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Biomedical subjects

H Akanuma

Publications and source records attributed to H Akanuma.

49 records · Page 3Linked to original sources
PubMed1985

Relation of blood glucose levels to the changes in plasma levels of various hydrolytic enzymes in diabetic patients.

Plasma levels of various hydrolytic enzymes in diabetic patients, as diagnosed by their blood glucose levels, were compared with those of control subjects. Most of the levels of the aminopeptidase activities examined were significantly increased, while those of angiotensin-converting enzyme (ACE) and esterase decreased, in diabetic patients. The results of multivariate analysis suggest that the increased aminopeptidase activities are related rather to secondary organ lesions than to the primary pancreatic lesion. It seems that the blood glucose level is not necessarily a good indicator of metabolic abnormalities underlying this pathological condition.

Acetylcholinesterase↗
PubMed1981

Reduced levels of plasma 1,5-anhydroglucitol in diabetic patients.

Anhydroglucitol in human plasma was studied by high pressure liquid chromatography, which was employed to simplify sampling procedures for the polyol assay and to avoid enzymatic treatment of the plasma for removal of glucose. Polyols in the column effluent were assayed by a flow fluorometric method, which involved two continuous successive reactions; periodate oxidation and the Hantzsch reaction. Plasma, 1 ml each, from 55 diabetic and 18 control subjects was deproteinized, concentrated, and subjected to the liquid chromatography. The results of this analysis indicated that the plasma anhydroglucitol level showed an inverse correlation with the plasma glucose level, while the correlation of glycerol and glucose levels was obscure.

Blood Glucose↗
PubMed1980

Ligand bindings of bovine carboxypeptidase B. II. Affinity chromatography and cooperative ligations.

Affinity chromatography was used to characterize the binding properties of carboxypeptidase B with its ligands. The affinity adsorbents employed included arginine directly attached to agarose beads, arginine attached to the same support through hydrophilic and hydrophobic spacers, and immobilized caproylphenylalanine. The enzyme showed marked retardation on all of the arginine columns but only slight retardation on the phenylalanine column. Several hydrophobic ligands in solution enhanced to some extent the enzyme retardation on columns having arginine directly attached to the solid support, while several amino- and guanidino-alkylcarboxylic acids (lysine and arginine analogs) greatly enhanced the enzyme retardation on the phenylalanine column and somewhat enhanced it on the other columns having hydrophobic spacer arms. These observations confirmed that the enzyme has twobinding sites for the soluble and immobilized ligands and that these two sites exhibit cooperative ligations. Binding constants of the enzyme for various soluble ligands were also calculated from their chromatographic effects and the resulting values were interpreted in terms of the cooperative action of the two bindings sites, i.e., one for the primary binding of basic amino acid analogs (SITE I) and the other for hydrophobic compounds (Site II). In this chromatographic study, however, such cooperation of the two sites was obscure when arginine, acylarginine, or alkylarginine was the ligand directing to Site I.

Animals↗
PubMed1980

Ligand bindings of bovine carboxypeptidase B. III. Hydrophobic activators in dipeptide hydrolysis.

Several hydrophobic compounds acted as activators in dipeptide (Bz-Gly-L-Arg-OH, Z-Gly-L-Phe-OH) hydrolysis by bovine carboxypeptidase B. These hydrophobic compounds include Bz-Gly-OH, Z-Gly-OH, Z-L-Phe-OH, and Z-L-Phe-GLy-OH. These compounds were indicated to bind to the secondary substrate binding sites which is proposed to be responsible for substrate activation kinetics in dipeptide hydrolysis. Of the compounds Z-L-Phe-OH alone acted also as a inhibitor at higher concentrations, indicating that it binds to both primary and secondary sites as the dipeptide substrates do. Comparison of the activation effects of the compounds employed indicated that hydrophobic interaction played an important role in binding to the secondary site. Substrate and modifier binding constants were also determined and the results indicated that modifier binding increased both affinity and catalytic rate constant of the primary site. On the other hand, Z-Gly-OH and Z-L-Phe-Gly-OH inhibited the hydrolyses of tri and tetrapeptide substrates. This observation suggests that the secondary site is contained in the extended active center which the enzyme possibly has.

Alcohols↗
PubMed1980

Ligand binding of bovine carboxypeptidase B. IV. Oligopeptide substrates and extended active center.

Catalytic and binding properties of bovine carboxypeptidase B were studied by kinetic and affinity chromatographic methods both using several oligopeptides as substrates or immobilized ligands. These oligopeptides contained either arginines or phenylalanines at carboxy termini as well as phenylalanyl residues in one of the other positions. The chromatographic studies showed that the phenylalanyl residues in endo-positions play a significant role in binding of the immobilized peptides to the enzyme, while the kinetics studies indicated further that the presence of an internal hydrophobic residue in a substrate was advantageous for the catalytic release of the carboxyl terminal residue from the substrate. These observations support the supposition that the enzyme has an extended active center which contains an extended hydrophobic binding site. Several hydrophobic compounds, which have been shown to act as activators in dipeptide substrate hydrolyses, showed inhibitory effect on hydrolyses of oligopeptide substrates. This observation suggests that these hydrophobic compounds bind to a portion of the hydrophobic site in the active center.

Animals↗
PubMed1979

Fatty acid alpha-hydroxylation and its relation to myelination.

alpha-Hydroxylation is an enzymatic reaction by which long-chain fatty acids are converted to their alpha-hydroxy derivatives. This reaction, in animals, can be detected only in developing brain and is the rate-determining step in the synthesis of hydroxycerebroside, which is an indispensable and abundant myelin lipid. In addition to a particulate fraction from brain, two cytoplasmic factors, one heat-stable and the other heat-labile, are required for alpha-hydroxylation. During the past eight years we have been investigating alpha-hydroxylation. Our progress is summarized and discussed here.

Aging↗
PubMed1979

Synthesis of ceramides and cerebrosides containing both alpha-hydroxy and nonhydroxy fatty acids from lignoceroyl-CoA by rat brain microsomes.

The conversion of [1-14C]lignoceroyl-CoA to nonhydroxy- and alpha-hydroxyceramides and cerebrosides by brain microsomes of developing rat in the presence of NADPH was investigated. A new technique of thin layer chromatography for the separation of these lipids and unreacted substrate was developed for this assay. The synthesis of nonhydroxy- and hydroxyceramides was significantly stimulated by the addition of heat-stable factor, a factor which is essential in the alpha-hydroxylation of free lignoceric acid (I. Singh and Y. Kishimoto, manuscript in preparation). The addition of sphingosine also stimulated the ceramide synthesis to a great extent. When the microsomes or heat-stable factor were crude, the ceramides formed were further converted to cerebrosides, apparently by UDP-galactose contamination. The purification of these subcellular components resulted in the formation of only ceramides which, in turn, were converted to cerebrosides by the addition of UDP-galactose. These observations indicate that hydroxyceramide is the precursor of hydroxycerebroside and is formed by alpha-hydroxylation of lignoceroyl-CoA and N-acylation of sphingosine. However, lignoceroyl-CoA, like free lignoceric acid, does not appear to be the immediate substrate of the alpha-hydroxylation.

Acyl Coenzyme A↗
PubMed1978

Simple hydrazidation method for carboxymethyl groups on cross-linked dextran.

A novel method for the use of CM-Sephadex in affinity chromatography is described. This method involves a carbodiimide-mediated synthesis of a lactone derivative, which is subsequently hydrazinolyzed to CM-Sephadex hydrazide. More than 90% of the carboxyl groups was converted to the hydrazide form by this method without any discernible degradation of the Sephadex beads. The hydrazide beads were further converted to the axide form and coupled with D-arginine and D-phenylalanine derivatives. These Sephadex derivatives contained more than 0.5 mmol of each amino acid per g dry beads and were found to be effective and specific adsorbents for carboxypeptidase B. This CM-Sephadex hydrazide should be useful in affinity chromatography because the resulting specific adsorbents involve stable amide coupling linkages and retain the physical properties of their precursor, Sephadex, which are favorable for column operation.

Amino Acids↗
PubMedDate not supplied

High performance liquid chromatographic determination of 1,5-anhydroglucitol in human plasma for diagnosis of diabetes mellitus.

This paper describes a high performance liquid chromatographic (HPLC) method for determining 1,5-anhydroglucitol in plasma, in which anion exchange chromatography and pulsed amperometric detection are used. Plasma samples deproteinized with trichloroacetic acid are passed through a three-layer column packed with (1) strongly basic anion (BO3(3-) form, the upper layer), (2) strongly basic anion (OH- form, the middle layer) and (3) strongly acidic cation (H+ form, the lower layer) exchange resins. 1,5-Anhydroglucitol is efficiently recovered in the flow-through fraction and interfering substances are completely removed by the column treatment. The analytical response of the method is linear with concentration to 40 mg/L, and it is possible to detect as little as 0.1 mg 1,5-anhydroglucitol per litre of plasma. Analytical recovery is between 96 and 103%, and there is good agreement between the results measured by our method and by a gas/liquid chromatographic method (r = 0.998). The method has been successfully used for the determination of very low 1,5-anhydroglucitol concentrations (less than 1 mg/L) in the plasma of diabetic patients.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

Determination of 1,5-anhydroglucitol in urine by high performance liquid chromatography and an enzyme sensor.

A simple high performance liquid chromatographic method combined with an enzyme sensor has been developed to measure 1,5-anhydroglucitol in urine. The enzyme sensor consists of a hydrogen peroxide electrode and a chitosan membrane of an immobilized pyranose oxidase. As the system does not resist interfering substances, urine samples are first purified by passing them through a two-layer column packed with (1) strongly basic anion (OH- form, the upper layer) and (2) strongly acidic cation (H+ form, the lower layer) exchange resins. 1,5-Anhydroglucitol is efficiently recovered in the flow-through fraction of the column. In this system, the minimum detectable concentration of 1,5-anhydroglucitol is 0.1 mg/L, and the measurable range extends from 0.1 to 60 mg/L. The coefficient of variation values of the within-day and day-to-day precisions are 3.0-6.5% and and 4.4-6.7% respectively, and there is good agreement between the results measured by our method and those obtained by the gas-liquid chromatographic/mass spectrometric method (r = 0.994). The method we have described here has been successfully used to elucidate a mechanism for the reducing 1,5-anhydroglucitol level in the serum and plasma of patients.

Biosensing Techniques↗