TY - JOUR
T1 - Albumin binding of unconjugated [3H]bilirubin and its uptake by rat liver basolateral plasma membrane vesicles
AU - Pascolo, Lorella
AU - Del Vecchio, Savino
AU - Koehler, Ronald K.
AU - Bayon, J. Enrique
AU - Webster, Cecile C.
AU - Mukerjee, Pasupati
AU - Ostrow, J. Donald
AU - Tiribelli, Claudio
PY - 1996/6/15
Y1 - 1996/6/15
N2 - Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([B(f)]) were calculated from UCB-HSA affinity constants (K'(f)) determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 μM, K'(f) (litre/mol) was inversely related to [HSA], irrespective of the [B(t)]/[HSA] ratio. K'(f) was 2.066 x 106 +(3.258 x 108/[HSA]). When 50 mM KCl was isoosmotically substituted for sucrose, the K'(f) value was significantly lower {2.077 x 106 +(1.099 x 108/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([B(f)] ≤ 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [B(f)], with K(m) values of 28 ± 7 and 57 ± 8 nM respectively (mean ± S.D., n = 3, P <0.001). The V(max) was greater for the electrogenic than for the electroneutral component (112 ± 12 versus 45 ± 4 pmol of UCB·mg-1 of protein·15s-1, P <0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K'(f) decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with K(m) values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [B(f)] using a K'(f) value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.
AB - Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([B(f)]) were calculated from UCB-HSA affinity constants (K'(f)) determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 μM, K'(f) (litre/mol) was inversely related to [HSA], irrespective of the [B(t)]/[HSA] ratio. K'(f) was 2.066 x 106 +(3.258 x 108/[HSA]). When 50 mM KCl was isoosmotically substituted for sucrose, the K'(f) value was significantly lower {2.077 x 106 +(1.099 x 108/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([B(f)] ≤ 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [B(f)], with K(m) values of 28 ± 7 and 57 ± 8 nM respectively (mean ± S.D., n = 3, P <0.001). The V(max) was greater for the electrogenic than for the electroneutral component (112 ± 12 versus 45 ± 4 pmol of UCB·mg-1 of protein·15s-1, P <0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K'(f) decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with K(m) values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [B(f)] using a K'(f) value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.
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M3 - Article
C2 - 8670181
AN - SCOPUS:0029739328
VL - 316
SP - 999
EP - 1004
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
IS - 3
ER -