Sheep Liver Enzyme Research Articles

Inhibition of sorbitol dehydrogenase by nucleosides and nucleotides

Sorbitol dehydrogenase inhibitors have been found to prevent, or alleviate, various secondary complications of diabetes mellitus. In the present study, the effects of nucleosides and nucleotides on the rate of sorbitol oxidation catalyzed by the sheep liver enzyme were studied by steady-state kinetics at pH 7.4. Various such compounds, including ATP and the 2′-deoxy-analogues of ATP, ADP and AMP, reversibly inhibit enzyme activity by formation of enzyme–coenzyme-inhibitor ternary complexes. In each case, no deviations from linearity were seen in the double-reciprocal plots using sorbitol or NAD+ as the varied substrate and there was a linear relationship between inhibitor concentration and the observed inhibitory effects. Sorbitol was docked into a model of the sheep SDH-NAD+ complex based upon the structure of the human SDH-NAD+ holoenzyme. The resulting structure of the ternary complex of sheep SDH, NAD+ and sorbitol (PMDB ID code PM 0078068) shows that the reactive C-2 hydroxyl group of sorbitol is oriented toward the 4′-position of the nicotinamide moiety of the coenzyme, and that the adjacent primary hydroxyl group of sorbitol interacts with the catalytic zinc. The results indicate that the ribose moiety of the inhibitor structures is an important determinant for the observed effects. Specifically, the 2′-position of the ribose ring exerts an effect with respect to inhibitor potency.

Ontogenesis of UDP-glucuronosyltransferase enzymes in sheep

UDP glucuronosyltransferases (UGTs) are important for the metabolism of many drugs. To understand the ontogeny of these enzymes in sheep ( Ovis aries), knowledge of their expression levels at different developmental stages is important. cDNA sequences of six UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9) were cloned in sheep liver by rapid amplification of cDNA ends. The mRNA and protein levels of the enzymes in sheep liver (adult n = 4; newborn n = 3; fetus n = 3) were determined using PCR and Western blots. mRNA levels in adult were very high followed by newborn and then fetus. However, no protein bands were detected on the Western blots of sheep hepatic microsomal proteins using several different antibodies against human UGTs. The effect of antenatal glucocorticoid administration was also studied by infusion of cortisol (0.45 mg/h; 80 h) to another group of fetuses (n = 5). This did not result in any significant changes in fetal mRNA levels. However, the correlation observed between the UGT enzymes and hepatocyte nuclear factor 4α (HNF4α) suggests a possible regulatory role for this transcription factor in sheep. We postulate that fetal and newborn lambs may have a reduced ability to metabolize drugs that are substrates of these enzymes.

Importance in Catalysis of the 6-Phosphate-binding Site of 6-Phosphogluconate in Sheep Liver 6-Phosphogluconate Dehydrogenase

The 6-phosphate of 6-phosphogluconate (6PG) is proposed to anchor the sugar phosphate in the active site and aid in orientating the substrate for catalysis. In order to test this hypothesis, alanine mutagenesis was used to probe the contribution of residues in the vicinity of the 6-phosphate to binding of 6PG and catalysis. The crystal structure of sheep liver 6-phosphogluconate dehydrogenase shows that Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 contribute a mixture of ionic and hydrogen bonding interactions to the 6-phosphate, and these interactions are likely to provide the majority of the binding energy for 6PG. All mutant enzymes, with the exception of T262A, exhibit an increase in K(6PG) that ranges from 5- to 800-fold. There is also a less pronounced increase in K(NADP), ranging from 3- to 15-fold, with the exception of T262A. The R287A and R446A mutant enzymes exhibit a dramatic decrease in V/E(t) (600- and 300-fold, respectively) as well as in V/K(6PG)E(t) (10(5) - and 10(4)-fold), and therefore no further characterization was carried out with these two mutant enzymes. No change in V/E(t) was observed for the Y191A mutant enzyme, whereas 20- and 3-fold decreases were obtained for the K260A and T262A mutant enzymes, respectively, resulting in a decrease in V/K(6PG)E(t) range from 3- to 120-fold. All mutant enzymes also exhibit at least an order of magnitude increase in 13C-isotope effect -1, indicating that the decarboxylation step has become more rate-limiting. Data are consistent with significant roles for Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 in providing the binding energy for 6PG. In addition, these residues also likely ensure proper orientation of 6PG for catalysis and aid in inducing the conformation change that precedes, and sets up the active site for, catalysis.

Characterization and modulation by drugs of sheep liver microsomal flavin monooxygenase activity.

The flavin monooxygenases (FMO) catalyse the NADPH and oxygen-dependent oxidation of a wide range of nucleophilic nitrogen-, sulfur-, phosphorus-, and selenium heteroatom-containing chemicals, drugs, and agricultural agents. In the present study, sheep liver microsomal FMO activity was determined by measuring the S-oxidation rate of methimazole and the average specific activity obtained from different microsomal preparations was found to be 3.8 +/- 1.5 nmol methimazole oxidized min(-1) mg(-1) microsomal protein (mean +/- SE, n = 7). The presence of 0.1% Triton X-100 in the reaction mixture caused an increase of specific sheep liver microsomal FMO activity towards methimazole to 6.1 +/- 1.4 nmol methimazole oxidized min(-1) mg(-1) microsomal protein (mean +/- SE, n = 6). Metabolism of imipramine and chlorpromazine was measured by following the oxidation of cofactor NADPH spectrophotometrically at 340 nm. Sheep liver microsomal FMO activity towards imipramine and chlorpromazine was found to be 10.7 and 12.3 nmol NADPH oxidized min(-1) mg(-1) microsomal protein, respectively. Characterization of sheep liver enzyme was carried out using methimazole as substrate and the maximum FMO enzyme activity was detected at 37 degrees C and at pH 8.0. The apparent K(m) value of sheep liver microsomal FMO for methimazole was 0.118 mM. Effects of the detergents Triton X-100, Cholate, and Emulgen 913, on FMO activity were determined and FMO activity was found to increase with the addition of detergents to the reaction medium. Sheep liver microsomal FMO-catalysed methimazole oxidation was inhibited by imipramine and chlorpromazine when these drugs were used at high concentrations. Western blot-immunochemical analysis revealed the presence of FMO3 in sheep liver microsomes.

Selective inhibition of Trypanosoma brucei 6-phosphogluconate dehydrogenase by high-energy intermediate and transition-state analogues.

Two series of compounds were designed to mimic the transition state and high-energy intermediates (HEI) of the enzymatic reaction of 6-phosphogluconate dehydrogenase (6PGDH). Sulfoxide analogues (7-11) were designed to mimic the transition state during the oxidation of the substrate to 3-keto-6-phosphogluconate, an enzyme-bound intermediate of the enzyme. Hydroxamate and amide derivatives of d-erythronic acid were designed to mimic the 1,2-cis-enediol HEI of the 6PGDH reaction. These two series of compounds were assayed as competitive inhibitors of the Trypanosoma brucei and sheep liver enzymes, and their selectivity value (ratio sheep/parasite) was calculated. The sulfoxide transition-state analogues showed weak and selective inhibition of the T. brucei enzyme. The hydroxamic derivatives showed potent and selective inhibition of the T. brucei 6PGDH with a Ki in the nanomolar range.

Surgical operation planning—an application for 3D-geometry specification

The synthesis and biological evaluation of three series of 6-phosphogluconate (6PG) analogues is described. (2R)-2-Methyl-4,5-dideoxy, (2R)-2-methyl-4-deoxy and 2,4-dideoxy analogues of 6PG were tested as inhibitors of 6-phosphogluconate dehydrogenase (6PGDH) from sheep liver and also Trypanosoma brucei where the enzyme is a validated drug target. Among the three series of analogues, seven compounds were found to competitively inhibit 6PGDH from T. brucei and sheep liver enzymes at micromolar concentrations. Six inhibitors belong to the (2R)-2-methyl-4-deoxy series (6, 8, 10, 12, 21, 24) and one is a (2R)-2-methyl-4,5-dideoxy analogue (29b). The 2,4-dideoxy analogues of 6PG did not inhibit both enzymes. The trypanocidal effect of the compounds was also evaluated in vitro against T. brucei rhodesiense as well as other related trypanosomatid parasites (i.e., Trypanosoma cruzi and Leishmania donovani).

Development of secure applications using small medical institutions

The synthesis and biological evaluation of three series of 6-phosphogluconate (6PG) analogues is described. (2R)-2-Methyl-4,5-dideoxy, (2R)-2-methyl-4-deoxy and 2,4-dideoxy analogues of 6PG were tested as inhibitors of 6-phosphogluconate dehydrogenase (6PGDH) from sheep liver and also Trypanosoma brucei where the enzyme is a validated drug target. Among the three series of analogues, seven compounds were found to competitively inhibit 6PGDH from T. brucei and sheep liver enzymes at micromolar concentrations. Six inhibitors belong to the (2R)-2-methyl-4-deoxy series (6, 8, 10, 12, 21, 24) and one is a (2R)-2-methyl-4,5-dideoxy analogue (29b). The 2,4-dideoxy analogues of 6PG did not inhibit both enzymes. The trypanocidal effect of the compounds was also evaluated in vitro against T. brucei rhodesiense as well as other related trypanosomatid parasites (i.e., Trypanosoma cruzi and Leishmania donovani).

Synthesis and biological evaluation of substrate-Based inhibitors of 6-phosphogluconate dehydrogenase as potential drugs against African Trypanosomiasis

The synthesis and biological evaluation of three series of 6-phosphogluconate (6PG) analogues is described. (2 R)-2-Methyl-4,5-dideoxy, (2 R)-2-methyl-4-deoxy and 2,4-dideoxy analogues of 6PG were tested as inhibitors of 6-phosphogluconate dehydrogenase (6PGDH) from sheep liver and also Trypanosoma brucei where the enzyme is a validated drug target. Among the three series of analogues, seven compounds were found to competitively inhibit 6PGDH from T. brucei and sheep liver enzymes at micromolar concentrations. Six inhibitors belong to the (2 R)-2-methyl-4-deoxy series ( 6, 8, 10, 12, 21, 24) and one is a (2 R)-2-methyl-4,5-dideoxy analogue ( 29b). The 2,4-dideoxy analogues of 6PG did not inhibit both enzymes. The trypanocidal effect of the compounds was also evaluated in vitro against T. brucei rhodesiense as well as other related trypanosomatid parasites (i.e., Trypanosoma cruzi and Leishmania donovani).

6-Phosphogluconate dehydrogenase: Structural symmetry and functional asymmetry

Abstract: 6-Phosphogluconate dehydrogenase catalyzes the oxidative decarboxylation of 6-phosphogluconate to ribulose 5-phosphate through an oxidation, a decarboxylation and a tautomerization. The two subunits in the crystals of the dimeric sheep liver enzyme have the same conformation, as the apoenzyme and with the substrate or coenzyme. An hypothesis is now advanced that in solution, during catalysis, the two subunits have a different alternating role and thus a functional asymmetry.

Molecular organization, catalytic mechanism and function of serine hydroxymethyltransferase — a potential target for cancer chemotherapy

Serine hydroxymethyltransferase, a pyridoxal-5′-phosphate dependent enzyme, catalyzes the retro-aldol cleavage of serine to yield glycine and the hydroxymethyl group is transferred to 5,6,7,8-tetrahydrofolate to generate 5,10-methylene-H 4-folate. The enzyme plays a pivotal role in channeling metabolites between amino acid and nucleotide metabolism. Dihydrofolate reductase and thymidylate synthase have been favorite targets for the development of anticancer drugs. However, development of resistance to drugs, due to a variety of reasons, has necessitated the identification of alternate targets for cancer chemotherapy and serine hydroxymethyltransferase is one such potential target. A detailed study of the kinetics of interaction of serine and folate analogs with this enzyme revealed several unique features that can be exploited for the design of new chemotherapeutic agents. The pathways for the reversible unfolding of the dimeric Escherichia coli and the tetrameric sheep liver enzyme, although different, revealed a requirement for the cofactor in the final step for generating an active enzyme. The gly A gene of Escherichia coli has been shown to code for this enzyme. Analysis of available gene sequences indicate that serine hydroxymethyltransferase is one of the most highly conserved proteins. The isolation of the cDNA clones for the enzyme and their overexpression in heterologous systems has enabled the probing of the molecular mechanisms of catalysis and the role of lysine, arginine and histidine in cofactor, substrate(s) binding and in maintaining the structure of the protein. Recently, the three-dimensional structure of the human liver serine hydroxymethyltransferase has been published. This, along with the information already available, provides a framework for the rational design of drugs targeted specifically towards this enzyme.

6-Phosphogluconate dehydrogenase: the mechanism of action investigated by a comparison of the enzyme from different species

The mechanism of action of 6-phosphogluconate dehydrogenase with the alternative substrate 2-deoxy 6-phosphogluconate was investigated using enzymes from sheep liver, human erythrocytes and Trypanosoma brucei. The three enzymes oxidize 2-deoxy 6-phosphogluconate, but only the sheep liver enzyme releases the intermediate 2-deoxy,3-keto 6-phosphogluconate. Kinetic comparison showed that an increase in the rate of NADP + reduction at high pH is due to increased release of the intermediate, rather than an increase in the overall reaction rate. 2-Deoxy,3-keto 6-phosphogluconate is decarboxylated by the erythrocyte and trypanosome enzymes but not the liver one in the absence of either NADPH or 6-phosphogluconate, which act as activators. The pH dependence of decarboxylation and the degree of activation suggest that 6-phosphogluconate is the activator which operates under normal assay conditions, while NADPH acts mainly by increasing the binding of the intermediate. The data suggest that the activity of 6PGDH is subjected to a two-way regulation: NADPH, which regulates the pentose phosphate pathway, inhibits the enzyme, while 6-phosphogluconate, levels of which rise when NADPH inhibition is removed, acts as an activator ensuring that 6-phosphogluconate is rapidly removed.

A 2.8 Å resolution structure of 6-phosphogluconate dehydrogenase from the protozoan parasiteTrypanosoma brucei: comparison with the sheep enzyme accounts for differences in activity with coenzyme and substrate analogues

The three-dimensional structure of 6-phosphogluconate dehydrogenase (6PGDH) from the parasitic protozoan Trypanosoma brucei has been solved at 2.8 Å resolution. This pentose phosphate pathway enzyme is NADP-dependent; NADPH generated in the reaction protects against oxidative stress. The enzyme crystallises in the space-group P3121 with a dimer in the asymmetric unit and cell dimensions a=b=135.13 Å, c=116.74 Å, α=β=90°, γ=120°. The structure has refined to R=18.6% (Rfree=27.3%) with good geometry.The amino acid sequence of T. brucei 6PGDH is only 35% identical to that of the sheep liver enzyme and significant activity differences have been observed. The active dimer assembles with the C-terminal tail of one subunit threaded through the other, forming part of the substrate binding site. The tail of T. brucei 6PGDH is shorter than that of the sheep enzyme and its terminal residues associate tightly with the second monomer. The three-dimensional structure shows this generates additional interactions between the subunits close to the active site; the coenzyme binding domain is thereby associated more tightly with the helical domain. Three residues, conserved in all other known sequences, are important in creating a salt bridge between monomers close to the substrate binding site. The differences could explain the 200-fold enhanced affinity observed for the substrate analogue 6-phospho-2-deoxy-d-gluconate and suggest targets for anti-parasite drug design.The coenzyme binding domain of 6PGDH has a β-α-β fold; while in most species the “fingerprint” sequence is GxAxxG, in the T. brucei enzyme it is GxGxxG. Additional interactions between the enzyme and the coenzyme bis-phosphate are likely in the parasite 6PGDH, accounting for greater inhibition (40-fold) of 2′5′-ADP. While the core of the T. brucei dimer was restrained during refinement, several conformational differences have been found between the monomers; those at the coenzyme binding site suggest the molecule could be asymmetric during the enzyme reaction.

Cloning, expression, purification, and characterization of the 6-phosphogluconate dehydrogenase from sheep liver.

The mRNA encoding the 51-kDa subunit of 6-phosphogluconate dehydrogenase (6PGDH) from sheep liver was reverse-transcribed and amplified. The resulting cDNA was reamplified in N-terminal and C-terminal segments and spliced to generate a full-length clone, and an internal cDNA fragment was also amplified. The full-length clone containing the complete coding sequence of the 6PGDH cDNA was sequenced and found to contain two mutations and two deletions in the internal region and two mutations outside of the internal region, an A to G point mutation at position 1407 that resulted in the amino acid change Gln 445 to Arg and a silent mutation at position 1426. The internal clone was sequenced and shown to be free of any mutations; therefore the internal piece was used to replace the same region in the full-length clone to correct the mutations in this region. The mutation at position 1407 which was outside of the internal region was corrected using site-directed mutagenesis. The cDNA with the correct codon was then subcloned into the bacterial expression vector pQE-30 and overproduced in Escherichia coli strain M15. A protein with a subunit molecular weight of 51,000 was expressed at a level of about 4.5% of the total soluble protein in M15 as judged by SDS/PAGE. Cloning into pQE-30 adds six histidines and a short linker to the N-terminus of the enzyme. The recombinant 6PGDH with His-tag was purified using the Ni-NTA affinity column supplied by Qiagen. The purification procedure resulted in a homogeneous protein by SDS/PAGE with 22.4-fold purification with an overall yield of 61%. The recombinant enzyme exhibits kinetic parameters within error identical to those measured for native sheep liver enzyme.

The effects of fascioliasis on the activities of some drug-metabolizing enzymes in desert sheep liver

Desert sheep experimentally or naturally infected with Fasciola gigantica were used to study the influence of infection on the activities of some drug-metabolizing enzymes found in the liver. The enzymes investigated were aminopyrine N-demethylase, aniline 4-hydroxylase and UDP-glucuronyltransferase. The experimental infection was confirmed histologically by detection of Fasciola eggs in faeces and by measuring the activities of sorbitol dehydrogenase (SD), glutamate dehydrogenase (GD) and aspartate aminotransferase (AST) in plasma during the course of the disease. Liver specimens from naturally infected sheep were obtained from the slaughter house. The activities of aminopyrine N-demethylase and aniline 4-hydroxylase were significantly decreased in sheep either naturally infected or during the acute stage of experimental fascioliasis (killed 5 weeks post-infection). The activity of UDP-glucuronyltransferase was decreased in naturally infected sheep and those killed 9 or 13 weeks post-experimental infection.

Serine Hydroxymethyltransferase from Mung Bean (Vigna radiata) Is Not a Pyridoxal-5′-Phosphate-Dependent Enzyme

Serine hydroxymethyltransferase from mammalian and bacterial sources is a pyridoxal-5'-phosphate-containing enzyme, but the requirement of pyridoxal-5'-phosphate for the activity of the enzyme from plant sources is not clear. The specific activity of serine hydroxymethyltransferase isolated from mung bean (Vigna radiata) seedlings in the presence and absence of pyridoxal-5'-phosphate was comparable at every step of the purification procedure. The mung bean enzyme did not show the characteristic visible absorbance spectrum of a pyridoxal-5'-phosphate protein. Unlike the enzymes from sheep, monkey, and human liver, which were converted to the apoenzyme upon treatment with l-cysteine and dialysis, the mung bean enzyme similarly treated was fully active. Additional evidence in support of the suggestion that pyridoxal-5'-phosphate may not be required for the mung bean enzyme was the observation that pencillamine, a well-known inhibitor of pyridoxal-5'-phosphate enzymes, did not perturb the enzyme spectrum or inhibit the activity of mung bean serine hydroxymethyltransferase. The sheep liver enzyme upon interaction with O-amino-d-serine gave a fluorescence spectrum with an emission maximum at 455 nm when excited at 360 nm. A 100-fold higher concentration of mung bean enzyme-O-amino-d-serine complex did not yield a fluorescence spectrum. The following observations suggest that pyridoxal-5'-phosphate normally present as a coenzyme in serine hydroxymethyltransferase was probably replaced in mung bean serine hydroxymethyltransferase by a covalently bound carbonyl group: (a) inhibition by phenylhydrazine and hydroxylamine, which could not be reversed by dialysis and or addition of pyridoxal-5' phosphate; (b) irreversible inactivation by sodium borohydride; (c) a spectrum characteristic of a phenylhydrazone upon interaction with phenylhydrazine; and (d) the covalent labeling of the enzyme with substrate/product serine and glycine upon reduction with sodium borohydride. These results indicate that in mung bean serine hydroxymethyltransferase, a covalently bound carbonyl group has probably replaced the pyridoxal-5'-phosphate that is present in the mammalian and bacterial enzymes.

Isolation and primary structure of the gene encoding fructose-1,6-bisphosphatase from Saccharomyces cerevisiae

The gene encoding Saccharomyces cerevisiae fructose-1,6-bisphosphatase ( FBP1) was isolated. Constructed fbpi::HIS3 null mutants were unable to grow with ethanol, and growth was restored after transformation with the cloned fbp gene. The gene codes for a protein of 347 amino acid residues with an M r of 38131. Homology with the pig kidney cortex and the sheep liver enzyme is 47.7% and 46.6%, respectively, within a central core of 328 amino acid residues. The cloned promoter size was 318 bp and allowed only low level expression of the gene. This indicates a positive activation site (UAS) upstream of the cloned DNA fragment.

The natural and unnatural diastereomers of leucovorin: aspects of their cellular pharmacology.

A mixture of the natural ((6S)-LV) and unnatural ((6R)-LV) diastereomers of leucovorin (LV, (6RS)-LV) is administered clinically in combination with 5-fluorouracil (FUra) to overcome drug resistance due to insufficient intracellular folate concentrations (1). Initially described as a growth factor for Pediococcus cerevesiae, LV has a S chirality at the 6 carbon of the tetrahydropterine ring (2). Following intravenous administration of a mixture of the (6S) and (6R) isomers of leucovorin to patients, plasmatic (6S)-LV disappears rapidly with a T0.5 of 30 min. while (6R)-LV has a slower clearance (To.5 of 7.5 hours) (1,3). The natural isomer is readily transported into cells where it has no known folate cofactor activity. Greenberg was the first to show that LV was converted to another folate involved in purine biosynthesis (4). The first enzymatic step involved was later found to be methenyltetrahy-drofolate synthetase (5-formyltetrahy-drofolate cyclodehydrase, EC 6.3.3.2) which catalyzes the irreversible and stereospecific ATP and Mg2+-dependent transformation of (6S)-LV to N5–10-methenyltetrahy-drofolate. Sheep liver enzyme activity was first partially purified (5) and the enzyme was more recently highly purified and characterized from Lactobacillus casei (6) and rabbit liver (7). We have recently obtained and characterized highly purified human liver methenyltetrahydrofolate synthetase (8) and will describe in the following paper its purification and characteristics. We next took advantage of the enzyme characteristics to prepare stereochemically pure (6R)-LV (9) and will present its transport characteristics and biological activity in human leukemic CCRF-CEM cells.

The influence of diet on the activity of certain lipogenic and gluconeogenic enzymes in sheep liver and adipose tissue

Conference Article| October 01 1987 The influence of diet on the activity of certain lipogenic and gluconeogenic enzymes in sheep liver and adipose tissue J. R. SCAIFE; J. R. SCAIFE 1Aberdeen School of Agriculture, 581 King Street, Aberdeen AB9 1UD, U.K. Search for other works by this author on: This Site PubMed Google Scholar N. S. JESSOP N. S. JESSOP *The Edinburgh School of Agriculture, West Mains Road, Edinburgh EH9 3JG, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1987) 15 (5): 827–828. https://doi.org/10.1042/bst0150827 Article history Received: April 01 1987 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation J. R. SCAIFE, N. S. JESSOP; The influence of diet on the activity of certain lipogenic and gluconeogenic enzymes in sheep liver and adipose tissue. Biochem Soc Trans 1 October 1987; 15 (5): 827–828. doi: https://doi.org/10.1042/bst0150827 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: PEPCK, phosphoenolpyruvate carboxykinase This content is only available as a PDF. © 1987 Biochemical Society1987 Article PDF first page preview Close Modal You do not currently have access to this content.

Regulation of carnitine palmitoyltransferase activity by malonyl-CoA in mitochondria from sheep liver, a tissue with a low capacity for fatty acid synthesis

The characteristics of inhibition of carnitine palmitoyltransferase (CPT) I by malonyl-CoA were studied for the enzyme in mitochondria isolated from sheep liver, a tissue with a very low rate of fatty acid synthesis. Malonyl-CoA was as potent in inhibiting the sheep liver enzyme as in inhibiting the enzyme in rat liver mitochondria. CPT I in guinea-pig liver mitochondria was also similarly inhibited. The inhibition showed the same time-dependent characteristics previously established for the rat liver enzyme. Methylmalonyl-CoA was as effective an inhibitor of CPT I as malonyl-CoA in sheep liver mitochondria, but did not affect CPT I activity in mitochondria from rat or guinea-pig liver. The concentrations of malonyl-CoA required to inhibit CPT I in sheep liver mitochondria in vitro were similar to those found in freeze-clamped sheep liver samples (about 7 nmol of malonyl-CoA/g wet wt.). In sheep liver cells the content of malonyl-CoA was only one-tenth of that observed in vivo when glucose only was added to the incubation medium. Inclusion of acetate and/or insulin increased the malonyl-CoA content about 10-fold, to values similar to those observed in vivo. The rate of fatty acid synthesis in sheep liver cells was about 1% of that observed in rat liver, but was correlated with the concentrations of malonyl-CoA in the cells under various incubation conditions. These observations are discussed in relation to (i) the regulatory role of malonyl-CoA in tissues that have a low capacity for fatty acid synthesis, and (ii) the utilization by sheep liver of propionate as a gluconeogenic precursor.

COMPARITIVE TOXICITY AND METABOLISM OF PYRROLIZIDINE ALKALOIDS IN RUMINANTS AND NONRUMINANT HERBIVORES

The toxicity of tansy ragwort (Sencio jacobaea) to several ruminant and nonruminant species was tested. Cattle and horses were susceptible (lethal dose of tansy ragwort (TR) was 4–8% of body weight) whereas sheep and goats were resistant (lethal dose of TR was 200–300% of body weight). Other nonruminant herbivoires (rabbits, gerbils, guinea pigs, hamsters) were resistant to TR. Incubation of TR in sheep rumen fluid did not alter its toxicity to rats. No 1-methylene pyrrolizidine alkaloid (PA) derivatives were detected in TR incubated in sheep rumen fluid. The activities of PA-metabolizing enzymes in sheep liver were not influenced by dietary TR. Key words: Pyrrolizidine alkaloids, ruminants, nonruminant herbivores