Plasma Membranes of the Rat Liver

Abstract

A method is described for the rapid isolation of a plasma membrane fraction containing a high concentration of intact bile canaliculi from the rat liver . Isolated bile canaliculi retain most of the ultrastructural features exhibited in the intact liver cell . The final fraction contains 5'-nucleotidase activity at approximately the same concentration as that in previous preparations of plasma membranes . In the presence of 0 .01 M Mg++, 5'-nucleotidase exhibits a double pH optimum at pH values of 7 .5 and 9 .5. The activities of glucose-6-phosphatase and alkaline phosphatase are present in low amounts . Cytochrome P-450 is not detectable . Na+-K+-activation of ATPase is observed to the extent of 20-36 0/, in about half of the assays. The availability of a method for preparation of intact bile canaliculi should prove useful for studying the biochemical events associated with the transport of bile constituents into canaliculi .

Relationship between bile flow and Na+, K+-adenosinetriphosphatase in liver plasma membranes enriched in bile canaliculi.

“…This is further confirmed by the relatively small mitochondrial and microsomal contamination as judged from the specific activity of the marker enzymes, succinate dehydrogenase and glucose-6-phosphatase, respectively (Table III). The activities of these marker enzymes are comparable to those obtained by the original procedure of Song et al (3,4,9), although we performed the washing procedures at 10,000g as compared to the originally described 1,500 g (9). Previous experiments in our laboratory have shown that mitochondrial contamination was not further diminished when all washes were performed at 1,500 g, but that the protein yield was significantly reduced (unpublished observations).…”

“…Previous experiments in our laboratory have shown that mitochondrial contamination was not further diminished when all washes were performed at 1,500 g, but that the protein yield was significantly reduced (unpublished observations). Thus, the use of a centrifugal field of 10,000 g for the second and third washing procedure permitted an increase in the yield of protein in the canalicular-enriched membrane fraction by a factor of about five as compared to the original procedure (3,4,9). A further factor explaining the increase in protein yield is the inclusion of Ca2+ into the homogenization medium as proposed by Ray (10).…”

Preparation of Aminoazo Dye Induced Rat Hepatoma Membrane Fractions Retaining Tumour Specific Antigen

“…Several authors have reported the use of the A-XII zonal rotor for rate dependent fractionation of the nuclear sediment of liver homogenates (reviewed by Hinton, 1972) and in liver fractionation-studies, the 1 mmol/l NaHCO3 (pH 7.6) homogenization medium, originally described by Neville (1960) for rat liver plasma membrane isolation, has been found to be satisfactory (Emmelot et al, 1964;Song et al, 1969;Evans, 1970). When applied to chemically induced rat hepatomata, however, nuclear disruption is promoted with the subsequent formation of a nucleoprotein gel, thus preventing, the isolation of a plasma membrane fraction (Emmelot and Bos, 1966;Price et al, 1972).…”

Monoclonal antibody 7H6 reacts with a novel tight junction-associated protein distinct from ZO-1, cingulin and ZO-2.

“…A bile canaliculus-rich fraction was prepared from the livers of 8-wk-old Fischer 344 (F344) rats (Charles River Japan, Inc., Yokohama, Japan) according to the method of Song et al (1969), with a slight modification. In each isolation, three rats were used.…”