Comparison of ornithine transcarbamylase from rat liver and intestine. Evidence for differential regulation of enzyme levels

Ornithine transcarbamylase (OTCase) was purified from the small intestine of rat and the properties of the gut enzyme were compared with those of the enzyme from liver. The enzymes from both sources bound to the transition-state analog inhibitor, delta-N-(phosphonoacetyl)-L-ornithine, immobilized on Sepharose and eluted with carbamyl phosphate as a homogeneous preparation. The specific activities of the pure enzymes were 966 mumol min-1 mg-1 and 928 mumol min-1 mg-1 from liver and gut respectively, and the molecular mass, based on electrophoretic mobility, was 38 000 Da. The isoelectric point of the enzymes from both sources was 7.3. The enzymes from both sources cross-react to the same extent with antibodies against the liver enzyme on Western transfers and the size of the mRNA was identical on Northern transfers probed with a cDNA for the liver enzyme. Although OTCase is apparently the same gene product in both liver and gut, the enzyme levels respond differently to alterations in the protein content of the diet. OTCase in liver increased from 0.76 mumol min-1 microgram-1 DNA on 15% casein to 1.3 mumol min-1 microgram-1 DNA on 60% casein (P less than 0.01) whereas in small intestine the level decreased from 8.8 nmol min-1 microgram DNA on 15% casein to 5.7 nmol min-1 microgram-1 DNA on 60% casein (P less than 0.05). When expressed on a fresh-weight basis, the enzyme activity in liver shows the characteristic increase with increasing protein, whereas the activity in gut does not. The connection between these differences in gene expression and the different physiological roles of OTCase in liver and gut is discussed.     

Arginine:glycine amidinotransferase. A comparative study in lizard and mouse tissues

Kidney transamidinase activity in the lizard (Calotes versicolor), like that in the mouse, showed the pH optimum at 7.4. The lizard enzyme was inhibited to a greater degree than the mouse enzyme at high concentrations (greater than 20 mM) of L-arginine and glycine. Kidney and liver in the lizard and kidney and pancreas in the mouse were the tissues with high transamidinase activity. While transamidinase activity was widely distributed in mouse tissues, the enzyme was found to be restricted only to a few tissues in the lizard. Hydrocortisone administration into male lizards did not significantly alter the transamidinase levels in kidney and liver.     

IMP-GMP 5'-nucleotidase in reptiles: occurrence and tissue distribution in a crocodile and three species of lizards

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 several tissues (liver, heart, kidney, spleen, stomach, lung and skeletal muscle) from constitutively uricotelic reptiles: a crocodile (Crocodylus siamensis), and three species of lizard (Furcifer oustaleti, Tupinambis rufescens and Varanus gouldi). The activities were markedly high in the livers: 3.0 units/g in the crocodile and 1.4-2.9 units/g in the lizards. These were similar to those previously reported for the livers from chicken and snakes (also constitutively uricotelic), and 4- to 10-fold higher than those in ammoniotelic or ureotelic vertebrates. These findings suggest that the high activity of IMP-GMP 5'-nucleotidase in the liver is a feature of constitutive uricotelism, and that the enzyme may participate in the production of uric acid as an end product of amino acid catabolism.     

The role of GTP in the regulation of two forms of AMP deaminase from chicken kidney

1. Two molecular forms of AMP deaminase have been revealed by phosphocellulose column chromatography of the chicken kidney extract. 2. The chromatographic, kinetic and regulatory properties of these two forms were similar to these of two enzyme forms previously found in the chicken liver, lizard liver and in rat small intestine. 3. GTP exerted different effect from MgGTP on the activity and kinetic parameters of both AMP deaminase I and II from chicken kidney.     

N-Acetyl-β-d-hexosaminidase component A. Different forms in human tissues and fluids

1. Hexosaminidase A of human serum was resolved into two components, a minor form with properties identical with those of the single hexosaminidase A component of human liver, and a major form with significantly different properties. 2. The major serum hexosaminidase A form was eluted from a DEAE-cellulose column at a lower salt concentration than that required to elute the liver form. 3. A multiple-pass technique was used to elute the major serum enzyme A from a Sephadex G-150 column before that of liver enzyme A. 4. Clostridium perfringens neuraminidase converted the major component of serum hexosaminidase A into a form that was held less tightly by DEAE-cellulose, but the minor component of the A enzyme of serum, and the A enzyme of liver were not affected. 5. The hexosaminidase A from tears was similar to the A enzyme from serum, whereas those from several human tissues and from urine and lymph were similar to the liver form. 6. The A enzyme from serum may be derived from the A enzyme from liver by glycosylation before secretion.     

The arylalkylamine-N-acetyltransferase (AANAT) acetylates dopamine in the digestive tract of goldfish: A role in intestinal motility

Melatonin has been found in the digestive tract of many vertebrates. However, the enzymatic activity of the arylalkylamine-N-acetyltransferase (AANAT) and the hydroxindole-O-methyltransferase (HIOMT), the last two enzymes of melatonin biosynthesis, have been only measured in rat liver. Therefore, the first objective of the present study is to investigate the functionality of these enzymes in the liver and gut of goldfish, analyzing its possible daily changes and comparing its catalytic properties with those from the retina isoforms. The daily rhythms with nocturnal acrophases in retinal AANAT and HIOMT activities support their role in melatonin biosynthesis. In foregut AANAT activity also show a daily rhythm while in liver and hindgut significant but not rhythmic levels of AANAT activity are found. HIOMT activity is not detected in any of these peripheral tissues suggesting an alternative role for AANAT besides melatonin synthesis. The failure to detect functional HIOMT activity in both, liver and gut, led us to investigate other physiological substrates for the AANAT, as dopamine, searching alternative roles for this enzyme in the goldfish gut. Dopamine competes with tryptamine and inhibits retinal, intestinal and hepatic N-acetyltryptamine production, suggesting that the active isoform in gut is AANAT1. Besides, gut and liver produces N-acetyldopamine in presence of acetyl coenzyme-A and dopamine. This production is not abolished by the presence of folic acid (arylamine N-acetyltransferase inhibitor) in any studied tissue, but a total inhibition occurs in the presence of CoA-S-N-acetyltryptamine (AANAT inhibitor) in liver. Therefore, AANAT1 seems to be an important enzyme in the regulation of dopamine and N-acetyldopamine content in liver. Finally, for the first time in fish we found that dopamine, but not N-acetyldopamine, regulates the gut motility, underlying the broad physiological role of AANAT in the gut.     

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.     

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.     

A comparative study of superoxide dismutase in various animal species

1. Superoxide dismutase activity has been determined in liver homogenates of five species. There were no significant differences in the activities of the enzyme in the rat, rainbow lizard, wall gecko and chicken. The activity was significantly lower in the fish. 2. The order of activity of the enzyme in the organs/tissues of the rat was liver greater than kidney greater than heart greater than skeletal muscle greater than brain greater than lung greater than spleen greater than spinal cord greater than retina greater than pancrease greater than lens greater than small intestine. 3. Inhibition studies with cyanide showed that the enzyme in the liver of the various animal species was inhibited by cyanide. 4. The developmental pattern for the enzyme showed no significant changes in the liver of the rat from birth and up to 7 weeks after. However, the activity increased at about 8 weeks and remained constant to adult life.     

Purification and physicochemical properties of NADPH-specific dihydropteridine reductase from bovine and human livers

A new type of dihydropteridine reductase [EC 1.6.99.10], which is specific for NADPH as the substrate in the reduction of quinonoid-dihydropterin to tetrahydropterin, was purified to homogeneity from bovine liver and human liver. The molecular weight of the enzyme was determined to be 65,000-70,000. The enzyme was composed of two subunits with identical molecular weight of 35,000; the amino terminal residue was determined to be valine. The isoelectric point of the enzyme was 7.05. The physicochemical properties of this enzyme were quite different from those of bovine liver NADH-specific dihydropteridine reductase [EC 1.6.99.7]. NADPH-specific dihydropteridine reductase did not cross-react with an antiserum raised against the NADH-specific dihydropteridine reductase, nor did the latter enzyme react with an antiserum to the former enzyme, indicating that the two enzymes have no common antigenic determinants. NADPH-specific dihydropteridine reductase from human liver was shown to have properties similar to those of the bovine liver enzyme.     

Oxygen consumption by mitochondria from an endotherm and an ectotherm

Comparisons of metabolic properties of mitochondria from an endothermic and an ectothermic vertebrate were performed. Oxygen (O2) consumption rates of liver mitochondria from laboratory mice and western fence lizard (Sceloporus occidentalis) were determined over a range of temperatures (10, 20, 30 and 37 degrees C) and in the presence of a variety of substrates. At 37 degrees C the O2 consumption rate of mouse mitochondria was 4-11 times higher than lizard mitochondria in the presence of five of eight substrates. This range of differences is similar to differences reported for O2 consumption of endothermic animals, tissues and cells over those of ectotherms. Thermal sensitivity of mitochondria was measured by calculation of Q10s for O2 consumption. Q10s were highest for mouse mitochondria overall. The range that showed the highest Q10s for the mouse mitochondria was 30-20 degrees C, whereas for the lizard mitochondria it was 20-10 degrees C. Thus, mitochondria from the ectotherm showed a lower degree of temperature sensitivity than did mitochondria from the endotherm. The preferred substrate for all mitochondria at all temperatures was succinate, but mouse mitochondria then showed some preference for alpha-ketoglutarate and citrate, whereas lizard mitochondria showed a preference for pyruvate and malate + pyruvate.     

黄河上游环境污染对花背蟾蜍抗氧化酶活性及丙二醛含量的影响

选择黄河上游污染程度较严重的兰州地区和相对无污染的刘家峡地区作为研究样点,比较分析了两地花背蟾蜍（Bufo raddei）肝脏和肾脏中抗氧化酶SOD、CAT和GPx活性及丙二醛（MDA）含量。与刘家峡地区相比,兰州地区花背蟾蜍肝脏的SOD活性升高,CAT和GPx活性极显著降低,肾脏GPx活性显著增高,肝脏和肾脏MDA含量明显高于刘家峡。结果表明,黄河上游环境污染使得花背蟾蜍体内氧化胁迫加重,导致其组织脂质过氧化程度升高。     

A comparative polyacrylamide gel electrophoretic study of arginase in vertebrate tissues

The electrophoretic behaviour of arginase in the tissue extracts of rat, beef, lizard and frog was studied by bidirectional polyacrylamide gel electrophoresis. The enzyme from rat liver and submaxillary gland migrated to the cathode with the activity concentrated in a single peak. Arginase from beef liver emerged as a single peak of anodal migration with a significant shoulder in the sample gel. Frog liver and kidney enzymes also appeared as single peaks with a distinct anodal movement. The activity in mammalian kidney and lizard liver and kidney resolved into two peaks of anodal migration suggesting the presence of two isoenzymes of arginase in these tissues.     

Comparative characterization of human and rat liver glycogen synthase.

Glycogen synthase from human liver was studied, and its properties were compared with those of rat liver glycogen synthase. The rat and human liver glycogen synthases were similar in their pH profile, in their kinetic constants for the substrate UDP-glucose and the activator glucose 6-phosphate, and in their elution profiles from Q-Sepharose. The apparent molecular weight of the human synthase subunit was 80,000-85,000 by gel electrophoresis, which is similar to that of the rat enzyme. In addition, antibodies to rat liver glycogen synthase recognized human liver glycogen synthase, indicating that the enzymes of these two species share antigenic determinants. However, there were significant differences between the two enzymes. In particular, the total activity of the human enzyme was higher than that of the rat. The human enzyme, purified to near homogeneity, had a specific activity of 40 U/mg protein compared with 20 U/mg protein for the rat enzyme. The active forms of the rat enzyme had greater thermal stability than those of the human enzyme, but the human enzyme was more stable on storage in various buffers. Last, amino acid analysis indicated differences between the enzymes of the two species. Since glycogen synthase is an important enzyme in liver glycogen synthesis, the characterization of this enzyme in the human will help provide insight regarding human liver glycogen synthesis.     

Gluconeogenesis in the kidney and liver slices of a lizard, Uromastix hardwickii

1. The liver and kidney of the lizard Uromastix hardwickii have much higher contents of carbohydrate than have been reported for the corresponding rat tissues. Most of this carbohydrate still remains in the tissue even after a long preincubation. 2. Kidney slices of this lizard release both glucose and other carbohydrates into the medium. Hence glucose release alone, as demonstrated for rats, cannot be used as a good criterion of gluconeogenesis in this lizard. Moreover, the results obtained by glucose release did not agree with those in which the total carbohydrate was estimated in the slice and medium. 3. l-Glutamate, l-aspartate, dl-valine, l-proline, l-cysteine, l-lactate and succinate stimulated gluconeogenesis in the kidney slices, whereas citrate, l-alanine, l-serine, glycine, l-arginine and l-leucine did not. In liver slices only l-glutamate increased gluconeogenesis. 4. New carbohydrate formation in the kidney and liver slices after incubation with various substrates indicated that gluconeogenesis as well as the amino acid metabolism in this animal may be somewhat different from that of mammals.     

Rat hepatoma (HTC) cell 6-phosphofructo-2-kinase differs from that in liver and can be separated from fructose-2,6-bisphosphatase

6-Phosphofructo-2-kinase was purified from rat liver and hepatoma (HTC) cells. The HTC cell enzyme had kinetic properties different from those of the liver enzyme (more sensitive to inhibition by citrate and not inhibited by sn-glycerol 3-phosphate) and was not a substrate of the cyclic-AMP-dependent protein kinase. Unlike the liver enzyme, which is bifunctional and phosphorylated by fructose 2,6-[2-32P]bisphosphate, the HTC cell enzyme contained no detectable fructose-2,6-bisphosphatase activity and phosphorylation by fructose 2,6-[2-32P]-bisphosphate could not be detected. HTC cell fructose-2,6-bisphosphatase could be separated from 6-phosphofructo-2-kinase activity by purification. Antibodies raised against liver 6-phosphofructo-2-kinase did not precipitate HTC cell fructose-2,6-bisphosphatase whose kinetic properties were completely different from those of the liver enzyme.     

Arginase from rat fibrosarcoma. Purification and properties

Arginase from rat fibrosarcoma was purified about 1900-fold and its properties were compared with those of the enzyme from liver and kidney. Arginase from fibrosarcoma was a neutral protein of molecular weight 120,000 with a K_m value of 11 mM for arginine. The activation energy was 7.2 kcal/mol and the pH optimum was 10. The fibrosarcoma enzyme was immunologically different from that of the liver. The arginase from fibrosarcoma closely resembled the arginase from the kidney in its electrophoretic, kinetic and immunological properties.     

Enzymes of ornithine metabolism in adult and developing rat intestine.

The levels of 11 enzymes, most of them involved in the metabolism of ornithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, ornithine aminotransferase, and ornithine transcarbamylase were compared with those in liver. Changes with age (late gestation of adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described. The results suggest that the proximal part of the intestine is well endowed with enzymes involved in the conversion of ornithine to proline as well as to citrulline. Fetal intestine is rich in proline oxidase and pyrroline-5-carboxylate reductase. The peak levels of ornithine aminotransferase found in intestine in the first 3 postnatal weeks were higher than seen in any other rat tissue. Some of the properties of arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase in small intestine were compared with those in liver. Isozymes of arginase in small intestine differed from those in liver; the kinetic properties of ornithine aminotransferase were similar in the two tissues. In intestine of 14-day-old rats, the ornithine aminotransferase reaction was reversible, forming ornithine from pyrroline-5-carboxylate. The intestinal pyrroline-5-carboxylate reductase was cold-labile as was the hepatic enzyme in rat.     

Enzymes of orithine metabolism in adult developing rat intestine

The levels of 11 enzymes, most of them involved in the metabolism of orithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, orithine aminotransferase, and orithine transcarbamylase were compared with those in liver. Changes with age (late gestation to adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described.The results suggests that the proximal part of the intestine is well endowed with enzymes involved in the conversion of ornithine to proline as well as to citrulline. Fetal intestine is rich in proline oxidase and pyrroline-5-carboxylate reductase. The peak levels of ornithine aminotraferase found in intestine in the first 3 postnatal weeks were higher than seen in any other rat tissue.Some of the properties of arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase in small intestine were compared with those in liver. Isozymes of arginase in small intestine differed from those in liver; the kinetic properties of ornithine aminotransferase were similar in the two tissues. In intestine of 14-day-old rats, the orithine aminotransferase reaction was reversible, forming ornithine from pyrroline-5-carboxylate. The intestinal pyrroline-5-carboxylate reductase was cold-labile as was the hepatic enzyme in rat.