Zinc metalloprotease activity in the cement precursor secretion of the barnacle, Chthamalus fragilis Darwin

Adult barnacles, Chathamalus fragilis, were removed carefully from the leaves and stems of marsh grass and floated base-up in algae-supplemented sea water. During the next 24-72 h, the animals secreted onto their exposed bases, a fluid, the cement precursor secretion (CPS), aliquots of which were collected in glass micropipets and pooled. The concentration of protein in pooled samples of CPS averaged 1.5 g +/- 0.42 protein/l of secretion. Protease activity was expressed as A(492) units/h/g of CPS protein. Aliquots of 5-45 l of pooled CPS samples, incubated in the presence of FTC-casein at 37 degrees C for 24 h, exhibited 8.31 x 10(-5) +/- 1.55 x 10(-5) DeltaA(492) units/hr/g of protein on average. Protease activity was optimized by the addition of 10 mM Ca(++) ions. Activity was detectable over a broad pH range, but was optimal around pH 8. Protease activity was inhibited up to 40% in the presence of 2.7 mM ethylenediaminetetraacetic acid (EDTA) and up to 97% in the presence of 2.5 mM 1,10-phenanthroline (OP) in the presence of 10 mM Ca(++) ions. Although low concentrations of Zn(++) ions (10 M) had little effect on protease activity, higher concentrations of Zn(++) ions (50 M to 15 mM Zn(++)) inhibited CPS protease activity. Protease activity was not inhibited by 1 mM phenylmethylsulfonylfluoride (PMSF), nor by 28 M E64, nor by 20 M leupeptin. At the present time, the protease activity in the barnacle CPS may best be characterized as a Ca-stimulated Zn-metalloprotease.