Functional polarity of the rat hepatocyte surface membrane. Isolation and characterization of plasma-membrane subfractions from the blood-sinusoidal, bile-Canalicular and contiguous surfaces of the hepatocyte

Abstract

1. Six rat liver plasma-membrane subfractions of different density and morphological, enzymric and chemical properties were prepared from homogenates by a combination of differential, rate-zonal and density-gradient centrifugation. They consisted of three vesicular 'light' subfractions of density 1.12-1.13 and three 'heavy' subfractions of density 1.16-1.18 containing membrane strips and intercellular junctions. 2. All six subfractions contained a basal adenylate cyclase activity. One of the 'light' subfractions that showed the highest glucagon-stimulated adenylate cyclase activity was identified as deriving from the blood-sinusoidal face of the hepatocyte. This subfraction, unlike the others, was contaminated by Golgi components, as indicated by its morphological properties and the presence of galactosyl-and sialyl-transferase activities. 3. All the six subfractions showed high activities of the following plasma-membrane marker enzymes: 5'-nucleotidase, alkaline phosphodiesterase (nucleotide pyrophosphatase), alkaline phosphatase, leucine naphthylamidase and Mg2+-activated adenosine triphosphatase. A 'light' subfraction that showed the highest specific activities of all the above marker enzymes, but lacked a glucagon-stimulated adenylate cyclase activity, was identified as deriving from the bilecanalicular face of the hepatocyte. 4. The 'heavy' subfractions, which showed generally the lowest activities of the above plasma-membrane enzyme markers, and were characterized by the presence of desmosomes and gap junctions, were taken to originate from the contiguous faces of the hepatocyte. 5. The protein composition of the six subfractions was generally similar, as shown by polyacrylamide-gel electrophoresis. Differences in the amounts of various protein and glycoprotein bands among the subfractions correlated with their morphology, enzymic composition and sialic acid content. 6. Hormonal and histochemical evidence supporting the identification of a bile-canalicular subfraction, a blood-sinusoidal subfraction and contiguous-face subfractions is discussed.

Bile‐Salt‐Binding Polypeptides in Plasma Membranes of Hepatocytes Revealed by Photoaffinity Labelling

“…Specific activities of the respective marker enzymes ( Table 1) also indicated that there was a low degree of contamination by non-plasma membrane material. The relatively high specific activities of nucleotide pyrophosphatase, alkaline phosphatase, and 5'-nucleotidase, however, confirm that this membrane subfraction is predominantly composed of vesicles originating from the bile canalicular membrane [35,38,40,50]. Neither the purity nor the yields of the different membrane subfractions were significantly improved by starving the rats for about 12 h before they were sacrificed.…”

“…The relatively low specific activity of glucose-6-phosphatase (Table I), a marker enzyme of endoplasmic reticulum [43,44], indicates that contaminations derived from this subcellular organelle are of little significance. The specific activities and the relative specific activities, with respect to the homogenate (Table 1) indicate that this subfraction of plasma membranes was highly enriched with sinusoidal surfaces [35,38].…”

“…A subfraction of plasma membranes from rat liver highly enriched with sinusoidal surfaces was isolated by an extension of the procedure of Touster and coworkers [29,38] as described by Wisher and Evans [35]. The subfraction obtained with an average yield of 0.8 mg protein/g rat liver has been shown by electron microscopy to consist predominantly of small vesicles (Fig.…”

“…The functional polarity of the plasma membrane of hepatocytes is correlated to a structural heterogeneity [35,36] rendering possible the isolation of distinct subfractions [35, 37 -401. A membrane subfraction of rat liver originating from the blood-sinusoidal surface of hepatocytes must comprise the polypeptides involved in bile salt uptake, whereas a subfraction predominantly composed of bile canalicular membranes contains those participating in secretion.…”

Affinity of integrin α1β1 from liver sinusoidal membranes for type IV collagen

“…Hepatic sinusoidal plasma membranes were isolated from liver homogenates on sucrose gradients as described [13].…”