DEVELOPMENTAL BIOLOGY 26, 393-399 (1971) The Appearance of cu-Amylase Activity during Gut Differentiation in Sand Dollar Plutei’ VICTOR D. VACQUIER, LAURENCE J. KORN, 2AND DAVID EPEL Marine Biology Research Division, Scripps Institution of Oceanography, La Jolla, California 92037 Accepted July 19, 1971 cY-Amylase activity appears during gut differentiation of plutei of the sand dollar Dendraster ex- centricus. The activity increases coincidentally with that of a j3-1,3-glucanase. The a-amylase ap- pears to have maltase activity. Glucose in the surrounding seawater depresses the increase in fl-glu- canase activity but does not affect the normal cr-amylase increase. This suggests that the glucose repression of 6-glucanase activity may be quite specific and not result from some general effect of glucose on metabolism. Sodium dodecyl sulfate-acrylamide gel electrophoresis resolves the larval cy-amylase into components of 52,000 and 76,000 molecular weight. The d-amylase from the intes- tines of adult sand dollars resolves as a single component of 57,006 molecular weight. The pH curves for adult and larval a-amylases are also different and it is probable that adult and larval rr-amy- lases are different proteins. INTRODUCTION /3-1,3-Glucanohydrolase (Bglucanase) activity appears coincidentally with gut dif- ferentiation in sand dollar plutei (Vacquier, 1971a). This temporal correlation and the finding that isolated larval intestines are enriched 1.5-fold in P-glucanase activity (Vacquier 1971b), suggest that the p-glu- canase may be a digestive enzyme. We postulated that other degradative enzymes involved in the digestive process of larvae may also appear at the time of gut differ- entiation. Here we report that an increase in the activity of an a-amylase (Fischer and Stem, 1960), as judged by the ability of larval ho- mogenates to release glucose from potato starch, also occurs during gut differentia- tion and follows the same pattern of increase as the previously described &glucanase. The /3-glucanase and cY-amylase activities have distinctly different heat inactivation curves. Although both enzymes release free glucose as an end product, the increase in a-amylase activity is unaffected by the presence of glucose under conditions where ‘Supported by NSF Grant 27139 to Professor David Epel. zPresent address: Department of Biological Sci- ences, Stanford University, Stanford, California 94305. the increase in &glucanase activity is se- verely depressed. Homogenates of sand dollar eggs, em- bryos, and larvae degrade maltose to glu- cose. This activity increases 3-fold during the differentiation of the gut. The increase in maltase appears to result from the (Y- amylase (i.e., the a-amylase has maltase activity). The a-amylase, like the Bglucanase, is stable and can be eluted in an active form from sodium dodecyl sulfate (SDS) acryl- amide gels. As was found with the fi- glucanase, the larval a-amylase exists as two electrophoretically distinct bands as opposed to a single band of activity in the gut of adult sand dollars. MATERIALS AND METHODS Embryos of the sand dollar Dendruster excentricus were raised at 22°C. The de- tails, including all volumes, of the cultur- ing, sampling, glucose assay procedures, and acrylamide gel electrophoresis tech- niques are identical to those previously de- scribed for the &glucanase (Vacquier, 1971a) and are summarized briefly for clarification. At various times during development, aliquots of cultures were removed, the lar- 393