Isoenzyme patterns in parenchymal and non-parenchymal cells isolated from regenerating and regenerated rat liver

Parenchymal and non-parenchymal cells were isolated from adult rat liver that had been fully regenerated after a 70% partial hepatectomy. The characteristics of the parenchymal cell preparations from regenerated rat liver indicated that they were a homogeneous population and comparable with parenchymal cells isolated from intact liver. The parenchymal cells from regenerated adult rat liver contain glucokinase, hexokinase, pyruvate kinase type I and aldolase B. The non-parenchymal cells contain hexokinase, pyruvate kinase type III and aldolase B. When cells were isolated at different times of the day from rats on controlled feeding schedules, variation of tyrosine aminotransferase activity and liver glycogen content were observed in the parenchymal cells in keeping with the reported diurnal oscillations found in whole liver extracts. When parenchymal cells were isolated from rats 48 and 72h after partial hepatectomy, different isoenzyme patterns were observed. These cells appeared to synthesize pyruvate kinase type III, a function that was assigned previously to non-parenchymal cells or to foetal rat liver hepatocytes.     

Different types of mitochondria in parenchymal and non-parenchymal rat-liver cells.

1. Intact and pure parenchymal and non-parenchymal cells were isolated from rat liver. The specific activities of several mitochondrial enzymes were determined in both parenchymal and non-parenchymal cell homogenates to characterize the mitochondria in these liver cell types. 2.In general the activities of mitochondrial enzymes were lower in non-parenchymal liver cells than in parenchymal cells. The specific activity of pyruvate carboxylase in non-parenchymal cells expressed as the percentage of that in parenchymal cells was onlu 2% for glutamate dehydrogenase 4.3% and for cytochrome c oxidase 79.4%. Monoamine oxidase, as an exception, has an equal specific activity in both cell types. 3. The activity ratio of pyruvate carboxylase at 10 mM pyruvate over 0.1 mM pyruvate is 3.35 for parenchymal cells and 1.50 for non-parenchymal cells. This indicates that non-parenchymal liver cells only contain the high affinity form of pyruvate carboxylase in contrast to parenchymal cells. 4. The ratio of glycerol-3-phosphate cytochrome c reductase over succinate cytochrome c reductase activity differs from parenchymal (0.01) and non-parenchymal cells (0.10). This might indicate that the glycerol-3-phosphate shuttle, which is important for the transport of reduction equivalents for cytosol to mitochondria is relatively more active in non-parenchymal cells than in parenchymal cells. 5. The activity pattern of mitochondrial enzymes in parenchymal and non-parenchymal cell homogenates indicates that these cell types contain different types of mitochondria. The presence of these different cell types in liver will therefore contribute to the heterogeneity of isolated rat liver mitochondria in which the mitochondria from non-parenchymal cells might be considered as "non-gluconeogenic".     

Glycolytic and gluconeogenic enzyme activities in parenchymal and non-parenchymal cells from mouse liver

1. Parenchymal cells have been prepared from mouse liver by enzymic and mechanical means. 2. The dry weights, protein and DNA contents of these cells have been determined. 3. Mouse liver ;M-' and ;L-type' pyruvate kinases have been prepared free of contamination with each other; their kinetic properties have been examined and a method has been developed for their assay in total liver homogenates. 4. Recoveries of phosphoglycerate kinase, lactate dehydrogenase and phosphofructokinase in enzymically prepared cells indicate that little, if any, cytoplasmic protein is lost during preparation. 5. Parenchymal cells exhibit a very substantial increase in the activity ratio of glucokinase to hexokinase over that in total liver homogenate; in three out of eight experiments, hexokinase activity was undetectable. 6. ;L-type' pyruvate kinase alone occurs in the parenchymal cell. Non-parenchymal cells are characterized by the presence of ;M-type' activity only. 7. Parenchymal cells contain both glucose 6-phosphatase and fructose 1,6-diphosphatase. The non-parenchymal fraction appears to contain fructose 1,6-diphosphatase, but is devoid of glucose 6-phosphatase. 8. No aldolase A was detectable in the whole liver. Aldolase B occurs in both parenchymal and non-parenchymal tissue. 9. Parenchymal cells prepared by mechanical disruption of mouse liver with 20% polyvinyl alcohol exhibit a similar enzyme profile to those prepared enzymically. 10. The methodology involved in the preparation of isolated liver cells is discussed. The importance of the measurement of several parameters as criteria for establishing the viability of parenchymal cells is stressed. 11. The metabolic implications of the results in the present study are discussed.     

All hexokinase isoenzymes coexist in rat hepatocytes.

The cellular distribution of hexokinase isoenzymes, N-acetylglucosamine Kinase and pyruvate kinases in rat liver was studied. Hepatocytes and non-parenchymal cells with high viability and almost no cross-contamination were obtained by perfusion in situ of the liver with collagenase, with the use of an enriched cell-culture medium in all steps of cell isolation. Separation of hexokinase isoenzymes was done by DEAE-cellulose chromatography, and enzyme activities were measured by a specific radioassay. Cytosol from isolated hepatocytes contained high-affinity hexokinases A, B and C, in addition to hexokinase D. The last-mentioned represented about 95% of total glucose-phosphorylating activity. Only hexokinase A was found associated t the particulate fraction. Isolated non-parenchymal cells contained only hexokinases A, B and C. N-Acetylglucosamine kinase was measured with a specific radioassay and was found as a single enzyme form in both hepatocytes and non-parenchymal cells, with higher activities in the former. Pyruvate kinase isoenzyme L was present only in the hepatocytes and isoenzyme K only in the non-parenchymal liver cells, confirming that they are good cellular markers.     

Identity and activities of lysosomal enzymes in parenchymal and non-parenchymal cells from rat liver.

1. Intact parenchymal and non-parenchymal cells were isolated from rat liver. The parenchymal cells were purified by differential centrifugation, while non-parenchymal cells were obtained free of parenchymal cell contamination by preferentially destroying the parenchymal cells with the aid of pronase (0.25%). 2. The ability to isolate pure intact parenchymal and non-parenchymal cells permitted the characterization and measurement of specific activities of various lysosomal enzymes, representing the main functional hydrolytic activities of the lysosomes in these distinct cell types. 3. Lysosomal enzymes catalysing the hydrolysis of the terminal carbohydrate moiety of glycoproteins and glycolipids were not particularly enriched in the non-parenchymal cells as compared to parenchymal cells. The ratio of the specific activities of non-parenchymal cells over parenchymal cells varied between 0.7 for N-acetyl-beta-D-hexoseaminidase to 2.1 for alpha-glucosidase. This suggests no specific role of the non-parenchymal cells in the hydrolysis of terminal carbohydrate moieties of glycoproteins and glycolipids. 4. The enzymes acid phosphatase and aryl sulphatase, representing the phosphate and sulphate hydrolyzing activities, were enriched in the non-paranchymal cells as compared to the parenchymal cells by a factor of 2.5. 5. The most important peptidase cathepsin D, representing protein breakdown capacity, is enriched in the non-parenchymal cells as compared to parenchymal cells by a factor 6.0, suggesting a possible specific function of non-parenchymal cells in protein breakdown. 6. The most enriched lysosomal enzyme, representing lipid hydrolysis, is acid lipase, which is enriched in the non-parenchymal cells with a factor of 10. 7. The distribution of lysosomal enzymes between parenchymal and non-parenchymal cells suggests different functional roles of the lysosomes in these cell types. It can be concluded that the non-parenchymal cells possess a set of lysosomal enzymes which makes them extremely suitable for a phagocytic and antimicrobial function in the liver.     

Identification of metallothionein in parenchymal and non-parenchymal liver cells of the adult rat.

Parenchymal and non-parenchymal cells were isolated from the livers of control, starved, Zn2+-injected and Cd2+-injected rats. Parenchymal cells were prepared by differential centrifugation after perfusion of the liver with collagenase. Non-parenchymal cells were separated from parenchymal cells by unit-gravity sedimentation and differential centrifugation. Yields of 2 x 10(8) non-parenchymal cells with greater than 95% viability and less than 0.2% contamination with parenchymal cells were obtained without exposing cells to Pronase. Metallothioneins-I and -II were identified in parenchymal cells and non-parenchymal cells from Zn2+-treated rats. The metallothionein contents of parenchymal cells, non-parenchymal cells and intact liver were quantified by a competitive 203Hg-binding assay. Administration of heavy-metal salts significantly increased the metallothionein content of both cell populations, although the concentration of the protein was approx. 2.5-fold greater in parenchymal cells than in non-parenchymal cells. Overnight starvation increased the metallothionein content of parenchymal cells without altering that of non-parenchymal cells. The potential significance of this differential response by different liver cell types with regard to the influence of Zn2+ on stress-mediated alterations in hepatic metabolism is discussed.     

Stability of hexokinases A, B and C and N-acetylglucosamine kinase in liver cells isolated from rats submitted to diabetes and several dietary conditions.

The effect of dietary and hormonal variations on the specific activities of hexokinase isoenzymes, N-acetylglucosamine kinase and pyruvate kinase isoenzymes in parenchymal and non-parenchymal liver cells was studied. Hexokinase D was markedly decreased in hepatocytes from animals fasted or fed on the carbohydrate-free diet as well as from diabetic rats, attaining a constant low level of about 17% of normal values. Pyruvate kinase L was also diminished in hepatocytes under the same experimental conditions. In contrast, the three high-affinity hexokinase isoenzymes A, B and C remained without variation in total amount or in their relative proportions in hepatocytes and non-parenchymal liver cells isolated from animals under the various conditions studied. N-Acetylglucosamine kinase activities also did not change either in parenchymal or in non-parenchymal liver cells under all conditions. The results are discussed in relation to the significance of N-acetylglucosamine kinase and the various hexokinase isoenzymes for the phosphorylation of glucose after dietary and hormonal manipulations.     

Characteristics of acid lipase and acid cholesteryl esterase activity in parenchymal and non-parenchymal rat liver cells

(1) Parenchymal and non-parenchymal cells were isolated from rat liver. The characteristics of acid lipase activity with 4-methylumbelliferyl oleate as substrate and acid cholesteryl esterase activity with cholesteryl[1-14C]oleate as substrate were investigated. The substrates were incorporated in egg yolk lecithin vesicles and assays for total cell homogenates were developed, which were linear with the amount of protein and time. With 4-methylumbelliferyl oleate as substrate, both parenchymal and non-parechymal cells show maximal activities at acid pH and the maximal activity for non-parenchymal cells is 2.5 times higher than for parenchymal cells. It is concluded that 4-methylumbelliferyl oleate hydrolysis is catalyzed by similar enzyme(s) in both cell types. (2) With cholesteryl[1-14C]oleate as substrate both parenchymal and non-parenchymal cells show maximal activities at acid pH and the maximal activity for non-parenchymal cells is 11.4 times higher than for parenchymal cells. It is further shown that the cholesteryl ester hydrolysis in both cell types show different properties. (3) The high activity and high affinity of acid cholesteryl esterase from non-parenchymal cells for cholesterol oleate hydrolysis as compared to parenchymal cells indicate a relative specialization of non-parenchymal cells in cholesterol ester hydrolysis. It is concluded that non-parenchymal liver cells in cholesterol ester hydrolysis. It is concluded that non-parenchymal liver cells possess the enzymic equipment to hydrolyze very efficiently internalized cholesterol esters, which supports the suggestion that these cell types are an important site for lipoprotein catabolism in liver.     

Microsomal and cytosolic epoxide hydrolases, the peroxisomal fatty acid beta-oxidation system and catalase. Activities, distribution and induction in rat liver parenchymal and non-parenchymal cells

A number of structurally unrelated hypolipidaemic agents and certain phthalate-ester plasticizers induce hepatomegaly and proliferation of peroxisomes in rodent liver, but there is relatively limited data regarding the specific effects of these drugs on liver non-parenchymal cells. In the present study, liver parenchymal, Kupffer and endothelial cells from untreated and fenofibrate-fed rats were isolated and the activities of two enzymes associated with peroxisomes (catalase and the peroxisomal fatty acid beta-oxidation system) as well as cytosolic and microsomal epoxide hydrolase were measured. Microsomal epoxide hydrolase, cytosolic epoxide hydrolase and catalase activities were 7-12-fold higher in parenchymal cells than in Kupffer or endothelial cells from untreated rats; the peroxisomal fatty acid beta-oxidation activity was only detected in parenchymal cells. Fenofibrate increased catalase, cytosolic epoxide hydrolase and peroxisomal fatty acid beta-oxidation activities in parenchymal cells by about 1.5-, 3.5- and 20-fold, respectively. The induction of catalase (2-3-fold) and cytosolic epoxide hydrolase (3-5-fold) was also observed in Kupffer and endothelial cells; furthermore, a low peroxisomal fatty acid beta-oxidation activity was detected in endothelial cells. Morphological examination by electron microscopy showed that peroxisomes were confined to liver parenchymal cells in untreated animals, but could also be observed in endothelial cells after administration of fenofibrate.     

Intrahepatic distribution of small unilamellar liposomes as a function of liposomal lipid composition

We investigated the intrahepatic distribution of small unilamellar liposomes injected intravenously into rats at a dose of 0.10 mmol of lipid per kg body weight. Sonicated liposomes consisting of cholesterol/sphingomyelin (1:1), (A); cholesterol/egg phosphatidylcholine (1:1), (B); cholesterol/sphingomyelin/phosphatidylserine (5:4:1), (C) or cholesterol/egg-phosphatidylcholine/phosphatidylserine (5:4:1), (D) were labeled by encapsulation of [3H]inulin. The observed differences in rate of blood elimination and hepatic accumulation (A much less than B approximately equal to C less than D) confirmed earlier observations and reflected the rates of uptake of the four liposome formulations by isolated liver macrophages in monolayer culture. Fractionation of the liver into a parenchymal and a non-parenchymal cell fraction revealed that 80-90% of the slowly clearing type-A liposomes were taken up by the parenchymal cells while of the more rapidly eliminated type-B liposomes even more than 95% was associated with the parenchymal cells. Incorporation of phosphatidylserine into the sphingomyelin-based liposomes caused a significant increase in hepatocyte uptake but a much more substantial increase in non-parenchymal cell uptake, resulting in a major shift of the intrahepatic distribution towards the non-parenchymal cell fraction. For the phosphatidylcholine-based liposomes incorporation of phosphatidylserine did not increase the already high uptake by the parenchymal cells while uptake by the non-parenchymal cells was only moderately elevated; this resulted in only a small shift in distribution towards the non-parenchymal cells. The phosphatidylserine-induced increase in liposome uptake by non-parenchymal liver cells was paralleled by an increase in uptake by the spleen. Fractionation of the non-parenchymal liver cells in a Kupffer cell fraction and an endothelial cell fraction showed that even for the slowly eliminated liposomes of type A endothelial cells do not participate to a measurable extent in the elimination process, thus excluding involvement of fluid-phase pinocytosis in the uptake process.     

Immunocytochemical localization of serine: pyruvate aminotransferase in peroxisomes of the human liver parenchymal cells

The localization of serine:pyruvate aminotransferase (SPT) in human liver was investigated by indirect immunoenzyme and protein A-gold techniques. By light microscopy, diaminobenzidine reaction product was present in cytoplasmic granules of the parenchymal cells. By electron microscopy, gold particles indicating the antigenic sites for SPT were exclusively confined to peroxisomes but not to mitochondria. By double labeling technique, both peroxisomal marker enzyme, catalase and SPT were detected in the same peroxisomes. Quantitative analysis of the labeling density showed that SPT is contained only in peroxisomes. The results indicate that in human liver most of SPT is contained in the peroxisomes.     

Immunocytochemical localization of serine: pyruvate aminotransferase in peroxisomes of the human liver parenchymal cells

Summary The localization of serine:pyruvate aminotransferase (SPT) in human liver was investigated by indirect immunoenzyme and protein A-gold techniques. By light microscopy, diaminobenzidine reaction product was present in cytoplasmic granules of the parenchymal cells. By electron microscopy, gold particles indicating the antigenic sites for SPT were exclusively confined to peroxisomes but not to mitochondria. By double labeling technique, both peroxisomal marker enzyme, catalase and SPT were detected in the same peroxisomes. Quantitative analysis of the labeling density showed that SPT is contained only in peroxisomes. The results indicate that in human liver most of SPT is contained in the peroxisomes.     

Pyruvate kinase isoenzyme transitions in cultures of fetal rat hepatocytes

Changes in the expression of two isoenzymic forms of pyruvate kinase in fetal hepatocyte cultures derived from 15- and 19-day gestation rats are studied by immunocytochemical localization of the respective antigens. Initially, in cultures established from 15-day gestation rats only the ‘embryonic’ form of the enzyme (M2-PK) is detected in all cells. Cells which stain positively for the liver specific form of the enzyme (L-PK) are not observed. After 2 days' culture, a significant number of cells have become positive for L-PK. All the positive cells have a morphology which is typical of liver parenchymal cells. However, the majority of parenchymal cells remain negative for L-PK while retaining M2-PK. In contrast, all cells which display a fibroblastic morphology, as well as clear epithelial cells are M2-PK positive, but L-PK negative. In 5-day-old cultures, all hepatocytes have become L-PK positive. Hepatocytes derived from 19-day gestation rat liver stain positively for L-PK on day 1 of culture in agreement with previously published biochemical data. A minor population of negative cells is non-parenchymal in appearance. All parenchymal cells are negative when the culture is stained with M2-PK specific antibody. Five days after the culture is established, many non-parenchymal cells are present. Such cells are L-PK negative and M2-PK positive and their presence in cultures derived from both 15- and 19-day gestation rats explains the persistence of M2-PK. This study reveals that during enzymic differentiation of fetal hepatocytes, all immature hepatocytes are initially capable of expressing M2-PK while they do not produce L-PK. During culture, a sub-population of these cells initiates synthesis of L-PK, indicating that only a fraction of the cells differentiate. At the same time, hepatocytes which do not stain for M2-PK appear, which suggests that cells which initiate L-PK synthesis have ceased to make M2-PK. Eventually all hepatocytes are L-PK positive and M2-PK negative, indicating that a switchover in expression of the pyruvate kinase isoenzymes has occurred.     

Polymerized albumin receptor on rat liver cells.

The polymerized albumin hypothesis was proposed for the mechanism of a hepatitis B virus (HBV) infection of human liver parenchymal cells on the basis that a receptor for polymerized albumin treated with glutaraldehyde was detected on isolated human liver parenchymal cells. However, some controversy exists regarding this hypothesis, because a receptor for formaldehyde-treated bovine serum albumin (f-BSA) has been found on liver non-parenchymal cells. Therefore, we characterized the uptake of polymerized rat serum albumin (p-RSA) and f-BSA by rat liver in vivo, and their bindings to liver cells in vitro. Most p-RSA and f-BSA was taken up by the liver after intravenous administration, and the uptake of p-RSA was inhibited by a 1,000-fold excess of f-BSA. In addition, more than 80% of p-RSA taken up by the liver was found in the non-parenchymal cells, and the remainder was found in the parenchymal cells. P-RSA as well as f-BSA could bind to isolated rat liver parenchymal and non-parenchymal cells. Furthermore, p-RSA and f-BSA could bind to isolated rat liver cell plasma membranes, and these bindings were completely inhibited by 1,000-fold excess of either f-BSA or p-RSA. These results indicate that there is a receptor, which can recognize both p-RSA and f-BSA, on not only rat liver non-parenchymal cells but also the parenchymal cells. It is also indicated that the receptor on the parenchymal cells as well as the non-parenchymal cells is involved in the in vivo uptake of p-RSA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of liver lipase. I. Evidence for several regulatory sites, studied in corticotrophin-treated rats

The activity of liver lipase, an enzyme that can be released from the liver by heparin, varies under several hormonal conditions. The site(s) at which regulation of the enzyme activity may occur was investigated in vitro. As a model, rats were used which had been treated with a corticotrophin analogue, to induce hypercortisolism, a condition in which liver lipase activity is lowered. Lipases isolated from heparin-containing perfusates of livers from ACTH or control rats were identical with respect to heat stability and specific activity as determined by immunotitration and binding to isolated non-parenchymal liver cells, indicating that the enzyme structure was not affected by the treatment. The secretion of liver lipase by isolated parenchymal liver cells was studied. During incubation of parenchymal cells derived from ACTH rats, less enzyme activity was found to be secreted when compared with hepatocytes isolated from control rats (ACTH rats, 2.30 +/- 0.2 mU/10(6) cells; control rats, 3.3 +/- 0.3 mU/10(6) cells). Liver lipase partially purified from control rats could be bound specifically to saturation by non-parenchymal cells, isolated from ACTH or control rats. Non-parenchymal cells from ACTH rats bound less lipase activity (29 mU/mg cell protein) than cells from control rats (50 mU/mg cell protein). This reduction in binding capacity seems to be due to a diminished number of binding sites, since the affinity based on Scatchard analysis and half-maximal binding was not different. These results suggest that the lowered liver lipase activity found during hypercortisolism may be due to an impaired synthesis and/or secretion of the enzyme by the parenchymal cells and to a reduced binding capacity of the non-parenchymal cells for liver lipase.     

Cadmium metabolism by rat liver endothelial and Kupffer cells.

The metabolism of cadmium was investigated in Wistar-rat liver non-parenchymal cells. Kupffer and endothelial cells, the major cell populations lining the sinusoidal tracts, were isolated by collagenase dispersion and purified by centrifugal elutriation. At 20 h after subcutaneous injection of the metal salt (1.5 mg of Cd/kg body weight), endothelial cells accumulated 2-fold higher concentrations of Cd than did Kupffer or parenchymal cells. Most of the Cd in non-parenchymal cells was associated with cytosolic metallothionein (MT), the low-Mr heavy-metal-binding protein(s). When MT was quantified in cytosols from cells isolated from control rats by a 203Hg competitive-binding assay, low levels were found to be present in Kupffer, endothelial and parenchymal cells. Cd injection significantly increased MT levels in all three cell types. The induction of MT synthesis was investigated in vitro by using primary monolayer cultures. The incorporation of [35S]cysteine into MT increased 47% over constitutive levels in endothelial-cell cultures after the addition of 0.8 microM-Cd2+ to the medium for 10 h. MT synthesis in Kupffer cells was not observed. The lack of MT synthesis by monolayer cultures of Kupffer cells in vitro was associated with a decreased capacity of these cells to accumulate heavy metals from the extracellular medium. This apparent decreased ability to transport metals did not reflect a general defect in either cellular function or metabolic activity, since isolated Kupffer cells incorporated [3H]leucine into protein at rates comparable with those shown by liver parenchymal cells and readily phagocytosed particles.     

Localization of cytosolic NADP-dependent isocitrate dehydrogenase in the peroxisomes of rat liver cells: biochemical and immunocytochemical studies.

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123-1131, 2001)     

Alteration in the capacities as well as in the zonal and cellular distributions of pyruvate kinase L and M2 in regenerating rat liver

Pyruvate kinase L (PKL), the glucoregulatory isoenzyme of adult parenchymal cells, and M2 (PKM2), the isoenzyme of proliferating and non-parenchymal cells, were measured, using a specific anti-PKL antibody for differentiation, in total liver homogenates, in isolated parenchymal and non-parenchymal cells as well as in microdissected periportal and perivenous liver tissue from regenerating rat liver after two-thirds partial hepatectomy. Moreover, the zonal distribution of PKL was studied using immunohistochemical techniques. In total liver homogenates PKL activity per g liver decreased after partial hepatectomy, while PKM2 increased. Total PKL activity per 100 g body weight was restored to preoperational levels much more slowly than liver weight. During liver regeneration parenchymal cells acquired high PKM2 besides PKL activity. The isoenzyme outfit of non-parenchymal cells remained unchanged. Microdissection studies showed that PKL lost its normal perivenous to periportal gradient after partial hepatectomy and became evenly distributed within the liver acinus. PKM2 did not retain its even distribution, it became predominant in the periportal zone. Immunohistochemical staining revealed that after partial hepatectomy PKL was present in all parenchymal cells in an atypical non-zonal heterogeneous distribution. Normal specific activities as well as zonal and cellular distributions of both pyruvate kinase isoenzymes were restored 14-21 d after partial hepatectomy. During regeneration after 2/3 partial hepatectomy the liver loses its glucostat function as corroborated in this study by the decrease of the glycolytic capacity via the glucoregulatory PKL; this change of function is accompanied by a loss of PKL-zonation. This finding corroborates the view that zonation of carbohydrate-metabolizing enzymes is required only when the liver functions as a glucostat. The increase of PKM2 and the appearance of a zonal PKM2 heterogeneity are in line with the pattern of hepatocyte proliferation after partial hepatectomy.     

Subcellular distribution of pyruvate (glyoxylate) aminotransferases in rat liver.

The distribution of pyruvate (glyoxylate) aminotransferases in the particulate fraction of rat liver homogenates was examined by centrifugation in a sucrose density graident. Aminotransferase activities towards serine, phenylalanine and histidine with pyruvate and those towards phenylalanine and histidine with glyoxylate were nearly identically distributed. Some 50-55% of the particulate activity was localized in the peroxisomes and the remainder in the mitochondria. Most of alanine-glyoxylate aminotransferase activity was localized in the mitochondria, with some activity in the peroxisomes. Glucagon injection resulted in increases of these enzyme activities in the mitochondria, but not in the peroxisomes.     

Difference spectra, catalase- and peroxidase activities of isolated parenchymal and non-parenchymal cells from rat liver

The reduced minus oxidized difference spectra from isolated parenchymal and non-parenchymal cells from rat liver indicate that the non-parenchymal cells contain a considerable amount of peroxidase. This interpretation is favoured by the more than 30 times higher specific activity of peroxidase (EC 1.11.1.7) in the non-parenchymal cells as compared to the parenchymal cells. The catalase (EC 1.11.1.6) activity in the non-parenchymal cells is 4 times lower than in the parenchymal cells. These results are consistent with an antimicrobial function of the non-parenchymal cells in liver.