inhibit it, suggesting charge interactions are crucial.

Purpose of the Study:

• To investigate the effect of physiological pH changes on the activity of the purified plasma-membrane Ca2+ pump from stomach smooth muscle.
• To determine if altering the ATPase protein's charge through pH modification impacts its function.

Main Methods:

• Purified plasma-membrane Ca2+ pump from stomach smooth muscle was studied.
• Enzyme activity was measured at varying pH levels (6.9 to 7.4) using a calcium buffer (BAPTA).
• Experiments involved phosphatidylcholine (PC) and various negatively charged phospholipids, as well as plasma-membrane-enriched fractions and inside-out vesicles.

Main Results:

• Increasing pH from 6.9 to 7.4 doubled ATPase activity at low calcium concentrations (0.3 microM) with various phospholipids.
• This stimulation was attributed to increased enzyme affinity for Ca2+, with no change in Vmax.
• Alkalinization also enhanced PMCA activity in membrane fractions and Ca2+ uptake in vesicles, indicating a cytoplasmic site of action.

Conclusions:

• Physiological pH changes significantly modulate the activity of the stomach smooth muscle Ca2+ pump.
• The enzyme's affinity for Ca2+ is sensitive to pH, suggesting charge-based regulation at the cytoplasmic interface.
• These findings underscore the role of the pump's charge environment in regulating calcium homeostasis.