Guinea Pig Enzyme Research Articles

Role of S-adenosylhomocysteine hydrolase in adenosine metabolism in mammalian heart.

S-Adenosylhomocysteine hydrolase of mammalian hearts from different species is exclusively a cytosolic enzyme. The apparent Km for the guinea-pig enzyme was 2.9 microM (synthesis) and 0.39 microM (hydrolysis). Perfusion of isolated guinea-pig hearts for 120 min with L-homocysteine thiolactone (0.23 mM) and adenosine (0.1 mM), in the presence of erythro-9-(2-hydroxynon-3-yl)adenine to inhibit adenosine deaminase, caused tissue contents of S-adenosylhomocysteine to increase from 3.5 to 3600 nmol/g. When endogenous adenosine production was accelerated by perfusion of hearts with hypoxic medium (30% O2), L-homocysteine thiolactone (0.23 mM) increased S-adenosyl-homocysteine 17-fold to 64.3 nmol/g within 15 min. In the presence of 4-nitro-benzylthioinosine (5 microM), an inhibitor of adenosine transport, S-adenosylhomocysteine further increased to 150 nmol/g. L-Homocysteine thiolactone decreased the hypoxia-induced augmentation of adenosine, inosine and hypoxanthine in the tissue and the release of these purines into the coronary system by more than 50%. Our findings indicate that L-homocysteine can profoundly alter adenosine metabolism in the intact heart by conversion of adenosine into S-adenosylhomocysteine. Adenosine formed during hypoxia was most probably generated within the myocardial cell.

Influence of Cholesterol and Fat Feeding on Hepatic Lysosomal Enzymes in Guinea Pigs

The activity of four lysosomal enzymes have been examined in liver cytoplasmic extract from guinea pigs fed three different diets: a) an ordinary diet, low in fat, high in carbohydrates; b) a semisynthetic diet containing 10% cottonseed oil (by weight) without and c) with 1% cholesterol. The cholesterol content in the liver was similar in control-fed and fat-fed animals, while there was a 10-fold increase in cholesterol + fat-fed animals, and most of this cholesterol was present as ester. We observed increased activity of beta-glucuronidase, beta-acetyl-glucosaminidase and cathepsin D during fat cholesterol feeding (diet c) while the activity of acid phosphatase decreased compared to control-fed animals. These findings probably mirror the increased hepatic accumulation of lipids and lipoproteins observed in cholesterol + fat-fed guinea pigs.

Erythrocyte membrane anion-sensitive Mg 2+-ATPase—Identity with monovalent cation sensitive Ca 2+-Mg 2+-ATPase

1. 1. Activation of Mg 2+-ATPase of rabbit and guinea-pig erythrocyte membrane by bicarbonate or chloride could be completely abolished by ethylene-glycol-bis-(β-aminoethylether)- N, N'-tetraacetic acid. The anion stimulation was actually an activation of contaminating Ca 2+ -stimulated Mg 2+-ATPase by monovalent cations associated with the anions. 2. 2. Guinea-pig red cell Ca 2+-Mg 2+-ATPase could be activated by both sodium and potassium while the rabbit enzyme was sensitive only to sodium. The concentrations of monovalent cations for half-maximal stimulation of Ca 2+-Mg 2+-ATPase are: k na+ = 40.8 mM, k k+ = 12.2 mM (guinea-pig); K Na+ = 13.3mM (rabbit). 3. 3. Potassium enhanced activation of rabbit erythrocyte membrane Ca 2+-Mg 2+-ATPase by red cell Ca 2+-Mg 2+-ATPase activator protein. With the guinea pig enzyme, neither sodium nor potassium enhanced activator stimulation of Ca 2+-Mg 2+-ATPase. 4. 4. Ca 2+-Mg 2+-ATPase of aged rabbit erythrocyte membrane responded to sodium but not to activator protein. 5. 5. Triton X-100 solubilized rabbit erythrocyte membrane Ca 2+-Mg 2+-ATPase has an apparent molecular weight of 371,000. It did not respond to the activator. 6. 6. One major and three minor proteins, visualized by SDS-polyacrylamide gel electrophoresis, were extracted from rabbit erythrocyte membrane by 50 μM chlorpromazine.

Biochemical basis for the resistance of guinea-pig and monkey to the carcinogenic effects of arylamines and arylamides.

1. Ag.l.c. assay for N-hydroxy-2-acetamidofluorene has been modified to measure both N-hydroxy-2-acetamidofluorene and N-hydroxy-2-aminofluorene. 2. Like guinea-pig, monkey N-hydroxylates both 2-aminofluorene and 2-acetamido-fluorene. The N-hydroxy metabolites are rapidly further metabolized even in the presence of inhibitors of deacetylase. The exact nature of this further metabolism is still unknown. Preliminary evidence indicates that, at least in the guinea-pig, 7-hydroxy-2-acetamidofluorene may be a metabolite of N-hydroxy-2-acetmidofluorene. 3. 3-Methylcholanthrene, 7,8-benzoflavone and miconazole, which have been shown to inhibit guinea-pig liver microsomal N-hydroxylase, do not significantly inhibit the monkey liver enzyme. 4. 2-Acetamidofluorene, which inhibits the guinea-pig liver microsomal N-hydroxylation of 2-aminofluorene in vitro, activates the enzyme from monkey liver. This activation, which is dose-dependent, appears to be allosteric. 5. Both guinea-pig and monkey are more efficient in N-hydroxylating 2-aminofluorene than 2-acetamidofluorene. The affinity (in term of apparent KM) of the guinea-pig liver enzyme is 4 times greater than the affinity of the monkey liver enzyme.

Effects of vitamin A deficiency on hepatic and extrahepatic mixed-function oxidase and epoxide-metabolizing enzymes in guinea pig and rabbit

Male guinea pigs and male rabbits were fed a vitamin A deficient diet for 9 weeks and for 12 weeks respectively. Hepatic levels of vitamin A were significantly reduced in the vitamin A deficient animals. The activities of some xenobiotic-metabolizing enzymes were measured in the liver, lung and small intestine. Aryl hydrocarbon hydroxylase, aniline hydroxylase, and 7-ethoxycoumarin deethylase activities were decreased in the vitamin A deficient guinea pig liver. However, in the guinea pig small intestine, aniline hydroxylase, 7-ethoxycoumarin deethylase, aminopyrine demethylase, and aryl hydrocarbon hydroxylase specific activities were increased. In rabbits, vitamin A deficiency decreased hepatic aniline hydroxylase and 7-ethoxycoumarin deethylase activities but increased intestinal aminopyrine demethylase activity. Enzyme activities in lung were not altered by vitamin A deficiency in guinea pig or rabbit. Microsomal epoxide hydrase and microsomal supernatant glutathione S-transferase activities in the three tissues of both species were not altered by vitamin A deficiency.

Comparison of hepatic epoxide hydratase from different species: Purification and immunochemical properties

1. 1. Purification of epoxide hydratase showed that an enzyme protein species of molecular weight approx. 50,000 daltons was obtained from all liver sources examined. 2. 2. Antiserum raised against purified rat liver epoxide hydratase forms immunoprecipitin complexes with the solubilized enzymes from human guinea pig and mouse livers. 3. 3. An immunoprecipitin line indicating complete identity was observed between rat and mouse epoxide hydratase, whereas human and guinea pig enzymes exhibited only partial-identity with the rat enzyme. 4. 4. The existence of different forms of epoxide hydratase in different species is discussed.

Characterization of cholesterol ester hydrolase activities in rabbit and guinea pig aortas

Cholesterol ester hydrolase activity was determined in preparations of rabbit and guinea pig aorta utilizing micellar and glycerol-dispersed cholesterol oleate substrates. Both substrate preparations demonstrated an acid pH optimum of 4–5 for the soluble and particulate rabbit media cholesterol ester hydrolase, suggesting a lysosomal origin for this activity. Approximately one-fifth of the total recovered activity was particulate. Particulate media preparations from guinea pig aorta also demonstrated cholesterol ester hydrolase activity at acid pH values with a definite optimum at pH 5 for the glycerol-dispersed substrate. However, in contrast to the rabbit media enzyme, activity was also observed at neutral pH with another optimum at pH 7. The supernatant enzyme from guinea pig media exhibited only a single pH optimum of 7. Cholesterol ester hydrolase activity from either rabbit or guinea pig media was not influenced by preincubation with cyclic AMP, ATP and protein kinase. The addition of chloroquine resulted in the inhibition of both the rabbit and guinea pig enzyme. Cholesterol ester hydrolase activity from rabbit and guinea pig media was also inhibited by phenyl methane sulfonyl fluoride; activity measured at pH 7 (guinea pig) was more sensitive to inhibition than activity measured at pH 5 (guinea pig and rabbit).

The development of hepatic drug-metabolizing enzymes in perinatal guinea pigs: a biochemical and morphological study.

The development of four functionally diverse, hepatic enzymes (p-nitroanisole O-demethylase, aniline hydroxylase, carboxyleterase, and glucuronyltransferase (with alpha-naphthol as the aglycone acceptor)) was studied in perinatal Hartley guinea pigs from 8 days prepartum to 28 days postpartum. A good correlation was observed between the activities measured in resuspended Ca2+-aggregated microsomes and the quantities of hepatic smooth endoplasmic reticulum visible by electron microscopic examination at the different stages of development. The study demonstrated that, postnatally, the guinea pig developed competent enzymatic systems as rapidly as did other laboratory species but that, prenatally, these same enzyme(s) systems were much further advanced than those in other species.

Purification of phosphodiesterase II from rat and guinea-pig intestinal mucosa

Phosphodiesterase II from extracts of intestinal mucosa of rat and guinea pig was purified by chromatography on DEAE-cellulose, CM-cellulose and agarose. The rat enzyme was purified 350-550-fold, with recoveries ranging up to 46%. The best purification of the guinea-pig enzyme was 15-fold, and the recovery was only 2.6%, the large loss occurring during chromatography on DEAE-cellulose and agarose. The poor results with the guinea-pig enzyme reflect the difficulty in obtaining a truly soluble material. Repeated sonication of the crude guinea-pig preparations yielded material that was initially soluble but tended to re-aggregate quickly. Purification of the rat phosphodiesterase II increased its thermostability, the temperature of half-inactivation being increased from 54degrees to 60degreesC. Both enzymes had a Km value of 4 X 10(-5) M with thymidine 3'-(2,4-dinitrophenyl) phosphate as substrate and showed similar pH optima for activity. Both enzymes were inhibited slightly in 0.1 M-MgC12 or 2M-urea and much more strongly in 2M-(NH4)2SO4 or 6M-NaC1. The guinea-pig enzyme was usually inhibited more than the rat enzyme. The Arrhenius plots of the two enzymes differed slightly in slope, but both were biphasic, showing breaks between 30degrees and 40degreesC. It was concluded that the two enzymes were markedly similar in behaviour and that the differences found were related to the different degrees of purification attained by the procedures described.

Development of Gluconeogenic Enzymes in the Newborn Guinea Pig

The activities of key gluconeogenic enzymes in the liver of newborn guinea pigs delivered vaginally at term were monitored as a function of time following birth. The activities of glucose-6-phosphatase and fructose-1,6-diphosphatase did not show a significant increase over the first 72 h of life, neither did the activity of mitochondrial phosphoenolpyruvate carboxykinase. The mitochondrial enzyme pyruvate carboxylase and the cytosolic phosphoenolpyruvate carboxykinase (PEPCK) both increased significantly in the first 24 h postpartum. Mitochondrial protein and succinate dehydrogenase activities showed only slight increases in the 72-hour period. Rapid depletion of liver glycogen was evident in these animals following birth, but severe hypoglycaemia was not evident. Mitochondrial and cytosolic PEPCK showed similar kinetic behaviour with respect to their affinities for oxalacetate and divalent metal cation Mn++, though the mitochondrial enzyme would accept Mg++ as the divalent metal in place of Mn++. The role of the compartmented PEPCK activities is discussed.

Properties of Benzpyrene Hydroxylase from Human Liver and Comparison with the Rat, Rabbit and Guinea-pig Enzymes

1. Benzpyrene hydroxylase of human liver biopsies and of livers from four inbred rat strains and from a non-inbred Sprague-Dawley rat strain, rabbit and guinea-pig was studied. 2. The human liver benzpyrene hydroxylase was similar to that of laboratory animals with respect to cofactor requirements, NADPH and O2, microsomal localization and inhibition by CO and N2. 3. Kinetic analysis of human adult benzpyrene hydroxylase indicated the presence of two enzymes, whereas human foetal liver contained only one enzyme hydroxylating 3,4-benzpyrene. Michaelis constants of human liver benzpyrene hydroxylase were slightly higher than Km values of animal liver enzymes. 4. Benzpyrene hydroxylase activity in human liver showed no sex difference, was enhanced by cigarette smoking and was decreased in patients with liver damage. 5. Variation of benzpyrene hydroxylase activity in human liver samples was about 6-fold in the 'control' group and 16-fold when all patients were considered. Variation of benzpyrene hydroxylase activity in inbred rat strains was less than 60% between different individuals and in the non-inbred rat, rabbit and guinea-pig the variation was 2- to 3-fold or less.

Enzymatic cleavage of carotenoids

1. 1.|Carotene 15,15′-dioxygenase (EC 1.13.11.21) has been isolated from the intestine of guinea pig and rabbit and purified 38- and 30-fold, respectively, but subjecting the intestinal homogenate to protamine sulfate treatment, (NH 4) 2SO 4 fractionation and acetone precipitation. 2. 2.|The guinea pig enzyme showed a pH optimum at 8.5, an optimum substrate concentration of 200 nmoles of β,β-carotene per 25 ml of reaction mixture, an apparent K m with β,β-carotene as substrate of 9.5 · 10 −6 M and a V 3.3 nmoles of retinal formation/mg protein per h. The reaction was linear upto 3 h and the reaction rate increased linearly with increase in enzyme protein concentration. The enzyme was activated by GSH and Fe 2+ and inhibited by sodium dodecylsulfate, sulfhydryl binding and iron chelating agents. 3. 3.|The reaction catalysed by guinea pig enzyme was strictly stoichiometric. 4. 4.|Rabbit enzyme showed a close similarity with guinea pig enzyme with respect to time course, optimum substrate concentration, activation by Fe 2+ and GSH, inhibition by sodium dodecylsulfate, iron chelating and sulfhydryl binding agents. However, it showed a slightly lower pH optimum (pH 7.8). 5. 5.|The enzyme from guinea pig and rabbit showed remarkable similarity with respect to cleavage of carotenoids. The enzyme from both the species was more specific for β,β-carotene but could also cleave a number of other carotenoids at the 15,15′-double bond. 6. 6.|10′-Apo-β-carotenal and 10′-apo-β-carotenol were readily cleaved compared with other apo-β-carotenals and apo-β-carotenols tested. 7. 7.|It has been conclusively shown for the first time that mono-ring substituted carotenoids are also cleaved at the 15,15′-double bond.

Phosphodiesterase II in epithelial cells from guinea-pig and rat small intestine

Phosphodiesterase II activity was determined by using a synthetic substrate, the 2,4-dinitrophenyl ester of thymidine 3'-phosphate. The enzyme activity was determined in fractions obtained by differential centrifugation of homogenates of epithelial cells from the small intestinal mucosa of guinea pigs and rats. In guinea-pig preparations phosphodiesterase II occurred with highest specific activity in those fractions rich in succinate dehydrogenase and acid phosphatase. A lysosomal location for the guinea-pig enzyme was indicated by its structure-linked latency and by its association with particles that under-went a characteristic decrease in equilibrium density when Triton WR-1339 was injected into the animals. With rat preparations a much greater proportion of the phosphodiesterase II activity was found in the soluble fraction after ultracentrifugation. The rat enzyme exhibited a lower degree of latency and administration of Triton WR-1339 had no effect. The rat enzyme further differed from that of the guinea pig in other respects; it was more labile at 60 degrees C, it exhibited a lower pH optimum and it had a higher molecular weight as determined by gel-filtration chromatography.

Guinea-pig liver tryptophan pyrrolase. Absence of detectable apoenzyme activity and of hormonal induction by cortisol and possible regulation by tryptophan

1. When assayed in fresh homogenates, guinea-pig liver tryptophan pyrrolase exists only as holoenzyme. It does not respond to agents that activate or inhibit the rat liver enzyme in vitro. Only by aging (for 30min at 5 degrees C) does the guinea-pig enzyme develop a requirement for ascorbate. 2. The guinea-pig liver enzyme is activated by the administration of tryptophan but not cortisol, salicylate, ethanol or 5-aminolaevulinate. 3. The tryptophan enhancement of the guinea-pig liver pyrrolase activity is prevented by 0, 34 and 86% by pretreatment with actinomycin D, cycloheximide or allopurinol respectively. 4. The guinea-pig liver tryptophan pyrrolase is more sensitive to tryptophan administration than is the rat enzyme. On the other hand, the concentrations of tryptophan in sera and livers of guinea pigs are 45-52% less than those in rats. 5. It is suggested that tryptophan may regulate the activity of guinea-pig liver tryptophan pyrrolase by mobilizing a latent form of the enzyme whose primary function is the detoxication of its substrate.

Enzymatic hydrolysis of Di-(2-ethylhexyl) phthalate by lipases

Di-(2-ethylhexyl) phthalate was hydrolyzed by lipases from a variety of rat tissues. Of the preparations studied, only glycerol-ester hydrolase (pancreatic lipase, EC 3.1.1.3) and sterol-ester -hydrolase (cholesteryl ester hydrolase, EC 3.1.1.13) were unable to hydrolyze the phthalate diester. However, out of 15 different tissue preparations, only the liver alkaline lipase preparation could hydrolyze mono-(2-ethylhexyl) phthalate at a significant rate. The quantitative data on rates of phthalate ester hydrolysis by intestinal enzymes of rat, mouse, hamster, and guinea pig suggested that orally ingested di-(2-ethylhexyl) phthalate would have little opportunity to be absorbed intact. Injected di-(2-ethylhexyl) phthalate, however, would probably survive long enough for all of the tested tissue lipases and lipoprotein lipases to play some role in the metabolism of this compound.

The effect of protein quality and ascorbic acid deficiency on stimulation of hepatic microsomal enzymes in guinea pigs

Poor quality protein supports and even potentiates some metabolic responses in DDT + lindane-treated guinea pigs. Ascorbic acid deficiency, on the other hand, impairs induction of most of the responses measured. Animals fed soy protein and gelatin exhibit the highest O-demethylase activity, hepatic glycogen content and urinary excretion of lindane and lindane metabolites. However, only soy protein permitted significant increases in glucaric acid excretion, hepatic cytochrome P-450 content and glucuronyl transferase. Furthermore, only the guinea pigs on soy protein had statistically significant increases in the metabolic stimulation induced by DDT and lindane in the casein-fed animals. Thus, the animals on the soy protein diet excreted significantly more lindane and lindane metabolites as well as more glucaric acid than the guinea pigs receiving casein.

Metabolic availability of vitamin C in the guinea-pig.

THE results of studies in which L-gulonolactone-1-14C, the precursor of L-ascorbic acid in the rat, was administered to guinea-pigs showed that the guinea-pig is apparently unable to synthesize vitamin C1–3. This has been attributed to the absence of gulonolactone oxidase from the liver microsomes of the guinea-pig; because this enzyme is the last one in the series for converting glucose to ascorbic acid it completely blocks endogenous synthesis. Synthesis of ascorbic acid has been demonstrated in rats: gulonolactone oxidase is present in their livers4–6. A defect, probably a conditional lethal mutation7, in the gene controlling the synthesis of this enzyme in guinea-pigs produces an inactive enzyme or its complete absence. They are therefore dependent on exogenous vitamin C. This genetic disease has been named hypoascorbemia because of the low levels of ascorbic acid in the blood which are pathognomonic of the condition8. Neither the sex nor the age of the animals in these experiments was stated, and there was no indication of the state of ascorbic acid tissue saturation or of the scorbutic condition of the guinea-pigs.

Regulation of gluconeogenesis in the guinea pig liver.

Because of differences in the pattern of enzyme activities involved in glycolysis and gluconeo‐genesis between rat and guinea pig liver, the regulation of gluconeogenesis in guinea pig liver was studied.The following results were obtained: Starvation increases the capacity for gluconeogenesis from pyruvate in vivo. Gluconeogenesis from l‐lactate and from pyruvate in isolated perfused livers is greater in the guinea pig than in the rat, whereas gluconeogenesis from other precursors is similar in both species. Long chain and medium chain fatty acids inhibit gluconeogenesis from l‐lactate in isolated perfused rat liver. In the guinea pig liver the difference does not result from a lower rate of fatty acid oxidation but from a greater reduction in the cytoplasmic NAD+/NADH system.During enhanced fatty acid oxidation in guinea pig liver and in rat liver, similar changes in the concentrations of both acetyl‐S‐CoA and CoA‐SH are observed.With high concentrations of pyruvate as the gluconeogenic precursor, both fatty acids and ethanol lead to a slight stimulation of glucose formation by supplying hydrogen equivalents to the cytosolic NAD+/NADH system. Both glucagon and dibutyryl‐Ado‐3′:5′‐P stimulate ureogenesis and ketogenesis in isolated perfused guinea pig liver. However, the stimulation of gluconeogenesis by glucagon and Ado‐3′:5′‐P normally observed in isolated perfused rat livers was not found in isolated perfused guinea pig livers. Partially purified pyruvate carboxylases from rat and guinea pig liver do not differ with respect to the apparent Km values for pyruvate and ATP, and also the apparent Ka values for acetyl‐S‐CoA at different pH values. At constant concentrations of MgATP2− or MnATP2− the pH‐optima for the guinea pig enzyme are significantly lower. 5‐Methoxyindole‐2‐carbonic acid inhibits gluconeogenesis in guinea pig liver in the same way as in rat liver. Quinolinate, on the other hand is far less inhibitory in guinea pig liver and does not inhibit at all in isolated perfused pigeon livers. Major species differences are found when the activities of the rate limiting enzymes involved in gluconeogenesis are measured under Vmax conditions. Pyruvate carboxylase activity is in the same range in rat and guinea pig liver; pyruvate kinase activity is considerably lower, phospho‐enolpyruvate carboxykinase activity is considerably higher in guinea pig liver. According to our results gluconeogenesis in guinea pig liver from lactate or pyruvate is mainly regulated by the concentration of pyruvate.The difference in the regulation of gluconeogenesis between rat and guinea pig liver probably results from a difference in the compartmentalization of phosphoenopyruvate carboxykinase. A higher rate of futile cycling between phosphoenolpyruvate and pyruvate in rat liver, due to the higher activity of pyruvate kinase in relation to the activities of phosphoenolpyruvate carboxykinase and pyruvate carboxylase, may also contribute to the observed differences.

Studies on the role of hydrolysed glucose cycloacetoacetate on the restoration of the activities of certain enzymes in scorbutic guinea pigs.

The activities of certain enzymes in scorbutic guinea-pigs was studied. There was a significant decrease in the activity of hepatic succinic dehydrogenase, serum alkaline phosphatase and blood catalase. The activity of adenosine triphosphatase increased markedly in scorbutic conditions. Hydrolysed glucosecycloacetoacetate (GCA) could maintain the activities of all these enzymes at their normal levels. A possible mechanism of the beneficial effect of GCA has been discussed.

The Influence of Ultraviolet Light on the Langer-Hans Cell Population and Its Hydrolytic Enzymes in Guinea Pigs

Electron microscopic (1-3) and histochemi-cal (4-10) investigations have provided significant information on Langerhans cells, but the nature of these intra-epidermal dendritic cells and their relationship to the melanocytes are still controversial. A study, therefore, was undertaken to examine Langerhans cells under conditions that are known to induce considerable changes within the pigmentary system and to compare the behavior of the two types of dendritic cells. The Langerhans cells of the guinea pig contain nucleoside phosphatases and aminopeptidase (8) and, they can be visualized selectively in tissue sections because malpighian cells and melanocytes fail to exhibit these enzymatic reactions (9, 10). The application of the histochemical method for adenosine triphosphatase (ATPase) to isolated epidermal sheets constitutes an elegant technic for the quantitation of Langerhans cells and provides a basis for experimental studies on this cell population (9).