Difluoromethylornithine irreversibly inactivates ornithine decarboxylase of Pseudomonas aeruginosa, but does not inhibit the enzymes of Escherichia coli.

DL-alpha-Difluoromethylornithine, an enzyme-activated irreversible inhibitor of eukaryotic ornithine decarboxylase and consequently of putrescine biosynthesis, inhibited ornithine decarboxylase in enzyme extracts from Pseudomonas aeruginosa in a time-dependent manner t1/2 1 min, and also effectively blocked the enzyme activity in situ in the cell. Difluoromethylornithine, however, had no effect on the activity of ornithine decarboxylase assayed in enzyme extracts from either Escherichia coli or Klebsiella pneumoniae. However, the presence of the inhibitor in cell cultures did partially lower ornithine decarboxylase activity intracellularly in E. coli. Any decrease in the intracellular ornithine decarboxylase activity observed in E. coli and Pseudomonas was accompanied by a concomitant increase in arginine decarboxylase activity, arguing for a co-ordinated control of putrescine biosynthesis in these cells.     

Purification of S-adenosylmethionine decarboxylase from soybean.

S-Adenosylmethionine decarboxylase (EC 4.1.1.19) was purified to homogeneity from the cytosol of soybean (Glycine max) axes by ammonium sulfate fractionation, DEAE-Sepharose and methylglyoxalbis(guanylhydrazone)-Sepharose 6B chromatographies. The enzyme was free from diamine oxidase activity. The molecular weight of the enzyme estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis was 66,000. The Km value for S-adenosylmethionine was 0.26 mM. The optimum pH and temperature were 7.5 and 40 degrees C. Neither putrescine nor Mg2+ affected the enzyme activity, but the enzyme was inhibited by spermidine, spermine, methylglyoxalbis(guanylhydrazone), sodium borohydride and phenylhydrazine. Agmatine was a novel inhibitor which inhibited S-adenosylmethionine decarboxylase and arginine decarboxylase, preventing the accumulation of decarboxylated S-adenosylmethionine and putrescine, respectively.     

Outer and inner membrane proteins compose an arginine-agmatine exchange system in Chlamydophila pneumoniae

Most chlamydial strains have a pyruvoyl-dependent decarboxylase protein that converts L-arginine to agmatine. However, chlamydiae do not produce arginine, so they must import it from their host. Chlamydophila pneumoniae has a gene cluster encoding a putative outer membrane porin (CPn1033 or aaxA), an arginine decarboxylase (CPn1032 or aaxB), and a putative cytoplasmic membrane transporter (CPn1031 or aaxC). The aaxC gene was expressed in Escherichia coli producing an integral cytoplasmic membrane protein that catalyzed the exchange of L-arginine for agmatine. Expression of the aaxA gene produced an outer membrane protein that enhanced the arginine uptake and decarboxylation activity of cells coexpressing aaxB and aaxC. This chlamydial arginine/agmatine exchange system complemented an E. coli mutant missing the native arginine-dependent acid resistance system. These cells survived extreme acid shock in the presence of L-arginine. Biochemical and evolutionary analysis showed the aaxABC genes evolved convergently with the enteric arginine degradation system, and they could have a different physiological role in chlamydial cells. The chlamydial system uniquely includes an outer membrane porin, and it is most active at a higher pH from 3 to 5. The chlamydial AaxC transporter was resistant to cadaverine, L-lysine and L-ornithine, which inhibit the E. coli AdiC antiporter.     

N-acetylglutamate 5-phosphotransferase of Pseudomonas aeruginosa. Catalytic and regulatory properties.

Some kinetic properties of N-acetylglutamate 5-phosphotransferase (ATP: N-acetyl-L-glutamate 5-phosphotransferase EC 2.7.2.8) purified approx. 2000-fold from Pseudomonas aeruginosa have been studied. The enzyme required Mg2+ for activity. Mn2+, Zn2+, Co2+, and Ca2+, in this order, could replace Mg2+ partially. The substrate specificity was narrow: N-carbamoyl-L-glutamate and N-formyl-L-glutamate were phosphorylated, but at a lower rate than N-acetyl-L-glutamate; N-propionyl-L-glutamate was almost inactive as a substrate. dATP, but neither GTP nor ITP, could be used instead of ATP. The enzyme had a broad pH optimum from pH 6.5 to 9. Feedback inhibition by L-arginine was markedly dependent on pH. Above pH 9 no inhibition was observed. L-Citrulline was three times less potent an inhibitor than L-arginine. The enzyme showed Michaelis-Menten kinetics, even at low concentration of the second substrate. The apparent Km was 2 mM for N-acetyl-L-glutamate (at 10 mM ATP) and approx. 3 mM for ATP (at 40 mM N-acetyl-L-glutamate). In the presence of L-arginine the rate-concentration curves for N-acetyl-L-glutamate became signoidal, while no cooperativity was detected for ATP. A method was developed allowing the determination of N-acetyl-L-glutamate in the nanomolar range by means of purified enzyme.     

Putrescine-sensitive (artifactual) and insensitive (biosynthetic) S-adenosyl-L-methionine decarboxylase activities of Lathyrus sativus seedlings.

The crude extracts of 3-day-old etiolated seedlings of Lathyrus sativus contained two S-adenosyl-L-methionine decarboxylase activities. The artifactual putrescine-dependent activity was due to the H2O2 generated by diamine oxidase (EC 1.4.3.6) of this plant system and was inhibited by catalase. This observation was confirmed by using an electrophoretically and immunologically homogeneous preparation of L. sativus diamine oxidase. In the presence of putrescine, diamine oxidase, in addition to S-adenosylmethionine, decarboxylated L-lysine, L-arginine, L-ornithine, L-methionine and L-glutamic acid to varying degrees. The decarboxylation was not metal-ion dependent. The biosynthetic S-adenosylmethionine decarboxylase (EC 4.1.1.21) was detected after removing diamine oxidase specifically from the crude extracts by employing an immunoaffinity column. This Mg2+-dependent decarboxylase was not stimulated by putrescine or inhibited by catalase. The enzyme activity was inhibited by semicarbazide, 4-bromo-3-hydroxybenzoylamine dihydrogen phosphate and methylglyoxal-bis (guanylhydrazone). It was largely localized in the shoots of the etiolated seedlings and was purified 40-fold by employing a p-hydroxymercuribenzoate/AH-Sepharose affinity column, which also separated the decarboxylase activity from spermidine synthase.     

具有谷氨酸脱羧酶的大肠杆菌固定化细胞

本文研究了用海藻酸钙包埋法制备含谷氨酸脱羧酶固定化细胞的方法以及研究了制备的条件和影响其制备的因素。该法具有包埋细胞活力回收高,方法简便等优点。比较研究了固定化细胞和自然细胞谷氨酸脱羧酶的一些生物化学性质。其中固定化细胞最适pH和pH稳定性增加,最适温度及热稳定性下降;表观米氏常数增大;二价金属离子Zn~(++)、Cu~(++)、Mg~(++)、Fe~(++),Sr~(++)程度不同的抑制酶活性,Ca~(++)激活固定化细胞酶活性,EDTA无抑制作用。对固定化细胞和自然细胞酶活力活化的研究中发现这两种细胞经蒸馏水保温处理后酶活性都上升,且自然细胞酶活的上升较固定化细胞大;而用底物溶液处理后,自然细胞无变化,固定化细胞酶活下降。     

Putrescine biosynthesis in Tetrahymena thermophila.

The putrescine-biosynthesis pathway in Tetrahymena thermophila was delineated by studying crude extracts prepared from exponentially growing cultures. A pyridoxal phosphate-stimulated ornithine decarboxylase activity competitively inhibited by putrescine was detected. CO2 was also liberated from L-arginine, but analyses by t.l.c. and enzyme studies suggested that the activity was not due to arginine decarboxylase, nor could enzyme activities converting agmatine into putrescine be detected. We conclude that the decarboxylation of L-ornithine is probably the only major route for putrescine biosynthesis in this organism during exponential growth.     

Characterization of an acid-dependent arginine decarboxylase enzyme from Chlamydophila pneumoniae

Genome sequences from members of the Chlamydiales encode diverged homologs of a pyruvoyl-dependent arginine decarboxylase enzyme that nonpathogenic euryarchaea use in polyamine biosynthesis. The Chlamydiales lack subsequent genes required for polyamine biosynthesis and probably obtain polyamines from their host cells. To identify the function of this protein, the CPn1032 homolog from the respiratory pathogen Chlamydophila pneumoniae was heterologously expressed and purified. This protein self-cleaved to form a reactive pyruvoyl group, and the subunits assembled into a thermostable (alphabeta)(3) complex. The mature enzyme specifically catalyzed the decarboxylation of L-arginine, with an unusually low pH optimum of 3.4. The CPn1032 gene complemented a mutation in the Escherichia coli adiA gene, which encodes a pyridoxal 5'-phosphate-dependent arginine decarboxylase, restoring arginine-dependent acid resistance. Acting together with a putative arginine-agmatine antiporter, the CPn1032 homologs may have evolved convergently to form an arginine-dependent acid resistance system. These genes are the first evidence that obligately intracellular chlamydiae may encounter acidic conditions. Alternatively, this system could reduce the host cell arginine concentration and produce inhibitors of nitric oxide synthase.     

Purification and properties of two molecular forms of arginine kinase from the adductor muscle of the scallop, Pecten maximus.

1. Two molecular forms of arginine kinase, AK1 and AK2 have been purified from the adductor muscle of the scallop, Pecten maximus. AK2 was retained on a DEAE-cellulose column at pH 7.5, but AK1 was not. 2. Both forms were monomeric (mol. wt. approximately 42,000) and showed the same pH optimum (7.5-8.0) in the direction of phosphoarginine synthesis. 3. AK1 had slower electrophoretic mobility at pH 8.3 towards the anode, higher lysine content, lower glutamate content, lower Km for L-arginine and higher Km for Mg(2+)-ATP than AK2. Unlike AK1, AK2 was strongly inhibited at high concentrations of Mg(2+)-ATP. 4. Both molecular forms cross-reacted with antisera raised against native as well as performic acid-oxidized lobster muscle arginine kinase. However, AK1 showed a greater affinity than AK2 to anti-lobster arginine kinase antibodies, particularly to those raised against the native enzyme.     

Arginine decarboxylase in Trypanosoma cruzi, characteristics and kinetic properties.

Polyamines are known to play an essential role in cell growth and differentiation. In animals, putrescine is mainly synthesized from ornithine by ornithine decarboxylase (ODC). In higher plants and in bacteria putrescine can also be synthesized from arginine by arginine decarboxylase (ADC). In this paper we report the presence of significant levels of ADC activity in crude extracts of Trypanosoma cruzi, RA strain epimastigotes. ADC activity was detected during a very narrow time range, corresponding to the early logarithmic growth phase. This activity was inhibited by DL-alpha-difluoromethylarginine, a specific irreversible inhibitor of ADC and activated by DL-alpha-difluoromethylornithine, a specific irreversible inhibitor of ODC. The reaction showed an absolute requirement for pyridoxal phosphate, dithiothreitol and Mg++. The enzyme half life was about 10 hrs., showed maximum activity at pH 7.9 and a Km for arginine of 5 mM. ADC activity was stimulated by fetal-calf-serum and inhibited by spermine, probably through a negative feed-back regulation on the levels of the enzyme. ODC activity was not detected. These results confirm our previous reports on the capability of T. cruzi, RA strain epimastigotes to synthesize putrescine from arginine via agmatine by ADC and point out differences on polyamine metabolism between the parasite and the mammalian host cell.     

Comparison of the biosynthetic and biodegradative ornithine decarboxylases of Escherichia coli.

Biosynthetic ornithine decarboxylase was purified 4300-fold from Escherichia coli to a purity of approximately 85% as judged by polyacrylamide gel electrophoresis. The enzyme showed hyperbolic kinetics with a Km of 5.6 mM for ornithine and 1.0 micronM for pyridoxal phosphate and it was competitively inhibited by putrescine and spermidine. The biosynthetic decarboxylase was compared with the biodegradative ornithine decarboxylase [Applebaum, D., et al. (1975), Biochemistry 14, 3675]. Both enzymes were dimers of 80 000-82 000 molecular weight and exhibited similar kinetic properties. However, they differed significantly in other respects. The pH optimum of the biosynthetic enzyme was 8.1, compared with 6.9 for the biodegradative. Both enzymes were activated by nucleotides, but with different specificity. Antibody to the purified biodegradative ornithine decarboxylase did not cross-react with the biosynthetic enzyme. The evolutionary relationship of these two decarboxylases to the other amino acid decarboxylases of E. coli is discussed.     

DL-a-Monofluoromethylputrescine is a potent irreversible inhibitor of Escherichia coli ornithine decarboxylase.

DL-alpha-Monofluoromethylputrescine (compound R.M.I. 71864) is an enzyme-activated irreversible inhibitor of the biosynthetic enzyme ornithine decarboxylase from Escherichia coli. This compound, however, has much less effect in vitro on ornithine decarboxylase obtained from Pseudomonas aeruginosa. These findings are in contrast with those previously found with the substrate analogue DL-alpha-difluoromethylornithine (compound R.M.I. 71782). The K1 of the DL-alpha-monofluoromethylputrescine for the E. coli ornithine decarboxylase is 110 microM, and the half-life (t1/2) calculated for an infinite concentration of inhibitor is 2.1 min. When DL-alpha-monofluoromethylputrescine is used in combination with DL-alpha-difluoromethylarginine (R.M.I. 71897), an irreversible inhibitor of arginine decarboxylase, in vivo in E. coli, both decarboxylase activities are inhibited (greater than 95%) but putrescine levels are only decreased to about one-third of control values and spermidine levels are slightly increased.     

适于非水相催化用细菌脂肪酶基本性质的研究

对假单胞菌产脂肪酶的基本酶学性质进行了研究．该酶水解油脂时的最适作用ｐＨ为９．０,最适作用温度为４５℃;在ｐＨ７．０～１０．０范围内稳定,在６０℃以下热稳定性良好．Ｋ＋、Ｃａ２＋、Ｍｇ２＋等金属离子对酶有明显的激活作用,而Ｈｇ２＋、Ｃｕ２＋、Ｓｎ２＋等重金属离子却对酶有较强的抑制作用;几种表面活性剂和胆汁盐均使酶发生不同程度的失活．该酶在水解油脂时表现出１,３－位置专一性,且对不同种类和来源的油脂水解作用速率不同     

Activities and properties of putrescine-biosynthetic enzymes in Vibrio parahaemolyticus

The biosynthetic pathways for putrescine (Put) in Vibrio parahaemolyticus were delineated by measuring activities of the enzymes which would be involved in its biosynthesis. Experiments with labeled arginine and ornithine revealed that both of these amino acids were converted into Put by intact cells. The activities of three enzymes, arginine decarboxylase (ADC), ornithine decarboxylase (ODC), and agmatine ureohydrolase (AUH), were detected in cell extracts. ADC and ODC of V. parahaemolyticus were similar in the following properties to the corresponding enzymes of Escherichia coli: 1) both decarboxylases showed a pH optimum at 8.25 and required pyridoxal phosphate and dithiothreitol for full activity; 2) while ODC was considerably activated by GTP, ADC was only slightly; 3) both decarboxylases were inhibited by polyamines; 4) ADC was inhibited by difluoromethylarginine, a potent inhibitor of bacterial ADC. However, in contrast to the corresponding enzymes of E. coli, the V. parahaemolyticus ADC showed no requirement for Mg2+, and the AUH was active over a wide pH range of 8.5-9.5 with a maximum at pH 9.0. Furthermore, in all 6 strains tested, the activity of ADC was obviously high compared with that of ODC, and AUH was present with a relatively high activity. Cultivation of these strains at a suboptimal NaCl concentration (0.5%) resulted in a pronounced increase in both ADC and AUH activities. These observations suggest that the important pathway for Put biosynthesis in V. parahaemolyticus is the decarboxylation of arginine by ADC and the subsequent hydrolysis of its product, agmatine, by AUH.     

The properties of arginine transport in vacuolar membrane vesicles of Neurospora crassa

We have measured the uptake of arginine into vacuolar membrane vesicles from Neurospora crassa. Arginine transport was found to be dependent on ATP hydrolysis, Mg2+, time, and vesicle protein with transported arginine remaining unmodified after entry into the vesicles. The Mg2+ concentration required for optimal arginine transport varied with the ATP concentration so that maximal transport occurred when the MgATP2- concentration was at a maximum and the concentrations of free ATP and Mg2+ were at a minimum. Arginine transport exhibited Michaelis-Menten kinetics when the arginine concentration was varied (Km = 0.4 mM). In contrast, arginine transport did not follow Michaelis-Menten kinetics when the MgATP2-concentration was varied (S0.5 = 0.12 mM). There was no inhibition of arginine transport when glutamine, ornithine, or lysine were included in the assay mixture. In contrast, arginine transport was inhibited 43% when D-arginine was present at a concentration 16-fold higher than that of L-arginine. Measurements of the internal vesicle volume established that arginine is concentrated 14-fold relative to the external concentration. Arginine transport was inhibited by dicyclohexylcarbodiimide, carbonyl cyanide m-chlorophenyl-hydrazone, and potassium nitrate (an inhibitor of vacuolar ATPase activity). Inhibitors of the plasma membrane or mitochondrial ATPase such as sodium vanadate or sodium azide did not affect arginine transport activity. In addition, arginine transport had a nucleoside triphosphate specificity similar to that of the vacuolar ATPase. These results suggest that arginine transport is dependent on vacuolar ATPase activity and an intact proton channel and proton gradient.     

Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate

Previous studies have shown that murine macrophages immunostimulated with interferon gamma and Escherichia coli lipopolysaccharide synthesize NO2-, NO3-, and citrulline from L-arginine by oxidation of one of the two chemically equivalent guanido nitrogens. The enzymatic activity for this very unusual reaction was found in the 100,000g supernatant isolated from activated RAW 264.7 cells and was totally absent in unstimulated cells. This activity requires NADPH and L-arginine and is enhanced by Mg2+. When the subcellular fraction containing the enzyme activity was incubated with L-arginine, NADPH, and Mg2+, the formation of nitric oxide was observed. Nitric oxide formation was dependent on the presence of L-arginine and NADPH and was inhibited by the NO2-/NO3- synthesis inhibitor NG-monomethyl-L-arginine. Furthermore, when incubated with L-[guanido-15N2]arginine, the nitric oxide was 15N-labeled. The results show that nitric oxide is an intermediate in the L-arginine to NO2-, NO3-, and citrulline pathway. L-Arginine is required for the activation of macrophages to the bactericidal/tumoricidal state and suggests that nitric oxide is serving as an intracellular signal for this activation process in a manner similar to that very recently observed in endothelial cells, where nitric oxide leads to vascular smooth muscle relaxation [Palmer, R. M. J., Ashton, D. S., & Moncada, S. (1988) Nature (London) 333, 664-666].     

Modulation of arginine decarboxylase activity from Mycobacterium smegmatis. Evidence for pyridoxal-5'-phosphate-mediated conformational changes in the enzyme

Arginine decarboxylase (arginine carboxy-lyase, EC 4.1.1.19) from Mycobacterium smegmatis, TMC 1546 has been purified to homogeneity. The enzyme has a molecular mass of 232 kDa and a subunit mass of 58.9 kDa. The enzyme from mycobacteria is totally dependent on pyridoxal 5'-phosphate for its activity at its optimal pH and, unlike that from Escherichia coli, Mg2+ does not play an active role in the enzyme conformation. The enzyme is specific for arginine (Km = 1.6 mM). The holoenzyme is completely resolved in dialysis against hydroxylamine. Reconstitution of the apoenzyme with pyridoxal 5'-phosphate shows sigmoidal binding characteristics at pH 8.4 with a Hill coefficient of 2.77, whereas at pH 6.2 the binding is hyperbolic in nature. The kinetics of reconstitution at pH 8.4 are apparently sigmoidal, indicating the occurrence of two binding types of differing strengths. A low-affinity (Kd = 22.5 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations and a high-affinity (Kd = 3.0 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations. The restoration of full activity occurred in parallel with the tight binding (high affinity) of pyridoxal 5'-phosphate to the apoenzyme. Along with these characteristics, spectral analyses of holoenzyme and apoenzyme at pH 8.4 and pH 6.2 indicate a pH-dependent modulation of coenzyme function. Based on the pH-dependent changes in the polarity of the active-site environment, pyridoxal 5'-phosphate forms different Schiff-base tautomers at pH 8.4 and pH 6.2 with absorption maxima at 415 nm and 333 nm, respectively. These separate forms of Schiff-base confer different catalytic efficiencies to the enzyme.     

Sequence-specific endonuclease Bgl I. Modification of lysine and arginine residues of the homogeneous enzyme.

The sequence-specific endonuclease Bgl I from Bacillus globigii (RUB561) has been purified to homogeneity as determined by denaturing polyacrylamide gel analysis. The active form of the enzyme is a single polypeptide with a molecular weight of 32,000. The enzyme requires Mg2+ in the reaction mixture and displays a broad pH and monovalent cation requirement. Bgl I is not sensitive to sulfhydryl reagents but was affected by reagents that modify lysine and arginine residues. When lysine residues were modified by pyridoxal 5'-phosphate, both binding and catalysis were diminished while modification of arginine residues by 2,3-butanedione inhibited the enzyme activity but had no effect on its binding properties.     

Purification and properties of 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase from two strains of Pseudomonas putida.

Growth on phenol of two strains of Pseudomonas putida biotype A, NCIB 10015 and NCIB 9865, elicits the synthesis of an enzyme that hydrolyzes 2-hydroxy-6-oxo-2,4-heptadienoate to 2-oxopent-4-enoate. The purified enzyme from Pseudomonas NCIB 10015 has a molecular weight of 118,000 and dissociates in sodium dodecyl sulfate to a species of molecular weight 27,700; the enzyme from Pseudomonas NCIB 9865 has a molecular weight of 100,000 and dissociates to a species of 25,000 molecular weight. The hydrolases from both strains have similar Km values, pH optima, and thermal labilities and attack the same range of substrates. Neither hydrolase was stimulated by Mg2+ or Mn2+, and both were inhibited by p-chloromercuribenzoate and iodoacetamide. Immunodiffusion studies with the purified enzymes and antibodies formed against them show some cross-reaction of Pseudomonas NCIB 9865 enzymes with antibodies to Pseudomonas NCIB 10015, but not vice versa.