Enzymic activities associated with the induction of chondrogenesis in vitro

Abstract

Chick embryo somites, Stages 16–17, when grown in vitro with embryonic notochord or spinal cord, form cartilage, in contrast to somites grown in the absence of notochord or spinal cord. In a study of this system of embryonic induction, cell-free extracts of both types of cultures were examined for the presence of ATP-sulfurylase (ATP:sulfate adenylyl-transferase, EC 2.7.7.4) and adenosine-5′-phosphosulfate kinase (ATP:adenylylsulfate 3′-phosphotransferase, EC 2.7.1.25). Both enzymes were demonstrated in the somites stimulated with notochord by the ability of the enzymic preparations to form 3′-phosphoadenosine-5′-phosphosulfate from ATP, Mg 2+ and 35SO 42 -. Formation of 3′-phosphoadenosine-5′-phosphosulfate was not detected in somites cultured without notochord. 3′-Phosphoadenosine-5′-phosphosulfate generated by extracts of cartilage-forming somites was characterized by electrophoretic and chromatographic behavior, lability to mild acid, ability to serve as substrate with rabbit-liver sulfokinase (3′-phosphoadenylylsulfate: phenol sulfotransferase, EC 2.8.2.1), and the formation of adenosine 5′-phosphosulfate after incubation with alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1). Cell-free extracts were also prepared from tissues which were isolated from the embryo but not cultured. These extracts of somites, notochords and spinal cords, and mesonephri had no demonstrable activity for ATP-sulfurylase and adenosine-5′-phosphosulfate kinase. Cell-free preparations from the whole trunk, however, showed considerable activity for these enzymes.