Tyrosine beta-glucosyltransferase in the tobacco hornworm, Manduca sexta (L): Properties, tissue localization, and developmental profile

Tyrosine beta-glucosyltransferase activity in larval tissues of Manduca sexta was determined by reversed-phase HPLC to quantify the product tyrosine glucoside [beta-D-glucopyranosyl-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 15,000 g 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 Ca2+ and Mn2+ were only slightly stimulatory at 5 mM but progressively inhibited TG synthesis at higher concentrations as did Co2+. 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 natural and synthetic mono- and diphenols were found to be weak to moderate inhibitors of the enzyme. In a study of tyrosine beta-glucosyltransferase activity during development, no TG synthesis 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 beta-glucosyltransferase activity occurs primarily in the fat body of fifth stadium larvae for synthesis of the pupal cuticle tanning precursor tyrosine glucoside.