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Alterations in Liver Plasma Membranes and their Possible Role in Cholestasis
Vol. 62, No.2
GASTROENTEROLOGY
Printed in U. S. A.
Copyright © 1972 by The Williams & Wilkins Co.
EDITORIAL ALTERATIONS IN LIVER PLASMA MEMBRANES AND THEm POSSIBLE ROLE IN CHOLESTASIS As with other membranes, the plasma membrane of the hepatic parenchymal cell (LPM) consists of protein, lipid, and carbohydrate 1, 2; however, there are several differentiating features with respect to other surface membranes: (1) There is a functional polarity of LPM with respect to sinusoidal, canalicular, and lateral surfaces. Histochemical staining confirms these differences and reveals canalicular staining for magnesium-activated adenosine triphosphatase 3 and alkaline phosphatase activities·; sinusoidal staining for cobalt-activated cytidine monophosphatase activity 5 and staining of both canalicular and sinusoidal surface for 5'-nucleotidase activity. 3 (2) The major components of the LPM undergo differential synthesis and heterogeneous degradation. For example, mean half-life for pulse-labeled LPM proteins (1.8 days) is faster than that for total liver protein (4 days) and similar to that observed for endoplasmic reticulum membrane proteins (2.1 days). 6 LPM phospholipids and carbohydrates have shorter mean t", than do LPM proteins 7 , 8; however, these latter studies have not eliminated the possibility of isotope exchange or differential reutilization rather than degradation. How are these constituents incorporaated into the plasma membrane? The incorporation of pulse-labeled proteins into LPM has been studied after injection of 3H-Ieucine into rats. 9. 10 Peak incorporReceived September 3, 1971. Address requests for reprints to: Dr. Francis R. Simon, University of Colorado Medical Center, Division of Gastroenterology, 4200 E. Ninth Avenue, Denver, Colorado 80220. Research from the authors' laboratory was supported by grants from the United States Public Health Service (AM 02019, 05384) and performed during tenure of Special Fellowship (AM40777) to Dr. Simon. 342
ation of radioactivity into LPM proteins occurred 4 to 6 hr later. Peak isotope specific activity was delayed with respect to endoplasmic reticulum (15 min) and mitochondria (5 to 10 min). Cyloheximide, an inhibitor of protein synthesis, was administered 5 min after injection of labeled amino acid; incorporation of pulse-labeled proteins into' LPM continued unaltered for 3 hr. These studies suggest that LPM proteins are synthesized elsewhere in the cell, probably in the endoplasmic reticulum, and transferred to the plasma membrane for incorporation. The transfer process does not appear to require new protein synthesis for continued incorporation into the surface membrane. A similar time sequence for incorporation of newly synthesized proteins into small intestinal brush borders has been observed. 11 Incorporation of labeled carbohydrate (14C-glucosamine) into microsomal and plasma membrane fractions follows a time course similar to that described for amino acids. 8 Studies of lipid incorporation into LPM have not been reported. Many questions remain unanswered concerning plasma membrane biogenesis. Are lipid, protein, and carbohydrate incorporated together? What is the relationship between protein synthesis and incorporation into LPM of lipid and carbohydrate? At least three possibilities exist for incorporation of newly synthesized proteins into LPM: (1) mixture in a soluble pool and subsequent incorporation; (2) assembly into complex lipoprotein, micelle-like units prior to incorporation; and (3) incorporation into complete membrane units. The last process implies that all proteins are synthesized and degraded simultaneously (i.e., "single-step" assembly) . The first two possibilities are more
February 1972
EDITORIAL
consistent with differential protein synthesis and degradation as described for the endoplasmic reticulum. 6, 12 The degradation rate of one LPM protein relative to another was determined using a double isotope technique to pulselabel proteins at two different time points in the same animal 6 ; two protein fractions separated from LPM had differing degradation 'rates. 1 In addition, LPM proteins are degraded proportional to submit size; that is, large proteins are degraded more rapidly than are small proteins. 13 Therefore, degradation rates of LPM proteins are heterogeneous; individual proteins are assembled and subseql)ently degraded at rates characteristic for each protein. Similar observations have been obtained in studies of intestinal brush border and hepatic endoplasmic reticulum proteins. 14 The existence of "carriers" postulated to influence transfer of organic molecules across the hepatic surface membrane remains conjectural. Studies of bacteria illustrate the role of specific protein carriers in translocation of substances, particularly sugars and amino acids, across membranes. The following have been characterized: (1) specific pericytoplasmic binding proteins,15 (2) specific membrane, lactose-binding protein (Mprotein),16 and (3) a complex system of soluble proteins and membrane substrate-specific proteins involved in group translocation of sugars.17 Direct evidence linking these proteins with transport results from studies using bacterial mutants. In an analogous manner, inheritable disorders of transport in mammals offer potentialities to investigate the components of various transport systems. Several inheritable disorders are found in mutant sheep which demonstrate abnormalities in the transfer of various organic anions from plasma into the liver, and from the liver into the bile canaliculus. Mutant Corriedale sheep have a disorder which is morphologically and functionally identical with the Dubin-Johnson syndrome in man. IS Mutant sheep have a deficiency in hepatic excretion of certain organic anions such as sulfobro-
343
mophthalein sodium, porphyrins, and cholecystographic agents, but have normal biliary excretion of organic cations and bile salts, the major organic anion in bile. IS It is postulated that specific organic anion transfer processes which are normally present in the canalicular membrane may be deficient in this disorder. Mutant Southdown sheep show delayed plasma clearance of many organic anions including sulfobromophthalein sodium, unconjugated bilirubin, indocyanine green, and bilesaltsY Bilirubin-14C turnover studies demonstrate decreased transfer from the plasma into the liver and increased efflux from liver to the plasma. 20 Preliminary studies demonstrated that the two organic anion cytoplasmic binding proteins, Y and Z, are present. 21 In mutant Southdown sheep, the abnormality may involve a specific sinusoidal membrane defect affecting transfer of organic anions from plasma into the liver cell. Competition between several organic anions for binding by LPM has been demonstrated in vitro.22 Specific membrane binding sites and possible "carrier" proteins may be altered in other states associated with defective transfer of organic anions across the liver parenchymal cell, such as post-hypophysectomy,23 post-thyroidectomy,23 the neonatal period, 24 and in experimental cholestasis. The possibility that alterations in LPM may contribute to the pathogenesis of cholestasis has been studied in rats. 25 LPM fractions were separated, identified, and purified after differential centrifugation of liver homogenates through sucrose gradients. 26 Two models of cholestasis were studied to identify alterations in LPM that may be common pathogenic features. Extrahepatic obstruction was produced by bile duct ligation, and intrahepatic cholestasis was produced following administration of ethynylestradiol. No difference in relative degradation rates of pulse-labeled LPM proteins was noted between control and cholestatic rats; however, specific canalicular proteins were altered. LPM magnesium-
344
EDITORIAL
adenosine triphosphatase and 5'-nucleotidase activities were reduced in both models of cholestasis. These changes probably represent decreased protein content because, for each enzyme, (1) V max was decreased and Km was unchanged; (2) in vitro inhibition by taurocholate did not correlate with changes in vivo, and (3) enzyme activity in normal LPM was not inhibited by mixing with LPM from rats with cholestasis. Preferential loss during preparation of canalicular membrane and enhanced recovery of sinusoidal membrane probably did not occur in cholestasis because alkaline phosphatase, a canalicular enzyme, had greatly enhanced activity, and cobaltstimulated cytidine monophosphatase, a sinusoidal enzyme, was unaltered. Cholestasis was associated with alterations in the apparent content of specific canalicular proteins which were independent of the model of cholestasis and, therefore, may be involved in the pathogenesis of bile secretory failure. The recent development of techniques for LPM isolation, membrane protein separation, and measurement of differential protein synthesis and degradation offer opportunities to investigate abnormalities in plasma membrane biogenesis as well as identification of specific membrane proteins having "carrier" function. In addition, the occurrence of bile secretory failure in newborn animals,24 after administration of various drugs, taurolithocholic acid,27 and manganese,28 as well as in several inheritable disorders in sheep, permits study in many different experimental models. The observation that, in cholestasis, phenobarbital administration increases bile flow, possibly by changing (Na +-K +) adenosine triphosphatase, and reduces serum bile salt and bilirubin concentrations 29 , 30 emphasizes the need for further study of membrane transport processes and their possible pharmacologic manipulation. Normal membrane biogenesis is just beginning to be understood. Developments in this field may be very important to future understanding
Vol . 62, No, 2
of the pathogenesis of many liver diseases, particularly cholestasis. FRANCIS R. SIMON Division of Gastroenterology University of Colorado Medical Center 4200 E. Ninth Avenue Denver, Colorado 80220 IRWIN M, ARIAS Divison GI-Liver Disease Albert Einstein College of Medicine Bronx Municipal Hospital Center Bronx, New York 10461 REFERENCES 1. Simon FR, Blumenfeld 00, Arias 1M: Two pro·
tein fractions from hepatic plasma membranes: Studies of their composition and differential turnover. Biochim Biophys Acta 219:349-360, 1970 2. Emmelot P, Bos CJ, Benedetti EL, et al: Studies on plasma membranes I. Chemical composition and enzyme content of plasma membranes isolated from rat liver. Biochim Biophys Acta 9:126-145,1964 3. Essner E, Novikoff AB, Masek B: Adenosinetriphosphatase and 5' -nucleotidase activities in the plasma membrane of liver cells as revealed by electron microscopy. J Biophys Biochem Cytol 4:711-715, 1958 4. Wachstein M, Meisel E: Histochemistry of hepatic phosphatases at a physiological pH with special reference to the demonstration of bile canaliculi. Am J Clin Pathol 27:13-23, 1957 5. Novikoff AB: Membrane-bound enzymes. Sixth International Congress of Biochemistry, vol 6. New York, 1964, p 609 6. Arias 1M, Doyle D, Schimke RT: Studies on the synthesis and degradation of proteins of the endoplasmic reticulum of rat liver. J Bioi Chern 244:3303-3315, 1969 7. Widnell CC, Siekevitz P: The turnover of the constituents of various rat liver membranes. J Cell Bioi 35:142A, 1967 8. Kawasaki T, Yamashina I: Metabolic studies of rat liver plasma membranes using D-(I-14C) glucosamine. Biochim Biophys Acta 225:234238, 1971 9. Ray TK, Lieberman I, Lansing AI: Synthesis of the plasma membrane of the liver cell. Biochem Biophys Res Commun 31:54-58, 1968 10. Beattie DS: The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes. J Membrane Bioi 1:383-401, 1969 11. James WPT, Alpers DH, Gerber JE, et al: The
February 1972
12.
13.
14.
15. 16.
17.
18. 19.
20.
EDITORIAL
turnover of disaccharidases and brush border proteins in rat intestines. Biochem Biophys Acta 230:194-203, 1971 Omura T, Siekevitz P, Palade GE: Turnover of constituents of the endoplasmic reticulum membranes of rat hepatocytes. J Bioi Chern 242: 2389-2396, 1969 Dehlinger PJ, Schimke RT: Size distribution of membrane proteins of rat liver and their relative rates of degradation. J Bioi Chern 246:25742583, 1971 Alpers DH, Goodwin C: Effect of size and anatomic location on the degradation rate of intestinal brush border proteins. Gastroenterology 60:760, 1971 Pardee AB: Membrane transport proteins. Science 162:632-637, 1968 Fox CF, Kennedy EF: Specific labeling and partial purification of the M protein, a component of the ~-galactoside transport system of Escherichia Coli. Proc Natl Acad Sci USA 54:891- 899, 1965 Roseman S: The transport of carbohydrates by a bacterial phosphotransferase system, Membrane Proteins. Little, Brown and Co, Boston, 1969, p 138-180 Alpert S, Mosher M, Shanske A, et al: Multiplicity of hepatic excretory mechanisms for organic anions. J Gen Physiol 53:238-247, 1969 Cornelius CE, Gronwall RR: Congenital photosensitivity and hyperbilirubinemia in Southdown sheep in the United States. Am J Vet Res 29:291-295, 1968 Mia AS, Gronwall RR, Cornelius CE: Bilirubin"C turnover studies in normal and mutant Southdown sheep with congenital hyperbilirubinemia. Proc Soc Exp Bioi Med 133:955- 959, 1970
345
21. Levi AJ, Gatmaitan Z, Arias 1M: Two hepatic cytoplasma protein fractions, Y and Z, and their possible role in the hepatic uptake of bilirubin, sulfobromophthalein and other anions. J Clin Invest 48:2156-2167, 1969 22. Cornelius C, Ben-Ezzer J, Arias 1M: Binding of sulfobromophthalein sodium (BSP) and other organic amino by isolated hepatic cell plasma membranes in vitro. Proc Soc Exp Bioi Med 124:665-667, 1967 23. Gartner LM, Arias, 1M: The hormonal regulation of hepatic bilirubin excretion, Bilirubin Metabolism. Edited by lAD Bouchier, BH Billings. Oxford and Edinburgh, Blackwell Scientific Publications, 1967, p 175- 182 24. Gartner LM, Arias 1M: The transfer of bilirubin from blood to bile in the neonatal guinea pig. Pediatr Res 3:171-180, 1969 25. Simon FJ, Arias 1M: Cholestasis produces alterations in specific bile canalicular proteins (abstr) . Gastroenterology 62:165, 1972 26. Neville DM : Isolation of an organ specific protein antigen from cell-surface membrane of rat liver. Biochim Biophys Acta 154:540-552, 1968 27. Javitt NB, Emerman S : Effect of sodium taurolithocholate on bile flow and bile acid excretion. J Clin Invest 47:1002-1014, 1968 28. Witzleben CL, Pitlick P, Bergmeyer J, et al: Acute manganese overload: A new experimental model of intrahepatic cholestasis. Am J Pathol 53:409-414, 1968 29. Berthelot P, Erlinger S, Dhumeaux D, et al: Mechanism of phenobarbital-induced hypercholeresis in the rat. Am J Physiol 219:809-813, 1970. 30. Admirand WH, Bauer K: Phenobarbital (PB): An effective form of therapy in primary biliary cirrhosis. J Clin Invest 50:1a, 1971
GASTROENTEROLOGY
Printed in U. S. A.
Copyright © 1972 by The Williams & Wilkins Co.
EDITORIAL ALTERATIONS IN LIVER PLASMA MEMBRANES AND THEm POSSIBLE ROLE IN CHOLESTASIS As with other membranes, the plasma membrane of the hepatic parenchymal cell (LPM) consists of protein, lipid, and carbohydrate 1, 2; however, there are several differentiating features with respect to other surface membranes: (1) There is a functional polarity of LPM with respect to sinusoidal, canalicular, and lateral surfaces. Histochemical staining confirms these differences and reveals canalicular staining for magnesium-activated adenosine triphosphatase 3 and alkaline phosphatase activities·; sinusoidal staining for cobalt-activated cytidine monophosphatase activity 5 and staining of both canalicular and sinusoidal surface for 5'-nucleotidase activity. 3 (2) The major components of the LPM undergo differential synthesis and heterogeneous degradation. For example, mean half-life for pulse-labeled LPM proteins (1.8 days) is faster than that for total liver protein (4 days) and similar to that observed for endoplasmic reticulum membrane proteins (2.1 days). 6 LPM phospholipids and carbohydrates have shorter mean t", than do LPM proteins 7 , 8; however, these latter studies have not eliminated the possibility of isotope exchange or differential reutilization rather than degradation. How are these constituents incorporaated into the plasma membrane? The incorporation of pulse-labeled proteins into LPM has been studied after injection of 3H-Ieucine into rats. 9. 10 Peak incorporReceived September 3, 1971. Address requests for reprints to: Dr. Francis R. Simon, University of Colorado Medical Center, Division of Gastroenterology, 4200 E. Ninth Avenue, Denver, Colorado 80220. Research from the authors' laboratory was supported by grants from the United States Public Health Service (AM 02019, 05384) and performed during tenure of Special Fellowship (AM40777) to Dr. Simon. 342
ation of radioactivity into LPM proteins occurred 4 to 6 hr later. Peak isotope specific activity was delayed with respect to endoplasmic reticulum (15 min) and mitochondria (5 to 10 min). Cyloheximide, an inhibitor of protein synthesis, was administered 5 min after injection of labeled amino acid; incorporation of pulse-labeled proteins into' LPM continued unaltered for 3 hr. These studies suggest that LPM proteins are synthesized elsewhere in the cell, probably in the endoplasmic reticulum, and transferred to the plasma membrane for incorporation. The transfer process does not appear to require new protein synthesis for continued incorporation into the surface membrane. A similar time sequence for incorporation of newly synthesized proteins into small intestinal brush borders has been observed. 11 Incorporation of labeled carbohydrate (14C-glucosamine) into microsomal and plasma membrane fractions follows a time course similar to that described for amino acids. 8 Studies of lipid incorporation into LPM have not been reported. Many questions remain unanswered concerning plasma membrane biogenesis. Are lipid, protein, and carbohydrate incorporated together? What is the relationship between protein synthesis and incorporation into LPM of lipid and carbohydrate? At least three possibilities exist for incorporation of newly synthesized proteins into LPM: (1) mixture in a soluble pool and subsequent incorporation; (2) assembly into complex lipoprotein, micelle-like units prior to incorporation; and (3) incorporation into complete membrane units. The last process implies that all proteins are synthesized and degraded simultaneously (i.e., "single-step" assembly) . The first two possibilities are more
February 1972
EDITORIAL
consistent with differential protein synthesis and degradation as described for the endoplasmic reticulum. 6, 12 The degradation rate of one LPM protein relative to another was determined using a double isotope technique to pulselabel proteins at two different time points in the same animal 6 ; two protein fractions separated from LPM had differing degradation 'rates. 1 In addition, LPM proteins are degraded proportional to submit size; that is, large proteins are degraded more rapidly than are small proteins. 13 Therefore, degradation rates of LPM proteins are heterogeneous; individual proteins are assembled and subseql)ently degraded at rates characteristic for each protein. Similar observations have been obtained in studies of intestinal brush border and hepatic endoplasmic reticulum proteins. 14 The existence of "carriers" postulated to influence transfer of organic molecules across the hepatic surface membrane remains conjectural. Studies of bacteria illustrate the role of specific protein carriers in translocation of substances, particularly sugars and amino acids, across membranes. The following have been characterized: (1) specific pericytoplasmic binding proteins,15 (2) specific membrane, lactose-binding protein (Mprotein),16 and (3) a complex system of soluble proteins and membrane substrate-specific proteins involved in group translocation of sugars.17 Direct evidence linking these proteins with transport results from studies using bacterial mutants. In an analogous manner, inheritable disorders of transport in mammals offer potentialities to investigate the components of various transport systems. Several inheritable disorders are found in mutant sheep which demonstrate abnormalities in the transfer of various organic anions from plasma into the liver, and from the liver into the bile canaliculus. Mutant Corriedale sheep have a disorder which is morphologically and functionally identical with the Dubin-Johnson syndrome in man. IS Mutant sheep have a deficiency in hepatic excretion of certain organic anions such as sulfobro-
343
mophthalein sodium, porphyrins, and cholecystographic agents, but have normal biliary excretion of organic cations and bile salts, the major organic anion in bile. IS It is postulated that specific organic anion transfer processes which are normally present in the canalicular membrane may be deficient in this disorder. Mutant Southdown sheep show delayed plasma clearance of many organic anions including sulfobromophthalein sodium, unconjugated bilirubin, indocyanine green, and bilesaltsY Bilirubin-14C turnover studies demonstrate decreased transfer from the plasma into the liver and increased efflux from liver to the plasma. 20 Preliminary studies demonstrated that the two organic anion cytoplasmic binding proteins, Y and Z, are present. 21 In mutant Southdown sheep, the abnormality may involve a specific sinusoidal membrane defect affecting transfer of organic anions from plasma into the liver cell. Competition between several organic anions for binding by LPM has been demonstrated in vitro.22 Specific membrane binding sites and possible "carrier" proteins may be altered in other states associated with defective transfer of organic anions across the liver parenchymal cell, such as post-hypophysectomy,23 post-thyroidectomy,23 the neonatal period, 24 and in experimental cholestasis. The possibility that alterations in LPM may contribute to the pathogenesis of cholestasis has been studied in rats. 25 LPM fractions were separated, identified, and purified after differential centrifugation of liver homogenates through sucrose gradients. 26 Two models of cholestasis were studied to identify alterations in LPM that may be common pathogenic features. Extrahepatic obstruction was produced by bile duct ligation, and intrahepatic cholestasis was produced following administration of ethynylestradiol. No difference in relative degradation rates of pulse-labeled LPM proteins was noted between control and cholestatic rats; however, specific canalicular proteins were altered. LPM magnesium-
344
EDITORIAL
adenosine triphosphatase and 5'-nucleotidase activities were reduced in both models of cholestasis. These changes probably represent decreased protein content because, for each enzyme, (1) V max was decreased and Km was unchanged; (2) in vitro inhibition by taurocholate did not correlate with changes in vivo, and (3) enzyme activity in normal LPM was not inhibited by mixing with LPM from rats with cholestasis. Preferential loss during preparation of canalicular membrane and enhanced recovery of sinusoidal membrane probably did not occur in cholestasis because alkaline phosphatase, a canalicular enzyme, had greatly enhanced activity, and cobaltstimulated cytidine monophosphatase, a sinusoidal enzyme, was unaltered. Cholestasis was associated with alterations in the apparent content of specific canalicular proteins which were independent of the model of cholestasis and, therefore, may be involved in the pathogenesis of bile secretory failure. The recent development of techniques for LPM isolation, membrane protein separation, and measurement of differential protein synthesis and degradation offer opportunities to investigate abnormalities in plasma membrane biogenesis as well as identification of specific membrane proteins having "carrier" function. In addition, the occurrence of bile secretory failure in newborn animals,24 after administration of various drugs, taurolithocholic acid,27 and manganese,28 as well as in several inheritable disorders in sheep, permits study in many different experimental models. The observation that, in cholestasis, phenobarbital administration increases bile flow, possibly by changing (Na +-K +) adenosine triphosphatase, and reduces serum bile salt and bilirubin concentrations 29 , 30 emphasizes the need for further study of membrane transport processes and their possible pharmacologic manipulation. Normal membrane biogenesis is just beginning to be understood. Developments in this field may be very important to future understanding
Vol . 62, No, 2
of the pathogenesis of many liver diseases, particularly cholestasis. FRANCIS R. SIMON Division of Gastroenterology University of Colorado Medical Center 4200 E. Ninth Avenue Denver, Colorado 80220 IRWIN M, ARIAS Divison GI-Liver Disease Albert Einstein College of Medicine Bronx Municipal Hospital Center Bronx, New York 10461 REFERENCES 1. Simon FR, Blumenfeld 00, Arias 1M: Two pro·
tein fractions from hepatic plasma membranes: Studies of their composition and differential turnover. Biochim Biophys Acta 219:349-360, 1970 2. Emmelot P, Bos CJ, Benedetti EL, et al: Studies on plasma membranes I. Chemical composition and enzyme content of plasma membranes isolated from rat liver. Biochim Biophys Acta 9:126-145,1964 3. Essner E, Novikoff AB, Masek B: Adenosinetriphosphatase and 5' -nucleotidase activities in the plasma membrane of liver cells as revealed by electron microscopy. J Biophys Biochem Cytol 4:711-715, 1958 4. Wachstein M, Meisel E: Histochemistry of hepatic phosphatases at a physiological pH with special reference to the demonstration of bile canaliculi. Am J Clin Pathol 27:13-23, 1957 5. Novikoff AB: Membrane-bound enzymes. Sixth International Congress of Biochemistry, vol 6. New York, 1964, p 609 6. Arias 1M, Doyle D, Schimke RT: Studies on the synthesis and degradation of proteins of the endoplasmic reticulum of rat liver. J Bioi Chern 244:3303-3315, 1969 7. Widnell CC, Siekevitz P: The turnover of the constituents of various rat liver membranes. J Cell Bioi 35:142A, 1967 8. Kawasaki T, Yamashina I: Metabolic studies of rat liver plasma membranes using D-(I-14C) glucosamine. Biochim Biophys Acta 225:234238, 1971 9. Ray TK, Lieberman I, Lansing AI: Synthesis of the plasma membrane of the liver cell. Biochem Biophys Res Commun 31:54-58, 1968 10. Beattie DS: The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes. J Membrane Bioi 1:383-401, 1969 11. James WPT, Alpers DH, Gerber JE, et al: The
February 1972
12.
13.
14.
15. 16.
17.
18. 19.
20.
EDITORIAL
turnover of disaccharidases and brush border proteins in rat intestines. Biochem Biophys Acta 230:194-203, 1971 Omura T, Siekevitz P, Palade GE: Turnover of constituents of the endoplasmic reticulum membranes of rat hepatocytes. J Bioi Chern 242: 2389-2396, 1969 Dehlinger PJ, Schimke RT: Size distribution of membrane proteins of rat liver and their relative rates of degradation. J Bioi Chern 246:25742583, 1971 Alpers DH, Goodwin C: Effect of size and anatomic location on the degradation rate of intestinal brush border proteins. Gastroenterology 60:760, 1971 Pardee AB: Membrane transport proteins. Science 162:632-637, 1968 Fox CF, Kennedy EF: Specific labeling and partial purification of the M protein, a component of the ~-galactoside transport system of Escherichia Coli. Proc Natl Acad Sci USA 54:891- 899, 1965 Roseman S: The transport of carbohydrates by a bacterial phosphotransferase system, Membrane Proteins. Little, Brown and Co, Boston, 1969, p 138-180 Alpert S, Mosher M, Shanske A, et al: Multiplicity of hepatic excretory mechanisms for organic anions. J Gen Physiol 53:238-247, 1969 Cornelius CE, Gronwall RR: Congenital photosensitivity and hyperbilirubinemia in Southdown sheep in the United States. Am J Vet Res 29:291-295, 1968 Mia AS, Gronwall RR, Cornelius CE: Bilirubin"C turnover studies in normal and mutant Southdown sheep with congenital hyperbilirubinemia. Proc Soc Exp Bioi Med 133:955- 959, 1970
345
21. Levi AJ, Gatmaitan Z, Arias 1M: Two hepatic cytoplasma protein fractions, Y and Z, and their possible role in the hepatic uptake of bilirubin, sulfobromophthalein and other anions. J Clin Invest 48:2156-2167, 1969 22. Cornelius C, Ben-Ezzer J, Arias 1M: Binding of sulfobromophthalein sodium (BSP) and other organic amino by isolated hepatic cell plasma membranes in vitro. Proc Soc Exp Bioi Med 124:665-667, 1967 23. Gartner LM, Arias, 1M: The hormonal regulation of hepatic bilirubin excretion, Bilirubin Metabolism. Edited by lAD Bouchier, BH Billings. Oxford and Edinburgh, Blackwell Scientific Publications, 1967, p 175- 182 24. Gartner LM, Arias 1M: The transfer of bilirubin from blood to bile in the neonatal guinea pig. Pediatr Res 3:171-180, 1969 25. Simon FJ, Arias 1M: Cholestasis produces alterations in specific bile canalicular proteins (abstr) . Gastroenterology 62:165, 1972 26. Neville DM : Isolation of an organ specific protein antigen from cell-surface membrane of rat liver. Biochim Biophys Acta 154:540-552, 1968 27. Javitt NB, Emerman S : Effect of sodium taurolithocholate on bile flow and bile acid excretion. J Clin Invest 47:1002-1014, 1968 28. Witzleben CL, Pitlick P, Bergmeyer J, et al: Acute manganese overload: A new experimental model of intrahepatic cholestasis. Am J Pathol 53:409-414, 1968 29. Berthelot P, Erlinger S, Dhumeaux D, et al: Mechanism of phenobarbital-induced hypercholeresis in the rat. Am J Physiol 219:809-813, 1970. 30. Admirand WH, Bauer K: Phenobarbital (PB): An effective form of therapy in primary biliary cirrhosis. J Clin Invest 50:1a, 1971