A high-affinity calcium-stimulated ATPase activity in the hen oviduct shell gland

Abstract

The hen oviduct shell gland is a highly active calcium-transporting epithelial tissue which is responsible for the mineralization of the egg shell. We have identified a calcium-stimulated ATPase present at high specific activity in membrane preparations from shell gland mucosal shavings. In the presence of optimal MgCl 2 (5 m m) and a Ca 2+ buffer, ATP hydrolysis was stimulated by addition of low concentrations of free Ca 2+ ( K 0.5 ~0.4 μm); but not by similar concentrations of Mn 2+, Zn 2+, Co 2+, or La 2+. This stimulation was specific for ATP; there was little or no effect of Ca 2+ on hydrolysis of ADP, AMP, GTP, ITP, or p-nitrophenyl phosphate. Calcium-stimulated ATPase activity was inhibited by chlorpromazine, trifluoperazine, and quercetin, as well as by sulfhydryl-blocking agents, but not by oligomycin or ouabain. No significant effect of calmodulin was observed. Finally, low concentrations of free Ca 2+ (10 to 100 μ m) in the presence or absence of Mg 2+ stimulated transfer of 32P from [γ- 32P]ATP to a 105,000 molecular weight shell gland membrane protein. This phosphoprotein was sensitive to hydrolysis by heating or by hydroxylamine treatment at acidic pH, and its formation was not inhibited by addition of K +. The specific activity of Ca 2+-ATPase in total membrane preparations from laying hen shell gland ranged from 80 to 150 nmol/min/ mg protein, similar to or greater than levels found in purified plasma membrane fractions from a variety of tissues. No significant activity was found in membrane preparations from the magnum or isthmus regions of the oviduct, which are not involved in egg shell calcification. The characteristics of the Ca 2+-ATPase, its high specific activity, and its preferential localization in the shell gland region of the oviduct suggest a role for an ATP-dependent calcium transport system in egg shell mineralization.