Beta-galactosidase decreases the binding affinity of the insulin-like-growth-factor-II/mannose-6-phosphate receptor for insulin-like-growth-factor II.
The insulin-like growth-factor-II/mannose-6-phosphate (IGF-II/Man6P) receptor binds two classes of ligands, insulin-like growth factors and lysosomal enzymes. We have examined the ability of the lysosomal enzyme, beta-galactosidase, to modulate the binding of 125I-IGF-II to the receptor. beta-Galactosidase purified from bovine testis was fractionated on a DEAF-Sephacel ion-exchange column. Column fractions were assayed for enzymatic activity and for ability to inhibit the binding of 125I-IGF-II to the IGF-II/Man6P 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 125I-IGF-II to the receptor. A pool of these fractions from the DEAE-Sephacel column inhibited 125I-IGF-II binding to pure receptor by 80% with the concentration required for half-maximal inhibition being 25 nM. The inhibition of binding by beta-galactosidase was completely blocked by simultaneous incubation with Man6P. Inhibition of the enzymatic activity of beta-galactosidase with D-galactonic acid gamma-lactone did not affect the ability of beta-galactosidase to inhibit the binding of 125I-IGF-II to the receptor. Scatchard analysis of IGF-II binding to pure receptor in the presence and absence of beta-galactosidase showed that beta-galactosidase decreased the binding affinity for IGF-II (Kd 0.26 nM versus 1.0 nM in the presence of 57 nM beta-galactosidase). We confirmed the observations of others that Man6P alone actually increases the binding of 125I-IGF-II to the IGF-II/Man6P receptor, but we found that this phenomenon was dependent upon the method of preparation of the IGF-II/Man6P receptor. Microsomal membrane preparations, solubilized membranes, and receptors purified on an IGF-II-Sepharose column all exhibited stimulation of 125I-IGF-II binding by Man6P, whereas receptors purified on lysosomal enzyme affinity columns showed little or no stimulation of 125I-IGF-II binding by Man6P. We conclude that beta-galactosidase decreases the binding affinity of the IGF-II/Man-6-P receptor for IGF-II by binding with high affinity to the Man6P-recognition site.
Kiess
W
W
Endocrinology Section, National Cancer Institute, National Institutes of Health, Bethesda.
Thomas
C L
CL
Sklar
M M
MM
Nissley
S P
SP
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005696:Galactosidases / Q000494:pharmacology*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D008297:Male; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013002:Somatomedins / Q000378:metabolism*; D013737:Testis / Q000201:enzymology; D001616:beta-Galactosidase / Q000494:pharmacology*
D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D013002:Somatomedins; D007335:Insulin-Like Growth Factor II; D005696:Galactosidases; D001616:beta-Galactosidase