Redistribution of Tosylarginine Methylester Hydrolase Activity after Fertilization of Sea Urchin (Arbacia punctulata) Eggs

Abstract

Low speed residues (750g) from unfertilized sea urchin egg homogenates contain an enzyme capable of hydrolysing tosylarginine methylester (TAME)¹. Enrichment of this particulate fraction by differential centrifugation, followed by discontinuous sucrose gradient centrifugation, made it possible to distinguish visually about seventeen bands within the gradient (Fig. 1). Each band was carefully removed with a Pasteur pipette and assayed for total TAME hydrolase activity. As shown in Fig. 1, most of the enzyme was in a single narrow zone (fraction 5). This fraction banded at the interface between 1.00 and 1.55 M sucrose, indicating that the particles contained within this region of the gradient had a density of 1.14–1.19. When a similar fractionating procedure was carried out with 5-min-old sea urchin embryos TAME hydrolase activity in the particulate fraction isolated by differential centrifugation decreased to about 30% of that found in unfertilized egg preparations. As the particulate fraction contains 80% or more of the total TAME hydrolase activity in unfertilized eggs, there must be considerable redistribution of this enzyme to account for the 70% loss in activity of this fraction after fertilization.