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

• Published in: Biochimica et biophysica acta. General subjects Vol. 923; no. 1; pp. 136 - 142
• Main Authors: Chatterjee, Subroto, Castiglione, Elizabeth
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 1987
• Subjects: Galactosyltransferase; Glucosylceramide; Lactosylceramide; Proximal tubular cell; UDPgalactose; Lactosylceramide; FH; Proximal tubular cell; PT; LDL; LacCer; UDPgalactose; Galactosyltransferase; GbOse 3Cer; GbOse 4Cer; Glucosylceramide; GlcCer
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/0304-4165(87)90136-X

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 Mn 2+/Mg 2+ (1:1) and a detergent mixture, Triton X-100/Cutscum (1:2, v/v), 0.1 mg/ml. The apparent K m values for glucosylceramide and UDP[ 14C]galactose were 3 μM and 0.5 μM, respectively. The V max 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.