TY - JOUR
T1 - UDPgalactose:glucosylceramide β1 → 4-galactosyltransferase activity in human proximal tubular cells from normal and familial hypercholesterolemic homozygotes
AU - Chatterjee, Subroto
AU - Castiglione, Elizabeth
N1 - Funding Information:
We are grateful to Drs. Subash Basu and Manju Basu, Department of Chemistry, University of Notre Dame, for giving us fundamental instructions for the assay of glycosphingolipid glyco-syltransferases and many valuable suggestions throughout the course of this study. This work was supported through an NIH grant RO-1-AM-31722.
PY - 1987/1/20
Y1 - 1987/1/20
N2 - 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.
AB - 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.
KW - (Proximal tubular cell)
KW - Galactosyltransferase
KW - Glucosylceramide
KW - Lactosylceramide
KW - UDPgalactose
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U2 - 10.1016/0304-4165(87)90136-X
DO - 10.1016/0304-4165(87)90136-X
M3 - Article
C2 - 3099851
AN - SCOPUS:0023659941
SN - 0304-4165
VL - 923
SP - 136
EP - 142
JO - BBA - General Subjects
JF - BBA - General Subjects
IS - 1
ER -