ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 230, No. 2, May 1, pp. 66%675,1984 Studies on the Mechanism of Castanospermine Inhibition of (Y- and ,&Glucosidases RICK SAUL, R. J. MOLYNEUX,* AND A. D. ELBEINI Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78.284,and *Natural Products Chemistry Research Unit, Western Regional Research Center, Agricultural Research Service, USDA, Be&&g, California 94710 Received December 1, 1983 Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is an indolizidine alkaloid that was isolated from the Australian plant, Cast&ospermum austrak. This alkaloid was found to be a potent inhibitor of lysosomal (Y- and @-glucosidases. In this report, the mechanism of inhibition of amyloglucosidase (an exo-1,4-a-glucosidase) and almond emulsin P-glucosidase was examined. Castanospermine proved to be a competitive in- hibitor of amyloglucosidase at both pH 4.5 and 6.0 when assayed with the p-nitrophenyl- a-D-glucoside. It was also a competitive inhibitor of almond emulsin @-glucosidase at pH 6.5, but in this case previous studies had shown that inhibition was of the mixed type at pH 4.5 to 5.0. Th pH of the incubation mixture had a marked effect on the inhibition. Thus, in all cases, castanospermine was a much better inhibitor at pH 6.0 to 6.5 than it was at lower pH values. The pK for castanospermine was found to be 6.09, indicating that the alkaloid was probably more active in the unprotonated form. This was also suggested by the fact that the N-oxide of castanospermine, while still a competitive inhibitor, was 50 to 100 times less active than was castanospermine, and its activity was not markedly altered by pH. These results probably explain why cas- tanospermine is a good inhibitor of the glycoprotein processing enzyme, glucosidase I, since this is a neutral enzyme. Castanospermine (1,6,7,8-tetrahydroxy- octahydroindolizine) is a plant indolizidine alkaloid that was isolated from the Aus- tralian plant, Ca.stonospermum au&ah3 (1). We previously reported that this alkaloid was a potent inhibitor of almond emulsin B-glucosidase, as well as of fibroblast ly- sosomal cy- and @-glucosidases (2). How- ever, castanospermine did not inhibit a number of other glycosidases, including (Y- mannosidase, (Y- or P-galactosidases, /3-N- acetylhexosaminidase, /3-glucuronidase, or Lu-L-fucosidase. Castanospermine was also found to be a potent inhibitor of glyco- protein processing by virtue of the fact that it inhibits glucosidase I. Thus, when influ- 1 To whom correspondence should be addressed. ‘Abbreviation used: GlcNAc, N-acetylglucosamine. enza virus was grown in canine kidney cells in the presence of the alkaloid, the viral hemagglutinin no longer had the normal complement of the complex types of oli- gosaccharides. Instead, more than 85% of the oligosaccharides were of the structures Glc3Man7-9GlcNAcz (3). The purpose of the present study was to examine the kinetics of castanospermine inhibition on both CY- and &glucosidases, and to determine whether this inhibition was of the competitive type. Castano- spermine proved to be a competitive in- hibitor of amyloglucosidase when tested at both pH 4.5 and at pH 6.0. However, with the @-glucosidase, competitive inhibition 0003-9861/84 $3.00 Copyright Q 1994 by Academic Press. Inc. All rights of reproduction in any form reserved. 668