Open Accessβ‐Galactosidase decreases the binding affinity of the insulin‐like‐growth‐factor‐II/mannose‐6‐phosphate receptor for insulin‐like‐growth‐factor II
Author(s) -
KIESS Wieland,
THOMAS Cheryl L.,
SKLAR Mark M.,
NISSLEY S. Peter
Publication year - 1990
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1990.tb15547.x
Subject(s) - receptor , mannose 6 phosphate , enzyme , insulin like growth factor , insulin like growth factor 2 receptor , mannose , chemistry , biochemistry , binding site , insulin like growth factor receptor , growth factor , incubation , insulin like growth factor 2 , somatomedin , mannose 6 phosphate receptor , biology , insulin like growth factor 1 receptor , lysosome
The insulin‐like growth‐factor‐II/mannose‐6‐phosphate (IGF‐II/Man6 P ) receptor binds two classes of ligands, insulin‐like growth factors and lysosomal enzymes. We have examined the ability of the lysosomal enzyme, β‐galactosidase, to modulate the binding of 125 I‐IGF‐II to the receptor. β‐Galactosidase purified from bovine testis was fractionated on a DEAE‐Sephacel ion‐exchange column. Column fractions were assayed for enzymatic activity and for ability to inhibit the binding of 125 I‐IGF‐II to the IGF‐II/Man6 P receptor. Enzyme fractions eluting at higher NaCl concentrations which had previously been shown to exhibit greater uptake by cells in culture, exhibited greater potency in inhibiting the binding of 125 I‐IGF‐II to the receptor. A pool of these fractions from the DEAE‐Sephacel column inhibited 125 I‐IGF‐II binding to pure receptor by 80% with the concentration required for half‐maximal inhibition being 25 nM. The inhibition of binding by β‐galactosidase was completely blocked by simultaneous incubation with Man6 P . Inhibition of the enzymatic activity of β‐galactosidase with d ‐galactonic acid γ‐lactone did not affect the ability of β‐galactosidase to inhibit the binding of 125 I‐IGF‐II to the receptor. Scatchard analysis of IGF‐II binding to pure receptor in the presence and absence of β‐galactosidase showed that β‐galactosidase decreased the binding affinity for IGF‐II ( K d 0.26 nM versus 1.0 nM in the presence of 57 nM β‐galactosidase). We confirmed the observations of others that Man6 P alone actually increases the binding of 125 I‐IGF‐II to the IGF‐II/Man6 P receptor, but we found that this phenomenon was dependent upon the method of preparation of the IGF‐II/Man6 P receptor. Microsomal membrane preparations, solubilized membranes, and receptors purified on an IGF‐II – Sepharose column all exhibited stimulation of 125 I‐IGF‐II binding by Man6 P , whereas receptors purified on lysosomal enzyme affinity columns showed little or no stimulation of 125 I‐IGF‐II binding by Man6 P . We conclude that β‐galactosidase decreases the binding affinity of the IGF‐II/Man‐6‐ P receptor for IGF‐II by binding with high affinity to the Man6 P ‐recognition site.