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At least 577 records · Page 32Linked to original sources

PubMed1993

Enzymatic rate assay of creatinine in serum and urine.

A two-step method for assaying creatinine in serum and urine samples, suitable with automated analyzers, is reported. Reagent 1, for the first step, contains a blanking system [creatine amidinohydrolase (CRTase), urease, glutamate dehydrogenase, NADPH, and 2-oxoglutarate] and a NADPH-regenerating system [Mg(2+)-dependent isocitrate dehydrogenase (ICD), MgCl2, and excess isocitrate]. Reagent 2, for the second step, contains the metal-chelating reagent trans-1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid (CyDTA) and a trigger system [creatinine amidohydrolase (CRNase)]. When a specimen is mixed with reagent 1, all the creatine, urea, and NH3 present are removed by the blanking and NADPH systems. On adding reagent 2, CyDTA inactivates ICD to inhibit the NADPH system. Simultaneously, the creatinine (1 mol) in the specimen is hydrolyzed into creatine by CRNase, and then releases NADP+ (2 mol) through the blanking system. Our optimized method can determine creatinine linearly up to 500 mg/L, with within-day CVs < 1.2% and day-to-day CVs < 2.7%.

Ammonia↗

PubMed1996

Functional analyses of active site residues of human lysosomal aspartylglucosaminidase: implications for catalytic mechanism and autocatalytic activation.

Aspartylglucosaminidase (AGA) is a lysosomal asparaginase that participates in the breakdown of glycoproteins by cleaving the amide bond between the asparagine and the oligosaccharide chain. Active AGA is an (alphabeta)2 heterotetramer of two non-identical subunits that are cleaved proteolytically from an enzymatically inactive precursor polypeptide. On the basis of the three-dimensional structure recently determined by us, we have here mutagenized the putative active site amino acids of AGA and studied by transient expression the effect of targeted substitutions on the enzyme activity and catalytic properties of AGA. These analyses support the novel type of catalytic mechanism, suggested previously by us, in which AGA utilizes as the nucleophile the N-terminal residue of the beta subunit and most importantly its alpha-amino group as a base that increases the nucleophilicity of the OH group. We also provide evidence for autocatalytic activation of the inactive AGA precursor and putative involvement of active site amino acids in the proteolytic processing. The data obtained on the structure and function of AGA would indicate that AGA is a member of a recently described novel class of hydrolytic enzymes (amidohydrolases) sharing a common structural determinant in their three-dimensional structure and whose catalytic mechanisms with an N-terminal nucleophile seem basically to be similar.

Aspartylglucosylaminase↗

PubMed1996

Purification and preliminary characterization of L-asparaginase from Erwinia aroideae NRRL B-138.

L-Asparaginase (L-asparagine amidohydrolase EC 3.5.1.1) from Erwinia aroideae NRRL B-138 has been purified to apparent homogeneity by ammonium sulphate precipitation, chromatography on sulfopropyl-sephadex C-50 and sephadex G-200 with 22% recovery and 567-fold purification. The enzyme obtained from sulfopropyl-sephadex C-50 was unstable and lost activity within a few hours. Addition of glycerol helped in restoring the activity of the enzyme. The enzyme has an apparent molecular mass of approximately 155 kDa and has four subunits of identical molecular mass of approximately 38 kDa. The K(m) for L-asparagine is 2.8 x 10(-3) M. Enzyme shows optimal activity at 45 degrees C and pH 8.2. Energy of activation as determined from Arrhenius plot was 9.1 kcal/mol. Substrate L-asparagine and analogue L-glutamine, D-asparagine and 6 diazo-5-oxo-L-norleucine provide full protection to the enzyme against thermal denaturation.

Asparaginase↗

PubMed1998

Aspartylglucosaminuria in a Canadian family.

Aspartylglucosaminuria (McKusick 208400) is a lysosomopathy associated with aspartylglucosaminidase (L-aspartamido-beta-N-acetylglucosamine amidohydrolase, EC 3.5.1.26) deficiency. It has been most frequently encountered in Finland, where the regional incidence may be as high as 1 in 3600 births. In North America it is very rare, having been reported in only 8 patients. We encountered 4 patients with aspartylglucosaminuria in a Canadian family of 12 siblings. The 4 siblings affected--2 brothers and 2 sisters--were apparently normal at birth; however, their developmental milestones, particularly speech, were slow, and they acquired only a simple vocabulary. Throughout life, there was a progressive coarsening of facial features; 3 had inguinal hernia and recurrent diarrhea; all became severely retarded and by the 4th decade showed evident deterioration of both cognitive and motor skills; 2 exhibited cyclical behavioural changes. Three of the siblings have died, at 33, 39 and 44 years of age. Two died of bronchopneumonia and 1 of asphyxiation following aspiration. In the urine of all 4 siblings, and in the 1 liver examined, we found 2-acetamido-1-N-(4-L-aspartyl)-2-deoxy-beta-D-glucosamine (GlcNAc-Asn) and alpha-D-mannose-(1,6)-beta-D-mannose-(1,4)-2-acetamido- 2-deoxy-beta-D-glucose-(1,4)-2-acetamido-1-N-(4-L-aspartyl)-2-deoxy-beta - D-glucosamine (Man2-GlcNAc2-Asn). Compared with the level of activity in controls, aspartylglucosaminidase activity was less than 2% in fibroblasts from 3 of the siblings, less than 0.5% in leukocytes from 1 sibling, and less than 1% in the liver of 1 sibling, whereas other acid hydrolase activities in these tissues were normal. Ultrastructural studies of skin showed that fibroblasts, endothelial cells and pericytes contained vacuoles with fine reticulo-floccular material. Glial and neuronal cells of the central nervous system showed similar inclusions as well as others composed of concentric or parallel membranous arrays intermingled with lipid droplets.

Acetylglucosamine↗

PubMed1976

Direct enzymatic determination of urea in plasma and urine with a centrifugal analyzer.

A direct enzymatic micromethod (sample volume, 3mul) has been adapted to the centrifugal analyzer (ENI-GEMSAEC) for measurement of urea in plasma and urine. The method is based on urease (urea amidohydrolase, EC3.5.1.5)/glutamate dehydrogenase [l-glutamate:NAD(P)+oxidoreductase (deaminating), EC1.41.3] coupled reactions, and uses a two-point fixed-time (t(1)=20s,t(2)=50s)kinetic scheme for monitoring the rate of comsumption of NADH at 340 nm. Sensitivity and precision of the method are excellent,and results compare well with those from a commonly used continuous-flow method.

Autoanalysis↗

PubMed1963