Distribution of glucose-6-phosphatase activity in normal,hyperplastic, and preneoplastic rat liver

The significance of glucose-6-phosphatase (G6P) expression by bile duct-like cells proliferating during hepatocarcinogenesis in the histogenesis of hepatocellular carcinoma is not clear. To this end, we measured the histochemical and biochemical activity of G6P in normal rat liver, and in rat livers in which bile duct-like proliferation was induced by either hyperplastic (bile duct ligation for 14 days or feeding alpha-naphthylisothiocyanate for 28 days) or neoplastic (feeding a choline-devoid diet containing 0.1% ethionine for 60 days) regimens. In normal, hyperplastic, and preneoplastic livers, G6P histochemical activity was confined to the hepatocytes; proliferated bile duct-like cells, like normal bile ducts, did not display visible G6P staining. When the enzyme activity was determined biochemically, however, hydrolysis of glucose-6-phosphate was observed in both parenchymal and nonparenchymal liver cells isolated from all experimental animals. In elutriated nonparenchymal fractions, G6P activity was directly proportional to the number of cells positive for gamma-glutamyl transpeptidase and cytokeratin no. 19 (markers of bile duct cells) and inversely proportional to the number of cells positive for vimentin (marker of mesenchymal cells). These results indicate that, while by light microscopy hepatic G6P histochemical activity is detectable only in the hepatocytes, the biochemical activity is also expressed in proliferating bile duct-like cells. However, the nonparenchymal activity is observed during both neoplastic and hyperplastic liver growth, thus indicating that the presence of this enzyme in bile duct-like cells proliferating during hepatocarcinogenesis should not necessarily be construed as supporting their stem cell nature nor their neoplastic commitment.     

Changes in the acinar distribution of some enzymes involved in carbohydrate metabolism in rat liver parenchyma after experimentally induced cholestasis

Extrahepatic cholestasis induced by ligation and transsection of the common bile duct caused a change in the parenchyma/stroma relationship in rat liver. Two weeks after ligation, the periportal zones of the parenchyma were progressively invaded by expanding bile ductules with surrounding connective tissue diverging from the portal areas. Parenchymal disarray developed and small clumps of hepatocytes or isolated hepatocytes were scattered within the expanded portal areas. These cells showed normal activity of lactate, succinate and glutamate dehydrogenase and may, therefore, be considered to be functionally active. After cholestasis the remainder of the liver parenchyma showed adaptational changes with respect to glucose homeostasis, as demonstrated by histochemical means. Glycogen stores disappeared completely whereas glycogen phosphorylase activity increased about ten fold. The increased glycogen phosphorylase activity and glycogen depletion indicate a greater glycogenolytic capacity in liver parenchyma after bile duct ligation to maintain as far as possible a normal plasma glucose concentration. The parenchymal distribution pattern of glucose-6-phosphatase activity did not change significantly after bile duct ligation. The isolated hepatocytes within the expanded portal tracts showed a high activity of this enzyme whereas the pericentral parenchyma was only moderately active. The distribution patterns of glucose-6-phosphate dehydrogenase and lactate dehydrogenase activity in the liver parenchyma were also largely unchanged after bile duct ligation, but the histochemical reaction for glucose-6-phosphate dehydrogenase activity demonstrated infiltration of the remainder of the parenchyma by non-parenchymal cells, possibly Küpffer cells and leucocytes as part of an inflammatory reaction. Under normal conditions the mitochondrial enzymes succinate and glutamate dehydrogenase show an opposite heterogenous distribution pattern in liver parenchyma. Following cholestasis both enzymes became uniformly distributed. The underlying regulatory mechanism for these different changes in distribution patterns of enzyme activities is not yet understood.     

Adenovirus-mediated gene transfer to nonparenchymal cells in normal and injured liver

Adenovirus-mediated gene transfer has become an important tool with which to introduce genetic material into cells. Available data emphasize efficient adenoviral transduction of parenchymal liver cells (i.e., hepatocytes) in both in vitro and in vivo model systems, typically in normal cells. The aim of this study was to evaluate gene transfer to nonparenchymal (and parenchymal) cells of the normal and injured rat liver. Hepatocytes, stellate cells, and endothelial cells were isolated by standard methods. Liver injury was induced by bile duct ligation or carbon tetrachloride administration. Cells were transduced in vitro with an adenovirus encoding beta-galactosidase (Ad.beta-gal) over a range of viral titers, and transduced cells were identified by detection of X-gal. In vivo transduction efficiency was studied in normal and injured livers using cell isolation techniques. Nonparenchymal cells were transduced with greater frequency than hepatocytes at all adenoviral titers tested, both in vitro and in vivo. After liver injury, adenoviral transduction was reduced for all liver cell types compared with that for cells from normal livers (at all virus titers). Notably, transduction efficiency remained greater in nonparenchymal cells than in hepatocytes after liver injury. This work implies that, to achieve comparable gene expression in the injured liver, higher adenoviral titers may be required, an important consideration as gene therapy in disease states is considered.     

Oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase activity for the detection of (pre)neoplasm in rat liver.

Oxygen insensitivity of the histochemical assay to detect glucose-6-phosphate dehydrogenase (G6PD) activity with NT as tetrazolium salt has been proved to be a powerful tool to discriminate various types of adenocarcinoma from normal tissues. Here we investigated whether this phenomenon can also be applied to differentiate between chemically induced hepatocellular (pre)neoplasms and normal liver tissue in rats. Residual activity (percentage of the amount of final reaction product that is generated in oxygen and that is generated in nitrogen) was 60% in (pre)neoplastic cells and 6% in normal liver parenchymal cells. This means that the oxygen insensitivity test is a useful tool to distinguish (pre)neoplasms from normal rat liver tissue. N-Ethylmaleimide, a blocker of SH groups, did not affect G6PD activity in (pre)neoplastic cells, whereas activity in normal cells was reduced by half. Therefore, the absence of essential SH groups in G6PD in (pre)neoplastic cells is held responsible for the oxygen insensitivity phenomenon. We conclude that oxygen insensitivity of the histochemical assay for G6PD activity is a fast, easy, and cheap tool to diagnose (pre)neoplasms in rat liver. Discrimination is likely to be based on altered properties of the enzyme in (pre)neoplastic cells. (J Histochem Cytochem 49:565-571, 2001)     

Cell of origin of distinct cultured rat liver epithelial cells, as typed by cytokeratin and surface component selective expression

The cell of origin of the nonparenchymal epithelioid cells that emerge in liver cell cultures is unknown. Cultures of rat hepatocytes and several types of nonparenchymal cells obtained by selective tissue dispersion procedures were typed with monoclonal antibodies to rat liver cytokeratin and vimentin, polyvalent antibodies to cow hoof cytokeratins and porcine lens vimentin, and monoclonal antibodies to surface membrane components of ductular oval cells and hepatocytes. Immunoblot analysis revealed that, in cultured rat liver nonparenchymal epithelial cells, the anti-rat hepatocyte cytokeratin antibody recognized a cytokeratin of relative mass (Mr) 55,000 and the anti-cow hoof cytokeratin antibody reacted with a cytokeratin of Mr 52,000, while the anti-vimentin antibodies detected vimentin in both cultured rat fibroblasts and nonparenchymal epithelial cells. Analyses on the specificity of anti-cytokeratin and anti-vimentin antibodies toward the various cellular structures of liver by double immunofluorescence staining of frozen tissue sections revealed unique reactivity patterns. For example, hepatocytes were only stained with anti-Mr 55,000 cytokeratin antibody, while the sinusoidal cells reacted only with the anti-vimentin antibodies. In contrast, epithelial cells of the bile ductular structures and mesothelial cells of the Glisson capsula reacted with all the anti-cytokeratin and anti-vimentin antibodies. It should be stressed, however, that the reaction of the anti-vimentin antibodies on bile ductular cells was weak. The same analysis on tissue sections using the anti-ductular oval cell antibody revealed that it reacted with bile duct structures but not with the Glisson capsula. The anti-hepatocyte antibody reacted only with the parenchymal cells. The differential reactivity of the anti-cytokeratin and anti-vimentin antibodies with the various liver cell compartments was confirmed in primary cultures of hepatocytes, sinusoidal cells, and bile ductular cells, indicating that the present panel of antibodies to intermediate filament constituants allowed a clear-cut distinction between cultured nonparenchymal epithelial cells, hepatocytes, and sinusoidal cells. Indirect immunofluorescence microscopy on nonfixed and paraformaldehyde-fixed cultured hepatocytes and bile ductular cells further confirmed that both anti-hepatocyte and anti-ductular oval cell antibodies recognized surface-exposed components on the respective cell types.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electron microscopical demonstration of glucose-6-phosphatase in native cryostat sections fixed with glutaraldehyde through semipermeable membranes.

The cytochemical demonstration of glucose-6-phosphatase (G6Pase) activity in native cryostat sections fixed with glutaraldehyde through semipermeable membranes is superior to conventional methods with regard to exact localization and lack of inactivation and diffusion of the enzyme, together with simultaneous excellent preservation of the tissue fine structure. In rat liver not only hepatocytes but also many bile duct epithelia and endothelia of arterioles and venules show a marked G6Pase activity in the membranes of the endoplasmic reticulum including the nuclear envelope.     

Histochemical and microbiochemical demonstration of reduced pyruvate kinase activity in thioacetamide-induced neoplastic nodules of rat liver

A new method for the histochemical demonstration of pyruvate kinase (PK) activity was developed using a semi-permeable membrane and ATP-dependent phosphorylation of glucose coupled with tetrazolium reduction via glucose-6-phosphate dehydrogenase (G6PD) in order to investigate normal liver tissue and neoplastic hepatic nodules induced by thioacetamide (TAA). A series of control reactions and comparison with microbiochemical analysis of microdissected lyophilised material were used to determine the specificity of the reaction. In agreement with earlier reports, an activity gradient in control liver decreasing from zone 3 to zone 1 was apparent both histochemically and after biochemical analysis. Liver neoplastic nodules induced by 25 weeks dietary thioacetamide administration and characterized by increased G6PD demonstrated a clear decrease in PK activity. In contrast, epithelial cells within areas of cholangiocellular tumour development were characterized by a strong increase. Comparison of the results with immunohistochemical and biochemical data from the literature indicate that the specific histochemical method described will be of great assistance in future assessment of disease and physiological alteration in activity of this key enzyme of glycolysis.     

Monoclonal antibodies directed against rat liver epithelial cell lines selectively recognize bile duct epithelium in livers of adult rats

Monoclonal antibodies directed against antigens on rat liver epithelial cell lines were prepared. Three antibodies, 4C3, 19C6, and 3C2, recognized surface antigens present (although in different quantities) on eight epithelial cell lines tested, irrespective of whether they were normal or transformed. For MAb 3C2, the primary antigen common to all but one cell line showed a Mr of 135 kD. In paraffin sections of liver tissue, two antibodies, 40 and 19C6, reacted exclusively with bile duct epithelium, whereas the MAb 3C2 additionally reacted with sinusoidal endothelium and the endothelium of the portal venules. In sections of livers from rats exposed to diethylnitrosamine, the MAb 19C6 selectively stained bile duct-like structures in cholangiomas, while other preneoplastic and neoplastic lesions were not stained. These results demonstrate that the monoclonal antibodies obtained may prove useful for investigating cell lineages related to propagable liver epithelial cell lines and suggest that these cells may be derived from terminal bile ductular cells.Abbreviations ABTS2 2,2azinobis(3-ethylbenzthiazolinesulfonic) acid - ARL adult rat liver - DEN diethylnitrosamine - FCS fetal calf serum - MAb monoclonal antibody - PAP peroxidase antiperoxidase     

Electron microscopical demonstration of glucose-6-phosphatase in native cryostat sections fixed with glutaraldehyde through semipermeable membranes

Summary The cytochemical demonstration of glucose-6-phosphatase (G6Pase) activity in native cryostat sections fixed with glutaraldehyde through semipermeable membranes is superior to conventional methods with regard to exact localization and lack of inactivation and diffusion of the enzyme, together with simultaneous excellent preservation of the tissue fine structure. In rat liver not only hepatocytes but also many bile duct epithelia and endothelia of arterioles and venules show a marked G6Pase activity in the membranes of the endoplasmic reticulum including the nuclear envelope. This work was kindly supported by the Deutsche Forschungsgemeinschaft An erratum to this article is available at .     

Alkaline phosphatase in cholestatic and cirrhotic rats. A biochemical and histochemical study

Alkaline phosphatase (ALP) was studied by enzyme histochemical methods and by biochemical quantitations in rat livers with chronic bile duct obstruction and experimental cirrhosis. The most evident ALP increase was histochemically found in portal tracts of rats with bile duct obstruction and localized to the walls of proliferating blood vessels. Furthermore, a slight canalicular membrane enzyme increase was histochemically found in both groups, most evident in cirrhosis, whereas the biochemical assay of ALP in serum and liver from both pathological groups showed 3 times higher values compared to controls. The portal tracts did not seem to contribute to the serum increase, since the rise of serum ALP was similar in chronic bile duct obstruction and in experimental cirrhosis without changes of the portal tracts. It is concluded that the increase ALP activity in serum from rats with bile duct obstruction and cirrhosis mainly has a hepatocytic origin.     

Bioactivation of the hepatocarcinogen N-hydroxy-2-acetylaminofluorene by sulfation in the rat liver changes during the cell cycle.

Sulfation activity towards N-hydroxy-2-acetylaminofluorene and 4-nitrophenol was determined in male rat liver cytosol at several time points after partial hepatectomy corresponding to G1-, S-, and M-phase. N-hydroxy-2-acetylaminofluorene sulfation activity decreased by 80% when hepatocytes entered the G1-phase. This lower activity was maintained during the S-phase and M-phase, but was restored when hepatocytes entered the G0-phase again. Sulfation activity towards 4-nitrophenol did not alter after hepatectomy. Various other cytosolic enzyme activities were determined after hepatectomy to investigate the specificity of the decrease in sulfation activity. Lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities were increased in the S- and M-phase by maximally 80% and 60%, respectively. Glutathione-S-transferase and glutamate-pyruvate transaminase activity did not alter during the cell cycle. These results indicate that sulfation of N-hydroxy-2-acetylaminofluorene in hepatocytes may depend on the phase of the cell cycle. The relevance of the finding is discussed in relation to the resistance of proliferating (pre)neoplastic hepatocytes to the toxic and mitoinhibitory effects of N-hydroxy-2-acetylaminofluorene.     

Histochemical and microbiochemical demonstration of reduced pyruvate kinase activity in thioacetamide-induced neoplastic nodules of rat liver

Summary A new method for the histochemical demonstration of pyruvate kinase (PK) activity was developed using a semi-permeable membrane and ATP-dependent phosphorylation of glucose coupled with tetrazolium reduction via glucose-6-phosphate dehydrogenase (G6PD) in order to investigate normal liver tissue nd neoplastic hepatic nodules induced by thioacetamide (TAA). A series of control reactions and comparison with microbiochemical analysis of microdissected lyophilised material were used to determine the specificity of the reaction. In agreement with earlier reports, an activity gradient in control liver decreasing from zone 3 to zone 1 was apparent both histochemically and after biochemical analysis. Liver neoplastic nodules induced by 25 weeks dietary thioacetamide administration and characterized by increased G6PD demonstrated a clear decrease in PK activity. In contrast, epithelial cells within areas of cholangio-fibrosis thought to be direct precursors for cholangioccllular tumour development were characterized by a strong increase. Comparison of the results with immunohistochemical and biochemical data from the literature indicate that the specific histochemical method described will be of great assistance in future assessment of disease and physiological alteration in activity of this key enzyme of glycolysis.Supported by the Deutsche Forschungsgemeinschaft. Dr. Malcolm A. Moore is the recipient of a guest research scientist fellowship from the German Cancer Research Center     

Hepatoblast-like cells enriched from mouse embryonic stem cells in medium without glucose, pyruvate, arginine, and tyrosine

In order to enrich hepatocytes differentiated from embryonic stem cells, we developed a novel medium. Since only hepatocytes have the activity of ornithine transcarbamylase, phenylalanine hydroxylase, galactokinase, and glycerol kinase, we expected that hepatocytes would be enriched in a medium without arginine, tyrosine, glucose, and pyruvate, but supplemented with ornithine, phenylanaline, galactose, and glycerol (hepatocyte-selection medium, HSM). Embryoid bodies were transferred onto dishes coated with gelatin in HSM after 4 days of culture. At 18 days after embryoid body formation, a single type of polygonal cell survived with an enlarged intercellular space and micorvilli. These cells were positive for indocyanine green uptake and for mRNAs of albumin, transthyretin, and alpha-feto protein, but negative for mRNAs of tyrosine aminotransferase, alpha1-antitrypsin, glucose-6-phosphatase, and phosphoenol pyruvate carboxykinase. Since cells in HSM were positive for cytokeratin (CK)8 and CK18 (hepatocyte markers) and for CK19 (a marker of bile duct epithelial cells), we concluded that they were hepatoblasts. They showed weaker expression of CCAAT/enhancer-binding protein (C/EBP)alpha than fetal liver (18.5 days of gestation) and expression of C/EBPbeta at a similar level to that of fetal liver. These data support our conclusion that HSM allows the selection of hepatoblast-like cells.     

Receptor-mediated introduction of pepstatin-asialofetuin conjugate into lysosomes of rat hepatocytes

Pepstatin was linked through a carboxyl group to asialofetuin (PS-ASF). An analysis by separation of hepatocytes from nonparenchymal cells showed that PS-ASF was taken up by hepatocytes, following intravenous injection into rats. After the injection of PS-ASF, pepstatin concentration in the liver reached a maximum at 2 h and then decreased. In an analysis by differential centrifugation of the liver homogenate from rats injected with PS-ASF, pepstatin showed a lysosomal type subcellular distribution pattern. Isolation studies of tritosomes clearly demonstrated the exclusive accumulation of pepstatin within the lysosomes of livers from rats given PS-ASF (at 2 h after administration). Pepstatin contained in tritosomes was in a free form, as determined by column chromatography of Sephadex G-15. The activity of cathepsin D in the livers was markedly inhibited in rats given PS-ASF. However, the treatment of rats with PS-ASF had no effect on the hepatic lysosomal degradation of endocytosed FITC-labeled asialofetuin (FITC-ASF). Introduction of PS-ASF into the hepatocytes was followed by the immediate and time-dependent excretion of free pepstatin into the bile. Quantification of pepstatin excreted into the bile revealed that the biliary excretion route can account for the disappearance of pepstatin from the liver.     

Prostaglandins: a possible mediator to inhibit hepatic drug metabolism in adjuvant arthritic rats

In the liver of adjuvant arthritic rats perfused with a hemoglobin-free buffer solution, the rate of metabolism of a model drug, 2,6-dichloro-4-nitroanisole, was approximately half that of the control, while the bile flow rate was normal. Granulation tissue extracts and arthritic rat serum had no effect on the activity of CNA metabolism in normal rat liver preparations. In the perfused normal rat liver, the rate of CNA metabolism was inhibited by addition of prostaglandin (PG) E1, PGE2, and PGF2 alpha, respectively, in a final concentration of 0.5 microM. The inhibition by PGE1 was increased in the concentration range from 0.1 to 2.5 microM. The bile flow rate was not affected by the added PGs. However, these PGs had no direct effect on the CNA demethylating activity of the isolated hepatocytes from normal rat liver in a high concentration of 10 microM. Serotonin stimulated slightly CNA metabolism and bile production in the perfused livers by the intermittent infusion, but was without effect in the isolated hepatocytes. Epinephrine and histamine had no significant effect on CNA metabolism in both liver preparations. A similar pattern of the inhibition of CNA metabolism by PGs was reproduced in the normal rat liver perfused with the medium containing the supernatant of the hepatic nonparenchymal cells incubated in the presence of PGE1. The involvement of liver sinusoidal cells as secretory cells in depression of hepatic drug metabolism has been discussed.     

Modulation of the activity of hepatic gamma-glutamyl transpeptidase, adenosine triphosphatase, placental glutathione S-transferase and adenylate cyclase by acute administration of lead nitrate

The effect of a single administration of lead nitrate on the activity of gamma-glutamyltranspeptidase (gamma-GT), adenosine triphosphatase (ATPase), the placental form of glutathione S-transferase (GST-P) and adenylate cyclase (AC), four enzymes widely used as phenotypic markers for preneoplasia, was investigated in the liver of male Wistar rats. The results of the histochemical enzymatic staining indicated that an acute treatment with lead nitrate induces the activity of gamma-GT, mainly in the hepatocytes located around zone I of the liver acinus, with a maximum seen between 72-96 hours. On the other hand, the activity of ATPase was found to be severely inhibited at 2-3 days after treatment, as shown by a strong decrease in the staining of the bile canaliculi of zones II and III. Immunohistochemical analysis revealed that lead nitrate administration also resulted in the appearance in most of the hepatocytes of GST-P, an enzyme whose activity is almost undetectable in normal rat liver, but is elevated in preneoplastic liver lesions. Finally, lead nitrate treatment resulted in an inhibition of AC activity which was maximal after 24 hours.     

Xanthine oxidoreductase is present in bile ducts of normal and cirrhotic liver

Xanthine oxidoreductase (XOR) is a widely distributed enzyme, involved in the metabolism of purines, which generates superoxide and is thought to be involved in free radical-generated tissue injury. It is present at high concentrations in the liver, from where it may be released during liver injury into the circulation, binding to vascular endothelium and causing vascular dysfunction. The cellular localization of the enzyme, essential to understanding its function, is, however, still debated. The present study has used a highly specific mouse monoclonal antibody to define the cellular distribution of XOR in normal and cirrhotic human liver. As shown previously, XOR is present in hepatocytes. However, the novel finding of this study is that XOR is present in bile duct epithelial cells, where it is concentrated toward the luminal surface. Moreover, in liver disease, proliferating bile ducts are also strongly positive for XOR. These findings suggest that the enzyme is secreted into bile, and this was confirmed by analysis of human and rat bile. Xanthine oxidase activity was 10 to 20-fold higher in liver tissue obtained from patients with liver disease, than in healthy liver. We conclude that XOR is expressed primarily in hepatocytes, but is also present in bile duct epithelial cells and is secreted into bile. Its role in bile is unknown but it may be involved in innate immunity of the bowel muscosa.     

Early effects in chemical-induced rat liver carcinogenesis: an immunohistochemical study following exposure to 0.04% AAF

To investigate effects that distinguish AAF from incomplete carcinogens, the rate of cell death (apoptosis) and cell proliferation was studied at early stages of AAF induced rat liver carcinogenesis. Male Wistar rats were fed 0.04% AAF in the diet for 2, 6 and 16 weeks and immunohistochemical markers were measured in the liver. The formation of initiated cells and preneoplastic foci was followed by staining for GST-P (glutathione-S-transferase). GST-P-positive foci were present from 6 weeks on. Apoptosis was increased in the periportal area and in preneoplastic foci at all time points. Cell proliferation was enhanced in the periportal area in oval cells and in bile duct-like cells particularly at 2 and 6 weeks and mainly in GST-P positive foci at 16 weeks. Notably, more cells always proliferated than were eliminated. Other apoptosis-related markers like p53 and FAS/Apo-1 could not be demonstrated in either normal hepatocytes, preneoplastic foci or in hepatocytes from treated animals. Scattered bcl-2 positive cells were present in livers at 16 weeks of treatment. The two cell growth and differentiation related proto-oncogenes c-FOS and c-JUN were increased in all treated animals at early stages. If feeding was stopped after 6 weeks, livers did not recover significantly within the following 10 weeks. The results support the complex effects of AAF in rat liver carcinogenesis. Chronic toxicity locally impairs the balance between cell proliferation and cell death and induces morphological alterations that promote the growth of initiated cells.     

Expression of multiple proteins structurally related to gamma-glutamyl transpeptidase in non-neoplastic adult rat hepatocytes in vivo and in culture.

Freshly isolated hepatocytes from normal adult rat liver do not express measurable gamma-glutamyl transpeptidase (GGT) mRNA in contrast to the significant GGT mRNA levels expressed by normal adult rat kidney and hyperplastic bile ductular tissue from bile duct-ligated rats. However, the induction of GGT activity in rat hepatocytes by two-thirds hepatectomy was accompanied by the appearance of a high level of GGT mRNA. We are now able to demonstrate that normal adult rat hepatocytes express 5 protein bands which cross-react with 2 different anti-rat kidney GGT antisera. The apparent molecular weights were 26.9, 58.0, 63.9, 73.5, and 83.4 kDa, respectively. Expression of the 26.9- and 58.0-kDa proteins strikingly parallels the pattern of induction of GGT enzymatic activity. This suggests that these 2 proteins correspond to the active dimeric enzyme previously described in kidney and neoplastic hepatocellular tissue. In normal hepatocytes, the 73.5-kDa protein represents 50% of the total GGT-immunoreactive protein, in contrast to kidney, where this band contains less than 4% of the GGT protein. The kinetics of expression of the 73.5-kDa protein upon induction of GGT activity in hepatocytes, as well as in culture turnover studies, suggests that this protein is a precursor form of the active enzyme, such as the described 78/79-kDa single-chain glycoprotein propeptide of GGT. It appears that in normal hepatocytes, this precursor is not processed to the same extent as in kidney or in hyperplastic bile ductular tissue.