Kiess W, Thomas C L, Sklar M M, Nissley S P
Endocrinology Section, National Cancer Institute, National Institutes of Health, Bethesda.
Eur J Biochem. 1990 May 31;190(1):71-7. doi: 10.1111/j.1432-1033.1990.tb15547.x.
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.
胰岛素样生长因子-II/甘露糖-6-磷酸（IGF-II/Man6P）受体可结合两类配体，即胰岛素样生长因子和溶酶体酶。我们研究了溶酶体酶β-半乳糖苷酶调节125I-IGF-II与该受体结合的能力。从牛睾丸中纯化的β-半乳糖苷酶在DEAF-琼脂糖离子交换柱上进行分级分离。对柱分级分离物进行酶活性测定以及抑制125I-IGF-II与IGF-II/Man6P受体结合能力的检测。在较高NaCl浓度下洗脱的酶分级分离物，先前已证明其在培养细胞中的摄取量更大，在抑制125I-IGF-II与受体结合方面表现出更强的效力。来自DEAE-琼脂糖柱的这些分级分离物的一个组分以80%的比例抑制125I-IGF-II与纯受体的结合，半数最大抑制所需浓度为25 nM。β-半乳糖苷酶对结合的抑制作用可通过与甘露糖-6-磷酸（Man6P）同时孵育而完全被阻断。用D-半乳糖酸γ-内酯抑制β-半乳糖苷酶的酶活性并不影响其抑制125I-IGF-II与受体结合的能力。对存在和不存在β-半乳糖苷酶时IGF-II与纯受体结合的Scatchard分析表明，β-半乳糖苷酶降低了对IGF-II的结合亲和力（在存在57 nMβ-半乳糖苷酶时，Kd为0.26 nM，而无β-半乳糖苷酶时为1.0 nM）。我们证实了其他人的观察结果，即单独的Man6P实际上会增加125I-IGF-II与IGF-II/Man6P受体的结合，但我们发现这种现象取决于IGF-II/Man6P受体的制备方法。微粒体膜制备物、溶解的膜以及在IGF-II-琼脂糖柱上纯化的受体，所有这些都表现出Man6P对125I-IGF-II结合的刺激作用，而在溶酶体酶亲和柱上纯化的受体则几乎没有或没有表现出Man6P对125I-IGF-II结合的刺激作用。我们得出结论，β-半乳糖苷酶通过与Man6P识别位点高亲和力结合，降低了IGF-II/Man-6-P受体对IGF-II的结合亲和力。
