Measurement of the kinetics of protein uptake by proximal tubular cells using an optical biosensor.
BACKGROUND
The affinity and specificity of protein reabsorption by proximal tubular cells have been investigated using techniques for monitoring endocytosis, demonstrating a high capacity but low affinity process. It is not known whether uptake is through binding to a single binding site/receptor with differing affinities, or if there are several classes of binding sites receptors, each specific for differing proteins or groups, such as, high or low molecular weight proteins.


METHODS
We have developed a novel technique for analyzing the kinetics of protein binding to tubular cells using a optical biosensor system. We have studied the binding of cultured LLCPK cells to albumin and RBP immobilized onto the sensor. By adding increasing concentrations of competing proteins [varying in molecular weight from 66,000 to 11,800 D and pI from 4.6 to 9.2 as represented by albumin, alpha1-microglobulin (alpha1M), retinol binding protein (RBP), cystatin C and beta2-microglobulin (beta2m)], specific and inhibitable cell binding was demonstrated.


RESULTS
Equilibrium constants, KA, could be calculated from the reciprocal of the protein concentration causing 50% inhibition in binding rate. These were: albumin = 8.0 x 10(4) M(-1), alpha1M = 2.0 x 10(5) M(-1), RBP = 2.7 x 10(4) M(-1), cystatin C = 2.0 x 10(4) M(-1), beta2m = 4.2 x 10(3) M(-1). There were no significant differences between the measured KA's whether RBP or albumin were immobilized on the surface.


CONCLUSIONS
All the proteins gave similar shaped inhibition profiles, suggesting that there is one binding site/receptor for all proteins studied, regardless of molecular weight or charge, but there are differing affinities for each protein.
Department of Clinical Biochemistry, St. Bartholomew's and The Royal London School of Medicine and Dentistry, England, United Kingdom.
H
H
Thakkar
P A
PA
Lowe
C P
CP
Price
D J
DJ
Newman
D000510:Alpha-Globulins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D015374:Biosensing Techniques; D004705:Endocytosis; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D007700:Kinetics; D018374:LLC-PK1 Cells; D008854:Microscopy, Electron; D055095:Optics and Photonics; D011506:Proteins / Q000378:metabolism*; D012177:Retinol-Binding Proteins / Q000378:metabolism; D012709:Serum Albumin / Q000378:metabolism; D013552:Swine; D001613:beta 2-Microglobulin / Q000378:metabolism
D000510:Alpha-Globulins; D011506:Proteins; D012177:Retinol-Binding Proteins; D012709:Serum Albumin; C016001:alpha-1-microglobulin; D001613:beta 2-Microglobulin