Motoo Nakajima

Streptavidin-Biotin Based Bioluminescent Enzyme Immunoassay Using Biotinylated Acetate Kinase and Recombinant Firefly Luciferase

Abstract Streptavidin-biotin system has been introduced to a bioluminescent enzyme immunoassay using biotinylated acetate kinase and recombinant firefly luciferase. The streptavidin-biotin based bioluminescent enzyme immunoassays (BLEIAs) for thyroid stimulating hormone (TSH) and human chorionic gonadotropin (hCG) were developed in which the complexes of biotinylated acetate kinase, streptavidin, and biotinylated antibody were used. In BLEIA for TSH, acetate kinase activity was determined by endpoint assay and for hCG the enzyme activity was measured by rate assay. The measurable ranges of TSH and hCG were 0.02 μIU/ml to 50 μIU/ml and 0.003 to 2 μIU/ml, respectively. The proposed streptavidin-biotinylated acetate kinase system was shown to be applicable to several sandwich type enzyme immunoassays employing biotinylated antibody with high sensitivity.

A new amidinohydrolase, methylguanidine amidinohydrolase from Alcaligenes SP. N-42

Me~~~~uan~dine is known to ~~~~~~1~~~ in body fluids of uremic patients [I ,2] and has been proved a uremic toxin [3]. It was found in several foods [4,S] and nitrosated under acidic conditions to give methylnitrosocyanamide with strong mutagenicity and carc~ogenicity f6]_ We attempted to isolate the microorganisms capable of producing methyiguanidine-decomposing enzyme which may have a potential use for clinical assay and/or detoxification. Here, the purification and iden~~ca~ion of a new enzyme, methylgu~id~~e ~idinohydrolase from ~~e~~~e~e~ sp. N42, which catalyzes specifically the hydrolysis of methylguanidine to yield methylamine and urea, is described.

Comparison of hydroxyl radical generation in patients undergoing coronary artery bypass grafting with and without cardiopulmonary bypass

We measured the hydroxyl radical (√OH) generation in fourteen patients undergoing coronary artery bypass grafting (CABG), of whom seven patients underwent on-pump CABG with cardiopulmonary bypass (CPB) and seven patients underwent off-pump CABG without CPB. To detect √OH generation, we measured the urinary excretion of √OH products of creatinine (Cr), creatol (CTL; 5-hydroxycreatinine) and methylguanidine (MG) with HPLC using the one point sampling and collected urine during and after the operation. The urinary CTL value corrected urinary Cr value of on-pump CABG significantly increased about 3–5 times from the beginning of CPB to 4 h after operation compared to the baseline value before CPB in both the collected urine and the one point sampling urine. The urinary MG/Cr value in both groups did not change significantly. Significantly increased √OH generation was found during and soon after on-pump CABG.

Molecular cloning and nucleotide sequence of the pyruvate kinase gene of an actinomycete Microbispora thermodiastatica

Abstract The gene for the thermostable pyruvate kinase of Microbispora thermodiastatica IFO 14046, a moderate thermophilic actinomycete, was cloned in Escherichia coli. This gene consists of an open reading frame of 1422 nucleotides and encodes a protein of 474 amino acids with molecular mass of 50 805 Da. The open reading frame was confirmed as the pyruvate kinase gene by comparison with the N-terminal amino acid sequence of the purified pyruvate kinase from M. thermodiastatica.

A new enzymatic assay of urinary guanidinoacetic acid.

We describe a new enzymatic determination of urinary guanidinoacetic acid (GAA) with guanidinoacetate kinase (ATP: guanidinoacetate N-phosphotransferase, EC 2.7.3.1), which does not require a blank to correct for endogenous constituents (ADP and pyruvate). In the first step, pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) and lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) were used to eliminate endogenous constituents (ADP and pyruvate) in the presence of phosphoenolpyruvate and NADH. In the second step, urinary GAA was phosphorylated in the presence of ATP by guanidinoacetate kinase to form phosphoguanidinoacetate and ADP. The resultant ADP was sequentially measured at 340 nm in a coupled reaction catalyzed by pyruvate kinase and lactate dehydrogenase. The standard curve was linear up to 20 mg/dl for standard solutions of GAA. Analytical recovery of GAA added to normal urines ranged from 97.0 to 103.2% (mean 100.7%). The within-run and between-run studies gave CV values of less than or equal to 3.6% and less than or equal to 4.8%, respectively. No significant interference by endogenous urinary compounds were observed with the proposed method under this study. The results obtained by the present method correlated well with those obtained by a high-performance liquid chromatographic method. This method is accurate and simple, and less time-consuming than those previously reported. We determined the concentrations of GAA in 24-h urine samples by the proposed method, and observed that the urinary excretion of GAA decreased markedly in patients with renal failure.

Enzymic Determination of Methylguanidine in Serum and Plasma of Hemodialysis Patients as a Marker for Hydroxyl Radicals

Methylguanidine (MG) is known to accumulate in body fluids of uremic and hemodialysis patients1–2 and has been proved to be a strong uremic toxin3. It was reported that erythrocyte deformability and Na+,K+— ATPase activity of erythrocyte membranes decreased in hemodialysis patients and there was a significant negative correlation between erythrocyte deformability and MG level4. Recent reports reveal that MG is converted from creatinine (CRN) by the action of various species of active oxygen, especially hydroxyl radicals as produced in the Fenton reaction5–6 and that free hemoglobin acts as a biological Fenton reagent to generate hydroxyl radicals7, and as an iron promoter in the Fenton reaction8. Since free radicals were shown to play an unfavorable role renal failure9–11, determination of MG in body fluids as a marker for hydroxyl radicals could prove useful in clinical practice.

Purification and some properties of guanidinoacetate amidinohydrolase produced by Corynebacterium sp.

Abstract Guanidinoacetate amidinohydrolase (EC 3.5.3.2) was purified from Cornebacterium sp. grown in a medium supplemented with guanidinoacetate, and some of its properties were investigated. The molecular weight of the enzyme was estimated to be 150,000 by gel filtration. SDS-polyacrylamide gel electrophoresis showed a single subunit component with a molecular weight of 38,000, suggesting that the enzyme is composed of four identical subunits. The isoelectric point of the enzyme was pH 5.8. The enzyme showed optimum activity at pH 9.0–9.5 and was stable at pH 6.0–10.5. 3-Guanidinopropionate and 4-guanidinobutyrate were respectively hydrolyzed 32% and 5% as fast as guanidinoacetate. The apparent K m for guanidinoacetate was 16 mM. Incubation of the enzyme by o -phenanthroline or 8-hydroxyquinoline resulted in almost complete inactivation. The activity of the inactivated enzyme was restored by incubation with Zn 2+ . p -Chloromercuribenzoic acid and iodine effectively inhibited the enzyme activity. Glycine was a competitive inhibitor, and n -alkyl amines such as n -octylamine, n -decylamine and n -dodecylamine were uncompetitive inhibitors.

Enzymic Determination of Methylguanidine in Urine

Methylguanidine is known to accumulate in the body fluids of uremic patients1 , 2 and has proved, to be a uremic toxin3. Determination of its concentration in body fluids would, therefore, be useful in clinical practice. Recently, the determination of methylguanidine has been carried out by means of automated high-performance liquid chromatography with a fluorometric detection method for guanidino compounds4. The guanidino compounds analyzer is well suited for the simultaneous determination of several guanidino compounds. However, it is very expensive and inappropriate for analyses of many samples. In clinical practice, the enzymic determination of metabolite concentration has been widely employed because of its simplicity and specificity.