Composition of Pteroylpolyglutamates (Conjugated Folates) in Guinea-Pig Liver and Their Formation from Folic Acid

1. The composition of guinea-pig liver folates and the biochemical route of formation of liver folates from injected tritium-labelled pteroylglutamic acid (folic acid) have been studied. 2. Endogenous folate was measured by microbiological assay with Lactobacillus casei and Streptococcus faecalis, with and without deconjugation of whole liver pteroylpolyglutamates (conjugated folates). Individual folate compounds were identified by microbiological assay after fractionation of liver folates by DEAE cellulose ion-exchange column chromatography. 3. Liver folate in the guinea-pig consists of about 84–87% reduced pteroylpolyglutamates with more than three glutamate moieties/molecule, about 12–15% reduced pteroyltriglutamates, about 1% reduced pteroyldiglutamates and only traces of reduced pteroylmonoglutamates. 4. About 53% of the liver folate consists of methylated derivatives. 5. Injected pteroylglutamic acid was first rapidly reduced and formylated or methylated. Glutamate moieties were then added, probably singly, to form di-, tri- and poly-glutamates. This was a relatively slow process with a hold-up at the triglutamate stage. 6. The proportion of the labelled pteroylglutamic acid in the polyglutamate form approximated to the proportion of endogenous folates in this form after 3–4 days. 7. The amount of radioactive folate in the liver increased progressively from 1 to 84 h after injection of a standard amount of radioactive pteroylglutamic acid.

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Subcellular Localization of Folate and Effect of Methotrexate on the Incorporation of Radioactive Folic Acid into Guinea-Pig Liver Folate

Clinical Science ◽

10.1042/cs0430815 ◽

1972 ◽

Vol 43 (6) ◽

pp. 815-822 ◽

Cited By ~ 14

Author(s):

R. Corrocher ◽

A. V. Hoffbrand

Keyword(s):

Folic Acid ◽

Guinea Pig ◽

Subcellular Localization ◽

Subcellular Distribution ◽

Mammalian Cells ◽

Pig Liver ◽

Pteroylglutamic Acid ◽

Tetrahydrofolic Acid ◽

Guinea Pig Liver ◽

Liver Folate

1. The subcellular distribution of endogenous folate and of injected [3H]folic acid has been studied in guinea-pig liver. 2. Endogenous folate was found to be concentrated in the mitochondrial and cell-sap fractions. 3. At 1 h after injection, labelled folic acid (pteroylglutamic acid) was concentrated in the microsomes (where it is possibly reduced to tetrahydrofolic acid). 4. At all subsequent times after injection of labelled pteroylglutamic acid (4, 24 and 84 h) radioactivity was concentrated in mitochondria and cell sap and it is, therefore, likely that conjugation of glutamates to form pteroylpolyglutamates occurs in one or both of these subcellular fractions in mammalian cells. 5. Methotrexate-treated animals converted at least some labelled pteroylglutamic acid into non-reduced pteroyl di-, tri- and poly-glutamic acids by 4 h after the injection. 6. Reduction of pteroylglutamic acid is, therefore, not essential for pteroylpolyglutamate formation in liver cells. 7. Methotrexate blocks the reduction of pteroyl di-, tri- and poly-glutamic acids, as well as of pteroylglutamic acid.

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Purification and properties of a new testosterone 17β-dehydrogenase (NADP+) from guinea-pig liver

Biochemical Journal ◽

10.1042/bj1630401a ◽

1977 ◽

Vol 163 (3) ◽

pp. 401-407 ◽

Cited By ~ 6

Author(s):

E Kaguera ◽

S Toki

Keyword(s):

Guinea Pig ◽

Column Chromatography ◽

Gel Filtration ◽

Deae Cellulose ◽

Supernatant Fraction ◽

Pig Liver ◽

Ring Junction ◽

Purification And Properties ◽

Androstane Derivatives ◽

Guinea Pig Liver

As a result of studies of guinea-pig live testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), a new testosterone 17beta-dehydrogenase was discovered. The new enzyme was purified to a single homogeneous protein from the 105 000 g-supernatant fraction of guinea-pig liver by (NH4)2SO4 fractional precipitation and two gel-filtration stages, DEAE-cellulose column chromatography and hydroxyapatite column chromatography. It was characterized by many properties. The enzyme has almost the same properties as the classical testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), with respect to cofactor requirement, pH optima for dehydrogenation, effect of phosphate ion on the NAD+-dependent reaction and molecular weight, but characteristic differences were observed in substrate-specificity between the two dehydrogenases. With various androstane derivatives, the configuration of the A/B-ring junction was closely connected with enzyme activity. 5alpha-Androstanes, such as 5alpha-androstane-3alpha,17beta-diol, 5alpha-androstane-3beta,17beta-diol and 17beta-hydroxy-5alpha-androstan-3-one, and 5beta-congeners, such as 5beta-androstane-3alpha,17beta-diol, 5beta-androstane-3beta,17beta-diol and 17beta-hydroxy-5beta-androstan-3-one, served as substrates for both the EC 1.1.1.64 enzyme and the new enzyme. The EC 1.1.1.64 enzyme oxidized testosterone more rapidly than did the new enzyme. These comparisons were based on the relative activities, apparent Km values and apparent Vmax values.

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Membrane-associated pyruvate kinase in developing guinea-pig liver

Biochemical Journal ◽

10.1042/bj2350103 ◽

1986 ◽

Vol 235 (1) ◽

pp. 103-110 ◽

Cited By ~ 1

Author(s):

S M Farrow ◽

C T Jones

Keyword(s):

Guinea Pig ◽

Pyruvate Kinase ◽

Membrane Fraction ◽

Deae Cellulose ◽

Kinetic Properties ◽

Microsomal Enzyme ◽

Hydrophobic Membrane ◽

Pig Liver ◽

Additional Protein ◽

Guinea Pig Liver

During analysis of pyruvate kinase distribution in developing guinea-pig liver it was observed that a substantial proportion of the activity remained associated with the microsomal membrane fraction (‘microsomes’). Although some of this could be removed by washing with sucrose, the majority required detergent treatment for liberation, and even then at least one-half remained attached to the microsomes. Estimates of the contribution of this fraction to total cell pyruvate kinase activity indicated that it was more than 50% of the total, and this is likely to be an underestimate because of the continued latency of the enzyme even in the presence of detergent. The susceptibility of the microsomal enzyme, whether released by detergent or sucrose washing, to inactivation by Triton X-100 suggested it to be different from the cytosolic enzyme, which was stable under such conditions. (The microsomal enzyme required the presence of additional protein, such as bovine serum albumin, to maintain stability.) This view was confirmed by DEAE-cellulose chromatography and particularly isoelectric focusing, where the microsomal enzyme was shown to consist of at least four forms, which were distinctly different from those in the cytosol. Those data and the kinetic properties of the four forms in the membrane fraction indicate that the microsomal pyruvate kinase could consist of four counterparts to the cytosolic isoenzyme forms. These results are discussed in relation to the two possible explanations for the phenomenon (not mutually exclusive): that the more hydrophobic membrane forms are precursors of the cytosolic enzyme and that they may be part of functional glycolytic pathway in the microsomes of developing liver.

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The identification of the folate conjugates found in rat liver 48 h after the administration of radioactively labelled folate tracers

Biochemical Journal ◽

10.1042/bj1860235 ◽

1980 ◽

Vol 186 (1) ◽

pp. 235-242 ◽

Cited By ~ 6

Author(s):

M J Connor ◽

J A Blair

Keyword(s):

Ion Exchange ◽

Rat Liver ◽

Paper Chromatography ◽

Column Chromatography ◽

Lactobacillus Casei ◽

Deae Cellulose ◽

Microbiological Assay ◽

Exchange Column ◽

Chromatographic Techniques ◽

Ion Exchange Column

About 70% of the radioactivity retained in the livers of rats dosed 48 h earlier with radioactively labelled folate was incorporated into two folate conjugates. The major derivative was purified and isolated by Sephadex G-15, DEAE-cellulose and DEAE-Sephadex ion-exchange column chromatography and paper chromatography. It was identified as 10-formylpteroylpentaglutamate by a combination of spectral, microbiological, chemical and chromatographic techniques. The minor conjugate, though less well characterized, exhibited similar properties and was assigned the structure 10-formylpteroyltetraglutamate. 10-Formylpteroylpentaglutamate (2.0nmol/g) and 10-formylpteroyltetraglutamate (0.25nmol/g) comprised about 20% of the total endogenous hepatic folate as determined by microbiological assay (Lactobacillus casei after conjugase treatment.

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Die Bedeutung der Homogenisationsart und -intensität für die Größe und Konstanz der Sauerstoffaufnahme von Meerschweinchen-Leberhomogenat / The Influence of Mode and Intensity of Homogenization on the Absolute Value and Stability of Oxygen Consumption of Guinea Pig Liver Homogenates

Zeitschrift für Naturforschung C ◽

10.1515/znc-1977-11-1205 ◽

1977 ◽

Vol 32 (11-12) ◽

pp. 908-912 ◽

Cited By ~ 5

Author(s):

H. J. Schmidt ◽

U. Schaum ◽

J. P. Pichotka

Keyword(s):

Oxygen Uptake ◽

Guinea Pig ◽

Measuring Time ◽

Pig Liver ◽

Absolute Value ◽

Modified Method ◽

Simultaneous Measurements ◽

Liver Homogenates ◽

The Absolute ◽

Guinea Pig Liver

Abstract The influence of five different methods of homogenisation (1. The method according to Potter and Elvehjem, 2. A modification of this method called Potter S, 3. The method of Dounce, 4. Homogenisation by hypersonic waves and 5. Coarce-grained homogenisation with the “Mikro-fleischwolf”) on the absolute value and stability of oxygen uptake of guinea pig liver homogenates has been investigated in simultaneous measurements. All homogenates showed a characteristic fall of oxygen uptake during measuring time (3 hours). The modified method according to Potter and Elvehjem called Potter S showed reproducible results without any influence by homogenisation intensity.

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Precision-cut Guinea-pig Liver Slices as a Tool for Studying the Toxicity of Volatile Anaesthetics

Alternatives to Laboratory Animals ◽

10.1177/026119299001800120.1 ◽

1990 ◽

Vol 18 (1_part_1) ◽

pp. 191-199

Author(s):

Hanan N. Ghantous ◽

Jeanne Fernando ◽

Scott E. Morgan ◽

A. Jay Gandolfi ◽

Klaus Brandel

Keyword(s):

Guinea Pig ◽

Rank Order ◽

Normal Liver ◽

Liver Slice ◽

Volatile Anaesthetics ◽

Pig Liver ◽

Liver Slices ◽

Guinea Pig Liver ◽

Krebs Henseleit Buffer

Cultured precision-cut liver slices retain normal liver architecture and physiological biochemical functions. Hartley male guinea-pig liver slices have proven to be a good model for studying the biotransformation and toxicity of halothane. This system was used to evaluate the biotransformation and toxicity of different volatile anaesthetics (halothane, enflurane, isoflurane and sevoflurane), and compare their effects to those of new anaesthetics (desflurane). Liver slices (250–300μm thick) were incubated in sealed roller vials, containing Krebs Henseleit buffer at 37°C under 95% O2:5% CO2 atmosphere. Volatile anaesthetics were delivered by volatilisation after pre-incubation for 1 hour to produce a constant concentration in the medium. Production of the metabolites, trifluroacetic acid and fluoride ion, was measured. Intracellular potassium ion content, protein synthesis and secretion were determined as indicators of viability of the slices. The rank order of biotransformation of anaesthetics by the liver slices was halothane >sevoflurane>isoflurane and enflurane>desflurane. The rank order of hepatotoxicity of these anaesthetics was halothane>isoflurane and enflurane>sevoflurane and desflurane. Halothane is the anaesthetic which is metabolised furthest and has the most toxic effect, while desflurane is the least metabolised anaesthetic and has the least toxicity. This in vitro cultured precision-cut liver slice system appears to be suitable for studying the biotransformation of volatile anaesthetics and correlating its role in the resulting toxicity.

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