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Acute ethanol exposure of rat isolated hepatocytes leads to a significant decrease (-30%) in cytosolic enzymatic activity of classic protein kinase C (PKC) isoforms, while immunoreactive protein level measured by Western Blot remains unaffected. The inactivation of classic cytosolic isoforms appears dependent on the modification of the enzyme function, probably due to ethanol metabolism. In fact, pretreatment with 4-methylpyrazole (4MP), an inhibitor of alcohol dehydrogenase, fully prevented such damage. After ethanol treatment, a decrease of about 40% in both enzymatic activity and immunoreactive protein level of novel PKC isoforms was evident both in the soluble and particulate fractions. Even if 4MP cell pre-treatment afforded protection in this case too, the inhibitory action of ethanol on novel PKC hepatocyte isoforms involves a proteolytic mechanism as shown by Western Blot analysis. The reproduction of PKC inactivation by ethanol in hepatocyte lysate excluded a role of peroxisomal hydrogen peroxide in the pathogenesis of the damage investigated. This damage was not reduced by addition of catalase to the lysate model system.

Acute ethanol administration induces significant modifications both in secretive and formative membranes of rat liver Golgi apparatus. The decrease in glycolipoprotein secretion and their retention into the hepatocyte contribute to the pathogenesis of alcohol-induced fatty liver. Molecular and cellular mechanisms behind the ethanol-induced injury of the liver secretory pathway are not yet completely defined. In this study on intact livers from ethanol-treated rats, the involvement of the Golgi compartment in the impairment of hepatic glycolipoprotein secretion has been correlated with changes in the expression level, subcellular distribution and enzymatic activity of protein kinase C (PKC) isoforms. Acute ethanol exposure determined a translocation of classic PKCs and delta isoform from the cytosol to cis and trans Golgi membranes, the site of glycolipoprotein retention in the hepatic cell. A marked stimulation of cytosolic epsilon PKC activity was observed throughout the period of treatment. The presence of activated PKC isozymes at the Golgi compartment of alcohol-treated rat livers may play a role in hepatic secretion and protein accumulation. Direct and indirect effects of ethanol consumption on PKC isozymes and Golgi function are discussed.

Isolated rat hepatocytes were exposed to increasing concentrations of ethanol. During exposure of cells to ethanol a moderate but significant modification in the level of hepatic PKC c-isoforms has been observed. The ethanol-induced effect on liver protein kinase C was reversed by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, indicating that the conversion of ethanol to acetaldehyde may be involved in the enzyme inactivation. The involvement of the alcohol metabolite in PKC modifications was confirmed by the exposure of hepatocytes or partially purified liver enzyme to acetaldehyde concentrations of pathological interest.

Rat intoxication with a single dose of the hepatotoxin carbon tetrachloride induces a significant modification of liver protein kinase C total activity which depends on the degree of the intrahepatocyte oxidative unbalance provoked by various concentrations of the haloalkane. Low carbon tetrachloride amounts stimulate total protein kinase C activity, while one order of magnitude higher amounts exert strong enzyme inhibition. The latter effect is due to an early inactivation followed with progress of time by a proteolytic degradation of the enzyme. A pathological recruitment of the calcium-dependent protein kinase C regulatory enzymes calpain and calpastatin appears responsible for protein kinase C loss. The prolonged excess of cytosolic calcium which characterizes the single high dose carbon tetrachloride poisoning also leads to inactivation of calpain II and calpastatin in a time-dependent manner.

The involvement of CCl4 biotransformation mechanism in decreasing the Protein Kinase C activity has been analyzed in hepatocytes isolated from phenobarbital-pretreated rats. A significant inhibition (55%) and an almost total disappearance (87%) of the enzyme activity were observed at 15 min and at 30 min incubation with CCl4, respectively. Cell preincubation with Trolox or desferrioxamine allowed a marked whilst not complete protection of both cytosolic and particulate Protein Kinase C activity. These results show that the CCl4 reactive metabolites play a primary role in hepatocyte Protein Kinase C impairment and suggest that besides lipid peroxidation other mechanisms -possibly a derangement of Ca2+ homeostasis- may be involved in this process.

The enzyme responsible for the conversion of "neutral" to "alkaline" fructose 1,6-bisphosphatase (EC 3.1.3.11) by removal of a 7000 dalton peptide (converting enzyme, Proteinase I) has been shown to be localized in rat liverlysosomes. Lysosomes also contain a specific proteinase (Proteinase II) that catalyzes the release of a small peptide from the NH2-terminus of the native subunits. In fasted rabbits Proteinase II is released into the cytoplasm, together with Cathepsin A, but Proteinase I remains associated with the lysosomal fraction. Increased osmotic fragility of liver lysosomes in fasted rabbits has also been observed, but this increased fragility does not result in the release of Proteinase I. The appearance of Proteinase II in the cytoplasm may be due either to its selective release from the lysosomes, without release of Proteinase I, or its localization in a different lysosomal fraction. Changes in lysosomal structure induced by fasting may play a dual role in : 1) the mobilization of amino acids for gluconeogenesis and 2) the modulation of activity of gluconeogenic enzymes.

Specific inhibitors of three lysosomal proteinases are present in the cytosolic and lysosomal compartments of rabbit liver. The cytosolic inhibitors, purified by chromatography on DEAE-Trisacryl and Sephadex G-75, show specificities toward cathepsin M, cathepsins B and L, and fructose 1,6-bisphosphatase converting enzyme (CE), respectively, and are designated IM, IB/L, and ICE. Inhibitors with similar specificities have been isolated from the intralysosomal compartment. Two of these inhibitors, IM and ICE, are also present in the lysosomal membranes. The lysosomal distribution parallels that of the respective proteinases. The inhibitors are polypeptides with molecular weights of 5,000-10,000 for the two forms of IB/L, 12,500 for IM, and 10,000-40,000 for the ICE species.

The accumulation of an inactive, immunologically crossreactive form of fructose-1,6-bisphosphate aldolase (EC 3.1.3.11) in livers of fasted rabbits has now been related to limited proteolysis at the COOH terminus. The extent of modification of this region of the molecule, determined by analysis of tyrosine residues in the peptides released by digestion with subtilisin, agrees with the observed decrease in the specific activity of the enzyme purified from livers of fasted rabbits. The following evidence supports the conclusion that the modified form is produced in vivo and not during the isolation of the enzyme from the liver homogenates: (i) liver homogenates prepared in isotonic sucrose contained negligible amounts of soluble lysosomal proteinases; (ii) the decreased aldolase activity after fasting was observed in the homogenates and no change in aldolase activity occurred when the homogenates were incubated for 2 hr at 37 degrees C; (iii) the modified enzyme was also isolated from the livers of fasted rabbits when leupeptin was injected intraportally before the animals were sacrificed or when the inhibitor was added to the homogenization solution. On the other hand, homogenization of livers in hypotonic medium resulted in release of lysosomal proteinases and also in decreases in catalytic activity and COOH-terminal modification of liver aldolase, similar to those observed in livers from fasted rabbits. We attribute the changes in activity and structure of aldolase isolated from livers of fasted rabbits to the action in vivo of cathepsin M.

The ability of rabbit liver aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphatate-lyase, EC 4.1.2.13) and rabbit liver fructose-1,6-bisphosphatase (Fru-P2ase; D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) to partition into the gel phase of Ultrogel AcA 34 is decreased in a mixture of the two enzymes. Titration experiments indicate that a 1:1 complex is formed. The value for the distribution coefficient of the complex corresponds to a molecular mass of 300,000 daltons, the value expected for a dimer containing one mole of each enzyme protein. Complex formation was not observed when either liver enzyme was replaced by the corresponding isozyme from rabbit muscle. The susceptibility of liver Fru-P2ase to limited proteolysis by subtilisin was reduced in the presence of liver aldolase, but not when the latter was replaced by muscle aldolase, suggesting that the conformation of Fru-P2ase is altered in the complex. Limited proteolysis of liver aldolase abolishes its ability both to form the heterodimer and to protect Fru-P2ase from modification by subtilisin.

Three lysosomal proteinases capable of catalyzing a limited modification of the gluconeogenic enzyme fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) have been purified from extracts of rabbit liver lysosomes. These have been designated "converting enzymes I, II, and III," respectively, based on the order of their elution from Ultrogel AcA34. The predominant form in lysosomes from livers of fed rabbits is converting enzyme III which has been identified as a mixture of cathepsins B and L. Fasting induces a 4- to 5-fold increase in the activity of converting enzyme II; in the livers of 96-hr-fasted rabbits, this form accounts for more than 70% of the total converting enzyme activity. The increase is largely in that fraction of converting enzyme II associated with lysosomal membranes; this increase is also seen in the activity expressed by intact lysosomes. The activity of intact lysosomes is not due to their increased fragility because other lysosomal enzyme activities remain latent. The effect of fasting on the activity of converting enzyme II is selective and greatly exceeds the 2-fold increase observed for other lysosomal enzymes.

The stoichiometry of complex formation between two lysosomal proteinases from rabbit liver, cathepsin M and fructose 1,6-bisphosphatase converting enzyme (CE), and their respective endogenous inhibitors was studied by the equilibrium gel penetration method. In each case the molecular weight of the complex was found to be the sum of the molecular weights of the proteinase and its inhibitor, indicating the formation of 1:1 complexes. From the reappearance of proteinase activity on dilution, it is concluded that complex formation is reversible. Localization of the proteinase activities on the outer surface of the lysosomes was confirmed in these experiments by the inhibition of this proteinase activity on addition of inhibitors to intact lysosomes. The digestion by subtilisin of rabbit liver aldolase and rabbit liver fructose 1,6-bisphosphatase, the endogenous substrates for the lysosomal proteinases, was unaffected by the inhibitors.

Two Ca2+-requiring proteinases have been purified from rabbit liver cytosol and shown to be present in isolated hepatocytes. They differ in relative molecular mass, with the major and minor forms, Mr = 150,000 and Mr = 200,000, accounting for 75 and 18% of the total cytosolic neutral proteinase activity, respectively. Both are recovered as inactive proenzymes that can be converted to the active, low-Ca2+-requiring proteinases by incubation with Ca2+ and substrate [S. Pontremoli, E. Melloni, F. Salamino, B. Sparatore, M. Michetti, and B. L. Horecker (1984) Proc. Natl. Acad. Sci. USA 81, 53-56. Each proenzyme is composed of two subunits, with molecular masses of 80 and 100 kDa, respectively. Activation of the proenzymes was found to correlate with their dissociation into subunits. The optimum pH for conversion of the proenzymes to the active proteinases in the presence of 5 mM Ca2+ and 2 mg/ml of denatured globin was approximately 7.5, and the same pH optimum was observed for the digestion of denatured globin by the activated proteinases. Following activation, each proteinase was observed to undergo autolytic inactivation at rates that were dependent on the concentration of both Ca2+ and the digestible substrate. A model is proposed for the activation of the proenzymes and the subsequent inactivation of the active proteinases.

Gluconeogenic conditions, such as administration of triamcinolone or alloxan diabetes, cause the following changes in the molecular structure and properties of rabbit liver fructose 1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11): (1) the appearance of traces (about 10%) of a lighter subunit; (2) loss of tryptophan from all of the subunits, including those that show no apparent change in molecular weight; (3) increase in requirement for the positive allosteric effector, histidine; (4) increase in amount of enzyme, but not its specific activity. These changes are identical to those induced by cold or fasting, and are related to increased activities of lysosomal proteases. The results suggest that lysosomes may act as mediators of gluconeogenic stimuli.

The activity of fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) in livers of fasted rabbits decreases to less than one-half the value found in livers of fed rabbits. However, the concentration of aldolase protein in the liver extracts, measured with a specific antibody, remains unchanged. More than twice as much antibody is required to neutralize the aldolase activity in liver extracts from fasted compared with fed rabbits. The results suggest that modification of liver aldolase occurs during fasting, resulting in loss of catalytic activity without loss of immunoreactivity.

Cathepsins M and B from rabbit liver lysosomes were separated by chromatography on Ultrogel AcA34 at low ionic strength and purified to homogeneity, and their catalytic and molecular properties were compared. Cathepsin M was relatively inactive with synthetic peptide substrates. Thus, it hydrolyzed benzoyl arginine naphthylamide at only one-fifth the rate observed with cathepsin B, and no activity was detected with Gly-Phe naphthylamide which is a relatively good substrate for cathepsin B. On the other hand, cathepsin M exhibited a preference for protein substrates. It was more active than cathepsin B in catalyzing the inactivation of the following enzymes: rabbit muscle or liver fructose-1,6-bisphosphate aldolases, rabbit liver fructose-1,6-bisphosphatase and pyruvate kinase, yeast glucose-6-phosphate dehydrogenase, and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. With glucagon as substrate, both enzymes showed similar peptidyl dipeptidase activities with some minor differences in peptide bond specificity. Cathepsins M and B are similar in size, with apparent molecular weights of 30,200 for cathepsin M and 28,800 for cathepsin B, and in amino acid composition and carbohydrate content. Each contains approximately 2-3 equivalents/mol glucosamine, 3 equivalents/mol mannose, and no fucose or galactosamine. They also show similar microheterogeneity in sodium dodecylsulfate-gel electrophoresis and isoelectric focusing; this microheterogeneity is probably related to differences in glycosylation. Extensive homology in primary structure for the two proteins was indicated by the similar patterns of peptides formed on digestion with trypsin.

During prolonged starvation, fructose 1,6bisphosphatase (EC 3.1.3.11) activity in rabbit liver and kidney shows a transient decrease during the first 36 hr, before rising at 96 hr to levels severalfold higher than those found in the livers of fed animals. Proteolytic activity appears in the 105,000 x g supernatant fraction within several hours of starvation, and continues to increase during the entire 96-hr period. On refeeding, the activities return to nearly the control levels within 24 hr. The catalytic properties of fructose 1,6-bisphosphatase isolated from the livers of fasted rabbits are similar to those of the enzyme from fed animals, but its structure is modified, since it no longer contains the single tryptophan residue located near the NH(2)-terminus in the native enzyme. Thus this tryptophan residue is not required for the neutral pH optimum. The structural changes and the transient decrease in activity may be related to the observed increase in "free" proteolytic activity.

Two neutral Ca2+-dependent proteinases, differing in molecular size, have been isolated from rabbit liver. Both are recovered as inactive proenzymes that can be converted to the active forms by high (0.1-1.0 mM) concentrations of Ca2+ in the absence of substrate or, in the presence of a protein substrate, by low (1-5 microM) concentrations of Ca2+. The activated proteinases required only 1-5 microM Ca2+ for maximal activity. Substrates hydrolyzed were denatured globin, globin, casein, and to a lesser extent, several extracellular proteins; no digestion was observed with several intracellular cytosolic enzymes tested. Only those proteins that served as substrates were capable of promoting conversion of the proenzymes to the active low-Ca2+-requiring proteinases.

Cathepsin M, which catalyzes inactivation of both rabbit liver fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) and rabbit liver fructose 1,6-bisphosphatase (Fru-P2ase; EC 3.1.3.11), has been characterized as a peptidyl peptidase. Modification of the COOH terminus of aldolase by cathepsin M or by Fru-P2ase converting enzyme 2 abolishes its ability to bind to phosphocellulose P11 and to form the complex with Fru-P2ase. On the other hand, modification of the COOH terminus of Fru-P2ase does not affect its interaction with aldolase. This property is lost, however, when Fru-P2ase is modified in the NH2-terminal region by the converting enzyme or by subtilisin. The results suggest that interaction of aldolase and Fru-P2ase may involve the exposed COOH-terminal region of the former and an exposed proteinase-sensitive region located between residues 57 and 67 of the latter.

Isolated rat hepatocytes exposed to CCl4 showed a dramatic decrease in [32P] incorporation into proteins which was evident as early as 5 min after the haloalkane addition. DEAE cellulose separation of protein kinases present in both particulated and cytosolic fractions of hepatocytes revealed that only the calcium and phospholipids dependent protein kinase C was affected by the treatment with CCl4, while kinases not requiring these factors for their activity were unmodified. Several 4-hydroxyunsaturated aldehydes known to be produced during CCl4-stimulated lipid peroxidation were found to inhibit protein kinase C at micromolar concentrations, suggesting the possibility that peroxidative events might be responsible for the impairment of protein kinase C during CCl4 intoxication.

A monoclonal antibody raised against rabbit liver cathepsin M binds to intact rabbit liver lysosomes. The binding is specific and is abolished by treating the lysosomes with trypsin, which has previously been shown to digest the membrane-bound cathepsin M [S. Pontremoli, E. Melloni, M. Michetti, F. Salamino, B. Sparatore, and B. L. Horecker (1982) Biochem, Biophys. Res. Commun. 106, 903-909]. Rabbit liver lysosomes are adsorbed onto Sepharose 4B coupled to anti-cathepsin M, but not to Sepharose 4B itself or to Sepharose coupled to a nonspecific antibody. The results confirm the location of membrane-bound cathepsin M on the outer surface of the lysosomal membrane.

An endogenous inhibitor of neutral Ca2+-dependent proteinases has been isolated from rabbit liver cytosol. The inhibitor is a heat-stable, 240-kDa, tetrameric protein. It is dissociated into its 60-kDa subunits by high concentrations of Ca2+ (0.1-1 mM), but not by lower concentrations in the physiological range. Inhibition of the 150-kDa proteinase of rabbit liver [Melloni, E., Pontremoli, S., Salamino, F., Sparatore, B., Michetti, M. and Horecker, B.L. (1984) Arch. Biochem. Biophys. 232, 505-512] requires the monomeric form of the inhibitor, and occurs only at the high concentrations of Ca2+ which also cause dissociation of the dimeric 150-kDa proteinase into its 80-kDa subunits. The molecular weight of the inactive proteinase-inhibitor complex was estimated by the equilibrium gel penetration method to be 140 kDa, suggesting that it contains one subunit of proteinase and one of inhibitor. The mechanism of interaction of the inhibitor with the 200-kDa proteinase at high concentrations of Ca2+ is identical to that observed for the 150-kDa proteinase, namely dissociation of both proteinase and inhibitor into subunits and formation of an inactive 160-kDa proteinase-inhibitor complex. However, unlike the 150-kDa proteinase, which does not interact with the inhibitor at low Ca2+ concentrations, the 200-kDa proteinase is also inhibited at low concentrations of Ca2+. Under these conditions, the high-molecular-weight complex (greater than 400 kDa) formed between the tetrameric inhibitor and the dimeric proteinase prevents conversion of the 200-kDa proenzyme to the active, low-Ca2+-requiring form.

Exposure of rabbits to cold or fasting results in marked increases in the number and size of liver lysosomes, associated with increases in the total and "free" proteolytic activity. Changes in lysosomal morphology also indicate increased activity and fragility. These effects of cold and fasting are correlated with decreases in the levels of liver fructose bisphosphatase (EC 3.1.3.11) activity and changes in the molecular properties of the purified enzyme, including the loss of a small peptide containing the only tryptophan residue in the peptide chain.

Seasonal variations in the properties of rabbit-liver fructose 1,6-bisphosphatase have now been linked to corresponding changes in the levels of proteolytic activity in the liver extracts. Incubation of native fructose 1,6-bisphosphatase with purified liver lysosomes causes a 3-fold increase in catalytic activity at pH 9.2, with a smaller, and variable, decrease in activity tested at pH 7.5. These changes in catalytic properties are accompanied by the appearance of a smaller subunit, as was previously reported for the enzyme treated with subtilisin. AMP, a negative modulator of fructose bisphosphatase activity, protects against this action of lysosomes. This proteolytic modification of fructose bisphosphatase by lysosomal enzymes may play a role in the modulation of gluconeogenesis.

Removal of the NH(2)-terminal region of fructose 1,6-bisphosphatase from rabbit liver by digestion with subtillisin, or changes in conformation in this region of the protein produced by exposure to low concentrations of urea, result in similar changes in catalytic and allosteric properties of the enzyme. These changes include shift of the pH optimum to more alkaline pH, and loss of sensitivity to inhibition by AMP. The conformation changes are monitored by changes in the fluorescence of the single tryptophan residue, which is located near the NH(2)-terminus. Thus, the tryptophan-containing peptide appears to determine the functional properties of the native enzyme.