Periportal and perivenous hepatocytes possess different amounts and activities of the rate-generating enzymes of carbohydrate and oxidative energy metabolism and thus different metabolic capacities. This is the basis of the model of metabolic zonation, according to which periportal cells catalyze predominantly the oxidative catabolism of fatty and amino acids as well as glucose release and glycogen formation via gluconeogenesis, and perivenous cells carry out preferentially glucose uptake for glycogen synthesis and glycolysis coupled to liponeogenesis. The input of humoral and nervous signals into the periportal and perivenous zones is different; gradients of oxygen, substrates and products, hormones and mediators and nerve densities exist which are important not only for the short-term regulation of carbohydrate metabolism but also for the long-term regulation of zonal gene expression. The specialization of periportal and perivenous hepatocytes in carbohydrate metabolism has been well characterized. In vivo evidence is provided by the complex metabolic situation termed the 'glucose paradox' and by zonal flux differences calculated on the basis of the distribution of enzymes and metabolites. In vitro evidence is given by the different flux rates determined with classical invasive techniques, e.g. in periportal-like and perivenous-like hepatocytes in cell culture, in periportal- and perivenous-enriched hepatocyte populations and in perfused livers during orthograde and retrograde flow, as well as with noninvasive techniques using miniature oxygen electrodes, e.g. in livers perfused in either direction. Differences of opinion in the interpretation of studies with invasive and noninvasive techniques by the authors are discussed. The declining gradient in oxygen concentrations, the decreasing glucagon/insulin ratio and the different innervation could be important factors in the zonal expression of the genes of carbohydrate-metabolizing enzymes. While it is clear that the hepatocytes sense the glucagon/insulin gradients via the respective hormone receptors, it is not known how they sense different oxygen tensions; the O2 sensor may be an oxygen-binding heme protein. The zonal separation of glucose release and uptake appears to be important for the liver to operate as a 'glucostat'. Thus, zonation of carbohydrate metabolism develops gradually during the first weeks of life, in part before and in part with weaning, when (in rat and mouse) the fat- and protein-rich but carbohydrate-poor nutrition via milk is replaced by carbohydrate-rich food. Similarly, zonation of carbohydrate metabolism adapts to longer lasting alterations in the need of a 'glucostat', such as starvation, diabetes, portocaval anastomoses or partial hepatectomy.
It is now well established that hepatocytes are the main liver cells responsible for the synthesis of plasma proteins produced by the liver. That these cells are not specialized in the production of the different plasma proteins is also well established. Presently the point still debated is whether a functional hepatocellular heterogeneity exists for plasma protein synthesis as for many other hepatocyte functions. Several physiological and pathological situations suggest that this heterogeneity takes place in the hepatocytes of two opposite hepatic lobular zones, the periportal and centrilobular zones. However, this zonal difference, which supposes different regulatory mechanisms, must be confirmed by techniques other than the now classical immunocytochemistry or the in situ hypbridization technique recently proposed for the demonstration of mRNAs in hepatocytes. Another hepatocellular heterogeneity, the intercellular heterogeneity, which can be observed in the same lobular zone, is more difficult to analyze, but shows that from hepatocyte to hepatocyte a variation exists in the synthesis of a given plasma protein.
An enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of bovine Cu,Zn-SOD. Accuracy of the ELISA and specificity of the antibody for cell-free extracts was established by: (1) measurement of antigen levels of bovine endothelial cell extracts reconstituted with pure antigen, and (2) immunoblotting with affinity purified antibody. The ELISA was highly sensitive and 0.05-0.10 ng of pure antigen could be accurately detected, which allowed the measurement of Cu,Zn-SOD in as few as 250 endothelial cells. With utilization of the ELISA for detection, DEAE-cellulose chromatography patterns of endothelial cell Cu,Zn-SOD overlapped those of pure bovine erythrocyte Cu,Zn-SOD. Exposure of cells in culture to 80% O2 for 48 h increased the relative abundance of the Cu,Zn-SOD as measured by the ELISA by 1.8-fold. Thus, endothelial cells in culture respond to hyperoxia by enhanced production of Cu,Zn-SOD protein. The ELISA developed in this study may be useful for assessing other factors that regulate cellular production of Cu,Zn-SOD.
6-Pyruvoyl tetrahydropterin synthase, the enzyme that catalyses the conversion of 7,8-dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, was purified 3,330-fold from human pituitary gland with an overall recovery of 30%. The native enzyme has a molecular mass of 68 kD and consists of four identical subunits of 16.5 kD. The pH optimum of the enzyme in Tris/HCl buffer is 7.5. The enzyme is dependent on Mg2+ and NADPH and has a Michaelis-Menten constant of 10 microM for its natural substrate, 7,8-dihydroneopterin triphosphate. The isoelectric point of the human enzyme is 4.3-4.6. The human pituitary gland enzyme is heat instable in contrast to the enzymes from human, rat and salmon liver, and Drosophila head. The amino acid composition showed remarkably high content of acidic amino acids Asp and Glu. The N-terminus was found to be blocked.
Hydrolysis of extracellular ATP and other nucleoside phosphates by A-431 human epidermoidal carcinoma cells was studied. The hydrolysis of extracellular ATP by these cells required either Mg2+ or Ca2+, and either cation could be replaced by Co2+, Fe2+, or Mn2+. Nucleoside triphosphates (ATP, GTP, CTP, UTP, and dTTP), but not nucleoside diphosphates, were hydrolyzed by the cells with K(m) and V(max) values similar to those for ATP (0.9- 1.1 mmol/l and 6-10 nmol Pi formed/10(6) cells, respectively). The hydrolysis of ATP was inhibited strongly by ATP-gammaS and AMPPNP, and weakly by AMPCPP and ADP-betaS, but not by AMPCPP or AMPCP. Since the hydrolysis of [gamma-P-32]ATP was inhibited by all these nucleoside triphosphates, the binding site for ATP is presumed lo be the same as that for the other nucleoside triphosphates. All these results indicate that ecto-ATPase activity associated with A-431 cells is due to ecto-nucleoside triphosphatase. The nucleotide specificity shown in the present study indicates that ecto-nucleoside triphosphatase associated with A-431 cells is a molecule different from P2-purinergic receptors which can be stimulated specifically with nucleoside phosphates like ATP, ADP, UTP, UDP, and GTP, but not by other nucleotides.
Three enzyme forms (CR1, CR2 and CR3) of carbonyl reductase were purified from chicken liver with using 4-benzoylpyridine as a substrate. CR1 was a dimeric enzyme composed of two identical 25-kD subunits. CR2 and CR3 were monomeric enzymes whose molecular weights were both 32 kD. CR1 exhibited 17 beta-hydroxysteroid dehydrogenase activity as well as carbonyl reductase activity in the presence of both NADP(H) and NAD(H). CR2 and CR3 had similar properties with regard to substrate specificity and inhibitor sensitivity. They could exhibit the activity only with NADPH and had no hydroxysteroid dehydrogenase activity. CR2 and CR3 cross-reacted with anti-chicken kidney carbonyl reductase antibody, though CR1 did not. The results suggest that CR1 is a hydroxysteroid dehydrogenase, and CR2 and CR3 are similar to each other and to the kidney enzymes.
Erythrocytes of 3 adult siblings with essential fructosuria contained 45-200 mumol/l fructose 3-phosphate (Fru-3-P), i.e. 3-15 times the concentration in normal controls. Sorbitol 3-phosphate was also increased, but to a lesser degree. An oral load with 50 g of fructose produced an additional 40 mumol/l increase of erythrocyte Fru-3-P after 5 h. The rate of Fru-3-P formation by red cells in vitro was normal. HbA1 and HbA1c were normal. The suspected pathogenetic role of Fru-3-P in diabetic complications is questioned.
Endopeptidase 24.11 (EC 3.4.24.11) enzymatic activity was spectrofluorimetrically measured in human urine, using a synthetic peptidic substrate. Urinary endopeptidase 24.11 output (U(endo)) was determined in 24-hour urine samples of 10 kidney transplant recipients during the first 2 weeks after surgery. In 9 patients, a large increase in U(endo) levels was noted during the 1st and/or the 2nd postoperative days (mean +/- SEM of peak U(endo) 624 +/- 122 mug/24 h, p = 0.0003 as compared to 239 +/- 20 mug/24 h in a healthy control population). This occurred whether patients received OKT3 (n = 6) or cyclosporine A (n = 3) as primary immunosuppression. U(endo) returned to normal between the 3rd and the 5th postoperative day. We conclude that renal transplantation is associated with an early and marked release of endopeptidase 24.11 in urine. This could be due to the potentially toxic effects of ischemia and/or immunosuppressive drugs on the proximal tubular epithelium. The clinical usefulness of urinary endopeptidase 24.11 as a marker of tubular injury remains to be assessed.
Lipid metabolism appears to be less zonated than carbohydrate and protein metabolism. Studies on the zonation of lipid metabolism have been centered in particular on fatty acid synthesis which, according to the concept of metabolic zonation, should be a predominantly perivenous process while fatty acid oxidation should be periportal. There are, however, conflicting data on the activity gradients of lipogenic enzymes as well as measurements of actual synthesis of fatty acid and very low density lipoprotein. Data obtained by microdissection show a 1.5- to 2-fold higher activity of acetyl-CoA carboxylase and citrate lyase in the perivenous zone in agreement with measurements of the actual rate of fatty acid synthesis in preparations of hepatocyte, enriched in periportal or perivenous cells. On the other hand, results obtained with the dual-digitonin-pulse perfusion technique demonstrate the opposite gradient in the form of a 2- to 3-fold higher specific activity of acetyl-CoA carboxylase in the periportal zone based on measurements of the acetyl-CoA carboxylase protein proper. This specific activity gradient, which applies to male and not female rats, disappears almost completely in the fasted-refed animal, were lipogenesis is strongly induced. In this review we attempt to rationalize these discrepancies in the results as methodological differences which in particular apply to the following parameters: (1) expression of results (reference substance); (2) selectivity of zonal sampling, and (3) differences in methodology of acetyl-CoA carboxylase measurements. It is concluded that these factors could account for the discrepancies, but further studies, in particular on the zonation acetyl-CoA carboxylase mRNA, are required in order to further understand the zonation of lipid metabolism and its possible role in the metabolic regulation of the liver.
A sensitive and precise automated assay of urinary lactate dehydrogenase (EC 1.1.1.27), alkaline phosphatase (EC 3.1.3.1) and gamma-glutamyltransferase (EC 2.3.2.2) is described. For this purpose, we used a BM/Hitachi System 704 model and reagents for automated analysis of serum enzymes from Boehringer Mannheim. However, the schedules of enzyme chemistry parameters recorded by the autoanalyzer and the spectrophotometric calibration are reprogrammed to meet requirements deriving from urine adoption and to optimize the enzyme assay in this unusual medium.
Results of our conclusive study on urinary enzyme stability during sample storage are reported. We measured alanine aminopeptidase (AAP) and N-acetyl-beta-D-glucosaminidase (NAG) in morning urines from 9 healthy normal subjects immediately after collection and throughout a 1-year storage at - 70 and - 20-degrees-C AAP proved to be quite stable at - 70-degrees-C (99.2% of the basal value at the end of the year). NAG is partially preserved (84.1% of the basal value) at - 70-degrees-C, but significantly decreased (50.4%) at - 20-degrees-C.
An enzyme activity which catalyzes the transfer of the sulfate group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to poly-Glu6,Ala3,Tyr1 (EAY; Mr 47,000) has been demonstrated in the antral and body mucosa of the rat stomach. The distribution of this tyrosylprotein sulfotransferase was similar to that of the Golgi marker enzyme, glycoprotein sulfotransferase, and its activity from body mucosa was 23% higher than that from the antrum. The optimum for tyrosylprotein sulfotransferase activity was obtained at pH 6.8, in the presence of 0.5% Triton X-100, 20 mmol/l MnCl2, 50 mmol/l NaF, 2 mmol/l 5'-AMP, and 1 mmol/l DTT, whereas Ca2+, Mg2+, Cu2+, Zn2+, EDTA, NEM, NaCl and Na2SO4 were inhibitory. The apparent K(m) of the sulfotransferase for EAY was 1.5 X 10(-6) mol/l and for PAPS 0.75 X 10(-6) mol/l. The enzyme was 28 times less susceptible to 2,6-dichloro-4-nitrophenol inhibition as compared to that required for phenol sulfotransferase inhibition. The tyrosine sulfation by the tyrosylprotein sulfotransferase was independent of the sulfation of carbohydrate residues in mucous glycoproteins and glycolipids, thus indicating that the identified sulfotransferase is specific for sulfation of the tyrosyl residues in the peptide core.
We measured the cholinesterase activity in morning urines from 63 insulin-dependent diabetics and 27 controls. The total esterase (TotE) activity (Ellman's method) has been divided into aliesterase (AliE), pseudocholinesterase and acetylcholinesterase by means of two inhibitors, eserine and quinidine. Diabetics were divided in 2 groups according to the urinary albumin/creatinine ratio (mg/mmol, < 2 in group 1, > 2 in group 2). The urinary cholinesterase behavior was correlated with that of a known tubular lysosomal hydrolase, N-acetyl-beta-D-glucosaminidase (NAG). Compared to normals, in addition to a significant increase in urinary NAG in diabetes (in group 2 more than in group 1), TotE and AliE were also significantly raised (+36% and 109% of the controls, in group 1 as much as in group 2).
Endopeptidase 24.11 (EC 3.4.24.11) enzymatic activity was spectrofluorimetrically measured in human urine, using a synthetic peptidic substrate. Urinary endopeptidase 24.11 output (Uendo) was determined in 24-hour urine samples of 10 kidney transplant recipients during the first 2 weeks after surgery. In 9 patients, a large increase in Uendo levels was noted during the 1st and/or the 2nd postoperative days (mean +/- SEM of peak Uendo 624 +/- 122 micrograms/24 h, p = 0.0003 as compared to 239 +/- 20 micrograms/24 h in a healthy control population). This occurred whether patients received OKT3 (n = 6) or cyclosporine A (n = 3) as primary immunosuppression. Uendo returned to normal between the 3rd and the 5th postoperative day. We conclude that renal transplantation is associated with an early and marked release of endopeptidase 24.11 in urine. This could be due to the potentially toxic effects of ischemia and/or immunosuppressive drugs on the proximal tubular epithelium. The clinical usefulness of urinary endopeptidase 24.11 as a marker of tubular injury remains to be assessed.
Rat kidney sialidase levels have been reported to be markedly altered in pathological states such as diabetes. This was associated with a modification of sialic acid levels. Therefore, it was interesting to study the variations of kidney sialidase and sialyltransferase activities and sialic acid content according to sex and age. This was carried out from birth to 210 days of age. The substrates used were sialyl alpha(2-3)[3H]-lactitol for sialidase activity, asialofetuin and [14C]-CMPNeu5Ac for sialyltransferase activity. In males sialidase activity increased until 32 days then slightly declined. In females, the activity increased and leveled off at 135 days of age. Higher sialidase activity was observed in females than in males from 56 days of age. Gonadectomy had no effect on this activity. In both sexes, sialyltransferase activity decreased markedly with age. This activity was higher in females than in males, whereas sialic acid levels varied only moderately with age and were slightly higher in females.
A kinetic study of the inhibition of several alkaline phosphatase (AP) isoenzyme activities by phenobarbital was carried out using p-nitrophenylphosphate (10 mM as a substrate at pH 9.8 in a 300-mM Hepes buffer. AP from bovine kidney, calf intestine, bovine liver, and rat bone was used. Over a phenobarbital concentration range of 20-400 mM, all these isoenzymes were inhibited in an uncompetitive manner with a K(i) of 200 mM for intestinal AP, and in a linear mixed-type manner for all the other isoenzymes tested. The K(i) values were 10, 40 and 55 mM for kidney, bone and liver AP, respectively. The use of 15 mM carbonate-bicarbonate or 400 mM diethanolamine buffer did not modify the degree of inhibition of intestinal AP activity. Dixon plots of the reciprocal of reaction velocity versus inhibitor concentration either at different substrate concentration or at different DEA concentration indicate uncompetitive inhibition for the intestinal enzyme. This in vitro inhibitory effect of phenobarbital is in contrast to its in vivo stimulating action on AP. However, in the whole animal, the effects of phenobarbital administration probably represent the sum of multiple effects.
In cultured amniotic cells from fetuses with Edward's syndrome (trisomy 18), the activities of two protein phosphatases, alkaline phosphatase and phosphotyrosine phosphatase, were measured. Comparison with normal fetal cells showed a different behavior for each enzyme. Alkaline phosphatase was significantly lowered while phosphotyrosine phosphatase remained at normal levels. The interest of these enzyme assays in the screening procedure of this severe chromosome defect is discussed.
A new method for the assay of maltase and sucrase is reported. The method makes use of mutarotase, hexokinase and glucose 6-phosphate dehydrogenase as ancillary enzymes. The reaction is linear at least up to a DELTAE/min of 0.13.
