Effect of benzodiazepines on 5'-nucleotidase activity in rat brain

Experiments on 330 rats were made to study the influence of benzodiazepines (diazepam, dormicum and phenazepam) on 5'-nucleotidase activity in brain homogenates. It was discovered that diazepam and dormicum in doses of 3 and 4 mg, phenazepam in doses of 3.75 and 5 mg per 200 g bw provoked a 16-20% reduction in 5'-nucleotidase activity. The maximal effect of diazepam (3 and 4 mg doses) was attained 1 h after intraperitoneal injection, that of dormicum (3 mg) 30 min and of phenazepam (5 mg) 1 h after intraperitoneal injection. It is assumed that benzodiazepines are involved in AMP metabolism.     

A simple microradioisotopic assay for 5'-nucleotidase activity. Application to central nervous tissues

A radiochemical method for measurement of 5′-nucleotidase is described in which the product, [¹⁴C]adenosine passes through a small alumina column and is separated from substrate 5′-AMP, which is completely retained. Optimal conditions for measurement of 5′-nucleotidase in particulate preparations of brain and spinal cord are described.     

Reduction of 5'-nucleotidase activity in rat thyroid and adenohypophysis following methylthiouracil treatment.

5' -Nucleotidase activity was determined in rat thyroid and some other organs employing a specific assay method. During the course of methylthiouracil (MTU) treatment, thyroid 5'-nucleotidase activity decreased significantly. This decrease was specific for this enzyme since the activity of neutral phosphatase did not change and the activity of alkaline phosphatase and Mg2+-activated adenosine triphosphatase increased markedly. The 5'-nucleotidase activity of the adenohypophysis also decreased following MTU treatment. This enzyme activity of the liver, heart and whole brain remained unchanged after the treatment. The role of this enzyme was discussed in relation to tissue growth and increased contents of RNA and DNA in the thyroid and adenohypophysis.     

Diurnal variation in 5'-nucleotidase activity in rat liver. A quantitative histochemical study

The diurnal variation of 5'-nucleotidase activity in periportal and pericentral areas of rat liver parenchyma has been determined with quantitative histochemical means. 5'-Nucleotidase activity was estimated using microdensitometry in cryostat sections after being incubated with a medium according to Wachstein and Meisel (1957). It appeared that 5'-nucleotidase activity was significantly higher in pericentral areas than in periportal areas throughout the daily cycle and showed a maximum at the end of the light period. It was concluded that 5'-nucleotidase activity may be related with the capacity to diminish messenger RNA resulting in protein breakdown.     

Histochemical demonstration of phospholipase B (lysolecithinase) activity in rat tissues.

A method has been developed for the histochemical demonstration of phospholipase B (lysolecithinase) of rat tissues. The enzyme attacks lysolecithin with liberation of 1 mole of glycerylphosphorylcholine and 1 mole of fatty acid. The recommended procedure involves use of 6-10 micro frozen sections, fixed in cold calcium-formol and incubated at 37 degrees C in Tris buffered medium at pH 6.6 containing 2.2 X 10(-3) M lysolecithin and 1% cobalt acetate. The fatty acid liberated by enzymatic hydrolysis is trapped as a cobalt precipitate and is then converted to a black-brown precipitate by treatment with dilute ammonium sulfide in cold isotonic saline. Equivalent amounts of fatty acid and glycerylphosphorylcholine are recovered by extraction and analysis of the incubated sections and of the incubation medium, thus proving that lysolecithin hydrolysis occurs under the proposed reaction conditions. Staining is reduced by treating the sections with copper ions, mercury compounds, alcohols, acetone and by heating at 60 degrees C prior to incubation with substrate. Lowering of the pH of the incubation medium has similar effect. These findings are interpreted as evidence of the enzymatic nature of the reaction. Cells exhibiting a positive staining are found in the lamina propria of the intestinal villi and crypts, in the red pulp of the spleen and in the interstitial tissue of lung, liver and thymus. Similar elements are present in bone marrow smears and in leukocyte preparations obtained by peritoneal lavage. The morphologic and staining characteristics of these cells correspond to those of the eosinophilic leukocytes. Physical and chemical agents (x-irradiation, corticosteroids) which sharply decrease the number of eosinophils also reduce the number of cells shown histochemically to hydrolyze lysolecithin. A correspondent diminution of phospholipase B activity of homogenates of the same tissues can be shown in vitro. Differences in tissue distribution and chemical properties distinguish the phospholipase B from less specific esterases and lipases.     

5'-nucleotidase activity in developing chick pectoral muscle.

The activity of the plasma membrane enzyme 5′-nucleotidase varies dramatically during the embryonic development of chick pectoral muscle. The specific activity is greatest at early stages of differentiation (8-day embryos), falls to a minimum on days 12–14, then rises again in older embryos. In cultured muscle cells obtained from embryonic chick muscle the 5′-nucleotidase activity is essentially absent. Muscle cells grown in the presence of bromodeoxyuridine, an inhibitor of muscle differentiation, contain enhanced levels of 5′-nucleotidase activity. These results indicate that 5′-nucleotidase may be absent in muscle fibers, but present in other cells of muscle tissue.     

A distinctive activity of 5'-nucleotidase for dTMP in rat liver mitochondria

The activities of 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 5'-Nucleotidase (5'Nase) and ATP-(Mg2+)-ase activities were compared with similar enzyme activities in the plasma membrane (PM) fraction, obtained from the same biological material. In the regenerating liver, 5'Nase for dTMP diminished its activity by 56% (24 h after partial hepatectomy) and 35 +/- 4% for all substrates in the PM fraction (48 h after operation). In mitochondria, 5'Nase for dTMP manifests sigmoidal substrate activity curve (in contrast with all substrates in the PM fraction and remaining substrates in mitochondria). In vivo 5-azacytidine (a) administered 1 h after partial hepatectomy, prevented changes of 5'Nase activity: (b) administered 24 or 48 h after partial hepatectomy, stabilized low 5'Nase activity (in mitochondria for dTMP, in the PM fraction for all substrates) and decreased ATP-(Mg2+)-ase activity by 51 and 31% in mitochondria and the PM fraction respectively.     

A simple, specific radiometric assay for 5'-nucleotidase.

A radiometric assay for 5′-nucleotidese (EC 3.1.3.5) has been developed, which is applicable for all 5′-nucleotide substrates. Various column materials and eluants were evaluated for their suitability in the separation of purine and pyrimidine bases and nucleosides produced in the reaction. Neutral alumina columns were found to be the best. The unadsorbed nucleosides and their bases could be quantitatively eluted with 0.1 m Tris-HCl, pH 7.4; subsequent elution of the 5′-nucleotide was then accomplished with 0.2 m sodium phosphate, pH 7.4. Differential measurement of 5′-nucleotidase can be accomplished in the presence of acid or alkaline phosphatases by inclusion of concanavalin A into the reaction mixture. It completely inhibits 5′-nucleotidase without effecting the phosphatases. The applicability of this assay has been demonstrated by studying the properties of 5′-nucleotidase present in a purified plasma membrane preparation from a rat tumor which is enriched with both 5′-nucleotidase and alkaline phosphatase.     

Acetoacetyl-CoA synthetase specific activity and concentration in rat tissues

An antibody against acetoacetyl-CoA synthetase purified from rat liver was raised in rabbits. Utilizing the binding of antibody-antigen complexes to a nitrocellulose membrane, a sensitive enzyme-linked immunosorbent assay was developed to estimate the enzyme concentration in rat tissues. The enzyme concentration (microgram immunoreactive protein/mg protein) in rat liver cytosol was increased about 3-, 1.8- and 7-fold by feeding rats diets containing 5% cholestyramine, 0.2% ML-236B (compactin), and 5% cholestyramine plus 0.2% ML-236B for 4 days, respectively, and decreased about 1.8-fold by fasting the animals or 1.3-fold by feeding them a diet containing 5% cholesterol. Changes in the enzyme activity were almost parallel to those in the enzyme concentration, suggesting the physiological role of this enzyme in cholesterol biosynthesis. Immunoblotting of the hepatic cytosol also confirmed that the increase in enzyme concentration on cholestyramine and/or ML-236B feeding was due to an increase in an enzyme protein the same as the purified enzyme and not the isozymic protein. Among various rat tissues examined, the concentrations of immunologically crossreactive enzyme were higher in lipogenic tissues, such as brain, adipose tissue and liver, than in other tissues. The enzymes in these three tissues were identical in molecular weight determined by gel filtration and immunoblotting.     

Monoclonal antibodies against 5'-nucleotidase from chicken gizzard. Evidence for species and tissue specific differences of 5'-nucleotidase

5'-Nucleotidase from chicken gizzard smooth muscle was purified to homogeneity and used as immunogen for generating monoclonal antibodies. From about 150 positive clones nine IgG producing hybridoma cell lines have been selected for further characterization and antibody preparation. The resulting antibodies bind 5'-nucleotidase from chicken smooth muscle, chicken skeletal muscle, and chicken heart muscle but not the enzyme from chicken liver or rat liver. It could clearly be demonstrated that the nine antibodies recognize different antigenic determinants. Four of these antibodies are strong inhibitors of the AMPase activity of 5'-nucleotidase. One antibody is a weak inhibitor and four other antibodies have no effect on its enzymic activity. One of the monoclonal antibodies was used for immunoaffinity purification of 5'-nucleotidase from chicken heart muscle and chicken skeletal muscle. Pure and active enzymes could be isolated from detergent extracts in one step with a 10 to 20-fold higher yield compared to classical purification procedures. The subcellular distribution of 5'-nucleotidase in chicken gizzard was investigated using indirect immunofluorescence. We found a staining of the plasma membrane of smooth muscle cells and endothelial cells by all of the nine antibodies with variations in the staining intensity.     

A simple, specific, radioisotopic assay for 5'-nucleotidase.

A method is presented using [¹⁴C]5′-AMP as a substrate for measuring 5′-nucleotidase activity in the presence of interfering phosphatases. An inhibitor of 5′-nucleotidase, α,β-methyleneadenosine diphosphate is utilized, and the enzyme activity is measured as the difference between total phosphatase activity and inhibitor-insensitive activity.     

Properties of 5'-nucleotidase in rat heart sarcolemma

The activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was examined in membrane fractions isolated by hypotonic shock-LiBr treatment (fraction HL) and sucrose gradient separation (fraction S) of rat ventricle homogenate. The enzyme activity in these two fractions differed significantly in several respects. In fraction HL, 5'-nucleotidase had a high affinity for AMP (Km 35 microM), and ATP was a potent competitive inhibitor. In contrast, the 5'-nucleotidase displayed by fraction S showed a low substrate affinity (Km 130 microM) and less sensitivity to ATP. Treatment of membranes with trypsin and neuraminidase markedly stimulated 5'-nucleotidase in fraction HL, whereas only a modest effect was observed in fraction S. Exposure of the membranes to Triton X-100 resulted in a 60% and 10% increase in the enzyme activity in fractions HL and S, respectively. The characteristic activity ratios of 5'-nucleotidase at 200 microM relative to 50 microM AMP in fractions HL and S were modified by alamethicin in an opposite way and became identical. Although concanavalin A almost completely inhibited the 5'-nucleotidase activity in both membrane preparations at a concentration of 2 microM, Hill plots of the data on concanavalin A inhibition revealed a coefficient of 2.2 for fraction S and 1.1 for fraction HL. The differences in 5'-nucleotidase activity of the two membrane fractions are considered to be due to differences in the orientation of the vesicles of the sarcolemmal preparations. These results suggest that two distinct catalytic sites for 5'-nucleotidase are present at the intra- and extracellular surface of the rat heart sarcolemma.     

Stimulation by glycerate 2,3-bisphosphate: a common property of cytosolic IMP-GMP 5'-nucleotidase in rat and human tissues

Glycerate 2,3-bisphosphate, a potent stimulator of the cytosolic 5'-nucleotidase which preferentially hydrolyzes IMP and GMP in human erythrocytes (Bontemps et al., 1988, Biochem. J. 250, 687-696), also stimulates the dephosphorylation of IMP in cytosol fractions of rat heart, liver, brain, kidney, spleen and erythrocytes, and of human polymorphonuclear leucocytes, mixed peripheral blood lymphocytes, platelets and fibroblasts. Depending on the cell type, stimulation by 5 mM glycerate 2,3-bisphosphate varied from 1.5- to 12-fold. Where investigated, glycerate 2,3-bisphosphate had an approx. 5-fold higher affinity for the enzyme than its other stimulator, ATP. These observations provide a useful tool to distinguish IMP-GMP 5'-nucleotidase from other 5'-nucleotidases, and suggest a common origin of the cytosolic IMP-GMP 5'-nucleotidase in various tissues.