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1.
J Am Chem Soc ; 128(11): 3838-47, 2006 Mar 22.
Article in English | MEDLINE | ID: mdl-16536560

ABSTRACT

Rapamycin, FK506, and FK520 are immunosuppressant macrolactone natural products comprised of predominantly polyketide-based core structures. A single nonproteinogenic pipecolic acid residue is installed into the scaffold by a nonribosomal peptide synthetase that also performs the subsequent macrocyclization step at the carbonyl group of this amino acid. It has been assumed that pipecolic acid is generated from lysine by the cyclodeaminases RapL/FkbL. Herein we report the heterologous overexpression and purification of RapL and validate its ability to convert L-lysine to L-pipecolic acid by a cyclodeamination reaction that involves redox catalysis. RapL also accepts L-ornithine as a substrate, albeit with a significantly reduced catalytic efficiency. Turnover is presumed to encompass a reversible oxidation at the alpha-amine, internal cyclization, and subsequent re-reduction of the cyclic delta1-piperideine-2-carboxylate intermediate. As isolated, RapL has about 0.17 equiv of tightly bound NAD+, suggesting that the enzyme is incompletely loaded when overproduced in E. coli. In the presence of exogenous NAD+, the initial rate is elevated 8-fold with a Km of 2.3 microM for the cofactor, consistent with some release and rebinding of NAD+ during catalytic cycles. Through the use of isotopically labeled substrates, we have confirmed mechanistic details of the cyclodeaminase reaction, including loss of the alpha-amine and retention of the hydrogen atom at the alpha-carbon. In addition to the characterization of a critical enzyme in the biosynthesis of a medically important class of natural products, this work represents the first in vitro characterization of a lysine cyclodeaminase, a member of a unique group of enzymes which utilize the nicotinamide cofactor in a catalytic manner.


Subject(s)
Ammonia-Lyases/metabolism , Pipecolic Acids/metabolism , Amino Acid Sequence , Ammonia-Lyases/antagonists & inhibitors , Ammonia-Lyases/genetics , Ammonia-Lyases/isolation & purification , Enzyme Inhibitors/pharmacology , Escherichia coli/genetics , Escherichia coli/metabolism , Kinetics , Molecular Sequence Data , Nipecotic Acids/pharmacology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sirolimus/metabolism
2.
Biochim Biophys Acta ; 1638(2): 173-8, 2003 Jul 14.
Article in English | MEDLINE | ID: mdl-12853123

ABSTRACT

Delta-aminolevulinic acid (ALA), precursor of heme, accumulates in a number of organs, particularly in liver of patients with acute porphyrias or lead intoxication. This study characterizes the involvement of bilirubin as an antioxidant in a chronic intoxication with ALA. Female Wistar rats were injected intraperitoneally a daily dose of 40 mg ALA/body wt., during 10 days. A marked increase in lipid peroxidation and a decrease in GSH content were observed 24 h after the last injection of ALA. The activities of liver antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase were also diminished. ALA synthase (ALA-S) and heme oxygenase-1 were induced. Both ALA dehydratase (ALA-D) and porphobilinogenase (PBG-ase) activities were inhibited. Administration of bilirubin (5 mmol/kg body wt.) 2 h before ALA treatment entirely prevented the effects of ALA. Co-administration of ALA and Sn-protoporphyrin IX (Sn-PPIX; 100 microg/body wt., i.p.), a potent inhibitor of heme oxygenase, completely abolished its induction and provoked a marked decrease in liver GSH levels as well as an increase in lipid peroxidation. These results add further support to the proposal assigning bilirubin a key protective role against oxidative damage here induced by ALA.


Subject(s)
Aminolevulinic Acid/toxicity , Antioxidants/pharmacology , Bilirubin/pharmacology , Liver/drug effects , Oxidative Stress/drug effects , 5-Aminolevulinate Synthetase/metabolism , Ammonia-Lyases/antagonists & inhibitors , Animals , Bilirubin/administration & dosage , Catalase/metabolism , Enzyme Induction , Female , Glutathione/metabolism , Glutathione Peroxidase/metabolism , Heme Oxygenase (Decyclizing)/metabolism , Lipid Peroxidation/drug effects , Liver/enzymology , Porphobilinogen Synthase/antagonists & inhibitors , Rats , Rats, Wistar , Superoxide Dismutase/metabolism
3.
Appl Microbiol Biotechnol ; 43(5): 901-7, 1995 Oct.
Article in English | MEDLINE | ID: mdl-7576557

ABSTRACT

3-Methylaspartase was purified 24-fold and crystallized from the crude extract of the cells of a facultative anaerobic bacterium from soil, strain YG-1002. The molecular mass of the native enzyme was about 84 kDa and that of the subunit was about 42 kDa. The pH optimum for the deamination reaction of (2S, 3S)-3-methylaspartic acid and those for the amination reaction of mesaconic acid were 9.7 and 8.5; its optimum temperature was 50 degrees C. The enzyme was stable at pH 5.5-11.0 and up to 50 degrees C. The enzyme required both divalent and monovalent cations such as Mg2+ and K+. The enzyme was inhibited by sulfhydryl reagents, metal-chelating reagents and some divalent cations. The enzyme catalyzed the reversible amination/deamination reactions between several 3-substituted (S)-aspartic acids and their corresponding fumaric acid derivatives. The enzyme preferentially acted on (2S, 3S)-3-methylaspartic acid and mesaconic acid in the deamination and the amination reactions respectively. The enzyme showed high similarities in several enzymological properties and N-terminal amino acid sequence with 3-methylaspartase from an obligate anaerobic bacterium Clostridium tetanomorphum.


Subject(s)
Ammonia-Lyases/isolation & purification , Ammonia-Lyases/metabolism , Bacteria, Anaerobic/enzymology , Amino Acid Sequence , Ammonia-Lyases/antagonists & inhibitors , Ammonia-Lyases/chemistry , Cations, Divalent/pharmacology , Cations, Monovalent , Chelating Agents/pharmacology , Crystallization , Enzyme Inhibitors , Enzyme Stability , Hydrogen-Ion Concentration , Isoelectric Point , Kinetics , Molecular Sequence Data , Molecular Weight , Sequence Analysis , Substrate Specificity , Sulfhydryl Reagents/pharmacology , Temperature
4.
Int J Biochem ; 26(12): 1377-81, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7890117

ABSTRACT

The biosynthesis of uroporphyrinogen III, the precursor of hemes, chlorophylls, corrins and related structures, is catalyzed by the porphobilinogenase system (PBGase), a complex of two enzymes, PBG-Deaminase (PBG-D) and Isomerase. Although the separate enzymes have been studied in some detail less work has been performed on the properties of the complex. In this study the kinetic behaviour of the enzyme PBGase in a normal yeast strain, D273-10B, and its derivative B231 has been investigated. Uroporphyrinogen formation was linear with time up to 2 hr at 37 degrees C. The enzyme complex shows classical Michaelis-Menten kinetics. From the double reciprocal plots kinetic parameters were estimated for PBGase and PBG-D. Porphyrins were found to be competitive inhibitors with respect to porphobilinogen (PBG) and these compounds appeared to act as inhibitors by forming dead-end complexes with the free enzyme. 5-Aminolevulinic acid (ALA) also inhibited PBGase and this inhibition was overcome by addition of levulinic acid (2 microM). These results indicate that ALA, is not an inhibitor but acts through its conversion into porphyrins which are the true inhibitors.


Subject(s)
Ammonia-Lyases/antagonists & inhibitors , Porphyrins/pharmacology , Saccharomyces cerevisiae/enzymology , Aminolevulinic Acid/pharmacology , Kinetics
5.
Int J Biochem ; 26(2): 259-62, 1994 Feb.
Article in English | MEDLINE | ID: mdl-8174760

ABSTRACT

1. The effect of URO I on the activity of ALA-D, PBGase, deaminase and URO-D, both in aerobiosis and anaerobiosis, was studied. 2. Photoinactivation of the enzymes was much lower in an anaerobic than in an aerobic atmosphere. 3. Dark inactivation in the absence of oxygen was lower than its presence. 4. Preincubation in the presence of ALA or PBG protected the enzymic activity of ALA-D, PBGase and deaminase against URO I-inactivation both under u.v. light and in the dark. 5. Photoinactivating action of URO I would be mediated by reactive oxygen species generated by the excited porphyrin after its absorption of light. Dark inactivation, in aerobiosis, can also be partly mediated by amino acid oxidation, although to a lesser extent than that observed under u.v. light.


Subject(s)
Darkness , Enzyme Inhibitors/pharmacology , Light , Uroporphyrins/pharmacology , Aerobiosis , Ammonia-Lyases/antagonists & inhibitors , Anaerobiosis , Humans , Hydroxymethylbilane Synthase/antagonists & inhibitors , Porphobilinogen Synthase/antagonists & inhibitors , Uroporphyrinogen Decarboxylase/antagonists & inhibitors
6.
Toxicol Lett ; 50(2-3): 137-42, 1990 Feb.
Article in English | MEDLINE | ID: mdl-2309232

ABSTRACT

The effect of lead on the activity of erythrocyte porphobilinogen deaminase (PBGD) in vivo and in vitro was investigated using blood specimens obtained from controls and lead-exposed workers. When lead nitrate was added to the incubation mixture at a final concentration of 10(-4) M, 83% inhibition of erythrocyte PBGD activity was found. However, in workers occupationally exposed to lead, no inhibition of erythrocyte PBGD activity was detected. This finding indicates that the erythrocyte PBGD test is not useful for evaluating exposure to lead in workers. In addition, the in vitro study confirmed that mercuric chloride strongly inhibits erythrocyte PBGD activity.


Subject(s)
Ammonia-Lyases/antagonists & inhibitors , Erythrocytes/enzymology , Hydroxymethylbilane Synthase/antagonists & inhibitors , Lead/toxicity , Nitrates/toxicity , Chromatography, High Pressure Liquid , Humans , In Vitro Techniques , Lead Poisoning/blood , Lead Poisoning/diagnosis , Lead Poisoning/enzymology , Mercuric Chloride/toxicity , Metals/toxicity
7.
J Enzyme Inhib ; 3(4): 303-10, 1990.
Article in English | MEDLINE | ID: mdl-2319333

ABSTRACT

The action of porphyrins, uroporphyrin I and III (URO I and URO III), pentacarboxylic porphyrin I (PENTA I), coproporphyrin I and III (COPRO I and COPRO III), protoporphyrin IX (PROTO IX) and mesoporphyrin (MESO), on the activity of human erythrocytes delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase in the dark and under UV light was investigated. Both photoinactivation and light-independent inactivation was found in all four enzymes using URO I as sensitizer. URO III had a similar action as URO I on porphobilinogenase and deaminase and PROTO IX exerted equal effect as URO I on delta-aminolevulinic acid dehydratase and uroporphyrinogen decarboxylase. Photodynamic efficiency of the porphyrins was dependent on their molecular structure. Selective photodecomposition of enzymes by URO I, greater specificity of tumor uptake by URO I and enhanced porphyrin synthesis by tumors from delta-aminolevulic acid, with predominant formation of URO I, underline the possibility of using URO I in detection of malignant cells and photodynamic therapy.


Subject(s)
Ammonia-Lyases/blood , Carboxy-Lyases/blood , Erythrocytes/enzymology , Hemeproteins/metabolism , Hydroxymethylbilane Synthase/blood , Porphobilinogen Synthase/blood , Porphyrins/pharmacology , Uroporphyrinogen Decarboxylase/blood , Ammonia-Lyases/antagonists & inhibitors , Ammonia-Lyases/radiation effects , Hemeproteins/antagonists & inhibitors , Hemeproteins/radiation effects , Humans , Hydroxymethylbilane Synthase/antagonists & inhibitors , Kinetics , Photochemistry , Porphobilinogen Synthase/antagonists & inhibitors , Porphobilinogen Synthase/radiation effects , Structure-Activity Relationship , Ultraviolet Rays , Uroporphyrinogen Decarboxylase/antagonists & inhibitors
8.
Biochim Biophys Acta ; 999(1): 52-7, 1989 Nov 09.
Article in English | MEDLINE | ID: mdl-2572277

ABSTRACT

Formiminotransferase-cyclodeaminase is stabilized and activated approx. 40% in the presence of low concentrations (equal or less than 0.2%) of Triton X-100, possibly because the average hydrophobicity (1.10 kcal per residue) and the frequency of large non-polar side-chains (0.34) of this protein are both somewhat higher than average. This stabilization enabled us to develop a new purification procedure for the enzyme using chromatography on Matrex Gel Orange A and heparin-Sepharose columns in the presence of Triton X-100. This procedure is easier, much more reproducible, and gives slightly higher yield than the previous method described by Drury, et al. Further investigations of the role of tetrahydropteroylpolyglutamates with formiminotransferase-cyclodeaminase reveal that the use of polyglutamylated folate substrates does not change the mechanism of the transferase reaction, but decreases the K(m) for formininoglutamate, the second substrate, more than 10-fold, bringing it closer to the expected physiological concentration.


Subject(s)
Ammonia-Lyases/isolation & purification , Folic Acid/analogs & derivatives , Liver/enzymology , Pteroylpolyglutamic Acids/metabolism , Amino Acids/analysis , Ammonia-Lyases/analysis , Ammonia-Lyases/antagonists & inhibitors , Ammonia-Lyases/metabolism , Animals , Chromatography, Gel , Formiminoglutamic Acid/metabolism , Glutamates/pharmacology , Glutamic Acid , Kinetics , Molecular Weight , Swine
9.
Biochem J ; 262(1): 119-24, 1989 Aug 15.
Article in English | MEDLINE | ID: mdl-2510713

ABSTRACT

A recombinant strain of Escherichia coli has been constructed that produces approx. 200 times the amount of hydroxymethylbilane synthase found in wild-type E. coli [Hart, Abell & Battersby (1986) Biochem. J. 240, 273-276]. Enzyme purified from this strain is shown to be permanently inactivated by pyridoxal 5'-phosphate/NaB1H3(3)H1. The inactivation is not complete despite the fact that approx. 1 mol of lysine residues is modified per mol of enzyme. Evidence is gained showing that (a) modification of one of two conserved lysine residues (Lys-55 or Lys-59) results in inactivation of hydroxymethylbilane synthase and (b) these lysine residues are present in or close to the active site.


Subject(s)
Ammonia-Lyases/antagonists & inhibitors , Hydroxymethylbilane Synthase/antagonists & inhibitors , Lysine/metabolism , Pyridoxal Phosphate/pharmacology , Amino Acid Sequence , Binding Sites , Escherichia coli/enzymology , Plasmids , Protease Inhibitors/metabolism
10.
Biochem Int ; 17(1): 103-9, 1988 Jul.
Article in English | MEDLINE | ID: mdl-3142474

ABSTRACT

Fumaraldehydic acid (FAA) induced a time-dependent inactivation of aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) from Escherichia coli at 30 degrees C and pH 7.4 following pseudo-first order kinetics. The rate of inactivation increased in proportion to the FAA concentration. In addition, the rate of inactivation increased, as the pH was increased. Determination of sulfhydryl groups showed that approximately one among 11 sulfhydryl groups was modified by FAA concomitant with the inactivation. L-Aspartate and fumarate protected the enzyme against FAA-inactivation, when Mg2+ ions were present. Unlike E. coli aspartase, P. fluorescens aspartase was not inactivated by FAA.


Subject(s)
Ammonia-Lyases/antagonists & inhibitors , Aspartate Ammonia-Lyase/antagonists & inhibitors , Fatty Acids, Monounsaturated/pharmacology , Binding Sites , Escherichia coli/enzymology , Fatty Acids, Monounsaturated/antagonists & inhibitors , Hydrogen-Ion Concentration , Kinetics , Pseudomonas fluorescens/enzymology , Sulfhydryl Compounds/analysis
11.
Biochem Int ; 16(3): 449-52, 1988 Mar.
Article in English | MEDLINE | ID: mdl-3289541

ABSTRACT

Aspartase of Escherichia coli was inhibited in a competitive manner by S-2,3-dicarboxyazirdine (DCAZ), an antibacterial substance against Aeromonas salmonesida. The inhibition constant (Ki) was 55 microM, which was as low as less than one tenth that of the Km value for the substrate, L-aspartate. In view of the fact that both aspartase and fumarase (J. Greenhut et al. (1985) J. Biol. Chem. 260, 6684-6686) were inhibited by DCAZ in competitive manners, common features of the reaction mechanism of the two enzymes were discussed.


Subject(s)
Ammonia-Lyases/antagonists & inhibitors , Aspartate Ammonia-Lyase/antagonists & inhibitors , Aziridines/pharmacology , Azirines/pharmacology , Escherichia coli/enzymology , Binding, Competitive , Kinetics
12.
C R Seances Soc Biol Fil ; 180(3): 302-8, 1986.
Article in French | MEDLINE | ID: mdl-2946374

ABSTRACT

The inhibitory activities of hydrazinoacetic acid (HAA), 2-hydrazinoethanol (HE) and ethyl hydrazino-acetate (EHA) are characterized and compared with those of L-or D-alpha-hydrazino-beta-phenylpropionic acid (L-or D-HPPA), 2-phenylethylhydrazine (PEH), hydrazine (H), alpha, beta-dihydrocinnamic acid (DHCA) and D-phenylalanine (D-Phe). Inhibitors can be arranged in order of increasing activity: HE less than EHA less than H less than D-Phe less than HAA less than D-HPPA less than DHCA less than PEH less than L-HPPA. Relations between activity and structure of inhibitors are discussed.


Subject(s)
Ammonia-Lyases/antagonists & inhibitors , Hydrazines/pharmacology , Mitosporic Fungi/enzymology , Phenylalanine Ammonia-Lyase/antagonists & inhibitors , Rhodotorula/enzymology , Kinetics , Phenylalanine Ammonia-Lyase/metabolism
13.
Biochem Biophys Res Commun ; 131(2): 557-63, 1985 Sep 16.
Article in English | MEDLINE | ID: mdl-3902011

ABSTRACT

Phenylalanine ammonia-lyase contained within permeabilized cells of Rhodosporidium toruloides was protected from proteolytic attack by trypsin, chymotrypsin and duodenal juice. The inactivation by the proteases was biphasic. The enzyme contained within the yeast cells had a similar Km for phenylalanine and Ki for cinnamic acid to the protein in free solution. Phenylalanine ammonia-lyase present in the yeast depleted duodenal juice of free phenylalanine, while the enzyme in free solution did not. The possibility of using permeabilized cells of R. toruloides as a vehicle for protecting orally ingested therapeutic enzymes from proteolytic inactivation is discussed.


Subject(s)
Ammonia-Lyases/antagonists & inhibitors , Basidiomycota/enzymology , Peptide Hydrolases/pharmacology , Phenylalanine Ammonia-Lyase/antagonists & inhibitors , Ustilaginales/enzymology , Cell Membrane Permeability , Chymotrypsin/pharmacology , Cinnamates/metabolism , Duodenum , Intestinal Secretions/enzymology , Kinetics , Phenylalanine/metabolism , Phenylalanine Ammonia-Lyase/metabolism , Trypsin/pharmacology
14.
Acta Biochim Pol ; 32(2): 131-43, 1985.
Article in English | MEDLINE | ID: mdl-4036447

ABSTRACT

L(-)-, and D(+)-enantiomers of 1-amino-2-phenylethylphosphonic acid (PheP), a phosphonic analogue of phenylalanine, inhibit the activity of L-phenylalanine ammonia-lyase (EC 4.3.1.5) of potato tuber tissue in vitro. The apparent type of inhibition depends on concentration of PheP; as the concentration of D-PheP is raised from 10(-5) M to 2.5 X 10(-3) M, the type of inhibition shifts from competitive through mixed and non-competitive to uncompetitive. L-PheP exerts either a competitive or mixed-type inhibition at low (10(-6)-10(-5) M) or moderate (5 X 10(-5)-2 X 10(-4) M) concentration. Ki for the concentration range of competitive inhibition were 6.5 X 10(-6) M, 5.3 X 10(-5)M and 1.6 X 10(-5) M for L-, D-, and D,L-PheP, respectively. These Ki values are valid for a relatively narrow range of L-Phe concentration (0.2-4 mM) as L-phenylalanine ammonia-lyase does not follow the Michaelis-Menten kinetics of the reaction.


Subject(s)
Ammonia-Lyases/antagonists & inhibitors , Organophosphorus Compounds/pharmacology , Phenylalanine Ammonia-Lyase/antagonists & inhibitors , Phenylalanine/analogs & derivatives , Kinetics , Organophosphonates/pharmacology , Organophosphorus Compounds/chemical synthesis , Phenylalanine/pharmacology , Plants , Stereoisomerism , Structure-Activity Relationship
15.
J Biochem ; 96(5): 1315-21, 1984 Nov.
Article in English | MEDLINE | ID: mdl-6396297

ABSTRACT

Aspartase purified from Escherichia coli W cells was inactivated by diethylpyrocarbonate following pseudo-first order kinetics. Upon treatment of the inactivated enzyme with NH2OH, the enzyme activity was completely restored. The difference absorption spectrum of the modified vs. native enzyme preparations exhibited a prominent peak around 240 nm. The pH-dependence of the inactivation rate suggested that an amino acid residue having a pK value of 6.6 was involved in the inactivation. These results indicate that the inactivation was due to the modification of histidine residues. L-Aspartate and fumarate, substrates for the enzyme, and the Cl- ion, an inhibitor, protected the enzyme against the inactivation. Inspection of the spectral change at 240 nm associated with the inactivation in the presence and absence of the Cl- ion revealed that the number of histidine residues essential for the enzyme activity was less than two. Partial inactivation did not result in an appreciable change in the substrate saturation profiles. These results suggest that one or two histidine residues are located at the active site of aspartase and participate in an essential step in the catalytic reaction.


Subject(s)
Ammonia-Lyases/antagonists & inhibitors , Aspartate Ammonia-Lyase/antagonists & inhibitors , Diethyl Pyrocarbonate/pharmacology , Formates/pharmacology , Histidine/analysis , Binding Sites , Chemical Phenomena , Chemistry , Escherichia coli/enzymology , Hydrogen-Ion Concentration
16.
Biochem J ; 222(1): 93-102, 1984 Aug 15.
Article in English | MEDLINE | ID: mdl-6433896

ABSTRACT

When hydroxymethylbilane synthase (porphobilinogen deaminase) from Euglena gracilis is incubated with pyridoxal 5'-phosphate at pH 7.0 and 0 degree C, it rapidly loses part of its activity. The proportion of activity that remains decreases as the concentration of the modifier increases up to approx. 2mM, above which no further significant inactivation occurs. Dialysis of the partly inactivated enzyme restores its activity, whereas reduction with NaBH4 makes the inactivation permanent. The maximum inactivation achievable from one cycle of the treatment with pyridoxal 5'-phosphate, then with borohydride, is 53 +/- 5%; taking this modified enzyme through second and third cycles causes further loss of activity. The enzyme from Rhodopseudomonas spheroides behaves similarly, but there are quantitative differences. Spectroscopic evidence indicates that the inactivation procedure modifies lysine residues, and labelling studies show that epsilon-N-pyridoxyl-L-lysine is a product when permanently inactivated enzyme is completely hydrolysed. Several lysine residues per molecule of the E. gracilis enzyme are modified by the treatment with pyridoxal 5'-phosphate and borohydride, but only one appears to be essential for enzymic activity, since porphobilinogen protects the enzyme against inactivation and then one fewer lysine residue per molecule of enzyme is affected. It is suggested that, during the biosynthesis of hydroxymethylbilane, the first porphobilinogen unit is covalently bound to the enzyme through the epsilon-amino group of the essential lysine.


Subject(s)
Ammonia-Lyases/antagonists & inhibitors , Hydroxymethylbilane Synthase/antagonists & inhibitors , Lysine/analysis , Pyridoxal Phosphate/pharmacology , Animals , Binding Sites , Borohydrides/pharmacology , Euglena gracilis/enzymology , Hydrogen-Ion Concentration , Kinetics , Oxidation-Reduction , Rhodobacter sphaeroides/enzymology , Uroporphyrinogen III Synthetase/antagonists & inhibitors
17.
Can J Biochem Cell Biol ; 62(1): 49-54, 1984 Jan.
Article in English | MEDLINE | ID: mdl-6732900

ABSTRACT

The in vitro inhibition of hydroxymethylbilane synthase (EC 4.3.1.8, uroporphyrinogen I synthetase) obtained from livers of Sprague-Dawley rats has been studied with a wide range of di- and tri-valent metal ions. After purification by cell lysis, heat treatment, and centrifugation, the stable, soluble enzyme yielded sigmoidal inhibition curves with increasing concentrations of each of the 16 test ions. Using the negative logarithm of metal concentration for 50% inhibition (the pM50 value), the metal ions could be classified according to their Klopman hardness values. Very soft ions including Hg2+, intermediate ions including Cr3+, and very hard ions including Al3+ all yielded large pM50 values indicating strong inhibition. In comparison to known metal-ion chemical behaviour, these three ions could indicate three different types of inhibitory binding sites at or near the active site: Hg2+ corresponding to sulfur in cysteine, Cr3+ corresponding to nitrogen in histidine, and Al3+ corresponding to oxygen in carboxyl groups. The presence of the first two sites is also indicated by the pH dependence of activity.


Subject(s)
Ammonia-Lyases/antagonists & inhibitors , Hydroxymethylbilane Synthase/antagonists & inhibitors , Liver/enzymology , Aluminum/pharmacology , Animals , Cations , Chromium/pharmacology , Kinetics , Male , Mercury/pharmacology , Rats , Rats, Inbred Strains
18.
Life Sci ; 34(2): 193-6, 1984 Jan 09.
Article in English | MEDLINE | ID: mdl-6694518

ABSTRACT

Purified rat hepatic uroporphyrinogen (UROgen) I synthase (URO-S) was inhibited by bilirubin or the ditaurine derivative. Inhibition was reversible and non-competitive to the substrate porphobilinogen (PBG). The inhibition constants (Ki values) for bilirubin and the conjugate were 1.5 microM and 0.26 microM respectively. Rats afflicted with hyperbilirubinemia caused by biliary obstruction had decreased levels of hepatic microsomal heme (58% of control) and cytochrome P-450 (60% of control) at day 3. Hepatic delta-aminolevulinic acid synthetase (ALAS) activity was increased (39% of control) at day 3.


Subject(s)
Ammonia-Lyases/antagonists & inhibitors , Bilirubin/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Heme/analysis , Hydroxymethylbilane Synthase/antagonists & inhibitors , Microsomes, Liver/drug effects , 5-Aminolevulinate Synthetase/metabolism , Animals , Humans , Hydroxymethylbilane Synthase/metabolism , Hyperbilirubinemia/physiopathology , Male , Microsomes, Liver/analysis , Microsomes, Liver/enzymology , Rats
19.
Eur J Cell Biol ; 29(2): 139-44, 1983 Jan.
Article in English | MEDLINE | ID: mdl-6832164

ABSTRACT

Mungbean (Vigna radiata (L.) Wilczek) seedlings grown for 9 days on filter paper soaked with 0.3 to 1 mM L-alpha-aminooxy-beta-phenylpropionic acid (AOPP), a potent inhibitor of L-phenylalanine ammonia-lyase, had a greatly reduced anthocyanin content, and the cell walls of the xylem vessels did not stain with the phloroglucinol/HCl or safranine/astrablue reagents indicating the absence of lignin-like material. Furthermore, vanillin was detectable in nitro-benzene-oxidized lignin preparations only from control seedlings, but not from AOPP-treated seedlings. Scanning electron microscopy of hypocotyl cross sections revealed collapsed xylem vessels in seedlings grown in the presence of AOPP indicating that lignin is required for resistance against the tensile forces in the conducting cells of the xylem. AOPP enhanced the growth of cultured cells of Lonicera prolifera Rehd. while it inhibited the production of extracellular material that gave a positive reaction with phloroglucinol/HCl.


Subject(s)
Ammonia-Lyases/antagonists & inhibitors , Lignin/biosynthesis , Phenylalanine Ammonia-Lyase/antagonists & inhibitors , Phenylalanine/analogs & derivatives , Anthocyanins/metabolism , Cell Division/drug effects , Phenylalanine/pharmacology , Plants/drug effects , Plants/metabolism , Plants/ultrastructure
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