Properties of latent and thiol-activated rat hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase and regulation of enzyme activity

The effect of the thiols glutathione (GSH), dithiothreitol (DTT), and dithioerythritol (DTE) on the conversion of an inactive, latent form (El) of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) to a catalyticaly active form (Ea) is examined. Latent hepatic microsomal HMG-CoA reductase is activated to a similar degree of activation by DTT and DTE and to a lower extent by GSH. All three thiols affect both Km and Vmax values of the enzyme toward HMG-CoA and NADPH. Studies of the effect of DTT on the affinity binding of HMG-CoA reductase to agarose-hexane-HMG-CoA (AG-HMG-CoA) resin shows that thiols are necessary for the binding of the enzyme to the resin. Removal of DTT from AG-HMG-CoA-bound soluble Ea (active enzyme) does not cause dissociation of the enzyme from the resin at low salt concentrations. Substitution of DTT by NADPH does not promote binding of soluble El (latent enzyme) to AG-HMG-CoA. The enzymatic activity of Ea in the presence of DTT and GSH indicates that these thiols compete for the same binding site on the enzyme. Diethylene glycol disulfide (ESSE) and glutathione disulfide (GSSG) inhibit the activity of Ea. ESSE is more effective for the inhibition of Ea than GSSG, causing a higher degree of maximal inhibition and affecting the enzymatic activity at lower concentrations. A method is described for the rapid conversion of soluble purified Ea to El using gel-filtration chromatography on Bio-Gel P-4 columns. These combined results point to the importance of the thiol/disulfide ratio for the modulation of hepatic HMG-CoA reductase activity.     

Coupling between cyclooxygenases and prostaglandin F(2alpha) synthase. Detection of an inducible,glutathione-activated,membrane-bound prostaglandin F(2alpha)-synthetic activity

Distinct functional coupling between cyclooxygenases (COXs) and specific terminal prostanoid synthases leads to phase-specific production of particular prostaglandins (PGs). In this study, we examined the coupling between COX isozymes and PGF synthase (PGFS). Co-transfection of COXs with PGFS-I belonging to the aldo-keto reductase family into HEK293 cells resulted in increased production of PGF(2alpha) only when a high concentration of exogenous arachidonic acid (AA) was supplied. However, this enzyme failed to produce PGF(2alpha) from endogenous AA, even though significant increase in PGF(2alpha) production occurred in cells transfected with COX-2 alone. This poor COX/PGFS-I coupling was likely to arise from their distinct subcellular localization. Measurement of PGF(2alpha)-synthetic enzyme activity in homogenates of several cells revealed another type of PGFS activity that was membrane-bound, glutathione (GSH)-activated, and stimulus-inducible. In vivo, membrane-bound PGFS activity was elevated in the lung of lipopolysaccharide-treated mice. Taken together, our results suggest the presence of a novel, membrane-associated form of PGFS that is stimulus-inducible and is likely to be preferentially coupled with COX-2.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase in minimal deviation hepatoma 7288C. Immunological measurements in hepatoma tissue culture cells.

The mechanism of action of serum lipoproteins and 25-hydroxycholesterol on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in hepatoma tissue culture (HTC) cells was investigated using antiserum against purified rat liver HMG-CoA reductase (Heller, R. A., and Shrewsbury, M. A. (1976)J. Biol. Chem. 251, 3815-3822). This antiserum cross-reacted with solubilized and membrane-bound HMG-CoA reductase from HTC cells. The enzymes from rat liver and HTC cells appeared antigenically identical. The increase in HMG-CoA reductase activity of HTC cells grown in medium which lacked serum lipoproteins was shown to be due to an increase in immunoprecipitable enzyme. In contrast, the 25-hydroxycholesterol suppression of reductase activity leads to a reduction in the antigenicity of the enzyme rather than a decrease in its number of molecules.     

Coupling between cyclooxygenases and prostaglandin F2α synthase: Detection of an inducible,glutathione-activated,membrane-bound prostaglandin F2α-synthetic activity

Distinct functional coupling between cyclooxygenases (COXs) and specific terminal prostanoid synthases leads to phase-specific production of particular prostaglandins (PGs). In this study, we examined the coupling between COX isozymes and PGF synthase (PGFS). Co-transfection of COXs with PGFS-I belonging to the aldo-keto reductase family into HEK293 cells resulted in increased production of PGF_2α only when a high concentration of exogenous arachidonic acid (AA) was supplied. However, this enzyme failed to produce PGF_2α from endogenous AA, even though significant increase in PGF_2α production occurred in cells transfected with COX-2 alone. This poor COX/PGFS-I coupling was likely to arise from their distinct subcellular localization. Measurement of PGF_2α-synthetic enzyme activity in homogenates of several cells revealed another type of PGFS activity that was membrane-bound, glutathione (GSH)-activated, and stimulus-inducible. In vivo, membrane-bound PGFS activity was elevated in the lung of lipopolysaccharide-treated mice. Taken together, our results suggest the presence of a novel, membrane-associated form of PGFS that is stimulus-inducible and is likely to be preferentially coupled with COX-2.     

Evidence for the Presence of the Ascorbate-Glutathione Cycle in Mitochondria and Peroxisomes of Pea Leaves

The presence of the enzymes of the ascorbate-glutathione cycle was investigated in mitochondria and peroxisomes purified from pea (Pisum sativum L.) leaves. All four enzymes, ascorbate peroxidase (APX; EC 1.11.1.11), monodehydroascorbate reductase (EC 1.6.5.4), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2), were present in mitochondria and peroxisomes, as well as in the antioxidants ascorbate and glutathione. The activity of the ascorbate-glutathione cycle enzymes was higher in mitochondria than in peroxisomes, except for APX, which was more active in peroxisomes than in mitochondria. Intact mitochondria and peroxisomes had no latent APX activity, and this remained in the membrane fraction after solubilization assays with 0.2 M KCl. Monodehydroascorbate reductase was highly latent in intact mitochondria and peroxisomes and was membrane-bound, suggesting that the electron acceptor and donor sites of this redox protein are not on the external side of the mitochondrial and peroxisomal membranes. Dehydroascorbate reductase was found mainly in the soluble peroxisomal and mitochondrial fractions. Glutathione reductase had a high latency in mitochondria and peroxisomes and was present in the soluble fractions of both organelles. In intact peroxisomes and mitochondria, the presence of reduced ascorbate and glutathione and the oxidized forms of ascorbate and glutathione were demonstrated by high-performance liquid chromatography analysis. The ascorbate-glutathione cycle of mitochondria and peroxisomes could represent an important antioxidant protection system against H2O2 generated in both plant organelles.     

Inactive 3-hydroxy-3-methylglutaryl-coenzyme A reductase in broken cell preparations of various mammalian tissues and cell cultures.

Preincubation of broken cell preparations from a variety of tissues and cell cultures resulted in an apparent increase in the level of 3-hydroxy-3-methylglutaryl-CoA reductase activity. However, apparent activation of the reductase in mouse liver, hepatomas and primary liver cell cultures was attributed largely to the loss, during the preincubation period, of an interfering enzyme, 3-hydroxy-3-methylglutaryl-CoA lyase. Among non hepatic cells and tissues (which did not contain appreciable lyase activity) the proportion of latent reductase was high in sonicates of fetal brain and in L cells and was independent of the level of total enzyme activity present. Activation of the reductase was blocked by hydroxymethylglutaryl-CoA and NADPH as well as by KF so that activation did not occur under the conditions of the enzyme assay. The enzyme was activated slowly at 4 degrees C, so that partial activation of the latent form occurred during isolation of the microsomal fraction by differential centrifugation. The reductase present in sonicates of cells with either a high or low proportion of the latent enzyme was inactivated by incubation with ATP and Mg2+. Suppression of reductase activity in L cell cultures by treatment with 25-hydroxycholesterol and an age-related decline in brain enzyme activity did not involve reversible conversion of the reductase to an inactive form.     

Reversible phosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase in Morris hepatomas

The reversible phosphorylation of microsomal 3-hydroxy-3-methylglutaryl CoA reductase in host liver and hepatoma 5123C has been investigated. The percentage of the total enzyme activity in vivo was similar in the normal liver, host liver and hepatoma 5123C. The inclusion of 30 mM EDTA and 10 mM mevalonic acid in assays of 3-hydroxy-3-methylglutaryl CoA reductase inactivation in vitro eliminated artifacts generated by the presence of mevalonate kinase. Inactivation of 3-hydroxy-3-methylglutaryl CoA reductase from normal liver, host liver and hepatoma occurred at a similar rate with similar half-times. We conclude that phosphorylation/dephosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase occurs in hepatomas and that the lack of dietary cholesterol feedback inhibition in the hepatomas is not a result of a defect in this particular aspect of the reversible phosphorylation system.     

Isolation from rat liver of a peroxisomal enzyme which converts molecular form 1 of biliverdin reductase into molecular form 3

Cobaltous chloride induced in rat liver an enzyme which converted biliverdin reductase molecular form 1 into the molecular form 3. This conversion involves the oxidation of two sulfhydryl groups of form 1 giving rise to a disulfide bond in form 3. The converting enzyme was isolated from the liver peroxisomal fraction (which was devoid of biliverdin reductase activity), and was absent in liver peroxisomes of control rats. The enzyme was solubilized by treatment of the peroxisomes with 0.1% sodium deoxycholate, and partially purified by DEAE-cellulose and Sephadex G-100 filtration. It is a NAD⁺ dependent enzyme which was inactivated by trypsing and heat treatments. It did not oxidize either reduced glutathione or cysteine. The converting enzyme had a molecular weight of about 54,000 daltons. The oxidation of biliverdin reductase molecular form 1 mediated by the converting enzyme did not affect the latter's molecular weight or activity.     

A reappraisal of leukocyte dehydroascorbate reductase

Dehydroascorbate reductase (glutathione:dehydroascorbate oxidoreductase, EC 1.8.5.1) activity was determined in human leukocyte homogenates using a direct spectrophotometric assay. Despite previous studies, using a less sensitive coupled assay, which reported that this enzyme was present in leukocytes, we found that neither neutrophil nor chronic lymphocytic leukemia lymphocyte extracts had detectable activity. Furthermore, when the product was quantitated by HPLC, protein-dependent generation could not be demonstrated. Mixing experiments with a partially purified enzyme preparation from spinach leaves provided no evidence for the presence of an inhibitor in neutrophil homogenates. These findings suggest that in human leukocytes, dehydroascorbate reduction does not occur enzymatically.     

Some features of cyclic adenosine monophosphate metabolism in mouse liver and hepatoma 22]

The levels of cyclic adenosine monophosphate (cAMP) and two forms of cAMP phosphodiesterase with low (PDE1) and high (PDE2) affinity for the substrate were determined in homogenates from mouse liver and transplanted hepatoma 22. The level of cAMP in the tumour is 3 times lower than that in liver. By te kinetic parameters (Vmax, Km, pH optimum) adenylate cyclase from tumour does not show any significant differences as compared to the liver enzyme; the enzyme from hepatoma is, however, more sensitive to activation by F- ions. The activities of adenylate cyclase in liver and tumour cells are the same. Phosphodiesterases of cAMP from tumour and liver cells are similar in their Km values (3,3-10(-4) M for PDE1 and 2-10(-6) M for PDE2); however, the maximal and real rates of cAMP hydrolysis in hepatoma are much higher than in liver. The fact that both cAMP phosphodiesterase activities have similar dependence on Mg2+ and Ca2+ concentrations, suggests that PDE1 is a latent form of PDE2. In tumour cells the equilibrium between these two forms is probably shifted towards the enzyme with high affinity for the substrate. The results suggest that a decreased cAMP level in hepatoma cells (as compared to the liver) is due to the activation of PDE2.     

Thiol-protein disulphide oxidoreductases. Assay of microsomal membrane-bound glutathione-insulin transhydrogenase and comparison with protein disulphide-isomerase.

1. Inhibition of endogenous microsomal NADPH oxidase by CO enables membrane-bound glutathione-insulin transhydrogenase (EC 1.8.4.2) to be assayed conveniently by a linked assay involving NADPH and glutathione reductase (EC 1.6.4.2). 2. The specific activity of the enzyme in rat liver microsomal preparations is of the order of 1 nmol of oxidized glutathione formed/min per mg of membrane protein. 3. The specific activity of the enzyme is comparable in rough and smooth microsomal fractions, and the activity is not affected by treatment with EDTA and the removal of ribosomes from rough microsomal fractions. 4. Membrane-bound glutathione-insulin transhydrogenase is not affected by concentrations of deoxycholate up to 0.5%, whereas protein disulphide-isomerase (EC 5.3.4.1) is drastically inhibited. 5. On these grounds it is concluded that, in rat liver microsomal fractions, glutathione-insulin transhydrogenase and protein disulphide-isomerase activities are not both catalysed by a single enzyme species.     

The autorelease of alkaline phosphatase from the plasma membrane during the incubation of cultured liver cell homogenates

When a rat hepatoma cell (R-Y121B) homogenate was incubated at 37 degrees C, 30-70% of the total alkaline phosphatase was released into the supernatant fluid from the precipitate fractions. The release reached a plateau level after 10 h of incubation at 37 degrees C. The optimum pH value for the release was 7.4. Alkaline phosphatase activity increased during the incubation of the cell homogenates, but this increase was independent of the enzyme release. Serum increased not only alkaline phosphatase activity in the cultured cells but also enzyme release in their homogenates. In addition, we examined a rat liver homogenate and the following 11 cell lines: 3 hepatoma cell lines, including the R-Y121B cell line, 4 liver cell lines, 2 human urinary bladder carcinoma cell lines, a kidney cell line, and a mouse adrenal tumor cell line. Only in the cultured liver cell line and hepatoma cell lines, 30-60% of the total enzyme was released into the soluble fraction from the precipitate fractions; the release was not observed in the other cell lines, nor in the rat liver homogenate. The release of alkaline phosphatase took place in both heat-stable and heat-labile alkaline phosphatases. Alkaline phosphatase, extracted from cell homogenates, showed two bands during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The mobilities of the two bands changed inversely with or without sodium dodecyl sulfate. In general, the alkaline phosphatase which showed slow mobility with sodium dodecyl sulfate was more readily released from the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered activation state of hydroxymethylglutaryl-coenzyme A reductase in liver tumors

Extensive studies have demonstrated that the normal inhibition of cholesterol synthesis by cholesterol feeding is decreased in all hepatomas studied in vivo. This loss of the normal feedback regulation of cholesterol synthesis has been shown to be due to the failure of cholesterol ingestion to inhibit the activity of hydroxymethylglutaryl (HMG)-CoA reductase. The basis for this absence of feedback control of cholesterogenesis is unknown. Studies to date have not demonstrated structural or kinetic differences between the HMG-CoA reductase of normal liver and hepatoma. The present study, however, demonstrates significant differences in the activation state of HMG-CoA reductase from normal liver and hepatoma. In normal liver only approximately 10-20% of the microsomal HMG-CoA reductase is in the dephosphorylated, active form while 80-90% is in the phosphorylated, inactive state. In contrast, in three different Morris hepatomas in vivo, from 53 to 73% of the HMG-CoA reductase is in the active state. That the increased activation state in hepatomas is a property of tumor tissue and is not solely due to rapid growth is demonstrated by the fact that in both fetal and regenerating liver an enhanced activation state of HMG-CoA reductase is not observed. Additionally, preincubation with magnesium and ATP results in the inhibition of HMG-CoA reductase both in tumor and in liver. Presumably, this decrease in HMG-CoA reductase activity is due to the phosphorylation of the enzyme. Similarly, the preincubation of tumor and liver microsomes with phosphatase results in an increase in HMG-CoA reductase activity presumably by the dephosphorylation of the enzyme to its active form. The relationship between the altered activation state of HMG-CoA reductase in hepatomas and the reduction in the feedback regulation of this enzyme in liver tumors remains to be explored.     

Mitochondrial thymidine kinase and ribonucleotide reductase from rat liver and rat hepatoma 27]

Fractions of heavy and light mitochondria are isolated from homogenates of homologous rat tissues (intact liver, regenerating liver within 24 hours after hepatectomy and 27 hepatoma) by means of differential centrifugation. It is found that tumour mitochondria have higher heterogeneity and lower buyoant density than mitochondria from normal hepatocytes. The activity of two enzymes of DNA precursors synthesis (ribonucleotide reductase and thymidine kinase) in subcellular fractions is demonstrated to correlate with the tissue growth rate. A single injection of cyclic AMP into hepatectomised rats resulted in the retardation of the regeneration process, and the activity of both enzymes reached its normal level in all the fractions studied after 24 hours after the operation. Thymidine kinase and ribonucleotide reductase are located mainly in the mitochondrial matrix, however, pronounced enzyme activity is observed also in membrane fractions. The activity of the enzymes in the fraction of external mitochondria membranes in rapidly growing tissues is 2--3 times as high as in the same fraction from normal rat liver.     

Regulation of squalene epoxidase in HepG2 cells

Regulation of squalene epoxidase in the cholesterol biosynthetic pathway was studied in a human hepatoma cell line, HepG2 cells. Since the squalene epoxidase activity in cell homogenates was found to be stimulated by the addition of Triton X-100, enzyme activity was determined in the presence of this detergent. Incubation of HepG2 cells for 18 h with L-654,969, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, increased squalene epoxidase activity dose-dependently. On the other hand, low density lipoprotein (LDL) and 25-hydroxy-cholesterol decreased the enzyme activity. These results demonstrate that squalene epoxidase is regulated by the concentrations of endogenous and exogenous sterols. The affinity of the enzyme for squalene was not changed by treatment with L-654,969. Cytosolic (S105) fractions, prepared from HepG2 cells treated with or without L-654,969, had no effect on microsomal squalene epoxidase activity of HepG2 cells, in contrast to the stimulating effect of S105 fractions from rat liver homogenate. Mevalonate, LDL, and oxysterol treatment abolished the effect of L-654,969. Simultaneous addition of cycloheximide and actinomycin D also prevented enzyme induction in HepG2 cells. From these results, the change in squalene epoxidase activity is thought to be caused by the change in the amount of enzyme protein. It is further suggested that squalene epoxidase activity is suppressed only by sterols, not by nonsterol derivative(s) of mevalonate, in contrast to the regulation of HMG-CoA reductase.     

Interaction of ferredoxin and ferredoxin-NADP reductase with thylakoids

Ferredoxin-NADP reductase accounts for about 50% of the NADPH diaphorase activity of spinach leaf homogenates. The enzyme is bound to thylakoid membranes, but can be slowly extracted by aqueous buffers. Ferredoxin-NADP reductase can be extracted from the membranes by a 1- to 2-min treatment with a low concentration of trypsin. This treatment completely inactivates NADP photoreduction but does not affect electron transport from water to ferredoxin. It is shown that the inactivation is due to solubilization of ferredoxin-NADP reductase: the activity can be restored by addition of a very large excess of soluble enzyme in pure form. When ferredoxin-NADP reductase is added as a soluble enzyme after extraction or inactivation (by a specific antibody) of the membrane-bound enzyme, NADP photoreduction requires a very large excess of this enzyme, and the apparent K_m for ferredoxin is also increased. These observations are discussed as related to the interactions of thylakoids with ferredoxin-NADP reductase.     

Solubilization and characterization of the leukotriene C4 synthetase of rat basophil leukemia cells: A novel,participate glutathione S-transferase

Rat basophil leukemia cell homogenates effectively catalyze the conversion of leukotriene A₄ to a mixture of leukotrienes C₄ and D₄ in the presence of glutathione. These homogenates also catalyze the formation of adducts of halogenated nitrobenzene with glutathione, as determined spectrophotometrically. While all the classical glutathione S-transferase activity resides in the soluble fraction of the homogenates, the thiol ether leukotriene-generating activity is found in the particulate fraction. This “leukotriene C synthetase” activity has been solubilized from a crude high-speed particulate fraction by means of the nonionic detergent, Triton X-100. The solubilized enzyme is incapable of converting 2,4-dinitrochlorobenzene to a colored product in the presence of glutathione. Nor will it react with 3,4-dichloronitrobenzene. On the other hand, under optimal conditions, this enzyme preparation is capable of generating about 0.1 nmol leukotriene C mg protein^?1 min^?1 in a reaction which continues in linear fashion for at least 10 min. This dissociation in substrate specificity, as well as differences in the inhibition profile, distinguish the enzyme activity in the particulate fraction from rat basophil leukemia cell homogenates from the microsomal glutathione S-transferase which has been described in rat liver homogenates, suggesting that this “leukotriene C synthetase” is a new and unique enzyme.     

Effect of concanavalin A on the activity of membrane-bound and detergent-solubilized Mg2+ -ATPase

Plasma membranes were isolated after binding liver and hepatoma cells to polylysine-coated polyacrylamide beads, and the effect of concanavalin A on the membrane-bound Mg2+ -ATPase and the Mg2+ -ATPase solubilized by octaethylene glycol monododecyl ether (C12E8) was studied. In the experiment of membrane-bound Mg2+ -ATPase, plasma membranes were pretreated with Concanavalin A and the activity was assayed. Concanavalin A stimulated the activity of both liver and hepatoma enzymes assayed above 20 degrees C. Concanavalin A abolished the negative temperature dependency characteristic of liver plasma membrane Mg2+ -ATPase. On the other hand, Concanavalin A prevented the rapid inactivation due to storage at -20 degrees C, which was characteristic of hepatoma plasma membrane Mg2+ -ATPase. With solubilized Mg2+ -ATPase from liver plasma membranes, the negative temperature dependency was not observed. Concanavalin A, which was added to the assay medium, stimulated the activity of the enzyme solubilized in C12E8 at a high ionic strength. However, Concanavalin A failed to show any effect on the enzyme solubilized in C12E8 at a low ionic strength. With solubilized Mg2+ -ATPase from hepatoma plasma membranes, Concanavalin A could not prevent the inactivation of the enzyme during incubation at -20 degrees C.     

Different developmental changes in latency for two functions of a single membrane bound enzyme: glucose-6-phosphatase activities as a function of age.

(1). The capacity for the synthesis of glucose 6-phosphate from PPi and glucose as well as for glucose-6-P hydrolysis, catalyzed by rat liver microsomal glucose-6-phosphatase, increases rapidly from low prenatal levels to a maximum between the second and fifth day, then slowly decreases to reach adult levels. When measured in enzyme preparations optimally activated by hydroxyl ions, the maximum neonatal activities were 4--5-fold higher than in adult animals and several-fold higher than had previously been observed for the unactivated enzyme. (2) The latencies of two catalytic activities associated with the same membrane-bound enzyme show strikingly different age-related changes. The latency of PPi-glucose phosphotransferase activity reaches high levels (60--80% latent) soon after birth and remains high throughout life, while the latency of glucose-6-P phosphohydrolase decreases with age. The phosphohydrolase is 2--3 times more latent in the liver of the neonatal animal than in the adult. (3). The well established neonatal overshoot of liver glucose-6-phosphatase is almost entirely due to changes in the enzyme in the rough microsomal membranes. The enzyme activity in the rough membrane reaches a maximum and then decreases after day 2, while that in the smooth membrane is still slowly increasing. Despite the great differences in absolute specific activities and in the pattern of early enzyme development between the rough and smooth microsomes, enzyme latency in the two subfractions remains parallel, glucose-6-P phosphohydrolase being only slightly more latent, while PPi-glucose phospho-transferase is much more latent in smooth than in rough membranes throughout life. (4). Kidney glucose-6-P phosphohydrolase and PPi-glucose phosphotransferase activities were found to change in a parallel fashion with age, showing a small neonatal peak between days 2 and 7 before rising to adult levels. Kidney phosphotransferase activity, like that of liver, remained highly latent throughout life. In contrast to liver, the glucose-6-P phosphohydrolase of kidney did not show a characteristic decrease in latency with age and in the adult remained appreciably more latent than in liver. (5). An improved method was devised for the separation of smooth microsomes from liver homogenates.     

High concentration of neutral endopeptidase (enkephalinase E.C. 3.4.24.11) in a malignant tumor: rat hepatoma 3924A

The activity of the membrane-bound neutral endopeptidase 24.11 was low in the normal liver (21 +/- 3 pmol/h/mg protein, mean +/- SE) but it increased 56-fold in rapidly-growing rat hepatoma 3924A. The identity of the enzyme in the tumor was established by immunoprecipitation and by using a specific inhibitor of neutral endopeptidase. The endopeptidase concentration in the differentiating and regenerating liver was lower than in normal tissue, 39 and 8% of the corresponding control. The activity of a plasma membrane marker enzyme carboxypeptidase M in the normal liver was 1.0 +/- 0.2 nmol/h/mg protein, it increased about 2-fold in the rapidly-growing hepatoma and in the differentiating liver, but was unchanged in regenerating liver. The function of the strikingly increased neutral endopeptidase activity in the rapidly growing hepatoma may relate to activation of autocrine or exocellular growth factors or to inactivation of cell proliferation-inhibitory factors. Such a biochemical change should confer selective advantages to the cancer cells.