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Complement-dependent cytotoxic antibodies in syngeneic serum from rats carrying transplanted aminoazo dye-induced hepatomas (D23 and D33) and from rats immunized with irradiated tumor cell or homogenates were studied by a short-term 51Cr release assay. The tumor-bearer sera (TBS) were subjected to chromatography on unsolubilized protein A and Sepharose 4B. The cytolytic activity of D23 TBS was recovered in the IgM molecular weight region, whereas no such activity was obtained in the IgG fraction. As judged from immunodiffusion experiments, the IgM molecular weight fraction did not contain any aggregated or complexed IgG. Moreover, in immunofluorescence tests against viable hepatoma cells, using a specific anti-rat IgG conjugate, the TBS were negative. Cross-testing of D23 and D33 TBS against the two hepatomas and cross-absorption of the sera with tumor plasma membranes revealed no tumor specificity in these cytotoxicity reactions. Furthermore, neither fetal nor early postnatal liver cells could absorb out the activity. Absorptions with adult liver plasma membranes, however, abrogated the cytotoxic activity, and even more effective in this respect were homogenates from kidney and small intestine, thus indicating that the cytotoxic antibodies are directed against 'normal' adult antigen(s) present also in tissues other than liver.

By a combination of affinity chromatography and immunoelectrophoretic methods, it was shown that antigens exhibiting several different enzyme activities (nucleoside di- and triphosphatase, NADH-tetrazolium reductase and also in some cases alkaline phosphodiesterase activities), solubilized by detergents from rat liver microsomes, carry sugar moieties that bind to Concanavalin A (Con A) coupled to Sepharose 4B. Similarly, some enzyme active antigens showing only one type of enzyme activity, bind to Con A-Sepharose 4B and can be specifically eluted with α-methyl- d -mannoside. However, two esterase- and two NADH-reductase-active antigens distinct from the multienzyme complexes do not interact with Con A. Thus, Con A binding not only provides a means of characterizing enzyme active antigens as glycoproteins containing certain sugar moieties, but it also gives a simple method for the separation of antigens with similar activities.

1. Two different microsomal fractions (rough tR, smooth tSa) and a plasma membrane fraction (tP) isolated from the 4-dimethylaminoazobenzene-induced rat hepatoma D23 were investigated with immunodiffusion methods combined with staining reactions for esterase, glycosidase and leucyl-β-naphthylamidase active antigens. 2. Two esterase active antigens, also present in liver membranes, were found in the hepatoma. One was precipitated from tSa and the other from tP extracts. The latter only hydrolyzed α-naphthyl acetate, while the former also attacked longer fatty acid ester substrates. 3. In contrast to liver plasma membranes the tP fraction contained all three glycosidases studied (β-glucuronidase, β-galactosidase andN-acetyl-β-glucosaminidase). These enzyme active antigens were also found in the tumour microsomes with varying distribution. 4. One leucyl-β-naphtylamidase active antigen was present in all fractions, while one additional antigen was only detected in liver microsomes.

1. A plasma membrane fraction (tP) and two submicrosomal fractions (rough tR, smooth tSa) were isolated from the 4-dimethylaminoazobenzene-induced rat hepatoma D23. Membrane extracts were tested in immunodiffusion and stained for various phosphatases, electron transport chains and catalase. 2. In the tP extract one 5′-nucleotidase (NMPase) active antigen, immunologically different from those present in liver, was found. tP is also contained two of the nucleoside tri- and diphosphatase (NTPase and NDPase) active antigens present in liver plasma membranes. The uridine diphosphatase (UDPase) active antigen (U1) characteristic for liver microsomes was only found in the tR fractions, while both tR and tSa contained an additional antigen with similar activity. 3. No hydroxylating capacity was found in any of the tumour fractions, not even after phenobarbital treatment, when 2-acetamidonaphthalene was used as substrate. However, some of the components of the liver electron transport chains, NADPH-cytochromec reductase, NADH- and NADPH-neotetrazolium (NT)-reductase and cytochromeb5 were present in the tR fraction. Furthemore, all tumour fractions contained one NADH-NT-reductase active antigen common to liver microsomes and plasma membranes. 4. One catalase active antigen was present in all liver and tumour fractions studied.

Antisera were prepared in rabbits against purified rat liver cytochrome b 5 and NADH-cytochrome c reductase, respectively. With the antiserum against cytochrome b 5 , the cytochrome could be precipitated in immunoelectrophoresis from rough and smooth liver microsomal membranes, kidney microsomes and outer mitochondrial membranes. The cytochrome was precipitable from the rough membranes on the 2nd prenatal day, from the smooth membranes on the day of birth and from the kidney microsomes on the 2nd postnatal day. The NADPH-cytochrome c reductase could be precipitated from the rough and smooth membranes and from kidney microsomes by the specific antiserum. It was precipitable from the rough and smooth membranes at birth and from kidney microsomes on the 2nd postnatal day. No NADPH-cytochrome c reductase was found in the mitochondrial membranes. None of the two antisera contained inhibiting antibodies against the catalytic action of the enzyme against which it was directed.

Esterase-active antigens present in male and female liver microsomes isolated from three different rat strains (Sprague-Dawley, Wistar and Dark Agouti) were characterized in crossed immunoelectrophoresis in combination with a zymogram method for esterase activity. No qualitative but some quantitative differencies were encountered between the sexes. Thus, two out of ten antigens were present in significantly lower and one in significantly higher concentrations in male than in female microsomes, demonstrating that although the overall esterase activity in liver may be similar for males and females, the concentration of the individual antigens does vary between the sexes. No qualitative or quantitative differences in the pattern of esterase-active antigens were found between the different strains.

Detergent-soluble antigens from rat liver microsomes and plasma membranes were separated by two-dimensional immunoelectrophoresis. Enzyme activities in the immunoprecipitates were assayed by different staining reactions. Nine different microsomal esterases were detected. Six of these, including one esterase shared with plasma membranes, were inhibited by 0.1 mM difluorophosphate, but not by 1 mM eserine. Plasma membranes and microsomes each contained at least ten nucleoside diphosphatase and triphosphatase-active antigens. Only three precipitates with these activities were detected when plasma membrane extracts were tested against anti-microsomal sera or microsomal extracts against anti-plasma membrane sera. Although the possible immunological identity of these antigens remains to be established these findings suggest that plasma membranes and microsomes have at most three of these nucleoside diphosphatases and triphosphatases in common. Seven microsomal nucleoside diphosphatases and triphosphatases were intimately associated with both acid phosphatase and NADH-neotetrazolium reductase activities. These complexes also contained phospholipids as shown by incorporation experiments either in vivo, or in vitro, with [14C]-choline or [14C]ethanolamine. Three microsomal UDPase-active antigens did not hydrolyze ATP or ADP and were not associated with acid phosphatase or NADH-neotetrazolium reductase activities. Furthermore, these antigens did not contain any of the labelled phospholipids. Lipid-containing multienzyme complexes of different composition were also detected in the plasma membranes. The nucleoside diphosphatases and triphosphatases in plasma membrane extracts were associated with NADH-neotetrazolium reductase activity and two of them also contained l-leucyl-β-naphthylamidase activity. p-Chloromercuribenzoate (0.1 mM) inhibited all NADH-neotetrazolium reductase activity in the plasma membrane and microsomal precipitates and 10 mM tartrate all acid phosphatase activity in the microsomal precipitates, leaving the other activities intact. This indicated that the various enzyme activities present in the multienzyme complexes depended on separate catalytic sites. The plasma membrane extracts contained three and the microsomal extracts two l-leucyl-β-naphthylamidase-active antigens, respectively. The latter two and one of the plasma membrane antigens were not found to be associated with any other enzyme activities or phospholipids, in contrast to the two plasma membrane antigens mentioned above.

Monospecific antisera were prepared against the most prominent arylamidase (alpha-aminoacyl-peptide hydrolase (microsomal), EC 3.4.11.2) active antigen in plasma membranes (the plasma membrane arylamidase) and lysomal content (the lysosomal content arylamidase), respectively. Plasma membrane extract and lysosomal content were allowed to react in crossed immunoelectrophoresis against their homologous antisera. The electrophoretic plates were washed extensively, dried and subsequently stained for arylamidase activity. The particular immunoprecipitates were thus identified and could be excised to be used for immunizations. The two resulting antisera precipitated the arylamidase used for immunization, but failed to be monospecific as they precipitated additional antigens. These antisera with restricted specificity against some plasma membrane and lysosomal content antigens, respectively, were used to produce immunoprecipitates intended for new attempts to prepare monospecific antisera by a second cycle of immunizations. A monospecific antiserum against the plasma membrane arylamidase was thus obtained, while a third cycle of immunizations was needed to get a monospecific anti-lysosomal content antiserum. The plasma membrane arylamidase showed ATPase activity also after precipitation with the monospecific antiserum, thus still retaining its characteristics as a multienzyme complex.

Detergent extracts of liver microsomes from drug-treated rats were analyzed semiquantitatively in crossed immunoelectrophoresis in combination with a zymogram technique for nonspecific esterase activity. One esterase-active antigen was quantitatively induced by phenobarbital as demonstrated with both antimicrosomal antiserum and a monospecific antiserum prepared against this antigen. No similar inducation of this or any other esterase-active antigen was detected after 3-methylcholanthrene treatment. With the monospecific antiserum, the antigen was also detected in other organs, capable of carrying out hydroxylation reactions, such as lung and kidney. It was, however, not induced by phenobarbital in these organs. Quantitative enzyme assays with acetanilide as substrate indicated that the phenobarbital-inducible esterase could also function as an amidase. Furthermore, from absorption studies, it was concluded that this antigen is located on the cytoplasmic side of the microsomal vesicles. Crossed immunoelectrofocusing experiments revealed that this esterase-active antigen consists of five subcomponents with different charge properties.

The subcellular distribution of arylamidase-active antigens in rat liver and in two chemically induced hepatomas (D23 and D33) was investigated. Soluble antigens or detergent-solubilized membrane antigens from isolated subcellular fractions were tested in fused rocket immunoelectrophoresis against antisera prepared against each of the fractions. The arylamidase active antigens were identified by means of a zymogram technique using L-leucine 2-naphthylamide as substrate. Two arylamidase-active antigens were shown to be shared between plasma membranes, microsomes, lysosomal membranes and lysosomal content of the hepatocytes. One of these occurred predominantly in the plasma membranes (the plasma membrane arylamidase) while the other was preferentially found in the lysosomal content (the lysosomal content arylamidase). Also a third arylamidase-active antigen was identified and was shown to be restricted to the microsomes and the lysosomal membranes (the microsomal/lysosomal arylamidase). The rat liver plasma membrane arylamidase-active antigen was also present in plasma membrane, microsomal and cell-sap fractions of both the hepatomas. However, in the hepatomas this antigen occurred predominantly in the microsomal fraction. The plasma membrane arylamidase was the only arylamidase-active antigen found in the hepatoma D33 while the plasma membrane and microsomal fractions of hepatoma D23 also contained another antigen with this activity. Neither the lysosomal content arylamidase nor the microsomal/lysosomal arylamidase could be detected in any of the hepatoma fractions.

Two immunologically different DT diaphorases were isolated on an affinity column containing Dicumarol as ligand from the cytosol fraction of rat liver. With a specific antiserum raised in rabbits against the DT diaphorase fraction eluted from the column, the two DT diaphorases were shown with immunodiffusion methods to be present in the microsomal and mitochondrial as well as in the cytosol fraction of rat liver. The NAD(P)H oxidizing activity in the immunoprecipitates containing the two DT diaphorases was found to be inhibited by 10 −4 m Dicumarol but not by 10 −3 m 2-pivaloyl-1,3-indandione or warfarin. With the anti-DT diaphorase antiserum the two DT diaphorases were also demonstrated to be present in other organs but with a somewhat different distribution. One of them appeared predominantly in kidney and heart and the other in lung, brain, testis, and spleen. The latter DT diaphorase was also found to be retained in four 3-methylcholanthrene-induced rat hepatomas. These findings indicate different physiological functions for the two DT diaphorases which at present are unknown.

Ten distinct immunoprecipitates with esterase activity were identified in the reaction between rat liver microsomes and rabbit anti-rat liver microsomal antisera in crossed immunoelectrophoresis. These esterases were further characterized by the use of inhibitors, different substrates, hormone induction experiments and cross-reactivity studies with microsomes from other organs. Two of the esterase active antigens were found to be present in 2–3-fold higher concentrations in microsomes from female than male liver. Administration of the female sex hormone estradiol to male rats caused an induction of these esterases to the female level. The microsomal esterases showed a preference to hydrolyze substrates with short fatty-acid chains. Only one esterase could attack acetyl- and butyrylthiocholine. It was also inhibited by 10−3M eserine and was thus considered to be a cholinesterase. All esterases except one were inhibited by 10−3M DFP. In cross-reactivity experiments it was shown that this DFP insensitive esterase was also present in lung and testes microsomes. Kidney microsomes had two other esterases in common with liver, while microsomes from intestine, ovaries and heart gave one weak and diffuse precipitate, not further characterized, with the rabbit anti-rat liver microsomal antiserum.

1. A transplanted hepatoma (D23) originally induced by 4-dimethylamino-azobenzene, was fractionated into three different microsomal subfractions (rough tR, smooth tSa and tSb) and a plasma membrane fraction (tP). 2. On electron micrographs the tR fraction consisted of vesicles mostly covred with ribosomes, while tSa, tSb and tP vesicles were of the smooth type. 3. The highest activity of the plasma membrane marker 5′-nucleotidase (NMPase) was found in tSb and tP. The activity in the tumour was, however, low in relation to liver. No significant activity of glucose-6-phosphatase or succinate-cytochrome c reductase were detected in any of the membrane fractions. 4. A 10-fold increase in adenosine-diphosphatase (ADPase) activity was found in the tSb fraction as compared to all other tumour and liver fractions. A similarly elevated activity in tSb was found with UDP and ATP as substrates. This activity was strongly inhibited by freezing.

By morphological and functional selection of cultured rat liver cells, a cloned strain (PAR-C 1) of epithelial-like cells was established in vitro and investigated by light and electron microscopy. The morphological evidence presented suggested a parenchymal origin of the PAR-C 1 strain, which after 7 months of in vitro propagation was composed of rapidly proliferating and moderately dedifferentiated cells containing large Golgi zones, numerous cytosomes, cytosegresomes (“autophagic vacuoles”) and multivesicular bodies, moderate numbers of mitochondria and cisternae of ER, and abundant free ribosomes. The parenchymal cell origin was suggested by the occurrence of particulate glycogen in the cytoplasmic ground substance and the presence of specialized cell junctions typical of epithelial cells.

Antigens from 3 submicrosomal rat liver fractions (R, Sa and Sb) were investigated for esterase activity in immunoelectrophoretic experiments with rabbit antisera against each of the subfractions. The esterases were further classified as carboxyl-, aryl-, acetyl- or choline esterases by their sensitivity to inhibition by 10 −3 –10 −4 M DFP, 10 −4 M eserine, 10 −2 –10 −4 M PCMB, heat and 10 M urea. The fractions R and Sa contained at least 3 acetyl esterases and 5 carboxyl esterases, while only 1 acetyl esterase and 2 carboxyl esterases were detected in the Sb-fraction. No aryl- or choline esterases were found in any of the fractions. Liver microsomes were shown to have one carboxyl esterase in common with kidney microsomes and one acetyl esterase in common with testes microsomes. By the use of different substrates, it was shown that short chain fatty acids were more easily attacked by the esterases of liver microsomes than long chain fatty acids. The activity of one esterase active antigen classified as carboxyl esterase and occurring in three variants of identical electrophoretic mobility, was strongly incresed in the R- and Sa-membranes by phenobarbital treatmet. In the Sb-membranes, this antigen is entirely missing. Two of the variants were also shown to be present in kidney microsomes where phenobarbital did not provoke any enhancement.