UDPgalactose:glucosylceramide β1 → 4-galactosyltransferase activity in human proximal tubular cells from normal and familial hypercholesterolemic homozygotes

Subroto Chatterjee, Elizabeth Castiglione

Research output: Contribution to journalArticle

Abstract

The activity of a galactosyltransferase (GalT-2) that catalyzes the transfer of galactose from uridinediphosphogalactose to glucosylceramide in cultured normal proximal tubular (PT) cells was characterized with respect to substrate saturation and metal ion requirements. Using a membrane-bound enzyme source, optimum activity was obtained in the presence of 1.0 mM Mn2+/Mg2+ (1:1) and a detergent mixture, Triton X-100/Cutscum (1:2, v/v), 0.1 mg/ml. The apparent Km values for glucosylceramide and UDP[14C]galactose were 3 μM and 0.5 μM, respectively. The Vmax values for glucosylceramide and UDP[U-14C]galactose were 0.12 nmol/mg protein per 2 h and 173 nmol/mg protein per 2 h, respectively. The purified 14C-labelled product comigrated with authentic lactosylceramide (LacCer) on TLC and HPLC analysis. The presence of a terminal β-[14C]galactosyl group in the enzymatic product was proved by its cleavage (79%) by β-galactosidase. Following the development of optimal assay conditions in normal PT cells, GalT-2 activity was next measured in urinary PT cells from homozygous familial hypercholesterolemic (FH) patients previously shown to accumulate large amounts of lactosylceramide. Urinary PT cells from familial hypercholesterolemic homozygous patients contained 35% higher GalT-2 activity as compared to control cells. We speculate that elevated GalT-2 acitivity may contribute to the storage of LacCer in FH-PT cells.

Original languageEnglish (US)
Pages (from-to)136-142
Number of pages7
JournalBBA - General Subjects
Volume923
Issue number1
DOIs
StatePublished - Jan 20 1987

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Keywords

  • (Proximal tubular cell)
  • Galactosyltransferase
  • Glucosylceramide
  • Lactosylceramide
  • UDPgalactose

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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