IMP-GMP 5'-nucleotidase in reptiles: occurrence in a turtle,a tortoise and three species of snakes

IMP-hydrolyzing activity, which is reactive with goose anti-pig lung IMP-GMP 5′-nucleotidase (c-N-II: EC.3.1.3.5) serum, was detected in extracts from liver, heart, kidney, spleen, stomach, skeletal muscle and lung from several species of reptiles: The values found in liver (U/mg protein) of one animal were: 4.5 for an ammono-ureotelic turtle (Trionyx sinensis japonicus); 3.7 for an ureo-uricotelic tortoise (Testudo elegans); 13–23 for three species of uricotelic snakes: Elaphe quadrivirgata, Elaphe conspicillata and Elaphe climacophora. These findings suggest that in the liver of snakes, c-N-II may participate in the production of uric acid as an end product of amino acid metabolism.     

Occurrence of IMP-GMP 5'-nucleotidase in three fish species: a comparative study on Trachurus japonicus,Oncorhynchus masou masou and Triakis scyllium

IMP-hydrolyzing activity, which is reactive with goose anti-pig lung IMP-GMP 5'-nucleotidase (EC.3.1.3.5) serum, was detected in extracts from various tissues of Trachurus japonicus (a marine teleost), Oncorhynchus masou masou (a freshwater teleost) and Triakis scyllium (an elasmobranch). Kinetic characteristics of the reactive enzymatic activity were similar to those of IMP-GMP 5'-nucleotidase from mammals and birds. In all species studied, the activity was highest in the liver (4-6 micromol of Pi released from IMP/min/mg of protein). The second highest activity was observed in the head portion of Oncorhynchus kidney (4 micromol of Pi released from IMP/min/mg of protein), which was twofold higher than that of its body portion. In all three species, the activity was lowest in white skeletal muscle among the tissues studied (0.1-0.3 micromol of Pi released from IMP/min/mg of protein), while the activity in red skeletal muscle was sixfold to 10-fold higher than that in white muscle.     

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.     

A comparative study on tissue distribution and metabolic adaptation of IMP-GMP 5'-nucleotidase.

1. Activity of a cytoplasmic 5'-nucleotidase which preferentially hydrolyzes IMP and GMP (IMP-GMP 5'-nucleotidase) was determined by a specific immunochemical method in two species of birds and two species of mammals. 2. The activity was markedly high in avian liver, and it increased two-fold in response to a high protein diet in chicken liver. 3. In mammals, the activity was high in testis and spleen. In the rat, the activities in liver, kidney and heart extracts increased by about 30% in response to the high protein diet, while they increased three-fold in regenerating liver. 4. Low activities were detected in skeletal muscles and in erythrocytes of all the species studied.     

Characterization of the membrane proteins of rat liver lysosomes. Composition, enzyme activities and turnover.

Lysosomes prepared from the livers of untreated rats and from the livers of rats injected with either Triton WR-1339 or dextran yielded membranes that were similar in both polypeptide composition and activities of ATPase and acid 5'-nucleotidase. The administration of Triton WR-1339 (and dextran) resulted in an increase in ATPase activity of liver homogenates that was associated with a parallel increase in the ATPase activity of the lysosomal membrane. On the other hand, plasma membranes appear to be different from lysosomal membranes with respect to polypeptide composition and enzyme activities. The ATPase activity of lysosomal membranes is not affected by ouabain and suramin, inhibitors of the plasma-membrane ATPase. The plasma-membrane alkaline 5'-nucleotidase has little activity at acid pH. Pulse-labelling of lysosomal membranes with [3H]fucose and with [3H]- and [14C]-leucine occurred rapidly, faster than labelling of plasma membranes. The labelling kinetics indicate that lysosomal membranes may be assembled independently of plasma membranes. These data suggest that, in liver, little bulk transport of plasma membrane to lysosomes takes place, and lysosomal-membrane proteins may not be derived from those of plasma membranes.     

Arginase isoforms in frog and lizard tissues

Isoforms of arginase in the liver and kidney tissues of the ureotelic frog (Rana tigerina) and uricotelic lizard (Calotes versicolor) were fractionated by DEAE-cellulose chromatography (pH 8.3). Four molecular forms, designated as A'1, A2, A3 and A4 based on the KCl concentration required for their elution from the ion-exchange column, were detected in lizard liver, while only two forms were found in lizard kidney (A3 and A4) and frog liver and kidney (A2 and A3). No major differences were found in the pH optimum, substrate affinity and molecular weight of the isoenzymes. The isoforms in lizard tissues were either totally unaffected or only partially immunoprecipitated by antibodies raised against rat liver and beef liver arginases, but those in frog tissues were significantly activated by the two antibodies. While the physiological importance of the presence of four isoforms in lizard liver remains enigmatic, different sets of isoenzymes were present in the liver of the two ureotelic vertebrates, rat and frog. Hence, it appeared that a given mode of nitrotelism was not associated with a specific set of isoenzymes. Also, the data were not consistent with the generally held view that a basic isoform of arginase served as a component of the urea cycle in liver and a neutral/slightly acidic form functions in the synthesis of proline, glutamate and polyamines in extra-hepatic tissues. The isoforms appeared to show considerable functional overlap.     

The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5''-nucleotidase.

An alkaline 5'-nucleotidase with properties similar to those of membrane-bound 5'-nucleotidase was recovered in soluble form in the postmicrosomal supernatant fraction (cytosol) of rat liver. The enzyme seems to constitute a quantitatively distinct fraction, since the activity in postmicrosomal supernatants was increased by a further 10% by additional homogenization of livers. Lysosomal acid phosphatase activity increased similarly, whereas other membrane-bound marker enzymes alkaline phosphatase, phosphodiesterase I and glucose-6-phosphatase showed no increase when homogenization of liver tissue was continued. Gel-permeation chromatography and pH-dependence studies indicated that enzyme activity in the supernatant fraction with 0.3 mM-UMP or -AMP as substrate at pH 8.1 was about 85 or 100% specific respectively. In regenerating liver the enzyme recovered in soluble form showed decreased specific activity, in contrast with alkaline phosphatase measured for comparison. The nucleotidase activity per mg of cytosolic protein was 2.1 nmol/min with AMP as substrate. The total activity measured in the postmicrosomal supernatant was 1.5% of the homogenate activity measured in the presence of detergent.     

Two forms of AMP deaminase from the lizard (Lacerta agilis) liver

Two molecular forms of AMP deaminase have been revealed by phosphocellulose column chromatography in the liver of uricotelic lizard. The calculated S0.5 value of the purified lizard liver AMP deaminase was 2.5 +/- 0.1 for the form I and 3.6 +/- 0.4 for the form II. Both forms of the enzyme were activated by ATP and ADP but the form II to a much higher extent. GTP activated only the form II and inorganic phosphate inhibited both forms. The occurrence of multiple forms of liver AMP deaminase in uricotelic species, as well as its difference from the mammalian enzyme regulation by GTP is suggested to be connected with the uricotelism in these animals.     

The subcellular location, maturation and response to increased plasma glucagon of Ruthenium Red-insensitive calcium-ion transport in rat liver

1. The subcellular distribution and maturation of Ruthenium Red-insensitive Ca(2+) transport activity were determined in livers of rats ranging in age from 3 days pre-term to 10 weeks of adult life and compared with those of glucose 6-phosphatase, 5'-nucleotidase and Ruthenium Red-sensitive Ca(2+) transport. Initial rates of Ruthenium Red-insensitive Ca(2+) transport were highest in those fractions enriched in glucose 6-phosphatase, i.e. the microsomal fraction; this fraction was devoid of Ruthenium Red-sensitive Ca(2+) transport activity. Although the heaviest fraction (nuclear) contained significant amounts of 5'-nucleotidase activity it was devoid of Ruthenium Red-insensitive Ca(2+) transport activity. 2. Foetal rat liver contain minimal amounts of Ruthenium Red-insensitive Ca(2+) transport activity, glucose 6-phosphatase and 5'-nucleotidase activities. These begin to be expressed concomitantly soon after birth; Ruthenium Red-insensitive Ca(2+) transport is maximal by 3 to 4 days and remains so for up to at least 10 weeks of adult life. Glucose 6-phosphatase also reaches a peak at 3-4 days, but then rapidly decreases to approach adult values. Maximal activity of 5'-nucleotidase in the microsomal and nuclear fractions is seen about 4-6 days after birth; this enzyme activity remains increased for up to about 10 days and then falls, but not as rapidly as glucose 6-phosphatase. It is tentatively suggested that the bulk of the Ruthenium Red-insensitive Ca(2+) transport is attributable to the system derived from the endoplasmic reticulum. 3. Administration of glucagon to adult rats enhances by 2-3-fold the initial rate of Ruthenium Red-insensitive Ca(2+) transport in the intermediate but not the microsomal fraction. The hormone-induced effect is fully suppressed by co-administration of puromycin, is dose-dependent with half-maximal response at approx. 1mug of glucagon/100g body wt. and time-dependent exhibiting a half-maximal response about 1h after administration of the hormone. 4. Ruthenium Red-insensitive Ca(2+) transport in the post-mitochondrial fraction of foetal liver also responds to the administration in situ of glucagon. The response, which also is prevented by co-administration of puromycin, is maximal in those foetuses nearing term. The suggestion is made that these effects of the hormone on Ruthenium Red-insensitive Ca(2+) transport are an integral part of the physiological network in the liver cell.     

Isolation and partial characterization of a bile canalicular plasma membrane fraction from normal and regenerating rat liver.

A bile canalicular membrane fraction was isolated from 24-hour regenerating rat livers, and its properties were compared to those of homologous fractions prepared from the livers of sham-operated and unoperated controls. These canalicular membrane fractions were found to be closely related in terms of their morphology, their purity, their yield, and their qualitative protein banding profiles on sodium dodecyl sulfate-polyacrylamide gels. However, when a rigorous examination of plasma membrane enzyme marker activities was made, the regenerating liver membranes were shown to possess an increased specific activity of alkaline phosphatase and lower levels of Mg2+ ATPase and 5'-nucleotidase in comparison with control specific activity values.     

Changes in some marker enzyme activities of liver plasma membranes during regeneration after partial hepatectomy in rats.

Liver plasma membranes were isolated from regenerating rat livers between 20 h and 10 days after partial hepatectomy in order to study the effect of partial hepatectomy on some membrane enzyme activities. Mg2+-ATPase (EC 3.6.1.4) activity, but not (Na+ + K+)-ATPase activity, decreased slightly at 2 days, whereas leucyl beta-naphthylamidase (EC3.4.1.1) and 5'-nucleotidase (EC3.1.3.5) activities increased considerably at 1-2 and 3-5 days, respectively. These changes were not parallel to a sharp increase in mitotic activity of liver cells which occurred at 36 h.     

Acylase distribution in animal and human tissues

Distribution of acylase in different tissues of nine species of animals was studied. The following types of nitrogen elimination were distinguished: ammoniatelic (fish), uricotelic (birds) and uriotelic (amphibians, mammalians). The enzymic activity was estimated in the tissues of the brain, lung, muscle, liver, kidney, spleen, pancreas, small intestine and blood serum. The acylase activity was found in the kidney, liver and pancreas. Its level in the kidney increases with the animal weight growth, the enzyme activity being observed only in the cortical layer.     

Molecular cloning of estrogen receptor alpha of the Nile crocodile

Estrogens are essential for normal reproductive activity in female and male vertebrates. In female reptiles, they are essential for ovarian differentiation during a critical developmental stage. To understand the molecular mechanisms of estrogen action in the Nile crocodile (Crocodylus niloticus), we have isolated cDNA encoding the estrogen receptor alpha (ERalpha) from the ovary. Degenerate PCR primers specific to ER were designed and used to amplify Nile crocodile cDNA from the ovary. The full-length Nile crocodile ERalpha cDNA was obtained using 5' and 3' rapid amplification cDNA ends (RACE). The deduced amino acid sequence of the Nile crocodile ERalpha showed high identity to the American alligator ERalpha (98%), caiman ER (98%), lizard ER (82%) and chicken ERalpha (92%), although phylogenetic analysis suggested profound differences in the rate of sequence evolution for vertebrate ER sequences. Expression of ERalpha was observed in the ovary and testis of juvenile Nile crocodiles. These data provide a novel tool allowing future studies examining the regulation and ontogenic expression of ERalpha in crocodiles and expands our knowledge of estrogen receptor evolution.     

Changes in plasma membrane enzyme activities during liver regeneration in the rat.

Changes in activities of plasma membrane enzymes during liver regeneration may be related to the maintenance of hepatic function or to the regulation of cell proliferation. Plasma membranes were isolated from rat livers at various times after partial hepatectomy, and the specific activities of alkaline phosphatase, (Na+ + K+)-ATPase, leucine aminopeptidase, 5'-nucleotidase, and adenylate cyclase (basal and with glucagon or epinephrine) were measured. Alkaline phosphatase and (Na+ + K+)-ATPase activity increased 3.6-fold and 2-fold respectively, during the first 48 h after partial hepatectomy. The time of onset and duration of change suggest that these increases in activity are involved in the maintenance of bile secretion. Decreases in leucine aminopeptidase activity at 48--108 h and in 5'-nucleotidase activity at 12--24 h were observed, which may be involved in the restoration of protein and accumulation of RNA. The basal activity of adenylate cyclase increased after partial hepatectomy. The response of adenylate cyclase to epinephrine showed a transitory increase between 36 and 108 h after surgery, while the response to glucagon was decreased by approximately 50% at all time points through 324 h after surgery. These changes in the hormone responsiveness of adenylate cyclase are similar to those previously observed in fetal and preneoplastic liver.     

Studies on the mechanism of the increase in serum alkaline phosphatase activity in cholestasis: significance of the hepatic bile acid concentration for the leakage of alkaline phosphatase from rat liver

In experimental bile obstruction the serum activities of the membrane-bound liver enzymes, alkaline phosphatase, 5'-nucleotidase and gamma-glutamyltransferase are greatly increased, whereas in the liver only the alkaline phosphatase activity is elevated. After partial hepatectomy or tetrachloride poisoning the alkaline phosphatase activity in the regenerating live is increased to the same extent as in cholestasis without an accompanying elevation in serum activity. The following results support the hypothesis of a bile salt-mediated solubilization of membrane-bound enzymes in cholestatic liver: (1) 30 min after bile duct ligation the total bile acids in the liver were increased 5-fold, 2 h later as much as 10-fold. After 1 day, the bile acid concentration was still 4 times above normal. (2) Isolated plasma membranes from normal and obstructed livers were incubated in vitro with increasing amounts of tri- and dihydroxycholanic acids. At a final concentration of 1 mmol/l taurochenodeoxycholate significant amounts of membrane-bound enzymes were released into the 12,000-g supernatant. (3) In the regenerating liver, where tissue phsophatase activity was high and serum phosphatase activity unchanged, the bile salt concentration was not increased.     

Studies on 5'-nucleotidase activity in blood serum, tissues and liver mitochondrial fraction of normal, hypo- and hyperthyroid rats.

Clinical and experimental studies have previously shown the effect of thyroid state on the activity of non-specific phosphohydrolases. This research was undertaken to determine the influence of thyroid hormones on the activity of specific phosphohydrolase-5'-nucleotidase. The activity of 5'-nucleotidase was estimated in blood serum, tissues (liver, kidney, lung, brain) and mitochondrial fraction of liver of normal, methylothiouracil-induced hypothyroid and thyroxin-induced hyperthyroid rats of Wistar strain. The experiments have shown that the hypothyroid state induced decrease of the activity of 5'-nucleotidase in all studied materials, and on the contrary, the hyperthyroidism induced the increase of the enzyme activity. The hypothetical mechanism of the effect of thyroid hormones on the processes of feed back regulation of oxydative phosphorylation and delivery of substrates and oxygen is presented. The key role of 5'-nucleotidase in this mechanism is discussed.     

广东罗坑自然保护区鳄蜥生境选择的季节性差异

2006年3-10月,在广东省罗坑鳄蜥自然保护区利用直接观察法对鳄蜥春、夏、秋3季的生境选择进行研究。春、夏、秋3季各测定了31、71、31个有鳄蜥样方以及50、90、51个对照样方的14种生态因子。利用生态因子对比分析和逐步判别分析确定各季节生境选择的主要影响因素。结果表明,春季影响鳄蜥生境选择的主要影响因素是溪沟底质中沙的含量和植被盖度,正确判别率为97.5%;夏季影响鳄蜥生境选择的主要影响因素是溪沟底质中沙的含量、植被盖度、溪流宽度和枯枝百分比,正确判别率为94.4%;秋季影响鳄蜥生境选择的主要影响因素是溪沟底质中沙的含量、植被盖度和可利用食物量,正确判别率为97.6%。在区分春、夏、秋3季鳄蜥生境选择方面有一系列生态因子发挥作用,依照贡献值的大小依次为可利用食物量、干扰距离、枯枝百分比、溪水流速、溪沟坡度和溪宽等6个因子,由这6个变量构成的方程对春、夏、秋3季鳄蜥生境的正确区分率达到76.7%。判别函数的分析表明,春、夏、秋3季鳄蜥的生境选择具有较高的差异性,以判别函数建立的判别分类图表明,春、夏季以及夏、秋季鳄蜥的生境选择重叠较多,而秋季与春季的重叠较少。     

Proton leak in hepatocytes and liver mitochondria from archosaurs (crocodiles) and allometric relationships for ectotherms

It has previously been shown that mitochondrial proton conductance decreases with increasing body mass in mammals and is lower in a 250-g lizard than the laboratory rat. To examine whether mitochondrial proton conductance is extremely low in very large reptiles, hepatocytes and mitochondria were prepared from saltwater crocodiles ( Crocodylus porosus) and freshwater crocodiles ( Crocodylus johnstoni). Respiration rates of hepatocytes and liver mitochondria were measured at 37 degrees C and compared with values obtained for rat or previously measured for other species. Respiration rates of hepatocytes from either species of crocodile were similar to those reported for lizards and approximately one fifth of the rates measured using cells from mammals (rat and sheep). Ten-to-thirty percent of crocodile hepatocyte respiration was used to drive mitochondrial proton leak, similar to the proportion in other species. Respiration rates of crocodile liver mitochondria were similar to those of mammalian species. Proton leak rate in isolated liver mitochondria was measured as a function of membrane potential. Contrary to our prediction, the mitochondrial proton conductance of liver mitochondria from crocodiles was greater than that of liver mitochondria from lizards and was similar to that of rats. The acyl composition of liver mitochondrial phospholipids from the crocodiles was more similar to that in mitochondria from rats than in mitochondria from lizards. The relatively high mitochondrial proton conductance was associated with a relatively small liver, which seems to be characteristic of crocodilians. Comparison of data from a number of diverse ectothermic species suggested that hepatocyte respiration rate may decrease with body mass, with an allometric exponent of about -0.2, similar to the exponent in mammalian hepatocytes. However, unlike mammals, liver mitochondrial proton conductance in ectotherms showed no allometric relationship with body size.     

A comparative study of bile acid CoA:amino acid:N-acyltransferase (BAT) from four mammalian species.

1. Bile acid CoA:amino acid:N-acyltransferase (BAT) was partially purified from dog, human, pig and rat livers. The interspecies variation in substrate specificity and kinetics were determined for glycine and taurine. 2. BAT activity from dog liver formed bile acid conjugates with taurine exclusively, whereas BAT activity from each of the other species formed conjugates with both taurine and glycine. 3. Biliary composition of glycine and taurine bile acid conjugates could partly be accounted for by substrate affinity (Km) and turnover number (Vmax) of BAT activity. 4. A monospecific anti-human BAT polyclonal antibody reacted on Western blot analysis with a 40 kDa band in a 100,000 g supernatant fraction from rat liver. 5. Immunoabsorption chromatography using an anti-human BAT antibody-Sepharose affinity column showed that both the immunoreactive protein band and BAT activity were removed from the 100,000 g supernatant fraction from human and rat livers.     

5'-Nucleotidase in rat liver lysosomal membranes is anchored via glycosyl-phosphatidylinositol

We have previously demonstrated that 5'-nucleotidase, known as a plasma membrane enzyme, is also distributed both in rat liver tritosomal membranes and contents (J. Biochem. 101, 1077-1085, 1987). When the lysosomal membranes isolated from rat livers were incubated with phosphatidylinositol-specific phospholipase C purified from B. thuringiensis, about 70% of 5'-nucleotidase activity was released from the membranes. Judging from the result by phase separation with Triton X-114, the enzyme solubilized by the phospholipase C digestion showed a hydrophilic nature such as that of the tritosomal contents. Immunoblot analysis showed that the molecular weight of 5'-nucleotidase released from the lysosomal membranes by the phospholipase C digestion was almost identical with that of the enzymes from the Tritosomal contents. The above results showed that the phosphatidylinositol-specific phospholipase C-like enzyme in the lysosomes may be responsible for the conversion of the lysosomal membrane-bound 5'-nucleotidase to the soluble form present in the lysosomal matrix.