Clarke B L, Oka J A, Weigel P H
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
J Biol Chem. 1987 Dec 25;262(36):17384-92.
The ability of rat hepatocytes to degrade internalized surface-bound 125I-asialoorosomucoid (ASOR) was determined by measuring the appearance of acid-soluble radioactivity at 37 degrees C. The degradation kinetics were biphasic in cells previously equilibrated at 37 degrees C for 1 h or cultured for 24 h. Degradation began immediately and was linear for at least 20 min after which the rate increased to a steady state value 3-4 times greater than the initial rate. We previously showed that hepatocytes have two functionally distinct populations of galactosyl receptors that mediate ligand dissociation by two kinetically different pathways (Weigel, P. H., Clarke, B. L., and Oka, J. A. (1986) Biochem. Biophys. Res. Commun. 140, 43-50). The activity of one receptor population, designated State 2 galactosyl receptors, can be reversibly modulated by incubating cells between 22 and 37 degrees C and is not expressed on the surface of freshly isolated cells. When 125I-ASOR was prebound to freshly isolated cells at 4 degrees C and degradation was assessed subsequently at 37 degrees C, the kinetics were monophasic, not biphasic. Degradation of the surface-bound 125I-ASOR began immediately and was greater than 90% complete by 6 h. Freshly isolated cells were incubated at temperatures between 22 and 37 degrees C, chilled to 4 degrees C, allowed to pre-bind 125I-ASOR, and then incubated at 37 degrees C. As the State 2 galactosyl receptor population increased, the kinetics of degradation became progressively more biphasic and the rate of the delayed degradation process increased. This effect could be reversed in cells in culture or in suspension by down-modulating surface receptor activity at temperatures below 37 degrees C; only the degradation process appearing after a 20-min lag was affected. Degradation in both pathways is an apparent first order process with identical rate constants (kappa = 0.006 min-1, t1/2 = 116 min). We conclude that there are two separate pathways by which asialoglycoproteins are degraded. The major "classic" pathway mediated by State 2 galactosyl receptors occurs after a 20-min lag and the minor pathway mediated by State 1 galactosyl receptors begins immediately with no detectable lag.
通过测量37℃时酸溶性放射性的出现情况，来确定大鼠肝细胞降解内化的表面结合型125I-去唾液酸糖蛋白（ASOR）的能力。对于先前在37℃平衡1小时或培养24小时的细胞，降解动力学呈双相性。降解立即开始，至少20分钟内呈线性，之后速率增加至稳态值，比初始速率大3 - 4倍。我们先前表明，肝细胞有两个功能不同的半乳糖基受体群体，它们通过两种动力学不同的途径介导配体解离（Weigel, P. H., Clarke, B. L., and Oka, J. A. (1986) Biochem. Biophys. Res. Commun. 140, 43 - 50）。其中一个受体群体，称为状态2半乳糖基受体，其活性可通过在22℃至37℃之间孵育细胞进行可逆调节，且在新鲜分离的细胞表面不表达。当在4℃将125I-ASOR预先结合到新鲜分离的细胞上，随后在37℃评估降解时，动力学呈单相性，而非双相性。表面结合的125I-ASOR的降解立即开始，到6小时时超过90%完成。将新鲜分离的细胞在22℃至37℃之间的温度下孵育，冷却至4℃，使其预先结合125I-ASOR，然后在37℃孵育。随着状态2半乳糖基受体群体增加，降解动力学逐渐变得更具双相性，延迟降解过程的速率增加。通过在低于37℃的温度下调表面受体活性，这种效应在培养或悬浮的细胞中可以逆转；只有在20分钟延迟后出现的降解过程受到影响。两种途径中的降解都是明显的一级过程，速率常数相同（κ = 0.006 min-1，t1/2 = 116分钟）。我们得出结论，去唾液酸糖蛋白有两种独立的降解途径。由状态2半乳糖基受体介导的主要“经典”途径在延迟20分钟后发生，由状态1半乳糖基受体介导的次要途径立即开始，无明显延迟。
