Biosynthesis of rat liver pyruvate kinase. Measurement of enzyme lifetime and the rate of synthesis at weaning.

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of immunoprecipitates of liver cytosol with anti-(L-type pyruvate kinase) serum revealed proteins of mol.wt. 56 000 and 42 000 in addition to the heavy and light chains. The ratio of the 56 000 mol.wt. to the 42 000 mol.wt. protein increased under dietary conditions that resulted in an increase in the apparent specific activity of hepatic pyruvate kinase. The 42 000 mol.wt. protein was removed from immunoprecipitates if the liver cytosol was partially purified by pH precipitation and (NH4)2SO4 fractionation before addition of the antiserum. This technique may be used to analyse the formation of pure L-type pyruvate kinase in liver. By using H14CO3-labelling, the t1/2 of L-type pyruvate kinase was estimated as 75 +/- 1.7 h in post-weaned high-carbohydrate-diet-fed rats. Before weaning there was little immunoreactive pyruvate kinase in rat liver cytosol. Induction began between 6 and 24 h after weaning and reached a maximum value 120 h after weaning. When clearly enhanced total pyruvate kinase activity was first observed at 24 h post-weaning, the apparent specific activity of hepatic pyruvate kinase was considerably lower than the specific activity of the pure isolated enzyme. When the induction of L-type pyruvate kinase was monitored by the incorporation of L-[4,5-3H]leucine, the maximum rate of synthesis occurred 24--48 h after weaning. After this period synthesis declined, indicating a relatively slow turnover of the enzyme once the enzyme concentration was established in the liver.     

Flux of the L-serine metabolism in rabbit, human, and dog livers. Substantial contributions of both mitochondrial and peroxisomal serine:pyruvate/alanine:glyoxylate aminotransferase.

L-Serine metabolism in rabbit, dog, and human livers was investigated, focusing on the relative contributions of the three pathways, one initiated by serine dehydratase, another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Under quasi-physiological in vitro conditions (1 mM L-serine and 0.25 mM pyruvate), flux through serine dehydratase accounted for only traces, and that through SPT/AGT substantially contributed no matter whether the enzyme was located in peroxisomes (rabbit and human) or largely in mitochondria (dog). As for flux through serine hydroxymethyltransferase and GCS, the conversion of serine to glycine occurred fairly rapidly, followed by GCS-mediated slow decarboxylation of the accumulated glycine. The flux through GCS was relatively high in the dog and low in the rabbit, and only in the dog was it comparable with that through SPT/AGT. An in vivo experiment with L-[3-3H,14C]serine as the substrate indicated that in rabbit liver, gluconeogenesis from L-serine proceeds mainly via hydroxypyruvate. Because an important role in the conversion of glyoxylate to glycine has been assigned to peroxisomal SPT/AGT from the studies on primary hyperoxaluria type 1, these results suggest that SPT/AGT in this organelle plays dual roles in the metabolism of glyoxylate and serine.     

Flux of the L-serine metabolism in rat liver. The predominant contribution of serine dehydratase.

L-Serine metabolism in rat liver was investigated, focusing on the relative contributions of the three pathways, one initiated by L-serine dehydratase (SDH), another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Because serine hydroxymethyltransferase is responsible for the interconversion between serine and glycine, SDH, SPT/AGT, and GCS were considered to be the metabolic exits of the serine-glycine pool. In vitro, flux through SDH was predominant in both 24-h starved and glucagon-treated rats. Flux through SPT/AGT was enhanced by glucagon administration, but even after the induction, its contribution under quasi-physiological conditions (1 mM L-serine and 0.25 mM pyruvate) was about (1)/(10) of that through SDH. Flux through GCS accounted for only several percent of the amount of L-serine metabolized. Relative contributions of SDH and SPT/AGT to gluconeogenesis from L-serine were evaluated in vivo based on the principle that 3H at the 3 position of L-serine is mostly removed in the SDH pathway, whereas it is largely retained in the SPT/AGT pathway. The results showed that SPT/AGT contributed only 10-20% even after the enhancement of its activity by glucagon. These results suggested that SDH is the major metabolic exit of L-serine in rat liver.     

Protein synthesis, RNA concentrations, nitrogen excretion, and metabolism vary seasonally in the Antarctic holothurian Heterocucumis steineni (Ludwig 1898)

Seasonal changes in protein and nitrogen metabolism have not previously been reported in any Antarctic suspension-feeding species that ceases feeding for extended periods in winter. To provide comparison with data reported on Nacella concinna, a species that continues to feed in winter, we have measured feeding activity; oxygen consumption; ammonia, urea, and fluorescamine-positive substance (FPS) excretion; O : N ratios; body wall protein synthesis; RNA to protein ratios; and RNA activity at three times during the year in an Antarctic suspension-feeding holothurian. Feeding activity ceased for 4 mo during winter, and oxygen consumption rates decreased from 8.79+/-0.43 micro mol h(-1) to 4.48+/-0.34 micro mol h(-1). Ammonia excretion also decreased during winter from 2,600+/-177 nmol N h(-1) to 974+/-70 nmol N h(-1), but urea excretion rates increased from 178+/-36 nmol N h(-1) to 281+/-110 nmol N h(-1), while FPS excretion rates remained unchanged throughout the year with a seasonal mean of 88+/-13 nmol N h(-1). Oxygen to nitrogen ratios ranged between 6 and 10, suggesting that proteins were used as the primary metabolic substrate. Body wall protein synthesis rates decreased from 0.35%+/-0.03% d(-1) in summer to 0.23% d(-1) in winter, while RNA to protein ratios decreased from 33.10+/-1.0 microg RNA mg(-1) protein in summer to 27.88+/-1.3 microg RNA mg(-1) protein in winter, and RNA activity was very low, ranging between 0.11+/-0.01 mg protein mg(-1) RNA d(-1) in summer and 0.06+/-0.01 mg protein mg(-1) RNA d(-1) in winter. Heterocucumis steineni shows a larger seasonal decrease in oxygen consumption and ammonia excretion between February (summer) and July (winter) than N. concinna, while the proportional decrease in protein synthesis rates is similar in both species.     

Ontogeny of hepatic enzymes involved in serine- and folate-dependent one-carbon metabolism in rabbits

Serine occupies a central position in folate-dependent, one-carbon metabolism through 5,10-methylenetetrahydrofolate (MTHF) and 5-formyltetrahydrofolate (FTHF). We characterized the ontogeny of the specific activity of key enzymes involved in serine, 5,10-MTHF, and 5-FTHF metabolism: methenyltetrahydrofolate synthetase (MTHFS), MTHF reductase (MTHFR), the glycine cleavage system (GCS), methionine synthase (MS), and serine hydroxymethyltransferase (SHMT) in rabbit liver, placenta, brain, and kidney. In liver, MTHFS activity is low in the fetus (0.36 +/- 0.07 nmol. min(-1). mg protein(-1)), peaks at 3 wk (1.48 +/- 0.50 nmol. min(-1). mg protein(-1)), and then decreases to adult levels (1.13 +/- 0.32 nmol. min(-1). mg protein(-1)). MTHFR activity is highest early in gestation (24.9 +/- 2.4 nmol. h(-1). mg protein(-1)) and declines rapidly by birth (4.7 +/- 1.3 nmol. h(-1). mg protein(-1)). MS is highest during fetal life and declines after birth. Cytosolic SHMT activity does not vary during development, but mitochondrial SHMT peaks at 23 days. GCS activity is high in the fetus and the neonate, declining after weaning. In placenta and brain, all activities are low throughout gestation. Cytosolic and mitochondrial SHMT activities are low in kidney and rise after weaning, whereas MTHFS is low throughout development. These data suggest that the liver is the primary site of activity for these enzymes. Throughout development, there are multiple potential sources for production of 5,10-MTHF, but early in gestation high MTHFR activity and low MTHFS activity could reduce 5,10-MTHF availability.     

Phosphorylation of L-type pyruvate kinase by a Ca2+/calmodulin-dependent protein kinase

Rat liver L-type pyruvate kinase was phosphorylated in vitro by a Ca2+/calmodulin-dependent protein kinase purified from rabbit liver. The calmodulin (CaM)-dependent kinase catalyzed incorporation of up to 1.7 mol of 32P/mol of pyruvate kinase subunit; maximum phosphorylation was associated with a 3.0-fold increase in the K0.5 for P-enolpyruvate. This compares to incorporation of 0.7 to 1.0 mol of 32P/mol catalyzed by the cAMP-dependent protein kinase with a 2-fold increase in K0.5 for P-enolpyruvate. When [32P]pyruvate kinase, phosphorylated by the CaM-dependent protein kinase, was subsequently incubated with 5 mM ADP and cAMP-dependent protein kinase (kinase reversal conditions), 50-60% of the 32PO4 was removed from pyruvate kinase, but the K0.5 for P-enolpyruvate decreased only 20-30%. Identification of 32P-amino acids after partial acid hydrolysis showed that the CaM-dependent protein kinase phosphorylated both threonyl and seryl residues (ratio of 1:2, respectively) whereas the cAMP-dependent protein kinase phosphorylated only seryl groups. The two phosphorylation sites were present in the same 3-4-kDa CNBr fragment located near the amino terminus of the enzyme subunit. These results indicate that the CaM-dependent protein kinase catalyzed phosphorylation of L-type pyruvate kinase at two discrete sites. One site is apparently the same serine which is phosphorylated by the cAMP-dependent protein kinase. The second site is a unique threonine residue whose phosphorylation also inactivates pyruvate kinase by elevating the K0.5 for P-enolpyruvate. These results may account for the Ca2+-dependent phosphorylation of pyruvate kinase observed in isolated hepatocytes.     

Content of amino acids in axons from the CNS of the lobster.

The contents of alanine, proline, glycine, GABA, glutamate, and aspartate were measured in four bundles of axons (designated areas A through D) from the circumesophageal connective of the lobster (Homarus americanus). The contents of these amino acids were also determined in individual axons within specific bundles and in the external sheath covering the circumesophageal connective. Within the nerve bundles the levels of aspartate were highest of the amino acids measured, ranging from 1.95 +/- 0.12 mumol/mg protein in area C to 7.55 +/- 0.54 mumol/mg protein in area B. On the other hand, GABA had the lowest value in the four bundles; its highest level was found in area C (0.083 +/- 0.006 mu mol/mg protein) and the lowest in area B (none detected). The content of glycine ranged from 1.63 +/- 0.14 (area C) to 2.52 +/- 0.32 mumol/mg protein in area A; that for glutamate ranged from 0.390 +/- 0.019 (area C) to 1.01 +/- 1.03 (area B). The contents of alanine and proline changed relatively little from bundle-to-bundle. The content of aspartate was the highest of any of the amino acids assayed in individual axons (with diameters in the range of 40 to 65 mu) dissected from areas B and C. Glycine had the next highest content followed in order by glutamate, proline, and alanine. GABA was not detected in these axons. With the exception of GABA (which could not be detected), aspartate had the lowest level (0.066 +/- 0.017) and glycine had the highest level (2.00 +/- 0.498 mumol/mg protein) in the external sheath covering the the circumesophageal connective.     

ATP-dependent degradation of a mutant serine: pyruvate/alanine:glyoxylate aminotransferase in a primary hyperoxaluria type 1 case

Primary hyperoxaluria type 1 (PH 1), an inborn error of glyoxylate metabolism characterized by excessive synthesis of oxalate and glycolate, is caused by a defect in serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT). This enzyme is peroxisomal in human liver. Recently, we cloned SPT/AGT-cDNA from a PH 1 case, and demonstrated a point mutation of T to C in the coding region of the SPT/AGT gene encoding a Ser to Pro substitution at residue 205 (Nishiyama, K., T. Funai, R. Katafuchi, F. Hattori, K. Onoyama, and A. Ichiyama. 1991. Biochem. Biophys. Res. Commun. 176:1093-1099). In the liver of this patient, SPT/AGT was very low with respect to not only activity but also protein detectable on Western blot and immunoprecipitation analyses. Immunocytochemically detectable SPT/AGT labeling was also low, although it was detected predominantly in peroxisomes. On the other hand, the level of translatable SPT/AGT-mRNA was higher than normal, indicating that SPT/AGT had been synthesized in the patient's liver at least as effectively as in normal liver. Rapid degradation of the mutant SPT/AGT was then demonstrated in transfected COS cells and transformed Escherichia coli, accounting for the low level of immunodetectable mutant SPT/AGT in the patient's liver. The mutant SPT/AGT was also degraded much faster than normal in an in vitro system with a rabbit reticulocyte extract, and the degradation in vitro was ATP dependent. These results indicate that a single amino acid substitution in SPT/AGT found in the PH1 case leads to a reduced half- life of this protein. It appears that the mutant SPT/AGT is recognized in cells as an abnormal protein to be eliminated by degradation.     

Alteration in liver pyruvate kinase protein and catalytic activity upon starvation and refeeding

Liver pyruvate kinase (L-type isozyme) was purified from the livers of rats fed a high carbohydrate, low protein diet for 4 days. The protein was homogeneous as judged by polyacrylamide-gel electrophoresis with and without added sodium dodecyl sulfate and as judged by high speed sedimentation and low speed equilibrium centrifugation. The specific activity of the purified protein was 190–220 international units (IU)/mg. A precipitating antiserum directed specifically against liver pyruvate kinase was obtained from rabbits and was used to determine the amount of liver pyruvate kinase protein present in the 80,000g supernatant fraction of rat liver homogenates in response to the dietary status of the animal. Rats maintained on a high carbohydrate, low protein diet for 4 days prior to sacrifice have at least 20 mg of precipitable liver pyruvate kinase protein per liver. Starvation of the animal results in a marked reduction in liver pyruvate kinase so that by 3 days of starvation less than 7 mg of liver pyruvate kinase protein per liver remains. Refeeding the animal a high carbohydrate, low protein diet results in a return of the liver pyruvate kinase protein to the prestarvation level of 20 mg per liver. The liver pyruvate kinase activity per liver varies in the same direction as does the liver pyruvate kinase protein but does not parallel the change in protein. Animals fed a high carbohydrate, low protein diet for 4 days have 60–70 IU/mg of liver pyruvate kinase protein whereas animals starved for periods exceeding 30 h have greater than 100 IU/mg of liver pyruvate kinase protein. Refeeding starved animals with a high carbohydrate, low protein diet initially causes a large increase in activity per milligram of liver pyruvate kinase protein followed by a return of this value to the prestarvation level. The observed rise in the ratio of activity per milligram of liver pyruvate kinase protein during starvation suggests a modification in the enzyme protein resulting either in an increase in the specific activity of the enzyme or in a decrease in the affinity of the enzyme for the antibody.     

Effects of luteolin on arylamine N-acetyltransferase activity in human liver tumour cells

The human liver tumour cell line (J5) was selected in order to evaluate whether or not luteolin affected arylamine N-acetyltransferase (NAT) activity. Using high performance liquid chromatography, the NAT activity for acetylation of arylamine substrates (2-aminofluorene and p-aminobenzoic acid) was determined. The cytosolic NAT activity in human liver tumour cells was 2.74+/-0.26 and 1.68+/-0.20 nmol/min/mg of protein for 2-aminofluorene and p-aminobenzoic acid, respectively. Luteolin displayed a dose-dependent inhibition to cytosolic NAT activity and intact human liver tumour cells. Time-course experiments showed that NAT activity measured from intact human liver tumour cells was inhibited by luteolin for up to 24 h. Using standard steady-state kinetic analysis, it was shown that luteolin was a possible noncompetitive inhibitor to NAT activity in cytosols. This report is the first to show how luteolin affects NAT activity in human liver tumour cells.     

Urate synthesis in the perfused chick liver

Urate synthesis was studied in a perfused chicken liver preparation. The perfused liver had an ATP/ADP ratio of 0.29+/-0.05(6) compared with 0.34+/-0.07(10) in liver obtained from chicks under ether anaesthesia. Lactate/pyruvate ratios were 9.4+/-1.7(5) in the perfused liver and 14.8+/-1.8(5) in the rapidly sampled liver. Urate synthesis was only marginally stimulated by glycine, glutamine, aspartic acid or NH(4)Cl, but significant increases were observed with phosphoribosyl pyrophosphate, aminoimidazolecarboxylic acid riboside, inosine, inosinic acid and xanthine. Urate synthesis from glycine, glutamine, NH(4)Cl, asparagine, alanine, histidine and a mixture of 21 amino acids was obtained on inclusion of insulin in the perfusion medium. Evidence for the inclusion of the carbon of histidine into uric acid was obtained. Aspects of the energy consumption associated with the conversion of excess of amino acid into uric acid are considered.     

Dual-digitonin-pulse perfusion. Concurrent sampling of periportal and perivenous cytosol of rat liver for determination of metabolites and enzyme activities.

A previously described digitonin-perfusion technique [Quistorff, Grunnet & Cornell (1985) Biochem. J. 226, 289-297], by which intracellular material of rat liver could be liberated, has been refined, now allowing release of cytosol of high purity from both periportal and perivenous parts of the same liver. The cytosolic fractions are obtained by perfusing the liver for short intervals (10-20 s) with digitonin (4-5 mg/ml), first in the normal perfusion direction and then, after an interval of 1-2 min, in the retrograde direction, the eluate being collected during and after both intervals. The technique is termed 'dual-digitonin-pulse perfusion'. The eluate fractions showed a peak specific activity of the cytosolic enzymes alanine aminotransferase (ALAT), lactate dehydrogenase (LDH) and pyruvate kinase (PK) of 3-5-fold higher than obtained in a biopsy from the same liver. For glutamine synthetase (GS) a 10-fold higher specific activity was obtained. Zonation, defined as the ratio of the specific activities in periportal and perivenous eluates, of ALAT, LDH and PK was 10, 1.7 and 0.70 respectively. Zonation of GS was less than 0.01. These factors may be modified by a slight zonation of cytosolic protein of 1.2-1.3. Peak concentrations in the eluate of ATP, ADP, Pi, NAD+ and glycerol 3-phosphate were 32.5 +/- 11.4, 19.9 +/- 4.3, 71.9 +/- 25.4, 2.41 +/- 0.83 and 6.84 +/- 2.74 nmol/mg of protein for periportal eluates. There was no difference between periportal and perivenous eluates except for glycerol 3-phosphate, which was significantly higher in perivenous eluates, 12.8 +/- 4.5 nmol/mg of protein.     

Phospholipid hydroperoxide accumulation in liver of rats intoxicated with carbon tetrachloride and its inhibition by dietary alpha-tocopherol

The formation and accumulation of phospholipid hydroperoxides, especially of phosphatidylcholine hydroperoxide (PCOOH), a primary peroxidation product of phosphatidylcholine (PC), in livers of carbon tetrachloride-intoxicated rats was investigated. PCOOH in liver and blood plasma was measured by a chemiluminescence-high-performance liquid chromatography procedure originally developed by Miyazawa et al. (Anal. Lett. 20, 915, 1987; Free Radical Biol. Med. 7, 209, 1989). Male Sprague-Dawley rats (120 g body wt., 5 weeks of age) were used in the experiments. The amount of PCOOH in the liver of control rats (CCl4-untreated) was 160 +/- 20 pmol/100 mg protein (mean +/- SD) and the PCOOH/PC molar ratio was 1.1 +/- 0.1 X 10(-5). In CCl4 (0.1 ml/100 g body wt.)-dosed rats, the liver PCOOH was 289 +/- 65 pmol/100 mg protein (PCOOH/PC = 2.4 +/- 0.4 X 10(-5], 764 +/- 271 pmol/100 mg protein (PCOOH/PC = 5.2 +/- 1.7 X 10(-5], and 856 +/- 165 pmol/100 mg protien (PCOOH/PC = 6.0 +/- 0.8 X 10(-5] at 6 h, 24 h, and 1 week after the dose, respectively. Under such conditions, the liver phosphatidylethanolamine hydroperoxide (PEOOH) level was not altered and the concentration was less than 100 pmol/100 mg protein even after the dose. The increments of liver PCOOH were suppressed 56% by the oral supplementation of DL-alpha-tocopherol (5 mg/100 g body wt./day) for a week before CCl4 administration. A relatively larger amount of PEOOH was found after stimulation of PC hydroperoxidation in the liver of rats with a large amount of CCl4 (0.25 ml/100 g body wt.) rather than with the small amount of CCl4 (0.1 ml/100 g body wt.).(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of delta-aminolevulinate: subcellular localization and purification of murine hepatic L-alanine: 4,5-dioxovaleric acid aminotransferase

1. L-Alanine: 4,5-dioxovaleric acid aminotransferase (DOVA transaminase) activity was measured in murine liver, kidney and spleen homogenates. 2. Among the organs examined, the specific activity of the enzyme was highest in kidney, followed by liver then spleen. 3. No differences in DOVA transaminase activity in kidney, liver and spleen homogenates were detected between mouse strains C57BL/6J and DBA/2J. 4. Based on enzyme activity, the capacity of DOVA transaminase to catalyze the formation of delta-aminolevulinic acid (ALA) in liver appeared much greater than the capacity of ALA synthase. 5. In DBA/2J animals, DOVA transaminase activity in liver mitochondrial fractions prepared by differential centrifugation was 24 nmol ALA formed/hr/mg protein compared with 0.63 nmol ALA formed/hr/mg protein for ALA synthase. 6. Cell fractionation analyses indicated that liver DOVA transaminase is located in the mitochondrial matrix. 7. The liver enzyme was purified from mitoplasts by chromatography on DEAE-Sephacel followed by affinity chromatography on L-alanine-AH-Sepharose. 8. The specific activity of the purified DOVA transaminase was 1600 nmol ALA formed/hr/mg protein. 9. The yield of the purification was ca 90 micrograms of protein per gram liver wet weight. 10. The purified enzyme had a subunit mol. wt of 146,000 +/- 5000 as determined by electrophoresis under denaturing conditions.     

O6-methylguanine-DNA methyltransferase activity in tissues and cells of the rat respiratory tract

A product of alkylating agents and DNA, O6-methylguanine (O6-MG), can mispair with thymine, resulting in initiation of a carcinogenic tissue response. O6-alkylguanine-DNA alkyltransferase (AGT) is an acceptor protein responsible for repairing O6-MG. The purpose of our experiments was to characterize in vitro AGT activity in tissues and cells in the respiratory tract, a target tissue for inhaled alkylating agents. Anatomically defined regions throughout the respiratory tract of male F344 rats were obtained. These included two regions of the lateral wall of the left and right nasal airway (maxilloturbinates and ethmoturbinates), trachea, extrapulmonary bronchi and peripheral lung. Alveolar type II cells were also used in these studies. Radioactive 3H-methylated DNA was synthesized for use in all experiments. Removal of [3H]methyl from O6-MG was measured by high-pressure liquid chromatography after incubation for up to 30 min of tissue and cell extracts with the [3H]DNA. With the exception of tracheal and bronchial extracts, all tissues and cells analyzed contained AGT activity, which was found to increase proportionally to the amount of protein added to reaction flasks. AGT activity in tracheal and bronchial extracts was only detected at the highest protein concentration used (1.5 mg protein/ml) and ranged from 10-15 fmol/mg protein. AGT activity was highest in the lung (integral of 75 fmol/mg protein) and a region of the nasal tissue, the ethmoturbinates (integral of 45 fmol/mg protein). AGT activity in the maxilloturbinates was about 50% less than the AGT activity measured in the ethmoturbinates. These data suggest that methylated DNA in specific regions of the rat respiratory tract should be readily repaired, albeit to different extents.     

Investigation of the relationship between nitric oxide metabolites' levels and adenosine deaminase activity in 7,12-dimethylbenz[a]anthracene induced mouse liver

7,12-Dimethylbenz[a]anthracene (7,12-DMBA) is a member of the polycyclic aromatic hydrocarbons with a severe carcinogenic effect. In this study, nitrate levels and ADA (Adenosine deaminase) activity in the liver homogenates of mice were measured and the effect of free radicals induced by 7,12-DMBA on inducible nitric oxide synthase (iNOS) and ADA activity were investigated. Antioxidant effects of melatonin were also compared. Three groups of mice were included in the study. The first served as control, the second was treated only with 7,12-DMBA and the third was treated with 7,12-DMBA + melatonin. Spectrophotometric methods were used at all measurements. Data were analyzed using Kruskal-Wallis Variance Analysis Test and Mann-Whitney U Test that were applied to the groups. The nitrate concentrations of mouse liver were as follows: 4.98 +/- 0.63 micro mol/L in the control group (n = 10), 8.23 +/- 1.58 micro mol/L (higher than control group, p < 0.05) in the 7,12-DMBA-treated group (n = 10), and 6.43 +/- 0.57 micro mol/L (lower than 7,12-DMBA-treated group, p < 0.05) in the 7,12-DMBA + melatonin-treated group (n = 10). Liver ADA activities were measured to be 4.14 +/- 0.674 U/L in the control group, 6.25 +/- 1.261 U/L (higher than control group, p < 0.05) in the 7,12-DMBA-treated group, and 4.93 +/- 0.916 U/L (lower than 7,12-DMBA-treated group, p < 0.05) in the 7,12-DMBA+melatonin-treated group. Differences between free nitrite levels were no significantly. Results demonstrated that nitrate levels and ADA activities were increased by means of free radicals induced by 7,12-DMBA. Melatonin inhibited the 7,12-DMBA induced increase that was observed in the activities of ADA enzyme and nitrate levels. It is concluded that determination of ADA activity and nitrate levels can be useful in the assessment of liver damage caused by toxic chemicals.     

Human wild-type alanine:glyoxylate aminotransferase and its naturally occurring G82E variant: functional properties and physiological implications

Human hepatic peroxisomal AGT (alanine:glyoxylate aminotransferase) is a PLP (pyridoxal 5'-phosphate)-dependent enzyme whose deficiency causes primary hyperoxaluria Type I, a rare autosomal recessive disorder. To acquire experimental evidence for the physiological function of AGT, the K(eq),(overall) of the reaction, the steady-state kinetic parameters of the forward and reverse reactions, and the pre-steady-state kinetics of the half-reactions of the PLP form of AGT with L-alanine or glycine and the PMP (pyridoxamine 5'-phosphate) form with pyruvate or glyoxylate have been measured. The results indicate that the enzyme is highly specific for catalysing glyoxylate to glycine processing, thereby playing a key role in glyoxylate detoxification. Analysis of the reaction course also reveals that PMP remains bound to the enzyme during the catalytic cycle and that the AGT-PMP complex displays a reactivity towards oxo acids higher than that of apoAGT in the presence of PMP. These findings are tentatively related to possible subtle rearrangements at the active site also indicated by the putative binding mode of catalytic intermediates. Additionally, the catalytic and spectroscopic features of the naturally occurring G82E variant have been analysed. Although, like the wild-type, the G82E variant is able to bind 2 mol PLP/dimer, it exhibits a significant reduced affinity for PLP and even more for PMP compared with wild-type, and an altered conformational state of the bound PLP. The striking molecular defect of the mutant, consisting in the dramatic decrease of the overall catalytic activity (approximately 0.1% of that of normal AGT), appears to be related to the inability to undergo an efficient transaldimination of the PLP form of the enzyme with amino acids as well as an efficient conversion of AGT-PMP into AGT-PLP. Overall, careful biochemical analyses have allowed elucidation of the mechanism of action of AGT and the way in which the disease causing G82E mutation affects it.     

In vitro phosphorylation and inactivation of rat liver acetyl-CoA carboxylase purified by avidin affinity chromatography

Acetyl-CoA carboxylase (EC 6.4.1.2) has been isolated from rat liver by an avidin-affinity chromatography technique. This preparation has a specific activity of 1.17 +/- 0.06 U/mg and appears as a major (240,000 dalton) and minor (140,000 dalton) band on SDS-polyacrylamide gel electrophoresis. Enzyme isolated by this technique can incorporate 1.09 +/- 0.07 mol phosphate per mol enzyme (Mr = 480,000) when incubated with the catalytic subunit of the cyclic AMP-dependent protein kinase at 30 degrees C for 1 h. The associated activity loss under these conditions is 57 +/- 4.0% when the enzyme is assayed in the presence of 2.0 mM citrate. Less inactivation is observed when the enzyme is assayed in the presence of 5.0 mM citrate. The specific protein inhibitor of the cyclic AMP-dependent protein kinase blocks both the protein kinase stimulated phosphorylation and inactivation of acetyl-CoA carboxylase. The phosphorylated, inactivated rat liver carboxylase can be partially dephosphorylated and reactivated by incubation with a partially purified protein phosphatase. Preparations of acetyl-CoA carboxylase also contained an endogenous protein kinase(s) which incorporated 0.26 +/- 0.11 mol phosphate per mol carboxylase (Mr = 480,000) accompanied by a 26 +/- 9% decline in activity. We have additionally confirmed that the rat mammary gland enzyme, also isolated by avidin affinity chromatography, can be both phosphorylated and inactivated upon incubation with the cyclic AMP-dependent kinase.     

Pyruvate Dehydrogenase Activity in Osmotically Shocked Rat Brain Mitochondria: Stimulation by Oxaloacetate

Pyruvate dehydrogenase complex activity (PDHC) measured by CO2 release isotopic assay has generally been much lower than activity measured by the spectrophotometric arylamine acetyltransferase assay (ArAT). Decarboxylation of [1-14C]pyruvate was measured in osmotically shocked rat brain cortical mitochondria. Activity is dependent on the concentration of the substrate pyruvate. Activity of 74.6 units +/- 12.3 SD (n = 22) was observed at 4 mM pyruvate (1 unit = 1 nmol pyruvate decarboxylated/min/mg protein). Activity was dependent on added NAD, CoA, and thiamine pyrophosphate, implying increased mitochondrial permeability after osmotic shock. Freeze/thaw with sonication of the mitochondrial preparation reduced PDHC activity to 11.5 units +/- 3.0 SD (n = 4). Oxaloacetate produced a marked stimulation of activity. The optimal assay contained 3 mM oxaloacetate, and without oxaloacetate activity fell to 15.4 units +/- 9.9 SD (n = 8). These studies highlight the importance of optimal substrate concentrations in the CO2 release isotopic PDHC method. Higher PDHC activity is found with intact mitochondria and thus activity values should be interpreted in the light of the presence or absence of intact mitochondria in individual preparations.