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PubMed1988

Purification and properties of S-adenosyl-L-methionine:nicotinic acid-N-methyltransferase from cell suspension cultures of Glycine max L.

A soluble enzyme which catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the nitrogen atom of pyridine-3-carboxylic acid (nicotinic acid) could be detected in protein preparations from heterotrophic cell suspension cultures of soybean (Glycine max L.). Enzyme activity was enriched nearly 100-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography to study kinetic properties. S-adenosyl-L-methionine:nicotinic acid-N-methyltransferase (EC 2.1.1.7) showed a pH optimum at pH 8.0 and a temperature optimum between 35 and 40 degrees C. The apparent KM values were determined to be 78 microM for nicotinic acid and 55 microM for the cosubstrate. S-Adenosyl-L-homocysteine was a competitive inhibitor of the methyltransferase with a KI value of 95 microM. The native enzyme had a molecular mass of about 90 kDa. The catalytic activity was inhibited by reagents blocking SH groups, whereas other divalent cations did not significantly influence of the enzyme reaction. The purified methyltransferase revealed a remarkable specificity for nicotinic acid. No other pyridine derivative was a suitable methyl group acceptor. To study a potential methyltransferase activity with nicotinamide as substrate, an additional purification step was necessary to remove nicotinamide amidohydrolase activity from the enzyme preparation. This was achieved by affinity chromatography on S-adenosyl-L-homocysteine-Sepharose thus leading to a 580-fold purified enzyme which showed no methyltransferase activity toward nicotinamide as substrate.

Hydrogen-Ion Concentration↗

PubMed1976

The effect of trinitrophenylation on subunit interactions in L-asparaginase.

L-Asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) from Escherichia coli B was modified by treatment with 2,4,6-trinitrobenzene-1-sulfonic acid at pH 7.5. The introduction of 13 trinitrophenyl groups into one mol of the tetrameric enzyme (TNP 13-asparaginase) results in a loss of 67% of the catalytic activity while the presence of 20 groups (TNP 20-asparaginase) reduces the enzymatic activity by 88%. The modified proteins are homogeneous as judged by disc gel electrophoresis and by the monodisperse boundary in the analytical ultracentrifuge having a sedimentation coefficient of 7.2 S. The rate of dissociation of the TNP 13-asparaginase is twice as fast and TNP 20-asparaginase three times as fast as that of unmodified asparaginase in 4 M urea. Trinitrophenylated subunits in 8 M urea can reassociate into the tetramer after removal of urea by dialysis or by dilution. hybridization of unmodified and TNP subunits indicates that that trinitrophenyl derivatives qualify as suitable variants for studying subunit interactions in oligomeric proteins.

Asparaginase↗

PubMed1976

Photocontrol of urease activity in spiropyran collagen membrane.

1. Collagen fibrils were modified with beta-1-[3,3-dimethyl-6'-nitrospiro-(indoline-2,2'-2H-benzopyran)] propionic anhydride. 2. Urease (urea amidohydrolase, EC 3.5.1.5) was immobilized in spiropyran collagen membrane. The activity of the urease-spiropyran collagen membrane was found to increase in the dark and then decrease with visible light irradiation. 3. The optimum pH of the urease-spiropyran collagen membrane under visible light was lowered in the dark. 4. The apparent Michaelis constant (K'm) of the urease-spiropyran collagen membrane in the dark was almost the same as that under visible light. The apparent maximum velocity was increased in the dark. 5. The diffusion coefficient of urea through the spiropyran collagen membrane in the dark was 1.4 times that under visible light. However, the increase of the diffusion rate was not responsible for the activity increase of the urease-spiropyran collagen membrane.

Collagen↗

PubMed1976

Purification and properties of L-asparaginase B from Acinetobacter calcoaceticus.

L-Asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) B from Acinetobacter calcoaceticus has been purified by precipitation with streptomycin, chromatography on DEAE-cellulose and CM-cellulose, gel filtration on Agarose and chromatography on phosphocellulose. The molecular weight of the enzyme was found to be 130 000. The enzyme was rather insensitive to pH changes between 7 and 9. The Michaelis constant was 3-10(-3) M. Hg2+, Cu2+, and Ni2+ as well as high ionic strength inhibited the activity of the enzyme, whereas citrate seemed to stimulate the activity. The enzyme catalyzed the deamination of L-glutamine to about the same extent as L-asparagine. The temperature stability of the enzyme is also reported. The enzyme had a weak tumor inhibitory power.

Acinetobacter↗

PubMed1977

The interaction of p-nitrophenyl carbamate with urease.

1. p-Nitrophenyl carbamate and thiourea have been shown to be substrates for urease (urea amidohydrolase, EC 3.5.1.5) 2. Urease has been shown to have a lower Km, 0.67 mM, with p-nitrophenyl carbamate than with urea, 2.0 mM. 3. The V of urease for the hydrolysis of urea, p-nitrophenyl carbamate and thiourea has been shown to be the same, indicating a common rate-limiting step. 4. A mechanism has been proposed for urease where the initial rate-limiting step is the release of a molecule of ammonia from the substrates.

Carbamates↗

PubMed1978

pH dependence of the kinetic parameters of L-asparaginase.

The concentration dependence of the rate of hydrolysis of L-asparagine by Escherichia coli L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) has been measured over the range pH 4.5 to pH 9.1 by a direct spectrophotometric assay at 220 nm and by a coupled assay utilizing glutamate dehydrogenase to detect the ammonia produced. The velocity of the hydrolysis reaction at saturating levels of substrate is independent of pH over this interval. The plot of V/km over the same interval is bell-shaped, being dependent on pKa values of 6.58 and 8.69. The higher pKa is attributed to the amino group of asparagine. The lower pKa is associated with the enzyme active site and is probably due to an imidazole group.

Asparaginase↗

PubMed1979

Activation of glutaminase by phosphoribosyl-pyrophosphate and its interference with the assay of phosphoribosylpyrophosphate amidotransferase.

Phosphate-dependent glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) from rat liver was found to be strongly activated by phosphoribosylpyrophosphate (P-rib-PP), the substrate of amidophosphoribosyltransferase (EC 2.4.2.14). Since the assay of the latter is based on the P-rib-PP-dependent conversion of glutamine to glutamate, the amidotransferase activities determined in crude tissue preparations were found to be too high. The interference of glutaminase, however, could be completely eliminated by its inactivation at 50 degrees C. Amidotransferase was not affected by the heat treatment. Because of the increased rate of the glutamate formation at this temperature, the incubation time of the assay could be significantly reduced.

Amidophosphoribosyltransferase↗

PubMed1980

Asparaginase II of Saccharomyces cerevisiae: inactivation during the transition to stationary phase.

Asparaginase II (L-asparagine amidohydrolase, EC 3.5.1.1) activity of cells from stationary phase cultures of Saccharomyces cerevisiae is very low. When these cells are inoculated into minimal medium, asparaginase II specific activity rises rapidly and reaches a maximum after 9-10 h. During the next 2.5-3 h, a rapid decrease in asparaginase II specific activity occurs. The enzyme does not appear to be secreted into the medium or to be reabsorbed into the cell. Addition of protease inhibitors at the time of maximum activity partially or totally prevents the loss of asparaginase II. L-1-Tosylamide-2-phenylethyl chloromethyl ketone decreases the rate of loss. The sulfhydryl reagents p-hydroxymercuribenzoate and iodoacetamide inhibit the loss of asparaginase II. However, addition of EDTA causes a further increase in activity. This increase is due to de novo protein synthesis. The effect of EDTA can be reversed by the addition of Zn2+. The most likely explanation for the rapid loss of asparaginase II is proteolytic degradation by a Zn2+-dependent, thiol protease or peptidase.

Asparaginase↗

PubMed1986

N-acylethanolamine phospholipid metabolism in normal and ischemic rat brain.

N-Acylethanolamine phospholipids accumulate in rat brain during post-decapitative ischemia. Small amounts of these phospholipids consisting primarily of diacyl and alkenylacyl species can be detected within 15 min of ischemia and they increase linearly for 60 min. This ischemia-induced synthesis is more pronounced in developing rat brain (approx. 5.0 nmol/h per mumol lipid P) than in adult brain (0.4 nmol). Pulse labeling experiments with subcellular preparations of 10-day-old rat brain indicate a precursor-product relationship between ethanolamine phospholipids and their N-acyl analogs. N-Acylation of endogenous substrates occurs with both microsomes and mitochondria, exhibits a pH optimum of 10 and requires 1 mM Ca2+ for maximal (0.2 mM Ca2+ for half maximal) activity. Cell-free preparations of both developing and adult rat brain contain a phosphodiesterase which hydrolyzes N-acylphosphatidylethanolamine to phosphatidic acid and N-acylethanolamine. The latter is further hydrolyzed to fatty acid and ethanolamine by an amidohydrolase. [1-3H]Ethanolamine, injected intracerebrally or intraperitoneally into 13- and 18-day-old rats, is incorporated into brain ethanolamine phospholipids. Since small amounts of radioactivity are also associated with N-acylethanolamine phospholipids 5 and 24 h after injection of the substrate, it appears that these phospholipids may occur at a very low level as a natural lipid constituent of rat brain.

Acylation↗

PubMed1996

The N-acylation-phosphodiesterase pathway and cell signalling.

Long-chain N-acylethanolamines (NAEs) elicit a variety of biological and pharmacological effects. Anandamide (20:4n-6 NAE) and other polyunsaturated NAEs bind to the cannabinoid receptor and may thus serve as highly specific lipid mediators of cell signalling. NAEs can be formed by phospholipase D-catalyzed hydrolysis of N-acylethanolamine phospholipids or by direct condensation of ethanolamine and fatty acid. So far, most of the latter biosynthetic activity has been shown to be the reverse reaction of the NAE amidohydrolase that catalyzes NAE degradation. Thus, increasing evidence supports the hypothesis that the N-acylation-phosphodiesterase pathway yields not only saturated-monounsaturated NAEs, but polyunsaturated ones, including anandamide, as well.

Animals↗

PubMed1979

Rapid assessment of endogenous creatinine in physiological samples analyzed by reversed-phase liquid chromatography with spectrophotometric detection.

An improved and direct analysis of endogenous creatinine levels using the reversed-phase mode of high performance liquid chromatography (RPLC) is described. The analysis is rapid, the sensitivity of the spectrophotometric detection is in the nanomole range, and the sample preparation requires only filtration to remove the particulate matter. Identification of chromatographic peaks was achieved by means of absorbance ratios, stopped-flow UV spectra and prechromatographic incubation of samples with creatinine amidohydrolase. Reported in this paper are representative chromatograms of samples of amniotic fluid, urine, and cerebrospinal fluid (CSF). The ranges of creatinine concentrations in 10 samples of amniotic fluid, urine and CSF were 0.774--1.850, 99--454.5 and 0.648--1.05 mg/100 ml, respectively. This highly specific and simple assay offers several improvements over previously used methods.

Amniotic Fluid↗

PubMed1984

A new enzymatic serum creatinine measurement based on an endogenous creatine-eliminating system.

We describe a new colorimetric determination of serum creatinine which does not require a blank to correct for endogenous creatine. In the first reaction, creatinase (creatinase amidinohydrolase EC 3.5.3.3) and sarcosine oxidase (sarcosine: oxygen oxidoreductase (demethylating) EC 1.5.3.1) were used in the enzymatic hydrolysis of endogenous creatine to produce hydrogen peroxide. In the presence of horseradish peroxidase, 2,4-dichlorophenolsulfonate (2,4-DCPS) was converted to a colorless polymer by hydrogen peroxide. In the second reaction, creatininase (creatinine amidohydrolase EC 3.5.2.10) and 4-aminoantipyrine (4-AA) were added, and only the creatine generated from creatinine by creatininase was hydrolyzed sequentially by creatinase and sarcosine oxidase to produce hydrogen peroxide. This newly-formed hydrogen peroxide was measured at 510 nm in a coupled reaction catalyzed by peroxidase, with 2,4-DCPS/4-AA as a chromogen. The standard curve was linear up to 20 mg/dl; 40 microliter of serum and 20 min were required for determination. Analytical recovery of creatinine added to either normal or abnormal sera averaged 98.5%. Within-day and day-to-day studies gave CV values of less than or equal to 2.9% and less than or equal to 4.8%, respectively. No significant interferences were observed with the proposed method. The results obtained by the present method correlated well with those obtained by the Jaffé procedure.

Ascorbic Acid↗

PubMed1981

Regional distribution of glycoasparagine storage material in the brain in aspartylglycosaminuria.

We have studied the regional distribution of glycoasparagine storage material in the brain in aspartylglycosaminuria, a condition characterized by inherited deficiency of lysosomal N-aspartyl-beta-N-acetylglucosamine amidohydrolase. Gaschromatographic measurements of the main accumulating glycoprotein-derived metabolite, N-acetylglucosaminyl-asparagine (GlcNAc-Asn), in 12 defined cerebral areas showed that GlcNAc-Asn is rather evenly distributed in the brain. The mean concentrations ranged from 0.454 mg/g wet tissue (corpus callosum) to 0.0610 mg/g (pons). The GlcNAc-Asn concentrations tended to be higher in grey matter areas than in white matter areas. GlcNAc-Asn was identified in the isolated neuronal fraction, but not in the myelin fraction, by mass-fragmentographic techniques. Electron-microscopic reexamination of a brain biopsy specimen revealed, in addition to the abundant presence of storage lysosomes in the neuronal perikarya, numerous cytoplasmic inclusions in brain capillary endothelial cells and pericytes as well as in occasional macrophages. The results indicate that the glycoasparagine storage material is not limited to expected cortical areas in aspartylglycosaminuria, but is distributed in a rather constant fashion in all cerebral grey and white matter areas studied.

Acetylglucosamine↗

PubMed1982

Chemical modifications of Escherichia coli L-asparaginase and their effect on plasma clearance rate and other properties.

Escherichia coli asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) has been modified by succinylation, acetylation and the attachment of N,N-dimethyl-1,3-propanediamine and glucuronic acid. The effect of these modifications on plasma clearance rates in mice and on other properties is compared to the effects of modification with lactose and N-acetylneuraminyl lactose studied previously. The t 1/2 values for the acylated enzyme samples (lower pI) were reduced, succinylated asparaginase sharply and the acetylated enzyme less so. The N,N-dimethyl-1,3-propanediamine-modified samples (increased pI) also had lower t 1/2 values, but samples modified with glucuronic acid (reduced pI) showed little change in clearance time. The main conclusion is that the increased t 1/2 value found in the previous work for N-acetylneuraminyl-lactosylated enzyme is not due to the decreased pI value of the modified enzyme, but must be attributed to interference by N-acetylneuraminyl-lactose residues, directly or indirectly, with the mechanism normally used by the mouse to clear itself of injected E. coli asparaginase.

Acetylation↗

PubMed1983

The effect of freeze-drying on the quaternary structure of L-asparaginase from Erwinia carotovora.

L-Asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) from Erwinia carotovora undergoes extensive dissociation from active tetramer to inactive monomers when freeze-dried. The monomeric state is stabilized by reconstitution of the freeze-dried enzyme with buffers of high pH and high ionic strength. Some compounds, particularly sugars and sugar derivatives, prevent dissociation on freeze-drying, whereas others, such as urea and chaotropic ions, increase dissociation. The effects of additives are not related to water retention. The dissociation is completely reversible on reconstitution at neutral pH, but the alkali-stabilized monomer only partially reassociates when the pH is brought back to neutrality.

Asparaginase↗

PubMed1989

On the role of histidine and tyrosine residues in E. coli asparaginase. Chemical modification and 1H-nuclear magnetic resonance studies.

The relative importance of tyrosine and histidine residues for the catalytic action of Escherichia coli asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) was studied by chemical modification and 1H-NMR spectroscopy. We show that, under appropriate reaction conditions, N-bromosuccinimide (NBS) as well as diazonium-1H-tetrazole (DHT) inactivate by selectively modifying two tyrosine residues per asparaginase subunit without affecting histidyl moieties. We further show that diethyl pyrocarbonate (DEP), a reagent considered specific for histidine, also modifies tyrosine residues in asparaginase. Thus, inactivation of the enzyme by DEP is not indicative of histidine residues being involved in catalysis. In 1H-nuclear magnetic resonance (NMR) spectra of asparaginase signals from all three histidine residues were identified. By measuring the pH dependencies of these resonances, pKa values of 7.0 and 5.8 were derived for two of the histidines. Titration with aspartate which tightly binds to the enzyme at low pH strongly reduced the signal amplitude of the pKa 7 histidyl moiety as well as those of resonances of one or more tyrosine residues. This suggests that tyrosine and histidine are indeed constituents of the active site.

Asparaginase↗

PubMed1985

Synthesis and pharmacology of nonmammalian angiotensins and their evolutionary development.

To understand how vertebrates utilize angiotensins during evolutionary development studies were undertaken to synthesize and/or characterize angiotensin-like peptides from nonmammalian species. These studies indicated the presence of a new L-asparaginase amidohydrolase type enzyme in eel plasma which deamidates L-asparagine residue at the amino terminus of the angiotensin peptides, thereby implying that l-asparaginyl decapeptide (rather than l-aspartyl decapeptide) is the natural form of angiotensin inherent in eel plasma. Pharmacological properties of the nonmammalian angiotensins compared with the synthetic analogs in one representative species of three distinct classes of vertebrates suggest that: in spite of variation in position 9 of the nonmammalian angiotensins I, the pressor activity of these peptides in rat and in dogfish shark is due to their conversion into the corresponding angiotensin II; in relatively more primitive stages of evolution, when vertebrates lived in salt water (e.g., dogfish shark) pressor action of exogenous angiotensin II appears to be due to the release of catecholamines (and not through direct vasoconstrictor effects, as in the mammalian species); and frog-skin angiotensin II has properties that may prove to be compatible with a role in the regulation of salt and water in amphibians.

Angiotensinogen↗

PubMed1990

Acylation of L-asparaginase with total retention of enzymatic activity.

Acylation of L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) with complete retention of catalytic activity was achieved. Several parameters of the acylation method, based on the binding of palmitoyl residues to epsilon-NH2 groups of protein, were optimized. The correlation between the acylation degree of L-asparaginase and the retention of catalytic activity was established. For a palmitoyl chloride/protein molar ratio ranging from 50 to 900, a degree of modification of 10 to 30% and a retention of catalytic activity of 98 to 60% respectively, was observed. Hydrophobicity of 30% acylated protein was correlated with turbidity in water and octanol and was compared with the native protein. Acylated protein incorporated into liposomes, showed an increase in catalytic activity in intact form as compared to the native enzyme. By the introduction of a sequential acylation cycle, an improvement of the degree of modification with a maximal value at 50% was obtained. Total retention of catalytic activity was achieved by acylation in the presence of 8 mM L-asparagine in a reactional medium.

Acylation↗