Pergamon 0965-1748(95)00060-7 Insect Biochem. Molec. Biol. Vol. 26, No. 1, pp. 49-57, 1996 Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0965-1748/96 $t5.00 + 0.00 Tyrosine/3-Glucosyltransferase in the Tobacco Hornworm, Manduca sexta (L.): Properties, Tissue Localization, and Developmental Profile SAAD A. AHMAD,* THEODORE L. HOPKINS,*~ KARL J. KRAMERt Received 15 February 1995; revised and accepted 28 June 1995 Tyrosine ~l-glucosyltransferase activity in larval tissues of Manduca sexta was determined by reversed-phase HPLC to quantify the product tyrosine glucoside [~-D-glucopyranosyi-O-L- tyrosine, (TG)] formed in incubation mixtures. Synthesis of TG occurred only in fat body preparations, with most of the enzyme activity (95%) in the 15,000g pellet of homogenates. Other tissues were devoid of activity but did support glucosylation of phenolic substrates other than tyrosine. Activity was greatest with uridine 5'-diphosphoglucose (UDPG) as the glucose donor. However, thymidine 5'-diphosphoglucose (dTDPG) afforded approximately one fourth the amount of tyrosine glucosylation provided by UDPG. Magnesium ions at concentrations up to 15 mM stimulated activity, whereas Ca 2÷ and Mn 2÷ were only slightly stimulatory at 5 mM but progressively inhibited TG synthesis at higher concentrations as did Co 2+. Maximal rates of glucosylation occurred in the pH range 7.5-9.0. The enzyme was highly specific for tyrosine, because no glucosylation occurred for a number of tyrosine derivatives. Several natu- ral and synthetic mono- and diphenols were found to be weak to moderate inhibitors of the enzyme. In a study of tyrosine ~-ghcosyltransferase activity during development, no TG syn- thesis occurred in enzyme preparations from first, second, third, or fourth larval instars, although glucosylation of p-nitrophenol and 4-hydroxycoumarin did occur. Activity was not detected in the fat body of newly ecdysed fifth instars, but low levels were observed 36 h later. The rate of tyrosine glucosylation continued to increase to a peak at 4 days, but then decreased to very low levels after cessation of larval feeding. Low activity was also observed in the pharate adult fat body about 1 day before eclosion. Therefore, tyrosine ~-glucosyitransferase activity occurs primarily in the fat body of fifth stadium larvae for synthesis of the pupal cuticle tanning precursor tyrosine glucoside. Conjugation /3-Glucoside Fat body Tobacco hornworm Manducasexta Tyrosine UDP- glucosyltransferase Sclerotization INTRODUCTION Tyrosine is the precursor of o-diphenolics and their quinonoid derivatives which are involved in the sclerotiz- ation or tanning and, often, pigmentation of insect cuticle (see reviews by Kramer and Hopkins, 1987; Hopkins and Kramer, 1991, 1992). Although tyrosine is sparingly sol- uble in aqueous media, concentrations in hemolymph from several insects indicate its abundance for cuticle tanning at levels several fold higher than its solubility *Department of Entomology, KansasStateUniversity, Manhattan,KS 66506 U.S.A. tU.S. GrainMarketing Research Laboratory, ARS, USDAManhattan, KS 66502 U.S.A. SAuthorfor correspondence. limit. In addition to this apparently free or weakly bound form of tyrosine, larvae of both Lepidoptera and Diptera have been shown to sequester it in even greater concen- trations as conjugates with glucose or phosphate. Thus, tyrosine is not only rendered more soluble as a conju- gated storage form, but is also protected from competing metabolic pathways until the time of larval-pupal or puparial transformation when hydrolysis regenerates free tyrosine for use in cuticle tanning. The types of tyrosine conjugates appear to be species specific. Either/3-gluco- side, phosphate ester, or/3-alanine peptidal conjugates of tyrosine are found among Diptera, but Lepidoptera seem to resort solely to the /3-glucoside form (Kramer and Hopkins, 1987). In the tobacco hornworm, Manduca sexta, the regu- lation of tyrosine glucoside (TG) synthesis and hydroly- 49