Biospecific adsorption of hepatic DT-diaphorase on immobilized dicoumarol. I. Purification of cytosolic DT-diaphorase from control and 3-methylcholanthrene-treated rats

Liver cytosolic DT-diaphorase has been purified from both control and 3-methylcholanthrene (MC)-treated rats, employing a new efficient affinity chromatography gel azodicoumarol coupled to divinyl sulfone cross-linked Sepharose 6B. A subsequent gel filtration on Sephacryl S-200 results in a homogeneous enzyme preparation with a yield of 50–55%. The enhanced DT-diaphorase activity observed in MC-treated rats is most probably due to an increase in enzyme concentration. This conclusion is based on the following results: (a) cycloheximide, an inhibitor of protein synthesis, prevents the increase in DT-diaphorase activity normally caused by MC; (b) purifications of DT-diaphorase from control and MC-treated rats result in enzyme preparations exhibiting closely similar specific activities; 15 times higher amounts of enzyme are obtained from MC-treated rats as compared to controls; (c) immunological identity between DT-diaphorase isolated from control and MC-treated rats are found with the Ouchterlony immunodiffusion method employing antisera raised against either type of enzyme preparation; (d) rocket immunoelectrophoresis reveals a severalfold higher specific content of DT-diaphorase in cytosol from MC-induced rats as compared to controls. The investigated physicochemical properties of DT-diaphorase are not altered after MC treatment of rats. The minimum molecular weight based on the flavin content of the enzyme is close to that obtained with SDS-slab gel electrophoresis, i.e., approximately 28,000, indicating that the dimer of DT-diaphorase contains two molecules of FAD. The molecular activities of DT-diaphorase with various electron acceptors have been investigated; no significant differences between DT-diaphorase isolated from control and MC-treated rats are found. However, the molecular activity of the enzyme with 2,6-dichlorophenolindophenol and menadione varies considerably from one preparation to another, irrespective of source. Employing fused rocket immunoelectrophoresis, it has been possible to detect at least three antigenic forms of DT-diaphorase with different reactivities toward electron acceptors such as 2,6-dichlorophenolindophenol and menadione. The possible existence of several forms of DT-diaphorase is discussed.     

Distribution of newly synthesized DT-diaphorase in rat liver

The distribution, synthesis transport, and glycosylation of rat-liver DT-diaphorase has been investigated. The enzyme could be isolated using specific antibodies, mainly from the soluble supernatant but also from microsomal vesicles, Golgi membrane, and mitochondria. 40% of the microsomal enzyme was located in the lumen or on the interior side of the membrane, the rest remaining as an integral non-extractable part of the membrane. Synthesis of DT-diaphorase takes place on both free and bound ribosomes, although it was found to be transported in a sequential manner from the rough to the smooth endoplasmic reticulum and also subsequently to the mitochondria. The rough and smooth microsomal DT-diaphorase contains covalently bound carbohydrate, but no sugar moiety could be detected bound to the cytoplasmic form of the enzyme.     

Purification by cibacron blue F3GA dye affinity chromatography and comparison of NAD(P)H:quinone reductase (E.C.1.6.99.2) from rat liver cytosol and microsomes

The dicoumarol-sensitive NAD(P)H:quinone reductase (E.C.1.6.99.2), often referred to as DT-diaphorase, has been purified from both the cytosolic and microsomal fractions from rat liver using a novel, highly efficient, two-step purification procedure utilizing immobilized Cibacron Blue F3GA dye affinity chromatography as the principal step. Under the conditions reported here, this dye affinity resin, generally recognized as preferentially binding nucleotide-dependent proteins, was highly selective in the recovery of up to 95% of the NAD(P)H:quinone reductase directly from the cytosol as a preparation which was often greater than 90% pure. Further purification by gel exclusion chromatography resulted in pure protein preparations with final recoveries approaching 80%. Similar results were obtained during the purification of this quinone reductase activity from microsomal extracts. Evidence is presented which suggests that the enzyme isolated from each cellular fraction are highly homologous, if not identical; data are consistent with genetic evidence.     

Identification of mitochondrial and microsomal phosphatidylserine synthase in Saccharomyces cerevisiae as the gene product of the CHO1 structural gene.

In Saccharomyces cerevisiae, the membrane-associated enzyme phosphatidylserine synthase (EC 2.7.8.8) is present in the mitochondria and the endoplasmic reticulum. The enzyme from both membrane fractions reacted with antiserum raised against a hybrid protein expressed from a TRPE-CHO1 fusion gene in Escherichia coli and was absent in a cho1 null mutant, strongly suggesting that both the mitochondrial and microsomal forms of phosphatidylserine synthase are the products of the CHO1 gene. The highest degree of purification of enzymatically active protein was 380- and 420-fold from the mitochondrial and the microsomal compartments, respectively. In both cases, the enzymatically active and immunoreactive material comigrated with a protein band of 30,000 apparent molecular weight. In the absence of protease inhibitors during the preparation of membranes, the enzyme underwent degradation to an enzymatically active protein of 23,000 apparent molecular weight.     

Subcellular distribution and properties of carbonyl reductase in guinea pig lung

On subcellular fractionation, carbonyl reductase (EC 1.1.1.184) activity in guinea pig lung was found in the mitochondrial, microsomal, and cytosolic fractions; the specific activity in the mitochondrial fraction was more than five times higher than those in the microsomal and cytosolic fractions. Further separation of the mitochondrial fraction on a sucrose gradient revealed that about half of the reductase activity is localized in mitochondria and one-third in a peroxidase-rich fraction. Although carbonyl reductase in both the mitochondrial and microsomal fractions was solubilized effectively by mixing with 1% Triton X-100 and 1 M KCl, the enzyme activity in the mitochondrial fraction was more highly enhanced by the solubilization than was that in the microsomal fraction. Carbonyl reductases were purified to homogeneity from the mitochondrial, microsomal, and cytosolic fractions. The three enzymes were almost identical in catalytic, structural, and immunological properties. Carbonyl reductase, synthesized in a rabbit reticulocyte lysate cell-free system, was apparently the same in molecular size as the subunit of the mature enzyme purified from cytosol. These results indicate that the same enzyme species is localized in the three different subcellular compartments of lung.     

In vitro synthesis of a putative precursor to the mitochondrial enzyme, carbamyl phosphate synthetase.

A simple and rapid procedure is described for purification of carbamyl phosphate synthetase from the matrix fraction of rat liver mitochondria. Antibodies to the enzyme were raised in sheep and purified from antiserum by affinity chromatography on enzyme-bound Sepharose columns. When membrane-free polyribosomes, isolated from a cytosolic fraction of rat liver, were incubated in a messenger-dependent rabbit reticulocyte protein-synthesizing system in the presence of [35S]methionine, the purified antibody precipitated a product of translation representing 0.2% of total trichloroacetic acid-insoluble radioactivity. It demonstrated mobility characteristics in sodium dodecyl sulfate-polyacrylamide gels expected for a polypeptide of molecular mass approximately 5500 daltons larger than the mature mitochondrial form of the enzyme (160,000 daltons). Proteolysis of both the mature and presumptive in vitro precursor forms of the enzyme yielded respective sets of peptide fragments which gave similar patterns upon gel electrophoresis. Excess mitochondrial enzyme effectively competed with the in vitro product for interaction with anti-carbamyl phosphate synthetase antibody.     

Expression of mammalian DT-diaphorase in Escherichia coli: purification and characterization of the expressed protein.

A full-length cDNA clone, pKK-DTD4, complementary to rat liver cytosolic DT-diaphorase [NAD(P)H:quinone oxidoreductase (EC 1.6.99.2)] mRNA was expressed in Escherichia coli. The pKK-DTD4 cDNA was obtained by extending the 5'-end sequence of a rat liver DT-diaphorase cDNA clone, pDTD55, to include an ATG initiation codon and the NH2-terminal codons using polymerase chain reaction (PCR). Restriction sites for EcoRI and HindIII were incorporated at the 5'- and 3'-ends of the cDNA, respectively, by the PCR reaction. The resulting full-length cDNA was inserted into an expression vector, pKK2.7, at the EcoRI and HindIII restriction sites. E. coli strain AB1899 was transformed with the constructed expression plasmid, and DT-diaphorase was expressed under the control of the tac promotor. The expressed DT-diaphorase exhibited high activity of menadione reduction and was inhibited by dicumarol at a concentration of 10(-5)M. After purification by Cibacron Blue affinity chromatography, the expressed enzyme migrated as a single band on 12.5% sodium dodecyl sulfate-polyacrylamide gel with a molecular weight equivalent to that of the purified rat liver cytosolic DT-diaphorase. The purified expressed protein was recognized by polyclonal antibodies against rat liver DT-diaphorase on immunoblot analysis. It utilized either NADPH or NADH as electron donor at equal efficiency and displayed high activities in reduction of menadione, 1,4-benzoquinone, and 2,6-dichlorophenolindophenol which are typical substrates for DT-diaphorase. The expressed DT-diaphorase exhibited a typical flavoprotein spectrum with absorption peaks at 380 and 452 nm. Flavin content determination showed that it contained 2 mol of FAD per mole of the enzyme. Edman protein sequencing of the first 20 amino acid residues at the NH2 terminus of the expressed protein indicated that the expressed DT-diaphorase is not blocked at the NH2 terminus and has an alanine as the first amino acid. The remaining 19 amino acid residues at the NH2 terminus were identical with those of the DT-diaphorase purified from rat liver cytosol.     

A novel, endogenous inhibitor of 7-ethoxyresorufin-O-deethylase activity isolated from liver cytosolic fractions of bream (Abramis brama L.)

The cytosolic supernatant of bream (Abramis brama L.) liver homogenates inhibits the 7-ethoxyresorufin-O-deethylase (EROD) activity of pike (Esox lucius) microsomal fractions. The inhibitor shows no activity against 7-ethoxycoumarin-O-deethylase and benzo(a)pyrene hydroxylase indicating a high isoenzyme specificity. The inhibiting component is a heat-sensitive substance (56 degrees C for 5') which is not self regenerating after subsequent cooling. It can be isolated from the cytosolic fraction using two combined steps of ion exchange chromatography. The purification factor is 500-fold with a recovery rate of 70%. SDS-PAGE of the purified fractions indicate that electrophoretic purity was not achieved. However, a prominent band at about 97 kDa was present in all fractions in a close intensity activity relationship. The molecular weight of the native form of the purified protein was determined to be 175 +/- 35 kDa using gel filtration on a Sephacryl S 300 HR column. So far the inhibitor can be characterized as a protein. It shows strong tendencies to aggregate due to lipophilic interactions. These interactions can be repressed by the addition of 1% sodium cholate. The inhibitor has an optimum activity at 25 degrees C and pH 8.0. The inhibitor does not correspond to any of the known cytosolic, endogenous inhibitors of EROD activities in fish, including proteases, cytosolic phosphatases, kinases and resorufin reductase (e.g. DT-diaphorase), although a non-dicoumarol (10 microM)-inhibited menadione oxidoreductase activity of up to 46.7 +/- 0.4 nmol/min per mg inhibitory protein was measured. Kinetic studies using Michaelis-Menten kinetics with purified inhibitor fractions prove a non-competitive mode of inhibition. As this kind of inhibitor is not described yet it is named CERODIP (cytosolic, EROD-inhibiting protein).     

Enhancement and inhibition of enzymes of heme metabolism by diethyl maleate in the rat kidney

The acylation of sn-glycerol 3-phosphate with palmityl-CoA was compared in mitochondria and microsomes isolated from rat liver. Polymyxin B, an antibiotic known to alter bacterial membrane structure, stimulated the mitochondrial glycerophosphate acyltransferase but inhibited the microsomal enzyme. When mitochondrial and microsomal fractions were incubated at 4–6 °C for up to 4 h, the mitochondrial enzyme remained virtually unchanged while the microsomal enzyme lost about one-half of its activity. Incubations at higher temperatures also revealed that the mitochondrial enzyme was comparatively more stable under the conditions employed. The mitochondrial acyltransferase showed no sensitivity to bromelain, papain, Pronase, and trypsin, all of which strongly inhibited the microsomal enzyme. The differential sensitivity to trypsin was observed in mitochondria and microsomes isolated from other rat organs. However, the liver mitochondrial glycerophosphate acyltransferase was inhibited by trypsin in the presence of either 0.05% deoxycholate or 0.1% Triton X-100. The trypsin sensitivity of the mitochondrial glycerophosphate acyltransferase in the presence of detergent was not due to the presence, in the mitochondrial fraction, of a trypsin inhibitor which became inactivated by Triton X-100 or deoxycholate. The results suggest that the catalytic site of mitochondrial glycerophosphate acyltransferase is not exposed to the cytosolic side and it is located in the inner aspect of the outer membrane.     

Demonstration of glyoxalase II in rat liver mitochondria. Partial purification and occurrence in multiple forms

Glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6), which has been regarded as a cytosolic enzyme, was also found in rat liver mitochondria. The mitochondrial fraction contained about 10-15% of the total glyoxalase II activity in liver. The actual existence of the specific mitochondrial glyoxalase II was verified by showing that all of the activity of the crude mitochondrial pellet was still present in purified mitochondria prepared in a Ficoll gradient. Subfractionation of the mitochondria by digitonin treatment showed that 56% of the activity resided in the mitochondrial matrix and 19% in the intermembrane space. Partial purification of the enzyme (420-fold) was also achieved. Statistically significant differences were found in the substrate specificities of the mitochondrial and the cytosolic glyoxalase II. Electrophoresis and isoelectric focusing of either the crude mitochondrial extract or of the purified mitochondrial glyoxalase II resolved the enzyme activity into five forms with the respective pI values of 8.1, 7.5, 7.0, 6.85 and 6.6. Three of these forms (pI values 7.0-6.6) were exclusively mitochondrial, with no counterpart in the cytosol. The relative molecular mass of the partially purified enzyme, as estimated by Superose 12 gel chromatography, was 21,000. These results give evidence for the presence of mitochondrial glyoxalase II which is different from the cytosolic enzymes in several characteristics.     

Changes in the phospholipid catabolism of mitochondria and microsomes during the development of rat liver.

The lipolytic activities of mitochondrial and microsomal fractions ('microsomes') isolated from foetal, suckling and adult rat liver were compared. The catabolism of endogenous phospholipids was followed by measuring the loss of phospholipids and the appearance of non-esterified fatty acids and lysophosphatides. The rate of mitochondrial phospholipid catabolism does not change significantly during development, but the rate of lipolysis of microsomal phospholipids increases 3-fold during development. Balance studies showed that, in mitochondria and microsomes of foetal, suckling and adult rat liver, fatty acid formation is greatly in excess of the fatty acids that can be accounted for by measuring phospholipid disappearance and lysophosphatide appearance. The hypothesis that this excess fatty acid formation resulted from the lipolysis of mitochondrial and microsomal triacylglycerols were tested and confirmed by preliminary experiments. Mitochondria and microsomes isolated from all developmental ages investigated had phospholipases with A1 and A2 activities. The degree of unsaturation of the fatty acids derived from the phospholipids of mitochondria did not vary significantly during development.     

Properties of cytosolic epoxide hydrolase purified from the liver of untreated and clofibrate-treated mice. Purification procedure and physiochemical characterization of the pure enzymes

Cytosolic epoxide hydrolase was purified from the liver of untreated and clofibrate-treated male C57Bl/6 mice. The purification procedure involves chromatography on DEAE-cellulose, phenyl-Sepharose and hydroxyapatite, takes two days to perform and results in a 120-fold purification and approximately 35% yield of the enzyme from untreated mice. The purified enzyme is a dimer with a molecular mass of 120 kDa, a Stokes' radius of 4.2 nm, a frictional ratio of 1.0 and an isoelectric point of 5.5. The subunits behave identically upon isoelectric focusing in 8 M urea and only one band with a molecular mass of 60 kDa is seen after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The form purified from clofibrate-treated mice had very similar properties and was apparently identical to the control form as judged by amino acid analysis and peptide mapping as well. These analyses also demonstrated that the cytosolic enzyme is clearly different from microsomal epoxide hydrolase isolated from rat liver. Furthermore, Ouchterlony immunodiffusion using antibodies raised in rabbits towards the control form of cytosolic epoxide hydrolase revealed identity between the two forms of cytosolic epoxide hydrolase, but no reaction with the microsomal epoxide hydrolase was observed. These findings indicate large structural differences between the cytosolic and microsomal forms of epoxide hydrolase in the liver.     

Human placental steryl-sulfatase. Enzyme purification, production of antisera, and immunoblotting reactions with normal and sulfatase-deficient placentas

The steryl-sulfatase of normal human placental microsomes was solubilized and enriched about 350-fold. Chromatography on Sepharose 6B of the purified enzyme preparation revealed a single protein peak which eluted according to an apparent molecular mass of 270 +/- 30 kDa; when electrophorized on sodium dodecyl sulfate polyacrylamide gel the sulfatase migrated according to a molecular mass of 64 +/- 4 kDa. Estrogensulfatase activity was co-purified with the steryl-sulfatase activity; obviously, both activities belong to the same enzyme species. The purified sulfatase was injected into three rabbits. Antisera produced by the rabbits yielded a single sharp immunoprecipitation line in Ouchterlony double diffusion experiments when tested with the isolated sulfatase or with a solubilized microsomal fraction of normal placentas. The activity of sulfatase preparations incubated with antiserum was precipitated by addition of polyethylene glycol followed by centrifugation; none of the antibodies reacting with the sulfatase therefore appeared to interfere with its enzymatic activity. Using these antisera, steryl-sulfatase protein could be detected by immunoblotting analysis in solubilized microsomal fractions of normal placentas but not in solubilized microsomal fractions of three steryl-sulfatase activity-deficient placentas. This finding argues in favour of human placental steryl-sulfatase deficiency being due to extremely diminished or absent enzyme protein in the placenta.     

Regulation of mitochondrial and microsomal phospholipid synthesis by liver fatty acid-binding protein.

Recently we have detected and partially purified a 15-kDa cytosolic L-alpha-lysophosphatidic acid (LPA)-binding protein (LPABP), which stimulates export of LPA from mitochondria (Vancura, A., Carroll, M. A., and Haldar, D. (1991) Biochem. Biophys. Res. Commun. 175, 339-343). Now we have purified this protein to homogeneity. By Western immunoblot analysis, amino acid sequence analysis, and binding characteristics we have shown that LPABP is identical with liver fatty acid-binding protein (L-FABP). This protein binds LPA, and stimulates mitochondrial and microsomal glycerophosphate acyltransferase (GAT) and the export of LPA from both the organelles. The mitochondrially synthesized LPA exported by L-FABP can be converted to phosphatidic acid by microsomes. L-FABP also stimulates microsomal conversion of LPA to phosphatidic acid but strongly inhibits this reaction in mitochondria. However, in the absence of L-FABP mitochondria predominantly synthesize PA. Taken together, these findings are suggestive that L-FABP plays a major role in mitochondrial and microsomal phospholipid metabolism by regulating both the synthesis and utilization of LPA.     

The markers of pig heart mitochondrial sub-fractions : I. - The dual location of NADPH-cytochrome c reductase in outer membrane and microsomes.

Evidence is presented about the dual location of NADPH-cytochrome c reductase in mitochondrial outer membranes as well as in microsomes, from pig heart. A high specific activity, was found in both fractions, even after their purification by washing, digitonin treatments, or passages on sucrose gradients. A large fraction of the total activity was associated with both mitochondria and microsomes. Mitochondrial outer membrane differs from microsomes by a low choline phosphotransferase activity and the absence of cytochrome P-450. The properties of mitochondrial and microsomal rotenone-insensitive NADH- and NADPH-cytochrome c reductases were studied. In microsomes, both activities have the same optimum pH (8.5) ; in contrast, in mitochondria they have a different one. The Km-NADPH were always much higher than those for NADH. In mitochondria the Km for NAD(P)H were dependent on cytochrome c concentration. The results show that the rotenone-insensitive NADH- and NADPH-cytochrome c reductases of mitochondria and microsomes have quite different behavior and do not appear to be supported by the same enzyme.     

Purification of human liver cytosolic epoxide hydrolase and comparison to the microsomal enzyme

Epoxide hydrolase (EC 3.3.2.3) was purified to electrophoretic homogeneity from human liver cytosol by using hydrolytic activity toward trans-8-ethylstyrene 7,8-oxide (TESO) as an assay. The overall purification was 400-fold. The purified enzyme has an apparent monomeric molecular weight of 58 000, significantly greater than the 50 000 found for human (or rat) liver microsomal epoxide hydrolase or for another TESO-hydrolyzing enzyme also isolated from human liver cytosol. Purified cytosolic TESO hydrolase catalyzes the hydrolysis of cis-8-ethylstyrene 7,8-oxide 10 times more rapidly than does the microsomal enzyme, catalyzes the hydrolysis of TESO and trans-stilbene oxide as rapidly as the microsomal enzyme, but catalyzes the hydrolysis of styrene 7,8-oxide, p-nitrostyrene 7,8-oxide, and naphthalene 1,2-oxide much less effectively than does the microsomal enzyme. Purified cytosolic TESO hydrolase does not hydrolyze benzo[a]pyrene 4,5-oxide, a substrate for the microsomal enzyme. The activities of the purified enzymes can explain the specific activities observed with subcellular fractions. Anti-human liver microsomal epoxide hydrolase did not recognize cytosolic TESO hydrolase in purified form or in cytosol, as judged by double-diffusion immunoprecipitin analysis, precipitation of enzymatic activity, and immunoelectrophoretic techniques. Cytosolic TESO hydrolase and microsomal epoxide hydrolase were also distinguished by peptide mapping. The results provide evidence that physically different forms of epoxide hydrolase exist in different subcellular fractions and can have markedly different substrate specificities.     

Purification and characterization of rat-liver cytosolic epoxide hydrolase

Rat liver cytosolic epoxide hydrolase has been purified and characterized. The enzyme was purified from tiadenol-induced rat liver 540-fold with respect to trans-stilbene oxide as a substrate. Similar purification was obtained with the substrates trans-beta-ethyl styrene oxide and styrene 7,8-oxide, the specific activities decreasing in the order trans-beta-ethyl styrene oxide greater than styrene 7,8-oxide greater than trans-stilbene oxide. The enzyme exerts highest activity at pH 7.4 Km and Vmax of the pure enzyme for trans-stilbene oxide were 1.7 microM and 205 nmol x min-1 x mg protein-1 respectively. With trans-stilbene oxide as a substrate, the inhibition by organic solvents (2.5% by vol.) increased in the order ethanol less than methanol less than acetone less than isopropanol = N,N-dimethyl formamide less than acetonitrile less than tetrahydrofuran. The native enzyme, with a molecular mass of 120 kDa, consists of two 61-kDa subunits. Digestion of rat liver cytosolic and microsomal epoxide hydrolase by three proteases resulted in markedly different peptide maps. Western-blot analysis with antiserum against rat liver cytosolic epoxide hydrolase revealed a single band with the purified enzyme, and with liver cytosol from control and clofibrate-induced rats. No cross-reactivity was observed with purified rat microsomal epoxide hydrolase or microsomes. A positive reaction at the same molecular mass was obtained with liver cytosol of mouse, guinea pig, Syrian hamster and New Zealand white rabbit but not with that of green monkey.     

Subcellular distribution of aromatase in human placenta and ovary.

The aromatization of androstenedione in human ovarian microsomes is inhibited by an antibody to porcine hepatic microsomal NADPH-cytochrome c reductase. Likewise, the antibody inhibits aromatization in mitochondria isolated from human ovaries and placentae. A given quantity of the antibody produces the same percent inhibition of aromatization in microsomes and mitochondria of both ovaries and placentae. These data, in addition to the low specific activity observed for the mitochondrial aromatase, indicate that aromatization in mitochondria probably results from microsomal contamination.     

Isolation, purification, and characterization of a rat liver mitochondrial protein disulfide isomerase

The isolation and purification to electrophoretical homogeneity and characterization of a protein disulfide isomerase from rat liver mitochondria is reported. The purified enzyme exhibits a single band on sodium dodecylsulfatepolyacrylamide gel electrophoresis with an apparent molecular weight of approximately 54 kDa. Comparatively, the microsomal form shows an apparent molecular weight of 57 kDa indicating that the two forms are slightly different. The antibody raised against the microsomal isoform does not recognize the mitochondrial enzyme. To characterize the enzyme, different classical methodologies utilized for protein disulfide isomerase estimation have been adopted. The isolated enzyme is active with all of them, indicating that it comprises all the features of a typical protein disulfide isomerase. At the mitochondrial level the enzyme appears mostly localized at the membrane level. Its potential involvement in mitochondrial membrane permeability control is also discussed.     

Characterization and distribution of protein kinase C in ovarian tissue

Although protein kinase C, an enzyme dependent on calcium, phospholipid and diacylglycerol, has been found in high levels in ovarian tissues, its biologic function is yet unknown. In initial studies on the role of this enzyme in regulating ovarian functions, we compared protein kinase C activity in subcellular fractions of porcine corpora lutea and medium follicles. Highest protein kinase C-specific activities were found in the cytosol, followed by microsomes and mitochondria for both follicles and luteal tissues. Solubilization of all membrane-containing fractions by 0.2% Triton X-100 was required for full expression (a 4-fold average increase) of protein kinase activity. Extraction of membrane fractions with 0.5 M NaCl or sonication in a hypotonic medium revealed that 90% of the total mitochondrial protein kinase C activity and 50% of the microsomal activity was tightly membrane-bound. Characterization of both cytosolic and Triton X-100 extracted membrane preparations of luteal tissue by diethylaminoethyl (DEAE)-cellulose chromatography revealed a single peak of protein kinase C activity eluting at 80 mM NaCl. Cytosolic fractions of corpora lutea contained 3 times more protein kinase C-specific activity than did cytosolic fractions of follicles. In contrast, mitochondria from medium follicles contained 30% more specific protein kinase C activity than did luteal mitochondria. These higher cytosolic levels of protein kinase C-specific activity in corpora lutea suggest that the enzyme may play an important role in the process of luteinization or in the regulation of luteal function.