Kinetic analysis of receptor-mediated endocytosis of g-csf
derivative, nartograstim, in rat bone marrow cells.
Kuwabara, Takashi, Satoshi Kobayashi, and Yuichi Sugiyama.
Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd.,
Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411, Japan, Faculty
of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku,
Tokyo 113, Japan
APStracts 3:0029E, 1996.
To elucidate the mechanism of the receptor-mediated clearance of
granulocyte colony-stimulating factor (G-CSF), we performed kinetic
analyses of the receptor-mediated endocytosis (RME) processes using a
human G-CSF derivative, nartograstim (NTG), and isolated rat bone
marrow cells. The first-order rate constants involved in RME
processes were obtained by computerized model-fitting of the time
courses of the ligand-receptor complex both on the cell surface and
in the cell interior, and the degradation products in the medium in
the pulse-chase experiment. They were also calculated based on a
kinetic model involving the ligand-concentration dependence of the
initial binding rate, the steady-state degradation rate and the
steady-state amounts of ligand both on the cell surface and in the
interior. The rate constants for the RME processes after receptor
binding determined in the different experiments were similar, that
is, the half-times for the dissociation, internalization and
degradation of the ligand-receptor complex were 770, 10-30 and 20
min, respectively. However, the association rate (Kon) obtained by
measuring the initial binding was 5-fold greater than that calculated
under steady-state conditions. These kinetic analysis support the
hypothesis that the internalization of the receptor may be
accelerated by ligand binding, causing down-regulation of the
receptor on the cell surface. These overall kinetic analyses based on
steady-state and non-steady-state data of the RME processes clarify
the dynamics of the interaction between NTG and its receptor.
Received 3 October 1995; accepted in final form 16 January 1996.
APS Manuscript Number E489-5.
Article publication pending Am. J. Physiol. (Endocrinol. Metab.).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 8 February 96