Biomedical subjects

S Satomura

Publications and source records attributed to S Satomura.

7 recordsLinked to original sources

PubMed1992

Renin assay using a fluorogenic substrate and high performance liquid chromatography.

Measurement of renin activity in human fluids using a fluorogenic substrate and high performance liquid chromatography (HPLC) is described. A nine amino acid peptide containing the fluorogenic residue, N-(2-pyridyl) glycine (Pg) is used as a substrate. The peptide sequence is homologous with the cleavage site of human angiotensinogen. This substrate is hydrolyzed by renin to generate fluorogenic and non-fluorogenic products. The amount of fluorogenic product is directly measured by reversed phase HPLC. Optimization of assay conditions and measurement of human serum renin levels are described. Assay results correlated well with those from radioimmunoassay. The method is simple, convenient, highly sensitive and can be used for routine clinical renin assays.

Amino Acid Sequence↗

PubMed1992

Analysis of lectin properties with membrane ultrafiltration and high-pressure liquid chromatography.

A convenient method for the analysis of the binding properties of lectin with fluorogenic sugar chains is described. A lectin (concanavalin A or Datura stramonium agglutinin) was mixed with pyridylaminated sugar chains in buffer and the free chains obtained were isolated by membrane ultrafiltration. The amount of free sugar chains in the filtrate was measured by high-pressure liquid chromatography. The binding constants with the sugar chains, reaction kinetics, and other properties of these lectins were easily investigated. The method is simple and could be used to study the characteristics of any lectin in native form.

Binding Sites↗

PubMed1991

Identification of the active site of human renin with use of new fluorogenic peptides.

Fluorogenic peptide substrates were synthesized in which amino acid residues corresponded to the C-terminal and the N-terminal sides of the site of human angiotensinogen cleaved by renin. Compared with the synthetic substrates of renin previously reported, these fluorogenic substrates had practical advantages in that their digestion products could be rapidly separated and sensitively detected by high-pressure liquid chromatography with a fluorescence detector. The recombinant human renin and human plasma split Leu-Val, which cleavage site is similar to that in human angiotensinogen. The kinetic parameters of the reaction of renin using these substrates were calculated. There seemed to be at least eight subsites in the active site of recombinant human renin, to judge from the enzyme-substrate binding characteristics. The two histidine residues (S5 and S'3) in the octapeptide His-Pro-Phe-His-Leu-Val-Ile-His were important in the enzyme action.

Amino Acid Sequence↗

PubMed1988

Alpha-amylase assay with use of a benzyl derivative of p-nitrophenyl alpha-maltopentaoside, BG5P.

p-Nitrophenyl O-(6-O-benzyl)-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)-alpha-D-glucopyranoside (BG5P) is hydrolyzed by both human salivary alpha-amylase (HSA) and human pancreatic alpha-amylase (HPA) to O-(6-O-benzyl)-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl- (1----4)-alpha-D-glucopyranose (BG3) and p-nitrophenyl alpha-maltoside (G2P). Glucoamylase and alpha-glucosidase cannot hydrolyze BG5P because of the modification of the OH group of the 6-position of the non-reducing-end glucose residue with the benzyl group. Taking advantage of these characteristics of the substrate, BG5P, we developed a method to assay the total alpha-amylase activity in human fluids using glucoamylase and alpha-glucosidase as the coupled enzymes. This method is simple and can be used as the standard method for routine clinical assays of alpha-amylase activity.

Chromatography, High Pressure Liquid↗

PubMed1988

Synthesis of p-nitrophenyl 6(5)-O-benzyl-alpha-maltopentaoside, a substrate for alpha amylases.

p-Nitrophenyl alpha-maltopentaoside, having a benzyl group on O-6 of the terminal (nonreducing) D-glucosyl group was prepared by use of a reductive ring-opening reaction. Highly regioselective reduction of p-nitrophenyl O-(2,3-di-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl)-(1----4)- tris[O-(2,3,6-tri-O-benzoyl-alpha-D-glucopyranosyl)-(1----4)]-2,3,6-tri- O- benzoyl-alpha-D-glucopyranoside by dimethylamine-borane and p-toluenesulfonic acid, followed by debenzoylation, gave p-nitrophenyl O-(6-O-benzyl-alpha-D-glucopyranosyl)-(1----4)-tris[O-alpha-D-glucopyran osyl- (1----4)]-alpha-D-glucopyranoside. An experiment was done on the mode of action of human pancreatic and salivary alpha amylases on this derivative. The compound is suitable as a substrate for the assay of alpha amylase when used with glucoamylase and alpha-D-glucosidase as coupling enzymes.

Carbohydrate Conformation↗

PubMed1986

Measurement of cyclomaltodextrin glucanotransferase activity by high-performance liquid chromatography using a fluorogenic substrate.

A mixture of p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranoside (FG5P) and p-nitrophenyl alpha-D-glucoside (GP) was incubated with cyclomaltodextrin glucanotransferase (CGTase) [EC 2.4.1.19]. Analysis of the digest by HPLC showed that the products were p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG4P) and p-nitrophenyl alpha-D-maltoside (G2P), and no other product could be detected. Based on the reaction, a sensitive method to assay for CGTase was developed.

Bacillus↗

PubMed1985

Kinetic assay of gamma-glutamyltransferase with use of bilirubin oxidase as a coupled enzyme.

We studied the kinetic measurement of gamma-glutamyltransferase (EC.2.3.2.2), coupling the reaction with that catalyzed by bilirubin oxidase (EC 1.3.3.5), which oxidizes and combines a phenylenediamine derivative with an aniline derivative to produce a green pigment. We measured the formation of the pigment kinetically (at lambda max745 nm, epsilon = 75 000 L mol-1 cm-1), with L-gamma-glutamyl-N-hydroxyethylaminoanilide as substrate and N-ethyl-N-hydroxy-3-sulfopropyl)-m-toluidine as a the coupling derivative. The within-run CV for measuring this reaction in samples of normal sera was 2.4%. A calibration plot of the change in absorbance per minute vs enzyme activity concentration showed good proportionality in the range of 0-1300 U/L. The results of this assay correlated well (r = 0.995) with those of the Boehringer method, in which L-gamma-glutamyl-3-carboxy-4-nitroanilide is the substrate. This new, highly sensitive procedure may be adapted to other assays involving phenylenediamine derivates as synthetic substrates.

Glycylglycine↗