Yoshio Shirokane

A novel enzymic determination of maltose

A novel enzymic determination of maltose with four enzymes (a new enzyme, maltose 1-epimerase [EC 5.1.3.-], maltose phosphorylase [EC 2.4.1.8], beta-phosphoglucomutase [EC 5.4.2.6], and glucose-6-phosphate dehydrogenase [EC 1.1.1.49]) is described. Maltose was rapidly and quantitatively determined within about 2 min by means of maltose 1-epimerase. The standard curve was linear up to 1.5 micromol/mL. The within-run and between-run studies gave precision (CV) values of < 2.0% and < 3.0%, respectively. No significant interferences by mono- and disaccharides were observed with the proposed method under this study. There was a good correlation (r = 0.997) between the results obtained by the enzymic and HPLC methods. This method fulfills the need for an accurate, specific and simple assay of maltose, and it is less time consuming than HPLC and enzymic methods previously reported.

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.

A novel enzyme, maltose 1-epimerase from Lactobacillus brevis IFO 3345

A novel enzyme, maltose 1‐epimerase (MER), that catalyzes the interconversion of α and β anomers of maltose was found in a cell‐free extract of Lactobacillus brevis IFO 3345, and MER was purified to homogeneity from the crude extract. The M r of the enzyme was estimated to be 43,000 and 45,000 by HPLC gel filtration and SDS‐PAGE, respectively. It showed optimum activity at pH 6.5–7.0. This novel enzyme catalyzed the conversion of β‐maltose more effectively than disaccharides such as α‐lactose and β‐cellobiose, whereas the relative velocities for β‐ and α‐d‐glucose were about one forth of that for β‐maltose.

Effect of Antibiotic Administration on Urinary Guanidinoacetic Acid Excretion in Renal Disease

It is known that there is a marked decrease in urinary guanidinoacetic acid (GAA) excretion in patients with uremia1–3. We have previously reported that urinary GAA (U-GAA) excretion is significantly decreased in experimental animals injected with 20 to 50 mg/kg of gentamicin4. Our further study also showed that such a reduction in GAA excretion is seen even in animals injected with 5 mg/kg, a relatively small dose of gentamicin5. These findings have led some to suggest that GAA would be useful for the early diagnosis of antibiotic-induced nephrotoxicity.

A new enzyme, maltobionate α-d-glucohydrolase, from alkalophilic Bacillus sp. N-1053

A new enzyme, maltobionate alpha-D-glucohydrolase, was purified to apparent homogeneity from a cell-free extract of alkalophilic Bacillus sp. N-1053 about 930-fold with a yield of 18% and some of its properties were investigated. The enzyme showed optimum activity at about pH 7.0, and was stable over the range of pH 6.0-9.5. The molecular weight was estimated to be 152,000 and 71,000 by HPLC gel filtration on TSKgel G3000SWXL and SDS-polyacrylamide gel electrophoresis, respectively. The enzyme hydrolyzed maltobionate more effectively than disaccharides such as maltose and maltitol or trisaccharides such as maltotrionate, maltotriose and maltotriitol, but showed no activity toward polysaccharides such as amylose, amylopectin and soluble starch. The reaction products from 1 mol of maltobionate were found to be 1 mol of beta-D-glucose and 1 mol of D-gluconate. The Km value for maltobionate was 1.63 mM and the Vmax/Km value for maltobionate was the largest among the substrates tested. The enzyme activity was almost completely inhibited by Hg2+, Ag+, iodine and N-bromosuccinimide, and also inhibited by p-nitrophenyl alpha-D-glucoside, maltose and maltitol.

Purification and some properties of β-phosphoglucomutase from Lactococcus lactis subsp. cremoris IFO 3427

Abstract β-Phosphoglucomutase (β-PGM, EC 5.4.2.6) was isolated to homogeneity from a cell-free extract of Lactococcus lactis subsp. cremoris IFO 3427 by chromatographies with QAE-Sephadex A-50, phenyl-Sepharose CL-4B, hydroxylapatite, and Bio-Gel A-1.5m. The enzyme was purified about 260-fold with a yield of 7.2% and a specific activity of 113 units/mg protein. The molecular weight was estimated to be 34,000 and 25,000 by HPLC gel filtration on TSKgel G3000SW XL and SDS-PAGE, respectively. activity around pH 7.0 and its optimum temperature was about 40°C. The enzyme was stable over a pH range from 5.0 to 9.5 and retained its activity up to 45°C. It was activated by four divalent cations (Co 2+ > Mn 2+ > Mg 2+ > Ni 2+ at 1.0 mM concentration). The K m value was 0.23 mM for β- d -glucose 1-phosphate. The enzyme activity was strongly inhibited by other divalent cations (Cu 2+ , Cd 2+ , Zn 2+ , and Hg 2+ ). ADP and ATP also greatly inhibited the enzyme activity, whereas AMP hardly did. α- d -Glucose 1-phosphate and d -glucose 6-phosphate were not potent inhibitors of the enzyme. A comparison of its characteristics with the properties of other known β-PGMs indicated that the β-PGM from Lactococcus lactis subsp. cremoris IFO 3427 is a new type of enzyme.

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.

A novel enzymic determination of maltobionate

Abstract A novel enzymic determination of maltobionate with three enzymes (a new enzyme, maltobionate α- d -glucohydrolase [EC 3.2.1.-], gluconate kinase [EC 2.7.1.12], and 6-phosphogluconic dehydrogenase [EC 1.1.1.44]) is described. It fulfilled the need for an accurate, specific, and rapid assay of maltobionate, as shown by the procedure, the linearity of calibration curve, and the analytical precision, and was simple as compared to an HPLC approach. There was a good correlation ( r = 0.998) between the results obtained by the enzymic and HPLC methods. As an application, the concentrations of maltobionate in the culture broth of Pseudomonas taetrolens IFO 3460 were determined by the proposed method, affording the maltobionate production from maltose in the course of cultivation of this strain.

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.