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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.

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.