Adenine nucleotide metabolism in relation to purine enzymes in liver,erythrocytes and cultured fibroblasts

To evaluate the regulation of adenine nucleotide metabolism in relation to purine enzyme activities in rat liver, human erythrocytes and cultured human skin fibroblasts, rapid and sensitive assays for the purine enzymes, adenosine deaminase (EC 2.5.4.4), adenosine kinase (EC 2.7.1.20), hypoxanthine phosphoribosyltransferase (EC 2.4.28), adenine phosphoribosyltransferase (EC 2.4.2.7) and 5′-nucleotidase (EC 3.1.3.5) were standardized for these tissues. Adenosine deaminase was assayed by measuring the formation of product, inosine (plus traces of hypoxanthine), isolated chromatographically with 95% recovery of inosine. The other enzymes were assayed by isolating the labelled product or substrate nucleotides as lanthanum salts. Fibroblast enzymes were assayed using thin-layer chromatographic procedures because the high levels of 5′-nucleotidase present in this tissue interferred with the formation of LaCl₃ salts. The lanthanum and the thin-layer chromatographic methods agreed with-in 10%.Liver cell sap had the highest activities of all purine enzymes except for 5′-nucleotidase and adenosine deaminase which were highest in fibroblasts. Erythrocytes had lowest activities of all except for hypoxanthine phosphoribosyltransferase which was intermediate between the liver and fibroblasts. Erythrocytes were devoid of 5′-nucleotidase activity. Hepatic adenosine kinase activity was thought to control the rate of loss of adenine nucleotides in the tissue.Erythrocytes had excellent purine salvage capacity, but due to the relatively low activity of adenosine deaminase, deamination might be rate limiting in the formation of guanine nucleotides. Fibroblasts, with high levels of 5′-nucleotidase, have the potential to catabolize adenine nucleotides beyond the control of adenosine kinase. The purine salvage capacity in the three tissues was erythrocyte > liver > fibroblasts. Based on purine enzyme activities, erythrocytes offer a unique system to study adenine salvage; fibroblasts to study adenine degradation; and liver to study both salvage and degradation.     

The measurement of cyclic GMP and cyclic AMP phosphodiesterases

Methods are described for measuring phosphodiesterases for cGMP and cAMP in the range of activity yielding 10⁻¹² to 10⁻⁸ mol of product. The 5′-GMP formed is measured by conversion to GDP with guanylate kinase. Amounts of GDP greater than 10⁻¹⁰ mol are measured directly with an enzyme system which results in stoichiometric oxidation of NADH. This is either determined by the decrease in fluorescence or the excess NADH is destroyed with acid and the NAD⁺ measured by its fluorescence in strong NaOH. With smaller amounts of GDP, sensitivity is amplified 1000-fold with the succinic thiokinase-pyruvate kinase cycle. In the case of cAMP diesterase, larger amounts of 5′-AMP are measured in the same way as 5′-GMP, except that adenylate kinase is substituted for guanylate kinase. With smaller amounts, the 5′-AMP is converted to ATP, and sensitivity is amplified with the adenylate kinase-pyruvate kinase cycle. As little as 20 ng dry weight of average brain is sufficient for accurate assay of the diesterase activity toward either cAMP or cGMP. When there is danger of significant destruction of AMP or GMP by tissue 5′-nucleotidase, this is prevented by adding GMP to the cAMP reagent, AMP to the cGMP reagent, or 5′-UMP to either reagent.     

Activity of adenosine and thymidine metabolism enzymes in the blood of cancer patients of various ages

The activity of metabolic enzymes, adenosine and thymidine, has been studied in the blood serum and lymphocytes of healthy people and oncological patients aged 23-80. An increase in the activity of thymidine kinase (EC 2.7.1.2), an enzyme of thymidine biosynthesis, was observed in the blood serum of oncological patients against a background of a sharp decrease in the activity of thymidine phosphorylase (EC 2.4.2.4), a catabolic enzyme. The revealed enzymic shifts have been observed in breast cancer patients after 36, in patients with the stomach cancer--after 46. It is found that an increase in the activity of adenosine deaminase (EC 3.5.4.4) and 5-nucleotidase of AMP (EC 3.1.3.5) in the blood serum of oncological patients is accompanied by a sharp decrease in the activity of these enzymes in lymphocytes.     

Activities of enzymes concerned with pyruvate and oxaloacetate metabolism in the heart and liver of developing sheep.

1. In order to assess whether the potential ability of heart ventricular muscle and liver to metabolise substrates such as alanine, aspartate and lactate varies as the sheep matures and its nutrition changes, the activities of the following enzymes were determined in tissues of lambs obtained at varying intervals between 50 days after conception to 16 weeks after birth and in livers from adult pregnant ewes: lactate dehydrogenase (EC 1.1.1.27), alanine aminotransferase (EC 2.6.1.2), pyruvate kinase (EC 2.7.1.40), pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxykinase (GTP)(EC 4.1.1.32), malate dehydrogenase (EC 1.1.1.37), aspartate aminotransferase (EC 2.6.1.1) and citrate (si)-synthase (EC 4.1.3.7). 2. In the heart a most marked increase in alanine aminotransferase activity was found throughout development. During this period the activities of citrate (si)-synthase, lactate dehydrogenase and pyruvate carboxylase also increased. There were no substantial changes in the activities of aspartate aminotransferase, malate dehydrogenase or pyruvate kinase. Pyruvate kinase activities were five times greater in the heart compared with those found in the liver. No significant activity of phosphoenolpyruvate carboxykinase (GTP) was detected in heart muscle. 3. In the liver the activities of both alanine aminotransferase and aspartate aminotransferase increased immediately following birth although the activity of alanine aminotransferase was lower in livers of pregnant ewes than in any of the lambs. As with alanine aminotransferase the highest activities of lactate dehydrogenase were found during the period of postnatal growth. No marked changes were observed in malate dehydrogenase or citrate (si)-synthase activities during development. A small decline in pyruvate kinase activity occurred whilst the activities of pyruvate carboxylase and phosphoenolpyruvate carboxykinase (GTP) tended to rise during development.     

A novel mechanism of soluble guanylate cyclase stimulation: time-dependent activation by bacterial lipopolysaccharide in rat fetal spleen cells

Small amounts of bacterial lipopolysaccharide (LPS) greatly increase cGMP levels in short term cultures of rat fetal liver and spleen cells in a dose and time dependent manner. To determine the role of guanylate cyclase in this response, a series of experiments was undertaken using either intact or broken fetal spleen cells, the most sensitive tissue evaluated to date. The phosphodiesterase inhibitor, 1-methyl-3-isobutyl-xanthine, potentiated the LPS-cGMP effect in cultures of these cells even at maximal doses of LPS. Moreover, after incubation of intact cells with LPS for 4 h, soluble guanylate cyclase (EC 4.6.1.2) activity was increased 2-fold, whereas particulate activity was unchanged. This increase in soluble activity was proportional to the dose of LPS, was synchronous with the elevation of cGMP levels, and was not associated with any change in cGMP-phosphodiesterase (EC 3.1.4.17) activity. In contrast to intact cells, neither total nor soluble guanylate cyclase activity was increased by the addition of LPS to spleen cell whole sonicate or cytosol for various times from 10 min to 3.5 h. These results suggest that the LPS-cGMP response is due to a persistent indirect stimulation of soluble guanylate cyclase activity that is both dose and time dependent.     

Enzyme electrode-based kinetic assays of enzyme activities.

Potentiometric enzyme electrodes for urea, AMP, and adenosine have been used for the kinetic assays of arginase (EC 3.5.3.1.), 3′:5′-cyclic-nucleotide phosphodiesterase (EC 3.1.4.17), and 5′-nucleotidase (EC 3.1.3.5), respectively. The initial rate of potential change, after addition of the enzyme to the substrate solution, is direetly proportional to the enzyme activity present. Arginase assays were found to be reproducible to a relative precision of 13% or better and applicable to the direct measurement of arginase activity in beef liver homogenates.     

The effect of high protein diet on the regulatory properties of AMP deaminase from chicken liver

Feeding high protein diet for 5 days caused a 3,5-fold and 2-fold increase of the activity of xanthine dehydrogenase (EC 1.2.1.37) and 5-nucleotidase (EC 3.1.3.31) respectively, in chicken liver. Six hours after feeding the high protein diet there was no change in either enzyme activity although a 3-fold increase in the level of serum uric acid was observed. High protein diet considerably decreased the activity of AMP deaminase at low, but not at high substrate concentration. The activity ratio, measured at 10.0 and 0.16 mM AMP increased from 14:1 (low protein diet) to 23:1 and 24:1 after 6 h and 5 days of high protein diet, respectively. It has been suggested that feeding birds a high protein diet may cause transformation of liver AMP deaminase (EC 3.5.4.6) from a low Km form toward a high Km form.     

Enhanced uridine kinase and RNA synthesis in regenerating rat liver after 5-azacytidine administration

The administration of 5-azacytidine to partially hepatectomized rats results in the increase of uridine kinase activity in cell-free liver extracts 24–72 hr after drug administration. At the same time the activity of uridine phosphorylase and of uridine 5′-nucleotidase is decreased, while uridinemonophosphate kinase and uridine 5′-triphosphatase are not affected. The repeated administration of 5-azacytidine leads to a further enhancement of uridine kinase which is 6- to 8-fold higher in 96-hr regenerating livers than in untreated controls. Simultaneously the enhanced incorporation of uridine into liver ribonucleic acids was observed. The metabolic alterations occurring in the liver at later phases after 5-azacytidine in vivo administration are discussed.     

INHIBITION OF 5''-NUCLEOTIDASE IN RAT BRAIN BY METHYLXANTHINES

—Rat brain 5′-nucleotidase (EC 3.1.3.5) is inhibited by methylxanthines such as theophylline. Inhibition of the 5′-nucleotidase by theophylline appears more efficient than the inhibition of cAMP phosphodiesterase by this drug. A similar inhibition is observed with caffeine, theobromine, 7′-methyl-xanthine and 1-methylxanthine.     

Enzymic basis for cyclic GMP accumulation in degenerative photoreceptor cells of mouse retina.

The activities of guanylate cyclase, guanosine 3', 5'-monophosphate (cyclic GMP) phosphodiesterase and 5'-nucleotidase were measured during postnatal development in retinas of control and C3H/HeJ mice. In control retina, each of these enzyme activities increases in conjunction with photoreceptor cell differentiation and maturation. In C3H retina, guanylate cyclase and 5-nucleotidase activities increase with photoreceptor cell development and decrease with photoreceptor cell death. However, the activity of a class of cyclic GMP phosphodiesterase which distinguishes the photoreceptor cells of control mice and those of several other species is not demonstrable in retina of C3H mice at any age. It is suggested that the deficiency in cyclic GMP phosphodiesterase activity may account for the accumulation of cyclic GMP which has been shown to occur in the C3H photoreceptor cells before they degenerate.     

Relationship between changes in free cholesterol and pyrophosphomevalonate decarboxylase activity during myelination

The patterns of cholesterol content in chick brain and liver were studied during embryonic development and compared with the variations in the specific activities of mevalonate-activating enzymes during the same period. Total cholesterol content in both embryonic chick brain and liver increased during incubation. The relative percentage of free cholesterol was always maintained over 85% in brain, while in liver this percentage decreased to less than 10% during the later days of incubation. A straight parallelism was observed between free cholesterol and pyrophosphomevalonate decarboxylase activity in the embryonic brain. On the other hand, the hepatic decarboxylase exhibited a lower specific activity than in brain and did not show significant variations throughout the same period of incubation. Changes in brain pyrophosphomevalonate decarboxylase activity were more pronounced than those observed in both mevalonate kinase and phosphomevalonate kinase activities, in spite of that the specific activity of decarboxylase was the lowest of the three mevalonate-activating enzymes, suggesting that this reaction is one rate-limiting step for cholesterogenesis during myelination.     

Activation of guanylate cyclase in cerebral cortex of rat by hydroxylamine.

Hydroxylamine actived guanylate cyclase in particulate fraction of cerebral cortex of rat. Activation was most remarkable in crude mitochondrial fraction. When the crude mitochondrial fraction was subjected to osmotic shock and fractionated, guanylate cyclase activity recovered in the subfractions as assayed with hydroxylamine was only one-third of the starting material. Recombination of the soluble and the particulate fractions, however, restored guanylate cyclase activity to the same level as that of the starting material. When varying quantities of the particulate and soluble fractions were combined, enzyme activity was proportional to the quantity of the soluble fraction. Heating of the soluble or particulate fraction at 55 degrees for 5 min inactivated guanylate cyclase. The heated particulate fraction markedly activated guanylate cyclase activity in the native soluble fraction, while the heated soluble fraction did not stimulate enzyme activity in the particulate. The particulate fraction preincubated with hydroxylamine at 37 degrees for 5 min followed by washing activated guanylate cyclase activity in the soluble fraction in the absence of hydroxylamine. Further fractionation of the crude mitochondrial fraction revealed that the factor(s) needed for the activation by hydroxylamine is associated with the mitochondria. The mitochondrial fraction of cerebral cortex activated guanylate cyclase in supernatant of brain, liver, or kidney in the presence of hydroxylamine. The mitochondrial fraction prepared from liver or kidney, in turn, activated soluble guanylate cyclase in brain. Activation of guanylate cyclase by hydroxylamine was compared with that of sodium azide. Azide activated guanylate cyclase in the synaptosomal soluble fraction, while hydroxylamine inhibited it. The particulate fraction preincubated with azide followed by washing did not stimulate guanylate cyclase activity in the absence of azide. The activation of guanylate cyclase by hydroxylamine is not due to a change in the concentration of the substrate GTP, Addition of hydroxylamine did not alter the apparent Km value of guanylate cyclase for GTP. Guanylate cyclase became less dependent on manganese in the presence of hydroxylamine. Thus the activation of guanylate cyclase by hydroxylamine is due to the change in the Vmax of the reaction.     

Increased responsiveness of the hepatic guanylate cyclase-guanosine 3',5'-monophosphate system to nitrosoguanidine following partial hepatectomy

When tested at concentrations producing submaximal responses, the N-nitroso carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (methylnitro-nitrosoguanidine) elicited a 2-fold greater increase in guanosine 3',5'-monophosphate (cyclic GMP) accumulation in slices and a 5-fold greater stimulation of guanylate cyclase activity in whole homogenates of rat liver examined 24 h after 75% hepatectomy compared to the corresponding methylnitro-nitrosoguanidine responses in sham-operated and unoperated controls. Enhanced methylnitro-nitrosoguanidine sensitivity of guanylate cyclase in whole homogenates of regenerating liver was attributable to altered responsiveness of the enzyme activity of the 100 000 X g soluble fraction, which contained 98% of the methylnitro-nitrosoguanidine responsive activity. Basal cyclic GMP accumulation and guanylate cyclase activities of these systems, and their responses to concentrations of methylnitro-nitrosoguanidine eliciting maximal stimulation were unchanged after partial hepatectomy or sham operation, compared to unoperated controls. The findings of (a) increased heme concentrations in the supernatant and the high molecular weight Sephadex G-25 fraction of sham operated, compared to regenerating liver, (b) suppression of methylnitro-nitrosoguanidine responsive activity after addition of exogenous hemoglobin to supernatants from regenerating liver, and (c) enhancement of the responsiveness of soluble guanylate cyclase from sham operated liver to submaximal methylnitro-nitrosoguanidine after reduction of endogenous heme content by in situ perfusion, all suggested that the difference in methylnitro-nitrosoguanidine action observed in control vs. regenerating liver are related to a lower heme-protein content of the latter. These results emphasize the importance of endogenous heme as a factor modulating the response of the hepatic guanylate cyclase system to methylnitro-nitrosoguanidine.     

The synthesis and turnover of 5′-nucleotidase in primary cultured hepatocytes

The synthesis and degradation of 5′-nucleotidase has been studied in rat hepatocytes. Primary cultures of rat hepatocytes were established with the cells showing evidence of polarity after 24–36 h in culture. After a 30 h lag period 5′-nucleotidase activity increased to a plateau level similar to the activity found in whole liver. The half life of the enzyme after reaching the plateau of activity was 22.8 h. Pulse-chase biosynthetic labelling studies of 5′-nucleotidase in the cultured hepatocytes using [³⁵S]methionine showed that the 5′-nucleotidase monomer was synthesised as an M_r 67 000 form which was converted to the mature M_r 72 000 form. [³⁵S]Methionine labelling studies in the presence of tunicamycin showed that the unglycosylated protein monomer was an M_r 57 000 form. The immature M_r 67 000 form of 5′-nucleotidase was sensitive to endoglycosidase H, whereas the mature form was sensitive only to endoglycosidase F. The data presented are consistent with 5′-nucleotidase in a polarised cell being synthesised and processed like other membrane glycoproteins, in contrast to earlier reports.     

Relationship between 5'-nucleotidase, adenosine deaminase, AMP deaminase, ATP-(Mg2+)-ase activities and dTMP kinase activity in rat liver mitochondria.

The activities of dTMP kinase (ATP-deoxythymidine monophosphate phosphotransferase, EC 2.7.4.9), 5'-nucleotidase (5'-ribonucleoside phosphohydrolase, EC 3.1.3.5), adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), AMP deaminase (AMP aminohydrolase, EC 3.5.3.6) and ATP-(Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3) were assayed in mitochondria of normal and regenerating rat liver. In regenerating mitochondria, the dTMP kinase activity increased 20 times, 5'-nucleotidase (5'Nase) activity for dTMP diminished by 65% and its activity for other nucleoside monophosphates did not change; adenosine deaminase activity for adenosine (AR) increased by 40%, but for deoxyadenosine (AdR) decreased by 70%. AMP deaminase and ATP-(Mg2+)-ase activities behaved similarly in mitochondria from regenerating liver, decreasing by 70 and 64% respectively. The changes of the amount of dTMP in mitochondria depend on enzyme activities which regulate the AdR concentration.     

Seizure susceptibility in the developing mouse and its relationship to glutamate decarboxylase and pyridoxal phosphate in brain.

The relationship between the susceptibility to convulsions, the content of pyridoxal 5'-phosphate and the activity of pyridoxal kinase (EC 2.7.1.35) and glutamate decarboxylase (EC 4.1.1.15) in brain, was studied in the developing mouse. Seizures were induced by pyridoxal phosphate-gamma-glutamyl hydrazone (PLPGH), a drug previously reported to reduce the levels of pyridoxal 5'-phosphate and as a consequence to inhibit the activity of glutamate decarboxylase in brain of adult mice. It was found that the seizure pattern, as well as the time of appearance of convulsions, differed between 2- and 5-day old mice and 10-day old or older mice, indicating a progressive increase in seizure susceptibility during development. In brain, pyridoxal kinase activity and pyridoxal 5'-phosphate levels were decreased by the administration of PLPGH at all ages studied, whereas glutamate decarboxylase activity was inhibited less than 25% in 2- and 5-day old mice, and about 50% thereafter. Parallelly, the activation of glutamate decarboxylase by pyridoxal 5'-phosphate added in vitro to control homogenates was less in 2- and 5-day old mice than in older animals. It is concluded that the increase in the susceptibility to seizures induced by PLPGH during development is probably related to the increase observed in the sensitivity of glutamate decarboxylase in vivo to a decrease of pyridoxal 5'-phosphate levels. The correlation between pyridoxal 5'-phosphate, glutamate decarboxylase, and seizure susceptibility seems to be established at about 10 days of age.     

Activation of liver guanylate cyclase by bile salts and contaminants in crude secretin and pancreozymin preparations.

Crude preparations of secretin or pancreozymin increased and at higher concentrations decreased guanylate cyclase (GTP pyophosphate-lyase, EC 4.6.1.2) activity from soluble and particulate fractions of rat liver homogenates. Partially purified and synthetic secretin were without effect as was the biologically active octapeptide fragment of pancreozymin. The active contaminants in these preparations survived boiling, saponification, and treatment with phospholipase A, trypsin and neuraminidase C. The activity was extractable with chloroform/methanol and did not survive ashing. Eight bile salt contaminants in crude secretin were obtained with thin-layer chromatography. Two of the contaminating bile salts that increased liver particulate guanylate cyclase activity were identified as taurodeoxycholate and either glycochenodeoxycholate or glycodeoxycholate; taurocholate was inhibitory. The sodium salts of cholate, deoxycholate, chenodeoxycholate and their glycine-or taurine-conjugated forms either increased or decreased particulate and soluble rat liver guanylate cyclase activity depending upon their concentration. Thus, the previously reported stimulatory and inhibitory effects of secretin and pancreozymin preparations on guanylate cyclase activity are probable attributable to their bile salt contaminants.     

Ectonucleotidase and acetylcholinesterase activities in silver catfish (Rhamdia quelen) exposed to different salinities

The purpose of this study was to investigate whether salinity adaptation can alter the purinergic (ecto-nucleoside triphosphate diphosphohydrolase; NTPDase and, 5′-nucleotidase) and cholinergic (acetylcholinesterase; AChE) systems in whole brain and blood tissue of the silver catfish, Rhamdia quelen. Silver catfish were gradually adapted to salinities of 0, 4 or 8 ppt and maintained at the experimental salinity for 10 days before brain and blood samples were collected. Blood AChE activity decreased significantly at 8 ppt and significant decreases in AChE activity were observed in the brain with salinity increases. ATP hydrolysis did not change between the groups. In contrast, ADP and AMP hydrolysis in silver catfish maintained at salinities of 4 and 8 ppt were significantly higher than those kept at 0 ppt. In conclusion, this study showed that there is an enhancement in the NTPDase (ADP hydrolysis) and 5′-nucleotidase activities in the brains of silver catfish exposed to increased salinity. Therefore, the activities of these enzymes can act as markers of salinity changes.     

Ontogeny of cyclic AMP-dependent protein phosphokinase during hepatic development of the rat.

The ontogeny of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) and cyclic AMP-binding activity in subcellular fractions of liver was examined during prenatal and postnatal development of the male rat. 1. Protein kinase activity and cyclic AMP-binding activity were found in the nuclear, microsomal, lysosomal-mitochondrial, and soluble liver fractions. 2. The protein kinase activity of the soluble (105 000 X g supernatant) fraction measured with histone F1 as substrate was stimulated by cyclic AMP. Cyclic AMP did not stimulate the protein kinase activity of the particulate fractions. 3. The protein kinase activity of all subcellular fractions increased rapidly from the activity observed in prenatal liver (3-4 days before birth) to reach maximal activity in 2-day-old rats. Thereafter, the protein kinase activity declined more slowly and regained the prenatal levels at 10 days after birth. 4. Considerable latent protein kinase activity was associated with liver microsomal fractions which could be activated by treatment of microsomes with Triton X-100. The latent microsomal protein kinase activity was highest in prenatal liver, at the time of birth, and 2 days after birth. During the subsequent postnatal development the latent microsomal protein kinase activity gradually declined to insignificantly low levels. 5. During the developmental period examined (4 days before birth to age 60-90 days) marked alterations of the cyclic AMP-binding activity were determined in all subcellular fractions of rat liver. In general, cytosol, microsomal, and lysosomal-mitochondrial cyclic AMP-binding activity was highest in 10-11 day-old rats. Nuclear cyclic AMP-binding activity was highest 3-4 days before birth and declined at birth and during the postnatal period. There was no correlation between the developmental alteration of cyclic AMP-binding activity and cyclic AMP dependency of the protein kinase activity in any of the subcellular fractions. This suggests that the measured cyclic AMP-binding activity does not reflect developmental alterations of the cyclic AMP-binding regulatory subunit of cyclic AMP-dependent protein kinase.