99mTc-neoglycoalbumin (NGA)-binding to human hepatic binding protein (HBP) in vitro.
1 Neoglycoalbumin (NGA) was synthesised by covalent coupling of 2-imino-2-methoxyethyl-1-thio-beta-D-galactopyranoside (IME-thiogalactose) to the primary amino groups of human serum albumin (HSA). NGA was purified by ultrafiltration and size exclusion h.p.l.c. (SEC). 99mTc-labelling was performed with and without SEC purification. 2 Estimation of 99mTc-NGA-binding to human hepatic binding protein (HBP) revealed a complex behaviour indicating saturable high- and low-affinity sites. The high-affinity binding capacity was 1.1 +/- 0.4 pmol mg-1 human liver plasma membrane protein, the low-affinity binding capacity was 6.2 +/- 1.8 pmol mg-1 liver plasma membrane protein. The apparent equilibrium dissociation constants were 2.4 +/- 1.2 and 18.4 +/- 4.8 nM, respectively. 3 Specific binding of 99mTc-NGA to human HBP in the presence of 100 microM unlabelled NGA, Ca++ and Mg++ at pH 7.5 and 37 degrees C reached 85 +/- 5% at equilibrium. The amount of ligand specifically bound increased with the amount of human liver membrane protein added. The concentration of unlabelled agonist necessary to displace 50% of ligand bound amounted to 100 nM.
Virgolini
I
I
Second Department of Nuclear Medicine, University of Vienna, Austria.
Angelberger
P
P
Müller
C
C
O'Grady
J
J
Sinzinger
H
H
D000418:Albumins / Q000378:metabolism*; D037263:Asialoglycoprotein Receptor; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism*; D015609:Organotechnetium Compounds / Q000378:metabolism*; D011485:Protein Binding
D000418:Albumins; D037263:Asialoglycoprotein Receptor; D002352:Carrier Proteins; D015609:Organotechnetium Compounds; C027122:hepatic sialoglycoprotein receptor; C036586:technetium Tc 99m neogalactoalbumin