Localization of prostaglandin synthetase in chicken epiphyseal cartilage.

Abstract

Prostaglandin synthetase activity in high-speed particulate fractions of chick epiphyseal cartilage has been characterized with respect to cofactor requirements, pH optimum, buffer-ion effects, types of prostaglandins formed, and the distribution of prostaglandin synthetase activity in zones of the epiphyseal plate. Direct homogenization of cartilage was found to be more efficacious than releasing chondrocytes by enzymatic digestion for preparation of prostaglandin synthetase, a homogenization time of 4 min yielding maximal activity. The optimal incubation medium contained 50 mM Tris buffer (pH 7.5), 2.5 mM epinephrine, 1 micronM hemoglobin, 3.25 mM glutathione, 200 microgram/ml enzyme protein, and 5 micronM substrate. Glutathione was effective only if present during homogenization. Rates of PGE2 biosynthesis were linear up to 15 min and then rapidly declined, indicative of self-deactivation. The low levels of PGF2alpha formed, and their decrease after 20 min incubation, suggests the possible presence of degradative enzymes. Prostaglandin synthetase was inhibited by aspirin, indomethacin, and vitamin E, but not vitamin K1. Cation concentrations in the physiological range had only modest effects on prostaglandin biosynthesis, and then only if present during tissue homogenization. In the presence of phosphate buffer, Ca2+ was somewhat inhibitory. Since in the absence of phosphate Ca2+ had no deleterious effect, it is probably that the inhibitory effect was caused by precipitation of calcium phosphate. Hypertrophic and calcified cartilage exhibited significantly higher prostaglandin synthetase activity than the proliferating and maturing zones. The increased synthesis of prostaglandins in the low layers of the growth plate may indicate a role of these factors in chondrocyte differentiation and/or calcification.