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PubMed1975

Kinetic enzymatic method for determining serum creatinine.

Creatinine amidohydrolase is used to measure serum creatinine in a totally enzymatic procedure. Creatine, produced by hydrolysis, is acted upon by creatine kinase, and then by pyruvate kinase and lactate dehydrogenase, to result in a change in absorbance at 340 nm. The amount of creatinine present is related to the rate of change in A340 and is determined from a standard curve. Absorbance and concentration are linearly related to 100 mg/liter and only 250 mul of serum is required. At 1.0 g/liter, heparin, oxalate, citrate, ethylenediaminetetraacetate, ascorbate, or glucose had no significant effect on the accurate determination of creatinine; higher concentrations (30 g/liter) had inhibitory effects on the test. Analytical recovery of creatinine added to either normal or abnormal sera averaged 102%. When results of this procedure and of the standard direct Jaffé test were compared, the latter were significantly higher. Unlike the Jaffé method, the present method of determining creatinine is rapid (about 10 min per test), subject to few or no interfering substances, and requires no serum deproteinization.

Amidohydrolases↗

PubMedDate not supplied

Neonatal screening for biotinidase deficiency.

Children with juvenile-onset multiple carboxylase deficiency lack biotinidase activity (biotinamide amidohydrolase, EC 3.5.1.12) in the liver and other tissues. Hence, little free biotin is metabolically available, resulting in seizures, acidosis, and serious neurological damage. As the absence of hepatic biotinidase activity is reflected in serum, assessment of biotinidase status can easily be made from a blood sample. A convenient qualitative procedure for screening infants has been employed in order to estimate serum levels of biotinidase in as little as 10 microliters of sample. This colorimetric procedure detects the formation of free p-aminobenzoate cleaved from the substrate, N-biotinyl-p-aminobenzoate at pH 6.0. The assay is easily performed and has a low incidence of false positive results. A kinetic assay for serum biotinidase has also been developed using biotinyl-p-nitroanilide (BpNA) as substrate. When 50 microliters of biotinidase positive serum was incubated with 0.2 mM BpNA in phosphate buffer at pH 6.0, an increase in absorbance was observed at 405 nm. The rate of change in absorbance was followed kinetically on the Roche Cobas BIO analyzer at 37 degrees C. Monitoring the increase in absorbance of para-nitroanilide every 60 seconds over 30 minutes demonstrated linearity from 10 to 30 minutes. In comparing results from this kinetic assay on 48 randomly selected sera with those obtained using a colorimetric procedure, a correlation coefficient of 0.85 was obtained. Several false positive results were observed in clearly lipemic sera.

4-Aminobenzoic Acid↗

PubMedDate not supplied

The enzymatic hydrolysis-activation of the adriamycin cardioprotective agent (+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane.

The cardioprotective agent ICRF-187 [(+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane, ADR-529] has shown a great deal of promise against what may be an iron-based adriamycin-induced cardiotoxicity. ICRF-187 likely exerts its actions through its rings-opened hydrolysis product, which has a structure similar to EDTA and like-wise strongly binds metal ions. The 105,000g soluble supernatant fraction of homogenates of porcine liver and kidney, but not of heart, enzymatically caused a ring-opening hydrolysis of ICRF-187 at the rate of 1.2, 1.4, and less than 0.15 nmol.(mg protein)-1.min-1, respectively. This enzymatic hydrolysis of ICRF-187 was completely abolished by 4-chlorobenzenesulfonamide, which suggested that dihydropyrimidine amidohydrolase (DHPase) might be, in large part, responsible for the ICRF-187 hydrolase activity. Bovine liver DHPase was isolated and was shown to enzymatically hydrolyze ICRF-187 with a Vmax of 14,900 nmol.(mg DHPase)-1.min-1, a value that exceeded that of its natural substrates. The KM for ICRF-187 of 6.7 mM was, however, much larger than that of its natural substrates. The enzyme kinetics were consistent with DHPase acting on ICRF-187 to form one-ring-opened hydrolysis product only. Thus, DHPase is not able to act on the one-ring-opened hydrolysis product to produce two-ring-opened product. The Ki for 4-chlorobenzenesulfonamide inhibition of DHPase was measured to be 26 microM.

Amidohydrolases↗

PubMed2006

[Cloning, expression and purification of D-carbamoylase from Sinorhizobium morelens S-5].

A N-carbamoyl-D-amino acid amidohydrolase gene (hyuC) from Sinorhizobium morelens S-5 was cloned by LA PCR, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the hyuC gene exhibited high homology to the amino acid sequences of D-carbamoylase from other sources. The gene could be highly expressed in Escherichia coli, and the recombinant enzyme was purified 16.1-fold to homogeneity with a yield of 21.2% by heat treatment and three steps of column chromatography. The results of gel filtration on Superdex 200 HR and SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a tetramer protein of identical 38-kDa subunits. The recombinant enzyme catalyzed the hydrolysis of N-carbamoyl alpha-amino acid to the corresponding free amino acid, and it was strictly D-specific. The enzyme showed broad substrate specificity, and exhibited high activity in the hydrolysis of N-carbamoyl-D-p-hydroxyphenylglycine as substrate. The enzyme did not hydrolyze N-carbamoyl-beta-alanine. The optimum pH and temperature of the enzyme were pH 7.0 and 60 degrees C, respectively. Enzyme activity was slightly improved by Ca2+ and Fe2+, and nearly not affected by metal chelators and sulfhydryl reagents. The enzyme showed high thermal and oxidative stability. These results show that the enzyme has great potential for industrial application.

Amidohydrolases↗

PubMed1986

Creatinine clearance: enzymatic vs Jaffé determinations of creatinine in plasma and urine.

We measured creatinine in plasma and urine samples from 17 normal subjects and 10 renally impaired subjects by four different methods: two enzymatic--Ektachem iminohydrolase and Boehringer Mannheim amidohydrolase--and two Jaffé reaction based--Beckman Astra 8 and Technicon AutoAnalyzer I. Creatinine clearances, standardized for body surface area, were also calculated. In both groups of subjects plasma creatinine values were significantly (p less than 0.05) lower, by 3 to 4 mg/L, when measured enzymatically than when measured by the Jaffé reaction. Additionally, creatinine clearances were significantly (p less than 0.05) greater by at least 30 mL/min when calculated from enzymatically measured creatinine values vs Jaffé method values for creatinine. The benefits of lack of interference with enzymatically measured creatinine concentrations and clearances should be assessed in relation to the lack of agreement with long-established (Jaffé) methods for determining creatinine (and inulin) clearances.

Amidohydrolases↗

PubMed1985

1H NMR evidence that almond "peptide: N-glycosidase" is an amidase. Kinetic data and trapping of the intermediate.

The enzyme from almond that catalyzes the hydrolysis of the N-glycosidic linkage between asparagine and the oligosaccharide chain of glycopeptides and glycoproteins has been variously termed an N-glycosidase and an amidase enzyme. Using turkey ovomucoid glycopeptide as a substrate for the enzyme, we followed the hydrolysis reaction by 1H NMR spectroscopy. These kinetic data revealed a rapid hydrolysis of the substrate but a delayed appearance of the final product. This implied that an intermediate, most likely a 1-aminooligosaccharide, was formed during the reaction. Identification of the intermediate as a 1-beta-amino-N-acetylglucosamine-oligosaccharide was achieved by trapping it as the 1-acetamido derivative using acetic anhydride and subsequent analysis by 1H NMR. The data conclusively demonstrate that the enzyme catalyzes the hydrolysis of the glycopeptide to form an aspartic acid-containing polypeptide and an intermediate oligosaccharide amine. The latter derivative is hydrolyzed nonenzymatically to yield the final carbohydrate product. Thus, the enzyme is in fact an amidohydrolase (amidase) and not an N-glycosidase. The trivial name glycopeptidylamidase is suggested.

Amidohydrolases↗

PubMed1984

Dihydroorotase from Escherichia coli. Purification and characterization.

Dihydroorotase (4,5-L-dihydroorotate amidohydrolase (EC 3.5.2.3], which catalyzes the reversible cyclization of N-carbamyl-L-aspartate to dihydro-L-orotate, has been purified to homogeneity from an over-producing strain of Escherichia coli. Treatment of 70 g of frozen cell paste produces about 7 mg of pure enzyme, a yield of about 35%. The native molecular weight, determined by equilibrium sedimentation, is 80,900 +/- 4,300. The subunit molecular weight, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 38,400 +/- 2,600, and by amino acid analysis is 41,000. The enzyme is thus a dimer and contains 0.95 +/- 0.08 tightly bound zinc atoms per subunit when isolated by the described procedure, which would remove any loosely bound metal ions. Isoelectric focusing under native conditions yields a major species at isoelectric point 4.97 +/- 0.27 and a minor species at 5.26 +/- 0.27; dihydroorotase activity is proportionately associated with both bands. The enzyme has a partial specific volume of 0.737 ml/g calculated from the amino acid composition and a specific absorption at 278 nm of 0.638 for a 1 mg/ml solution. At 30 degrees C, the Michaelis constant and kcat for dihydro-DL-orotate (at pH 8.0) are 0.0756 mM and 127 s-1, respectively; for N-carbamyl-DL-aspartate (at pH 5.80), they are 1.07 mM and 195 s-1.

Amidohydrolases↗

PubMed1984

Kinetic approach for the enzymic determination of creatinine.

By exploiting the kinetics of NADH consumption in the enzyme system creatinine amidohydrolase + creatine kinase + pyruvate kinase + lactate dehydrogenase, one can determine creatinine in serum in the range of 1 to 90 mg/L. By using the conditions defined here, this determination can be made with a single measurement of the rate of disappearance of NADH. The analytical rate measurement is made at a fixed time (2 min) after the sample is introduced into the enzyme solution. For this fixed time, the interferences of creatine and pyruvate are eliminated. Results so obtained for creatinine in human blood serum samples correlated well (r = 0.98) with those obtained by the classical Jaffé-reaction method. Run-to-run reproducibility (CV) was 3%, and the limit of detection was 1 mg/L.

Amidohydrolases↗

PubMedDate not supplied

[New isolation procedure for swine kidney acylase. Kinetics of Co2+, Mn2+, Ni2+ and Cd2+-enzymes].

A new procedure for the isolation of highly purified acylamino acid amidohydrolase from hog kidney is described which allows the preparation of the enzyme with a recovery of about 45%, a 200 fold purification and a spec. activity of 350-500 U. The essential Zn2+ of the enzyme was exchanged for Co2+, Ni2+, Mn2+ and Cd2+, and the kinetic parameters KM, kcat and kcat/KM of the different enzyme species for a series of acetyl-L-amino acids were determined.

Amidohydrolases↗

PubMed1983

Multi-enzyme membrane electrodes for determination of creatinine and creatine in serum.

An enzyme electrode system for the determination of creatinine and creatine was developed by utilizing three enzymes: creatinine amidohydrolase (CA), creatine amidinohydrolase (Cl), and sarcosine oxidase (SO). These enzymes were co-immobilized onto the porous side of a cellulose acetate membrane with asymmetric structure, which has selective permeability to hydrogen peroxide. Two kinds of multi-enzyme electrodes were constructed by combining a polarographic electrode for sensing hydrogen peroxide and an immobilized CA/Cl/SO membrane or Cl/SO membrane for creatinine plus creatine or creatine, respectively. The multi-enzyme electrodes responded linearly up to 100 mg of creatinine and creatine per liter in human serum. Response time was 20 s in the rate method and the detection limit was 1 mg/L. Only 25 microL of serum sample is required. Analytical recoveries, precisions, and correlations with the Jaffé method were excellent, and the multi-enzyme electrodes were sufficiently stable to perform more than 500 assays. No loss of activity of immobilized enzymes was observed after nine months of storage at 4 degrees C in air.

Amidohydrolases↗

PubMed1980

Accumulation of glycoprotein-derived metabolites in neural and visceral tissue in aspartylglycosaminuria.

The storage material in various tissues from patients with inherited deficiency of lysosomal aspartylglucosamine amidohydrolase (aspartylglycosaminuria) was characterized by gas chromatographic-mass spectrometric analyses of intact and specifically degraded derivatives. The major storage compound in cerebral gray and white matter, spinal cord, sciatic nerve, spleen, kidney, skeletal muscle and thyroid gland was identified as 2-acetamido-l-N-(4'-L-aspartyl)-2-deoxy-beta-D-glucopyranosylamine (GlcNAc-Asn). Thin-layer chromatography revealed an additional glycoasparagine band in spleen, kidney, thyroid, and brain. The compound was purified from spleen, and its structure is tentatively proposed to be D-mannopyranosyl-(1-6)-D-mannopyranosyl (1-4)-2-acetamido-2-deoxy-D-glucopyranosyl-(1-4)-2-acetamido-l-N-(4'-L-aspartyl )-2-deoxy-beta-D-glucopyranosylamine (Man2-GlcNAc2-Asn). The results show that the major glycoasparagine storage product in both neural and extraneural tissues in aspartylglycosaminuria is GlcNAc-Asn. Furthermore, a more complex glycoasparagine, presumably Man2-GlcNAc2-Asn, accumulates in smaller amounts in tissues of patients with aspartylglycosaminuria.

Acetylglucosamine↗

PubMed1993

Rat liver imidase.

Imidase, an enzyme variously identified as dihydropyrimidinase (EC 3.5.2.2), hydantoinase, dihydropyrimidine hydrase, and dihydropyrimidine amidohydrolase, has been purified to electrophoretic homogeneity from rat liver. Although a component in the chain of pyrimidine catabolism, imidase is capable of serving in a broader role that includes detoxication of xenobiotics. The enzyme catalyzes the hydrolytic cleavage of imides that range from the linear to the heterocyclic and that include hydantoins, dihydropyrimidines, and phthalimide. For some substrates, the reaction is experimentally reversible. The pH activity curves are a function of the pKa of the individual substrate's imino group, with cleavage favored at a pH near the respective pKa value. There is evidence for stereoselectivity and for stereospecificity. A mechanism is proposed for the enzyme-catalyzed reaction.

Amidohydrolases↗

PubMed1993

Human aminoacylase-1. Cloning, sequence, and expression analysis of a chromosome 3p21 gene inactivated in small cell lung cancer.

Human aminoacylase-1 (N-acyl-L-amino-acid amidohydrolase, EC 3.5.1.14; ACY1) is a homodimeric zinc-binding enzyme that catalyzes the hydrolysis of N alpha-acylated amino acids. ACY1 has been assigned to chromosome 3p21.1, a region reduced to homozygosity in small cell lung cancer (SCLC), and has been reported to exhibit reduced or absent expression in SCLC cell lines and tumors. Two human cDNA libraries and one human genomic DNA library were screened with a previously isolated partial ACY1 cDNA to isolate a full-length transcript. Sequence analysis of clones from each of these libraries resulted in an ACY1 cDNA of 1438 base pairs with an open reading frame of 1224-base pairs coding for a putative protein of 408 amino acids with a predicted molecular mass of 45,882 Da. Sequence analysis revealed no homologies to previously reported cDNA or protein sequences and establishes ACY1 as the first member of a new family of zinc-binding enzymes to be so characterized. The subcellular location of ACY1 has been established as cytosolic by flow cytometry. Southern and northern analyses of ACY1 in SCLC cell lines failed to demonstrate any gross abnormalities of the ACY1 structural gene or instances of absent or aberrantly sized mRNA, respectively.

Amidohydrolases↗

PubMed1979

[Formamidase--microheterogeneity, catalytic properties and inhibitors (author's transl)].

Formamidase from rat liver proved to be microheterogenous. After preparative isoelectric focusing in density gradient columns, two peaks of formamidase with identical substrate specificity were identified. By analytical focusing in thin layers of polyacrylamide or Sephadex G-75 SF, even five bands could be separated. Their isoelectric points were 4.75, 4.78, 4.82, 4.92 (main band) and 5.11, but their Michaelis constants did not differ significantly (54 to 62 mumol/l). An identical molecular weight of 34700 +/- 3200 for all bands was determined by disc electrophoresis. This value was confirmed by sedimentation analyses (so20,w = 3.00 S) and electrophoresis in the presence of sodium dodecyl-sulfate (Mr 34900 +/- 2300), which only gave a single band. The homogeneity was also confirmed by electrophoresis in the presence of 6M urea. Repeated disc electrophoresis of focusing under native conditions with single, isolated formamidases again resulted in different bands which were identified, not only by Coomassie Blue, but also by their hydrolytic cleavage of naphthyl acetate. Formamidase showed neither proteolytic nor asparagine-amidohydrolase activity and oligosaccharide conjugates were not detectable. Ampholytes, buffer ions, pH and peroxodisulfate did not affect the heterogeneity. "Initial burst" measurements with diethyl(4-nitrophenyl) phosphate yielded an equivalent weight of 36,300. Formylkynurenine reduced this inhibition very effectively. Thus, an extraordinary reactive serine residue appeared to be located in the catalytic site of formamidase. A participation of sulfhydrylgroups in the inactivating reaction of arsenite was excluded although two such groups were detected by 5,5'-dithiobis(2-nitrobenzoic acid). N-Bromosuccinimide reacted primarily with one of the nine tryptophan residues without loss of enzymatic activity, but a 18.6-fold excess of this reagent resulted in a complete loss of activity. The reaction rates of the most effective inhibitors and of the protective action of formylkynurenine were determined. Thus, formamidase must clearly be distinguished from typical serine esterases and proteases.

Amidohydrolases↗

PubMed1996

Comparison of Jaffé rate assay and enzymatic method for the measurement of creatinine clearance.

To evaluate the relationship between Jaffé rate assay (Jaffé assay) and creatinine amidohydrolase enzymatic method (enzymatic method), we measured serum and urine creatinine concentrations in 100 serum and 100 urine samples by two methods in patients with renal disease. Comparison of Jaffé (X) and enzymatic (Y) measurements of serum and urine creatinine levels revealed a high correlation (r2 = 0.9991 in serum, r2 = 0.9995 in urine). The creatinine concentrations in serum and urine determined by Jaffé assay were significantly (p < 0.01) higher than those assayed by the enzymatic method. The relationship between Jaffé and enzymatic analyses of creatinine clearance (Ccr) was obtained mathematically using regression lines (Y = 0.977X - 0.199 in serum, Y = 0.999X - 1.872 in urine). Ccr values obtained by both methods were almost the same at high serum creatinine levels. However, Ccr determined by Jaffé assay was much lower than that obtained by the enzymatic method when the serum creatinine concentration was under 2.0 mg/dl. We present here the equation for conversion of clearance values determined by both methods to obtain an accurate evaluation of renal function in many clinical studies.

Amidohydrolases↗

PubMed1997

A new PCR system for Agrobacterium tumefaciens detection based on amplification of T-DNA fragment.

The design of the PCR system presented in this work is based on the knowledge of the molecular character of the crown gall disease. The virulence of Agrobacterium tumefaciens requires the presence of a big (up to 235,000 bp) plasmid Ti (pTi-tumour inducing plasmid). This plasmid carries the so-called T-DNA fragment (T-DNA-transferred DNA), which integrates into cell chromosomes of the infected plants and subsequently changes the plant morphology nad metabolism. In cannot be excluded that after T-DNA integration the presence of Agrobacterium is not necessary for the development of pathological changes. Thus, T-DNA is the only sign that must be present both in virulent bacteria and in infected plants in any stadium of the disease and even before the infection. This is why T-DNA was chosen as the target region for PCR amplification. Primers flanking a 220 bp fragment of one of the conservative regions responsible for Agrobacterium pathogenicity, namely tms2 gene coding for indolacetamide amidohydrolase (the second step of auxin biosynthesis) were designed as the optimal for PCR amplification. The PCR amplification reactions were performed for matrixes isolated from cultures of reference strains giving one predicted product for each sample. First attempts of T-DNA detection in infected soils and plants were performed. We hope that the presented new PCR system for Agrobacterium tumefaciens detection will help to fight the crown gall disease in the nearest future.

Agrobacterium tumefaciens↗

PubMed1998

Molecular evolution of hydantoinases.

The complete amino acid sequence of the hydantoinase from Arthrobacter aurescens DSM 3745 has been derived by automated Edman degradation. This is the first ever reported amino acid sequence of a non-ATP-dependent hydantoinase, which hydrolyzes 5'-monosubstituted hydantoin derivatives L-selectively. A homology search performed in protein and nucleic acid databases retrieved only distantly related proteins. All of these are members of the recently described protein superfamily of amidohydrolases related to ureases (Holm and Sander, Proteins 28: 72-82, 1997). Phylogenetic analysis revealed that the novel hydantoinase forms a new branch separate from other hydantoin cleaving enzymes like dihydropyrimidinases (EC 3.5.2.2) and allantoinases (EC 3.5.2.5). Our results suggests that the enzymes of this protein superfamily have evolved from a common ancestor and therefore are the product of divergent evolution. We show further that the enclosed gene families developed very early in evolution, probably prior to the formation of the three domains, Archaea, Eukarya and Bacteria. Hydantoinases related to ATP-dependent N-methylhydantoinases (EC 3.5.2.14) or 5-oxoprolinases (EC 3.5.2.9) do not belong to this superfamily.

Amidohydrolases↗

PubMed1998

Luminometric single step urea assay using ATP-hydrolyzing urease.

An automatic enzyme kinetic luminometric method for determination of small quantities of urea in biological fluids and in microdialysates is presented. The method is based on the ATP-hydrolyzing urease reaction [urea amidohydrolase (ATP-hydrolyzing); EC 3.5.1.45], monitored by a luciferin-luciferase ATP reaction. The assay range is 100 pmol to 50 nmol with a detection limit of 5 micromol/L in the sample, compared with detection limits of 0.1 mmol/L in earlier spectrophotometric methods. To reduce the non-urea-dependent ATPase activity (v(blank)) and to increase the urea-dependent activity, 1,2-propanediol was included. Assay conditions were optimized by multivariate analysis. Recoveries of urea added to blood dialysate and plasma were 96-103%. No analytical interference of common metabolites, drugs, or other additives was observed. The total CVs (6 days and six concentrations, 1.2-21.8 mmol/L) were 3.6-8.5%. The results obtained with the present assay were highly correlated for dialysate (r = 0.979) and for plasma (r = 0.978) with those obtained by a spectrophotometric kit method with slopes of 1.02-1.03 and intercepts of 0.08-0.23 mmol/L.

Adenosine Triphosphate↗