Studies on mammalian glucoamylases with special reference to monkey intestinal glucoamylase

1. Highly purified preparations of glucoamylase were obtained from liver, spleen and intestine of the monkey. The enrichment factor was lower for intestine (60-fold) compared with that of liver (1200-fold) and of spleen (2000-fold) but the final specific activities were of a similar magnitude. 2. The liver and spleen enzymes had maximum activity at pH4.8 whereas the intestinal enzyme showed an optimum at pH5.8. The K(m) values for both starch and maltose with spleen and liver enzymes were higher than for the intestinal enzyme. With the intestinal enzyme, the V(max.) values were higher for both starch and maltose than those of the spleen and liver enzymes. 3. Gel filtration on Sephadex G-200 under identical conditions revealed that liver and spleen enzymes emerge from the columns much later than the intestinal enzyme. 4. Evidence is presented that the glucoamylase activity of the intestinal mucosa is exhibited by the maltase II fraction. 5. Tris, pentaerythritol and turanose inhibited glucoamylase from all the three tissues, but turanose inhibited the spleen and liver enzymes to a higher degree than the intestinal enzyme.     

Development of phenylalanine hydroxylase activity in rat kidney

The developmental pattern of phenylalanine hydroxylase was studied in rat kidney and compared with that of liver from the same animal. Traces of activity were observed from 19 to 21 days of gestation in the liver and on Day 20 and 21 of gestation in the kidney. Significant amounts of activity were noticed on Day 22 of gestation. Kidney on Day 21 of gestation showed slightly higher values than that seen in corresponding liver. Fetal liver and kidney showed about 30% and 90% activity of newborn animals, respectively. Both synthetic cofactor and organic reductant were necessary for optimal activity of liver and kidney enzymes. pH studies showed an optimum at pH 7.0 in both liver and kidney. Storage at ?15 °C resulted in loss of activity in both liver and kidney to the same extent at a given time. No evidence of inhibition of either liver or kidney enzyme by phenylalanine was noticed up to a concentration of 4 μmoles per assay. Heat denaturation studies at 50 °C showed the kidney enzyme to be slightly less stable than the liver enzyme though a similar pattern was observed in both tissues at all ages studied.     

Mammalian Brain Alkaline Phosphatase: Expression of Liver/Bone/Kidney Locus Comparison of Fetal and Adult Activities

The alkaline phosphatases (EC 3.1.3.1) are determined by at least three gene loci, which can be sharply distinguished one from another by their sensitivity to inhibition with various amino acids and peptides and by ther-mostability. Alkaline phosphatase is present in the brains of guinea pig, rat, mouse, hamster, squirrel, rabbit, cat, sheep, cow, tamarin, baboon, and man. The gene locus coding for alkaline phosphatase in all these brains is the liver/ bone/kidney locus, as indicated by thermostability studies and by inhibition studies with L-phenylalanine, L-homoarginine, and L-phenylalanylglycylglycine. The average brain alkaline phosphatase activity is about 35% of the average for the livers and only 7.2% and 4.4% of the average kidney and placental activities, respectively. During growth and development, brain alkaline phosphatase activity decreases in the mammals studied. The amount of change is tissue- and species-dependent.     

Comparison of two pig intestinal brush border peptidases with the corresponding renal enzymes.

Intestinal dipeptidyl peptidase IV and gamma-glutamyltransferase were compared to the corresponding kidney enzymes with respect to immunological and electrophoretic properties. The influences of selected effectors on the two enzymes were also studied. The two kidney peptidases exhibited the reaction of total identity with the corresponding intestinal enzymes in immunodiffusion. Furthermore, the intestinal dipeptidyl peptidase IV and gamma-glutamyl transferase showed the same inhibition patterns as the corresponding kidney enzymes and the acceptor specificity of the intestinal gamma-glutamyl-transferase was found to be identical to that of the kidney enzyme. The electrophoretic mobilities of dipeptidyl peptidase IV from the two organs differed greatly. The difference was almost abolished by treatment with neuraminidase, suggesting that the variation in mobility was due to different contents of sialic acid. It is suggested that the intestinal brush border peptidases, dipeptidyl peptidase IV and gamma-glutamyltransferase, are closely related to the corresponding enzymes obtained from the kidney.     

Substrate inhibition of rat liver and kidney arginase with fluoride

Fluoride is an uncompetitive inhibitor of rat liver arginase. This study has shown that fluoride caused substrate inhibition of rat liver arginase at substrate concentrations above 4 mM. Rat kidney arginase was more sensitive to inhibition by fluoride than liver arginase. For both liver and kidney arginase preincubation with fluoride had no effect on the inhibition. When assayed with various concentrations of L-arginine, rat kidney arginase did not have Michaelis-Menten kinetics. Lineweaver-Burk and Eadie-Hofstee plots were nonlinear. Kidney arginase showed strong substrate activation at concentrations of L-arginine above 4 mM. Within narrow concentrations of L-arginine, the inhibition of kidney arginase by fluoride was uncompetitive. Fluoride caused substrate inhibition of kidney arginase at L-arginine concentrations above 1 mM. The presence of fluoride prevented the substrate activation of rat kidney arginase.     

The appearance, properties, and functions of gluconeogenic enzymes in the liver and kidney of the guinea pig during fetal and early neonatal development.

The changes in the activity and properties of the four gluconeogenic enzymes have been followed during development of the guinea pig. Pyruvate carboxylase was almost exclusively mitochondrial and kinetically identical to the adult liver enzyme and did not appear in significant activity until after day 50 when it rose to values several times higher than those in the adult liver, then fell after birth. Little activity was detected in the fetal kidney. Phosphoenolpyruvate carboxylase appeared in the fetal liver from day 30 on, both in the mitochondrial and cytoplasmic fractions. The cytoplasmic enzyme was kinetically and chromatographically identical to the mitochondrial enzyme of the fetal and maternal liver. After birth the activity of the cytoplasmic enzyme increased and that of the particulate enzyme fell. Fetal kidney activity appeared several days before birth. Fructose 1,6-diphosphatase and glucose 6-phosphatase appeared in the fetal liver and kidney after day 40; the former showed no postnatal change while the latter rose 10-fold after birth. Fetal liver fructose 1,6-diphosphatase was more sensitive to AMP and fructose 1,6-diphosphate inhibition but was chromatographically indistinguishable from the maternal liver enzyme. Despite the presence of the gluconeogenic enzymes, gluconeogenesis and glyconeogenesis were not detected in the fetal liver until 7–9 days before birth. While the synthesis of glyceride-glycerol from 3-carbon compounds was detected from 35–40 days onwards and some of the gluconeogenic enzymes participate in that pathway, gluconeogenesis was not detected in the fetal kidney.     

LYSOSOMAL ENZYMES OF RAT INTESTINAL MUCOSA

Six intracellular hydrolases known to be associated with lysosomes in rat liver were found in rat intestinal mucosa. The extent to which they were particulate-bound and the degree of enzyme release when the particulate fractions were suspended in hypotonic media followed the same pattern in both mucosa and liver. The specific activities of the mucosa enzymes were either comparable to or slightly smaller than those of the liver enzymes. These results suggest that the mucosa hydrolases belong to lysosome-like particles. However, differential fractionation of the mucosa indicated that the particles from the mucosa sediment at lower centrifugal forces than do those from the liver and are more heterogeneous in size, bearing a closer resemblance to kidney lysosomes. Possible physiological functions of particulate-bound digestive enzymes in intestinal mucosa are discussed.     

In vitro inhibition of alkaline phosphatase activities from intestine, bone, liver, and kidney by phenobarbital.

A kinetic study of the inhibition of several alkaline phosphatase (AP isoenzyme activities by phenobarbital was carried out using p-nitrophenylphosphate (10 mM) as a substrate at pH 9.8 in a 300-mM Hepes buffer. AP from bovine kidney, calf intestine, bovine liver, and rat bone was used. Over a phenobarbital concentration range of 20-400 mM, all these isoenzymes were inhibited in an uncompetitive manner with a Ki of 200 mM for intestinal AP, and in a linear mixed-type manner for all the other isoenzymes tested. The Ki values were 10, 40 and 55 mM for kidney, bone and liver AP, respectively. The use of 15 mM carbonate-bicarbonate or 400 mM diethanolamine buffer did not modify the degree of inhibition of intestinal AP activity. Dixon plots of the reciprocal of reaction velocity versus inhibitor concentration either at different substrate concentration or at different DEA concentration indicate uncompetitive inhibition for the intestinal enzyme. This in vitro inhibitory effect of phenobarbital is in contrast to its in vivo stimulating action on AP. However, in the whole animal, the effects of phenobarbital administration probably represent the sum of multiple effects.     

Comparative studies of vertebrate and invertebrate pyruvate kinases.

1. Electrophoretic patterns showed that among the vertebrates studied, L isozyme was present in rat and frog liver and also in rat kidney. 2. M2 is a frequent component in vertebrate tissues, giving support to the proposal that M2 is the ancestral form differentiating to other isozymes. 3. The above trend cannot simply apply to invertebrates. 4. Muscle pyruvate kinases from various animals were inhibited by the particular phosphagen present in their muscle. 5. The inhibition may have an important role in the regulation of glycolysis in muscle.     

Carnitine synthesis and uptake into cells are stimulated by fasting in pigs as a model of nonproliferating species

In rodents, fasting increases the carnitine concentration in the liver by an up-regulation of enzymes of hepatic carnitine synthesis and novel organic cation transporter (OCTN) 2, mediated by activation of peroxisome proliferator-activated receptor (PPAR) α. This study was performed to investigate whether such effects occur also in pigs which like humans, as nonproliferating species, have a lower expression of PPARα and are less responsive to treatment with PPARα agonists than rodents. An experiment with 20 pigs was performed, which were either fed a diet ad-libitum or fasted for 24 h. Fasted pigs had higher relative mRNA concentrations of the PPARα target genes carnitine palmitoyltransferase 1 and acyl-CoA oxidase in liver, heart, kidney, and small intestinal mucosa than control pigs, indicative of PPARα activation in these tissues (P<.05). Fasted pigs had a higher activity of γ-butyrobetaine dioxygenase (BBD), enzyme that catalyses the last step of carnitine biosynthesis in liver and kidney, and higher relative mRNA concentrations of OCTN2, the most important carnitine transporter, in liver, kidney, skeletal muscle, and small intestinal mucosa than control pigs (P<.05). Fasted pigs moreover had higher concentrations of free and total carnitine in liver and kidney than control pigs (P<.05). This study shows for the first time that fasting increases the activity of BBD in liver and kidney and up-regulates the expression of OCTN2 in various tissues of pigs, probably mediated by PPARα activation. It is concluded that nonproliferating species are also able to cover their increased demand for carnitine during fasting by an increased carnitine synthesis and uptake into cells.     

Serum and tissue alkaline phosphatases in pigs

The alkaline phosphatases from serum, liver, bone and intestine of pigs were separated by starch and polyacrylamide gel electrophoresis. Treatments with neuraminidase, urea, heat, L-homoarginine and L-phenylalanine were performed. Variants of serum alkaline phosphatases were derived from different tissues and hence must be under the control of at least two different loci. Within the intestinal phosphatases, polymorphic electrophoretic patterns were observed among 195 animals.     

Prostaglandin metabolism. II. Identification of two 15-hydroxyprostaglandin dehydrogenase types.

Homogenates of several mammalian tissues were measured by radioimmunoassay for 15-hydroxyprostaglandin dehydrogenase activity. Two types of enzyme activity were detected. One, which used NAD-plus as cofactor much more effectively than NADP-lus, was found in monkey lung, heart, liver, kidney, and spleen and in chicken heart and dog lung. A second type, which uses NADP-plus as a cofactor more effectively than NAD-plus, was found in monkey and human brain and red blood cells and in swine kidney. These two types of 15-hydroxyprostaglandin dehydrogenase were partially purified from monkey brain and chicken heart. In addition to different cofactor requirements, the two partially purified enzymes could be distinguished by chromatographic properties, their relative affinities for prostaglandin I2 and F2alpha, and their sensitivities to inhibition by reduced pyridine nucleotides, thyroid hormones, and prostaglandin B2.     

Role of alkaline phosphatase in phosphate uptake into brush border membrane vesicles from human intestinal mucosa

In order to elucidate the physiological function of intestinal alkaline phosphatase, the characteristics of human intestinal alkaline phosphatase bound to brush border membrane vesicles were compared under optimal and physiological pHs. The Km value of this enzyme towards p-nitrophenylphosphate at the physiological pH was lower than that at the optimal pH. At the physiological pH, phosphate, arsenate and vanadate competitively inhibited the alkaline phosphatase activity, as they did at optimal pH, and the K1 values of these inhibitors at the physiological pH were also lower than those at the optimal pH. The effects of various inhibitors and antibody to human intestinal alkaline phosphatase on phosphate uptake into brush border membrane vesicles were investigated. The results indicated that phosphate uptake was affected by various inhibitors and the antibody to human intestinal alkaline phosphatase, but L-homoarginine, levamisole, and ouabain had no effect. From the above findings, it is strongly suggested that human intestinal alkaline phosphatase may function as a phosphate binding protein at low phosphate concentrations under physiological conditions.     

In vivo inhibition of aspartate aminotransferase in mice by L-hydrazinosuccinate

L-Hydrazinosuccinate, which has been shown to be a slow-, tight-binding inhibitor of aspartate aminotransferase (EC 2.6.1.1) in vitro, was tested as an inhibitor in vivo of the enzyme as well as other pyridoxal enzymes. Intraperitoneal administration to mice at a dose of 0.6 mmol/kg rapidly decreased aspartate aminotransferase activities in liver and kidney cytosols to a minimal level lower than 10% of the original, and no appreciable reversal of the inhibition was observed after 24 h; at lower doses the activities were significantly recovered during the same period following an initial marked decrease. Of the other pyridoxal enzymes tested, alanine aminotransferase in liver was the most sensitive to the inhibitor. It was initially inhibited as severely as aspartate aminotransferase, but the inhibition was reversed considerably faster. Aspartate aminotransferase activities in brain and heart were less severely affected than those in liver and kidney; they were less markedly lowered initially and were substantially recovered after 24 h. Consistent with the observed organ specificity, heated extracts from brain and heart in the mice administered with the inhibitor showed relatively weak inhibitory activities in vitro to aspartate aminotransferase purified from pig heart, while the extracts from liver and kidney were strongly inhibitory.     

Activities of glutathione-S-transferase and ethoxycoumarin-O-deethylase in tissues of camels, sheep, goats and rats.

1. The activities of the drug metabolizing enzymes ethoxycoumarin-O-deethylase, glutathione-S-transferase, and protein concentrations were measured in vitro in the liver, kidney and duodenal mucosa of camels, sheep, goats and rats. 2. Enzyme activities were generally higher in the liver than in the kidney and duodenal mucosa in the four species studied. 3. The activities of ethoxycoumarin-O-deethylase and glutathione-S-transferase in liver of male kids were about one third and half of that in adult male goats, respectively. In the kidney and duodenal mucosa of male kids, the activity of glutathione-S-transferase was about 70% and 53% of that in the mature male goat, respectively. In the latter tissues, however, there was no detectable activity of ethoxycoumarin-O-deethylase. 4. In general, goats and sheep had similar activities of the two enzymes which were significantly higher than those found in camels and rats. 5. Some sex-related differences were noted in the activity of the two enzymes studied. Female sheep had significantly higher hepatic glutathione-S-transferase than the male: while the enzyme activity in the kidney and duodenal mucosa of male goats was significantly higher than in females. Male rats had higher hepatic ethoxycoumarin-O-deethylase activity than females.     

Serum and tissue alkaline phosphatases in pigs*

The alkaline phosphatases from serum, liver, bone and intestine of pigs were separated by starch and polyacrylamide gel electrophoresis. Treatments with neuraminidase, urea, heat, L-homoarginine and L-phenylalanine were performed. Variants of serum alkaline phosphatases were derived from different tissues and hence must be under the control of at least two different loci. Within the intestinal phosphatases, polymorphic electrophoretic patterns were observed among 195 animals.     

Mouse thymoma cell line expresses a gluconeogenic enzyme, fructose 1,6-bisphosphatase

Fructose 1,6-bisphosphatase was observed in a thymic lymphoma cell line, WEH17.1 (11.5 +/- 0.8 munits/mg cytosol protein). Only a trace amount of the enzyme activity was observed in normal thymus tissue. The WEH17.1 enzyme had a pH optimum at around 7.5. The AMP-concentration giving 50% inhibition of the activity was about 73 microM. That of the crude mouse liver enzyme was 35 microM. The antibodies against the liver and intestinal enzymes cross-reacted with the WEH17.1 enzyme with a lower affinity than the liver enzyme. Immunoblot showed that the subunit molecular weight of the WEH17.1 enzyme was the same as that of the liver enzyme.     

Kinetic analyses of the pyruvate dehydrogenase complex from Candida 107 (NCYC 911).

Candida 107 (NCYC 911) accumulates up to 45% of the biomass as triglycerides under conditions of nitrogenous substrate limitation in the medium. In oilseeds and adipocytes, lipid accumulation is preceded and accompanied by increased activity of key enzymes such as pyruvate dehydrogenase. However, in Candida 107, the activity of this complex was greatly reduced during lipogenesis. The initial velocity patterns were in accordance with a Hexa Uni Ping Pong mechanism. The Km values for the various substrates were similar to those found for the yeast Saccharomyces cerevisiae, but much higher than those reported for the mammalian enzyme. Product inhibition studies indicated that the Ki for acetyl coenzyme A and NADH were higher than those reported for other yeasts. The values for Ki were similar to those found for the liver enzyme, whereas the enzyme complex from heart had much lower Ki values for products. It has been suggested that in the heart and kidney, pyruvate dehydrogenase is regulated by product inhibition whereas in the liver this does not appear to be the mechanism. Therefore, it is probable, that like the liver enzyme, pyruvate dehydrogenase from Candida 107 may not be regulated by product inhibition.     

Effect of chlorpyrifos on thiobarbituric acid reactive substances, scavenging enzymes and glutathione in rat tissues

The present study showed that exposure of chlorpyrifos, O,O'-diethyl-O-3,5,6-trichloro-2-pyridyl phosphorothionate (CPF), a widely used pesticide in rats caused significant inhibition of acetylcholinesterase (AChE) activity in different tissues viz., liver, kidney and spleen. CPF exposure also generated oxidative stress in the body, as evidenced by increase in thiobarbituric acid reactive substances (TBARS), decrease in the levels of superoxide scavenging enzymes viz., superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in liver, kidney and spleen at all doses. Malondialdehyde levels were increased by 14%, 31% and 76% in liver, 11%, 31% and 64% in kidney and 32%, 75% and 99.9% in spleen when 50 mg, 100 mg and 200 mg/kg body wt. CPF was administered for three days. SOD and CAT activities were decreased in liver, kidney and spleen, while GPx activity showed slight increase in kidney at 50 mg and 100 mg dose, and decreased on further increase in dose of CPF. Liver and spleen showed dose-dependent decrease in GPx activity. The levels of reduced glutathione (GSH) was decreased, while oxidized glutathione (GSSG) was increased, thus a marked fall in GSH/GSSG ratio was observed in all tissues. A maximum decrease of 83% was observed in liver, followed by kidney and spleen, which showed 78% and 57% decrease, respectively in group given 200 mg/kg CPF. The levels of glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR) were also decreased in liver and kidney, while spleen showed increase at lower doses, but decrease at high dose of CPF. The data provide evidence for induction of oxidative stress on CPF exposure.     

Enzyme activities in plasma, kidney, liver, and muscle of five avian species

Activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatine phosphokinase (CPK), and lactate dehydrogenase (LDH) were determined in plasma, kidney, liver, and muscle from five species of captive birds. Few differences occurred in plasma activities between sexes but considerable differences occurred between species. All five enzymes were detected in each of the tissues sampled. Relative enzyme activities in liver, kidney, and muscle were similar for each species. CPK activity was much higher in muscle than in liver or kidney and, of the five enzymes studied, may be the best indicator of muscle damage. Most of the other enzymes were more evenly distributed among the three tissues, and no organ-specific enzyme could be identified for liver or kidney. Because of interspecific variations in plasma enzyme activities, it is important to establish baseline values for each species to ensure accurate interpretation of results.