Abstract
Lysosomal enzymes contain 6-phosphomannosyl moieties which mediate their translocation to lysosomes. This recognition marker is synthesized by the sequential action of UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase and alpha-N-acetylglucosaminyl phosphodiesterase. A new assay for the N-acetylglucosaminylphosphotransferase, using alpha-methylmannoside as acceptor, is presented. Using this assay, we partially purified the transferase and examined its substrate specificity. The transferase exhibited a very high affinity toward lysosomal enzymes (apparent Km values of less than 20 microM) and was greater than 100-fold more efficient (Vmax/Km) when using lysosomal enzymes as acceptors as compared to nonlysosomal glycoproteins that contain high mannose oligosaccharide units. Heat denaturation of the lysosomal enzymes resulted in the loss of acceptor activity. The model compounds alpha-methylmannoside and Man5--8GlcNAc were poor acceptors. We propose that this enzyme catalyzes the initial, determining step by which synthesized acid hydrolases are distinguished from other newly synthesized glycoproteins and thus are eventually targeted to lysosomes.
Highlights
We found that human placental P-hexosaminidase A, human placental &hexosaminidase B, human hepatic &galactosidase, and partially purified porcine hepatic P-hexosaminidases A and B were all comparable in acceptor activity to the a-N-acetylglucosaminidase
We found that theenzyme can transfer N-acetylglucosamine l-[32P]phosphate to a-methylmannoside and this forms the basis for a sensitive, specific, and facile assay
The data presented in this paper demonstrate that UDPN-acetylg1ucosamine:lysosomaelnzyme N-acetylglucosamine
Summary
Partial Purification of N-acetylglucosaminylphosphotransferase-Our previous assay for N-acetylglucosaminylphosphotransferase activity depended on the transfer of Nacetylglucosamine 1-[”’Plphosphate to endogenous glycopro-. Accep- N-Acetylglucostor site aminylphosphoconcen- transferase tration activity tein acceptors [10]. In order to purify the enzyme it was necessary to develop an assay which is independent of endogenous acceptors. We found that theenzyme can transfer N-acetylglucosamine l-[32P]phosphate to a-methylmannoside and this forms the basis for a sensitive, specific, and facile assay (see “Experimental Procedures”). Using this assay to follow activity, the transferase was solubilized from rat liver smooth membranes with 0.3%Lubrol PX and partially purified by DEAE-cellulose chromatography.
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