Hog Liver Research Articles

N-Terminus determination: FAD and NADP binding domain mapping of hog liver flavin-containing monooxygenase by tandem mass spectrometry

Highly purified hog liver flavin-containing monooxygenase was sequentially denatured, reduced, carboxymethylated, and digested with endoproteinase Glu-C. The purified peptides were subjected to mass spectrometric analysis and the amino acid sequence of selected fragments was determined by tandem mass spectrometry. The amino acid sequence of the first 12 residues of the N-terminus was: Ac-Ala-Lys-Arg-Val-Ala-Ile-Val- Gly-Ala-Gly-Val-Ser-Gly . The amino acid sequence determined for another peptide was: Lys-Ser-Val-Leu-Val-Val- Gly-Met-Gly-Asn-Ser-Gly -Thr-Asp-Ile-Ala-Val-Glu. The results provide direct evidence for the structure of the N-terminal modification of the protein and for the existence of the FAD and NADP binding domains of Gly-X-Gly-X-X-Gly.

In vitro synthesis of nitroxide free radicals by hog liver microsomes

The in vitro biooxidation of 4-hydroxy-2,2,6,60tetramethylpiperidine (TEMP), 4-hydroxy-2,2,4,4-tetyramethyl-1,3-oxazolidine (TEMO) and diphenylamine (DPA)_by hog liver microsomes to their respective nitroxide free radicals, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 2,2,4,4-tetramethyl-1,3-oxazolidine-1-oxyl (TEMOO), and diphenylnitroxide (DPNO) has been investigated. For extending the life span of the liver microsomes, a calcium alginate immobilization procedure was used. The biooxidation rates of the above amines to their respective nitroxide metabolites were measured by means of oxygen uptake at 37°C and pH 7.4. N-octylamine was found to be an activator in the biooxidation of the amines. The formation of the nitroxide radicals was identified by E.S.R. spectroscopy.

Inhibition of mammalian folylpolyglutamate synthetase and human dihydrofolate reductase by 5,8-dideaza analogues of folic acid and aminopterin bearing a terminal L-ornithine.

Six new 5,8-dideaza analogues of folic acid and aminopterin containing a terminal L-ornithine residue were prepared by using multistep synthetic sequences. Each was evaluated as an inhibitor of hog liver folylpolyglutamate synthetase and human dihydrofolate reductase. Structural modifications at positions 2, 4, 5, and 10 were included to help define structure-activity relationships for compounds of this type. The compound N alpha-(4-amino-4-deoxy-5-chloro-5,8-dideazapteroyl)-L-ornithine (3f) was identified as the most potent inhibitor of mammalian folylpolyglutamate synthetase reported thus far (Ki congruent to 2 nM). Its 4-oxy counterpart, N alpha-(5-chloro-5,8-dideazapteroyl)-L-ornithine, was only 5-fold less inhibitory than 3f toward folylpolyglutamate synthetase but was found to be a much weaker inhibitor of dihydrofolate reductase than 3f.

Dihydrofolate synthetase and folylpolyglutamate synthetase: direct evidence for intervention of acyl phosphate intermediates.

The transfer of 17O and/or 18O from (COOH-17O or -18O) enriched substrates to inorganic phosphate (Pi) has been demonstrated for two enzyme-catalyzed reactions involved in folate biosynthesis and glutamylation. COOH-18O-labeled folate, methotrexate, and dihydropteroate, in addition to [17O]-glutamate, were synthesized and used as substrates for folylpolyglutamate synthetase (FPGS) isolated from Escherichia coli, hog liver, and rat liver and for dihydrofolate synthetase (DHFS) isolated from E. coli. Pi was purified from the reaction mixtures and converted to trimethyl phosphate (TMP), which was then analyzed for 17O and 18O enrichment by nuclear magnetic resonance (NMR) spectroscopy and/or mass spectroscopy. In the reactions catalyzed by the E. coli enzymes, both NMR and quantitative mass spectral analyses established that transfer of the oxygen isotope from the substrate 18O-enriched carboxyl group to Pi occurred, thereby providing strong evidence for an acyl phosphate intermediate in both the FPGS- and DHFS-catalyzed reactions. Similar oxygen-transfer experiments were carried out by use of two mammalian enzymes. The small amounts of Pi obtained from reactions catalyzed by these less abundant FPGS proteins precluded the use of NMR techniques. However, mass spectral analysis of the TMP derived from the mammalian FPGS-catalyzed reactions showed clearly that 18O transfer had occurred.

Two Types of Lipases in Hydrolysis of Polyester

Using three kinds of polyesters, polycaprolactone-diol (I), poly(hexamethylene adipate) (II), and a copolyester of I and II, the effects of the molecular weight () of polyester on their enzymatic hydrolysis were examined. The degrees of hydrolysis of the copolyester by Rhizopus arrhizus and R. delemar lipases were much higher than those of the two homopolymers over a wide range of . On the other hand, the degrees of hydrolysis of the copolyester by Candida cylindracea and hog pancreas lipases and hog liver esterase were between, or near, those of the two homopolyesters. R. delemar lipase could hydrolyze the polyester moiety of polyurethane which hog pancreas lipase could not attack. However, no difference in the hydrolysis products between R. delemar and hog pancreas lipases could be detected. The effects of the melting points of polyesters on their hydrolysis by lipases are discussed.

Facile N-oxygenation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by the flavin-containing monooxygenase. A convenient synthesis of tritiated [methyl-3H]-4-phenyl-2,3-dihydropyridinium species.

A rapid, efficient procedure useful for the radiosynthesis of [Me-3H]-MPDP+ ([methyl-3H]-4-phenyl-2,3-dihydropyridinium species) is described. Hog liver microsomes or the highly purified flavin-containing monooxygenase from hog liver quantitatively biotransforms [Me-3H]-MPTP to its corresponding radiolabeled N-oxide. For the small-scale synthesis required for radiolabeling procedures, this enzymatic process is superior to H2O2-mediated N-oxygenation of MPTP. In the presence of 0.5 mM NADPH, 4.5 mM n-octylamine, and 2 microCi [Me-3H]-MPTP, the only product detected in extracts from incubations performed with hog liver microsomes or purified hog liver flavin-containing monooxygenase is [Me-3H]-MPTP N-oxide. [Me-3H]-MPTP N-oxide is almost completely converted to [Me-3H]-MPDP+ by the action of trifluoroacetic anhydride. This procedure has the advantage of using a commercially available tritiated starting material, efficient transformations, and easily accomplished purification to afford a rapid synthesis of [Me-3H]-MPDP+.

Fluorometric procedures for measuring triglyceride concentrations in small amounts of tissue and plasma.

It has been previously shown that triglycerides can be specifically hydrolyzed by lipase from Rhizopus arrhizus in the presence of hog liver esterase and sodium dodecyl sulfate. The glycerol produced can then be measured by sequential reactions with glycerokinase, pyruvate kinase, and lactate dehydrogenase: glycerol and ATP are converted to glycerol-3-phosphate and ADP by glycerokinase; the ADP reacts with phosphoenolpyruvate and pyruvate kinase to yield pyruvate; the pyruvate is converted to lactate with lactate dehydrogenase, and the cofactor NAD+ is simultaneously reduced to NADH. This report describes procedures by which either the disappearance of NADH or the appearance of NAD+ was determined fluorometrically, with 10- to 100-fold greater sensitivity than by spectrophotometry. In addition, enzymatic cycling of NAD+ was used to increase the sensitivity of the assay over 1000-fold, and thereby provided accurate measurement of less than 1 ng of triglyceride. Results obtained from the three fluorometric methods were highly correlated with an automated periodate oxidation method using serum samples and lipid extracts of muscle tissue.

Distribution of Nicotinamide Methyltransferase and Fundamental Properties of the Enzyme in Hog Liver

Distribution of Nicotinamide Methyltransferase and Fundamental Properties of the Enzyme in Hog Liver

Two types of lipases in hydrolysis of polyester.

Using three kinds of polyesters, polycaprolactone-diol (I), poly(hexamethylene adipate) (II), and a copolyester of I and II, the effects of the molecular weight (Mn) of polyester on their enzymatic hydrolysis were examined. The degrees of hydrolysis of the copolyester by Rhizopus arrhizus and R. delemar lipases were much higher than those of the two homopolymers over a wide range of Mn. On the other hand, the degrees of hydrolysis of the copolyester by Candida cylindracea and hog pancreas lipases and hog liver esterase were between, or near, those of the two homopolyesters. R. delemar lipase could hydrolyze the polyester moiety of polyurethane which hog pancreas lipase could not attack. However, no difference in the hydrolysis products between R. delemar and hog pancreas lipases could be detected. The effects of the melting points of polyesters on their hydrolysis by lipases are discussed.

A convenient radiometric assay for flavin-containing monooxygenase activity

A rapid, convenient assay to determine the activity of the flavin-containing monooxygenase is described. The method is based on direct analysis of quenched incubation mixtures by thin-layer chromatography and utilizes tritiated dimethylaniline as the substrate. The synthesis of the radiolabeled substrate is described. The usefulness of dimethylaniline N-oxide formation as a measure of flavin-containing monooxygenase activity was assessed using the purified hog liver enzyme, hog liver microsomes, and liver microsomes from untreated and phenobarbital-pretreated rats.

Mammalian folylpoly-gamma-glutamate synthetase. 1. Purification and general properties of the hog liver enzyme.

Folylpolyglutamate synthetase was purified 30,000-150,000-fold from hog liver. Purification required the use of protease inhibitors, and the protein was purified to homogeneity in two forms. Both forms of the enzyme were monomers of Mr 62,000 and had similar specific activities. The specific activity of the homogeneous protein was over 2000-fold higher than reported for partially purified folylpolyglutamate synthetases from other mammalian sources. Enzyme activity was absolutely dependent on the presence of a reducing agent and a monovalent cation, of which K+ was most effective. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to tetrahydrofolate with the concomitant stoichiometric formation of MgADP and phosphate. Under conditions that resembled the expected substrate and enzyme concentrations in hog liver, tetrahydrofolate was metabolized to long glutamate chain length derivatives with the hexaglutamate, the major in vivo folate derivative, predominating. Enzyme activity was maximal at about pH 9.5. The high-pH optimum was primarily due to an increase in the Km value for the L-glutamate substrate at lower pH values, and the reaction proceeded effectively at physiological pH provided high levels of glutamate were supplied.

Mammalian folylpoly-gamma-glutamate synthetase. 4. In vitro and in vivo metabolism of folates and analogues and regulation of folate homeostasis.

The regulation of folate and folate analogue metabolism was studied in vitro by using purified hog liver folylpolyglutamate synthetase as a model system and in vivo in cultured mammalian cells. The types of folylpolyglutamates that accumulate in vivo in hog liver, and changes in cellular folate levels and folylpolyglutamate distributions caused by physiological and nutritional factors such as changes in growth rates and methionine, folate, and vitamin B12 status, can be mimicked in vitro by using purified enzyme. Folylpolyglutamate distributions can be explained solely in terms of the substrate specificity of folylpolyglutamate synthetase and can be modeled by using kinetic parameters obtained with purified enzyme. Low levels of folylpolyglutamate synthetase activity are normally required for the cellular metabolism of folates to retainable polyglutamate forms, and consequently folate retention and concentration, while higher levels of activity are required for the synthesis of the long chain length derivatives that are found in mammalian tissues. The synthesis of very long chain derivatives, which requires tetrahydrofolate polyglutamates as substrates, is a very slow process in vivo. The slow metabolism of 5-methyltetrahydrofolate to retainable polyglutamate forms causes the decreased tissue retention of folate in B12 deficiency. Although cellular folylpolyglutamate distributions change in response to nutritional and physiological modulations, it is unlikely that these changes play a regulatory role in one-carbon metabolism as folate distributions respond only slowly. 4-Aminofolates are metabolized to retainable forms at a slow rate compared to folates. Although folate accumulation by cells is not very responsive to changes in folylpolyglutamate synthetase levels and cellular glutamate concentrations, cellular accumulation of anti-folate agents would be highly responsive to any factor that changes the expression of folylpolyglutamate synthetase activity.

Studies on substrate specificity of the hog liver flavin-containing monooxygenase: Anionic organic sulfur compounds

The influence of anionic groups on interaction of nucleophilic sulfur compounds with the purified hog liver flavin-containing monooxygenase was evaluated from kinetic constants obtained with various dithiobenzoates, thiolbenzoates, and thiolalkylcarboxylic acids. All compounds tested bearing a single negative charge localized on sulfur were excellent substrates but derivatives with a carboxylic acid group one or two carbons removed from the heteroatom exhibited low or no substrate activity. The effect of a carboxylic acid group more distal from sulfur appeared to depend on steric factors that are not well defined. For instance, none of the carboxylic acids (C 2-C 8) bearing a single thiol on the terminal carbon were oxygenated at detectable rates, whereas dihydrolipoic acid appeared to be a substrate although the concentration required for half-maximal activity was quite high (approximately 2mM). Lipoic acid was a much better substrate ( K m = 0.12 mM), and kinetic constants obtained with lipoic acid analogues suggest that position of the negative charge relative to the dithiolane ring is critical, since increasing the length of the side chain increased the K m . None of the alicyclic disulfides or sulfides containing one or more carboxylic acid groups showed detectable substrate activity. However, the more lipophilic sulfur-containing fatty acids inhibited the enzyme which may mask their potential substrate activity.

Mammalian folylpoly-gamma-glutamate synthetase. 3. Specificity for folate analogues.

A variety of folate analogues were synthesized to explore the specificity of the folate binding site of hog liver folylpolyglutamate synthetase and the requirements for catalysis. Modifications of the internal and terminal glutamate moieties of folate cause large drops in on rates and/or affinity for the protein. The only exceptions are glutamine, homocysteate, and ornithine analogues, indicating a less stringent specificity around the delta-carbon of glutamate. It is proposed that initial folate binding to the enzyme involves low-affinity interactions at a pterin and a glutamate site and that the first glutamate bound is the internal residue adjacent to the benzoyl group. Processive movement of the polyglutamate chain through the glutamate site and a possible conformational change in the protein when the terminal residue is bound would result in tight binding and would position the gamma-carboxyl of the terminal glutamate in the correct position for catalysis. Steric limitations imposed on the internal glutamate residues that loop out and additional steric constraints imposed by binding of different pterin moieties would be expected to effect slight conformational changes in the protein and/or the terminal glutamate and would explain the decrease in on rate and catalytic rate with increased polyglutamate chain length, and the differential effect of one-carbon substitution on the catalytic rate with polyglutamate derivatives. The 4-amino substitution of folate increases the on rate for monoglutamate derivatives but severely impairs catalysis with diglutamate derivatives. Pteroylornithine derivatives are the first potent and specific inhibitors of folylpolyglutamate synthetase to be identified and may act as analogues of reaction intermediates.(ABSTRACT TRUNCATED AT 250 WORDS)

Sulfoxidation of albendazole by a cytochrome P450-independent monooxygenase from rat liver microsomes.

The in vitro biological oxidation of albendazole to albendazole sulfoxide by rat liver microsomes has been studied. This reaction corresponds to a NADPH-dependent enzymatic system, characterised by Km and Vm values of 53.6 microM and 0.59 nmole/mg protein per min. The rate of sulfoxidation by liver microsomes of rats treated with phenobarbital, B-naphthoflavone, Aroclor 1254 and 3-methylcholanthrene was not increased. SKF 525A and metyrapone did not inhibit albendazole sulfoxidase. Thiobenzamide and tranylcypromine decreased sulfoxidation to 48 and 52% of control values. The inhibition by tranylcypromine was competitive. Purified flavin adenine dinucleotide (FAD)-containing monooxygenase from hog liver microsomes catalysed sulfoxidation of albendazole (V = 0.52 nmole/nmole enzyme per min). The present data demonstrate that sulfoxidation of albendazole in the rat liver is not catalysed by a cytochrome P450-dependent monooxygenase and suggest that albendazole is a substrate for FAD-containing monooxygenase (FMO).

Resistance to enzymic hydrolysis as a parameter in drug potency

The enzymic hydrolysis of hydrocortisone 17- and 21-butyrates and hydrocortisone-21-acetate are studied. The results show that the 17-ester is essentially resistant to hog liver and mouse skin esterases while the 21-esters are highly susceptible thus confirming earlier results obtained with betamethasone valerates. The usefulness of these observations in the design of topically active drugs and prodrugs is discussed.

In vitro study of the biological activity of RNAs after incubation of hog liver, heart and brain tissue at room temperature

The biological activity of RNA, isolated from tissue which was incubated for 1, 3, or 6 hours at room temperature (simulation of post-mortem conditions), was preserved. However, the different organs used differ from each other. When liver is used, qualitative differences in the in vitro translation products are observed, after one hour incubation at room temperature, whereas when heart and brain are used these differences are not observed. We have also shown that relatively small amounts of post-mortem tissue is sufficient for RNA extraction. When using frozen tissue it is absolutely necessary to add RNase inhibitors during thawing to reduce the loss of biological activity.

8] Mevalonate kinase

This chapter discusses mevalonate kinase. Mevalonate kinase has been found in a wide variety of sources. Some of these are yeast autolysate, pig and rabbit liver extracts, superovulated rat ovaries, pumpkin seedlings, rubber latex, larva of the flesh fly, green leaves and etiolated cotyledons of French beans, Pinus pinaster seedlings and extracts of Agave americana. Kinase activity has also been found in Pinus radiata seedlings, orange juice vesicles, dark-grown cultures of Kalanchoe crenata, Staphylococcus aureus, and melon cotyledons. Mevalonate kinase has been partially purified from yeast, hog liver, rabbit liver, Cucurbita pepo seedlings, and Hevea brasiliensis (rubber) latex, and it has been purified to homogeneity from hog liver. This enzyme reacts stereospecifically with 3R-mevalonate and MgATP to produce R-mevalonate 5-phosphate, as indicated in the following reaction. Mevalonate kinase activity can also be assayed radiochemically. The amount of phosphorylated derivative formed in an incubation mixture from DL-[2A4C] mevalonate can be determined by paper or thin-layer chromatography and subsequent assay for radioactivity.

Bioreversible phosphate protective groups: Synthesis and stability of model acyloxymethyl phosphates

Six bis(acyloxymethyl) esters of phenyl phosphate and benzyl phosphate and three acyloxymethylbenzylphenyl phosphates were prepared. Debenzylation of benzyl analogs gave respective free acids which were isolated as cyclohexylammonium salts. Stability of bis(acyloxymethyl)phenyl phosphates were studied in various aqueous buffers, hog liver esterase, and mice plasma.

Studies on the metabolic activation of β-keto nitrosamines: Mechanisms of DNA methylation by N-(2-oxopropyl)- N-nitrosourea and N-nitroso- N-acetoxymethyl- N-2-oxopropylamine

At pH 7.35, N-(2-oxopropyl)- N-nitrosourea (OPNU) reacted with calf thymus DNA to yield O 6-methylguanine, 7-methylguanine and 3-methyladenine. Kinetic measurements of the base catalyzed decomposition of OPNU and the extent of methylation of DNA by OPNU suggested that methylnitrosourea is not formed as an intermediate product. Diazoacetone, acetic acid and methanol were identified as products of decomposition of OPNU at pH 7.35. Reaction of OPNU with N-methylmaleimide yielded the product resulting from 1,3-dipolar cycloaddition of diazomethane. Hydrolysis of N-nitroso- N-acetoxymethyl- N-2-oxopropylamine (NAMOPA) in the presence of hog liver esterase also produced diazoacetone, acetic acid and methanol. Enzymatic hydrolysis of NAMOPA in the presence of DNA produced O 6-methylguanine, 7-methylguanine and 3-methyladenine. These results suggest that OPNU undergoes base-catalyzed decomposition and NAMOPA undergoes enzymatic hydrolysis to yield the same intermediate, 2-oxopropyldiazotate. This diazotate then reacts either by protonation followed by loss of water to form diazoacetone, or by internal nucleophilic attack by the diazotate oxygen on the carbonyl carbon to form an oxadiazoline intermediate which then collapses to form acetate and the methylating agent diazomethane. These reaction schemes are used to suggest the mechanism by which N-nitroso-2-oxopropylpropylamine methylates hepatic DNA in vivo.