Active calcium transport by porcine thyroid microsomes

Area of Science:

• Cell Biology
• Biochemistry
• Endocrinology

Background:

• Cytosolic calcium (Ca2+) levels are critical for cellular function.
• Endoplasmic reticulum plays a role in calcium storage and release.
• Understanding calcium transport mechanisms is essential for cell physiology.

Purpose of the Study:

• To investigate the presence and characteristics of an ATP-dependent Ca2+ uptake system in porcine thyroid microsomes.
• To identify the molecular components responsible for Ca2+ transport.
• To elucidate the role of this system in maintaining cellular Ca2+ homeostasis.

Main Methods:

• Differential centrifugation and sucrose density gradient centrifugation to isolate microsomes.
• Electron microscopy to visualize microsomal vesicles and calcium oxalate precipitation.
• Enzyme activity assays (Ca2+-dependent ATPase) and [gamma-32P]ATP phosphorylation studies.
• Dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to identify phosphoproteins.

Main Results:

• Porcine thyroid microsomes exhibit ATP-dependent Ca2+ uptake, indicating active transport.
• Oxalate enhances Ca2+ uptake by forming calcium oxalate precipitates within vesicles.
• A 105-kDa phosphoprotein, sensitive to hydroxylamine, was identified as a Ca2+ pump ATPase.
• Ca2+ transport and ATPase activity showed similar Ca2+ dependence, with half-maximal activation around 1 microM.

Conclusions:

• Porcine thyroid microsomes possess an ATP-dependent Ca2+ pump (Ca2+ ATPase) in the endoplasmic reticulum.
• This pump actively translocates Ca2+ into the microsomal lumen, contributing to low cytosolic Ca2+ concentrations.
• The identified Ca2+ pump shares properties with those found in other cell types, suggesting a conserved mechanism for calcium homeostasis.