Human liver glycoprotein sialyltransferase

Jack A. Alhadeff, George Cimino, Aaron Janowsky, John S. O'Brien

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

Sialyltransferase (CMP-N-acetylneuraminate:d-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) has been studied using the two glycoprotein substrates asialofetuin and asialo-ovine submaxillary mucin (A-OSM) in fresh and frozen human adult and fetal livers. Enzymatic activity for both substrates is stimulated by Mg2+ (10-25 mM) and Triton X-100 (0.4-0.6%, v/v). Freeze-thaw, heat denaturation, stability on storage, additive and substrate competition studies suggest the presence of at least two forms of human liver glycoprotein sialyltransferase. Subcellular fractionation studies indicate that the major parts of both sialyltransferase activities are associated with membraneous fractions but that a greater percentage of A-OSM than asialofetuin activity is associated with these fractions. Subcellular fractionation studies using O.8% (v/v) Triton X-100 in the extracting buffer greatly diminishes the percentage of both asialofetuin and A-OSM sialyltransferase activity found in the mitochondrial and microsomal pellets. Kinetic characterization of sialyltransferase in a liver homogenate, resuspended 48 200 × g pellet and 48 200 × g supernatant suggested the possibility of two asialofetuin sialyltransferases. The pH optimum for homogenate and supernatant enzymes with respect to asialofetuin was 7.2 with a suggested second optimum at 8.5. The pellet enzyme had only one optimum at pH 7.2. Michaelis constants with regard to asialofetuin were 0.08, 0.06 and 0.22 mM in homogenate, resuspended pellet and supernatant, respectively. The homogenate had a Km of 5 μM for CMP-N-acetylneuraminic acid (CMP-NANA). Isoelectric focusing indicated that at least eight isoelectric forms of sialyltransferase with pI values ranging from pH 5.0 to 8.6 are present in the homogenate; several isoelectric forms were preferentially associated with the pellet or supernatant. All the data suggest at least two forms of liver glycoprotein sialyltransferase. One form is thermolabile, loses activity on freezing and storage, and is associated with membraneous fractions and A-OSM activity. The second form is less thermolabile, more stable to freezing and storage, less associated with membraneous fractions and associated more with asialofetuin activity. Furthermore, kinetic characterization of sialytransferase activity in liver homogenate, resuspended pellet and supernatant suggests the presence of two asialofetuin sialyltransferases.

Original languageEnglish (US)
Pages (from-to)307-321
Number of pages15
JournalBBA - Enzymology
Volume484
Issue number2
DOIs
StatePublished - Oct 13 1977

ASJC Scopus subject areas

  • Medicine(all)

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