To clarify the mechanism of a possible fatty acid (FA)-induced inhibition of pseudocholinesterase (pchE, EC 3. 1. 1. 8), the interaction or binding of pchE with FA was studied lusing ultrafiltration and dialysis techniques and circular dichroism and ultraviolet spectrometries. The binding study showed that pchE activity was inhibited by FA's (capric acid or lauric acid) containing 10 or 12 carbon atoms, which bind to the pchE molecule, but not by FA's (caproic acid and caprylic acid) which do not bind with pchE. Dialysis of pchE solution with lauric acid containing [1-¹⁴C] lauric acid showed the irreversibility of both lauric acid binding to the pchE molecule and lauric acid-induced inhibition of pchE activity. Circular dichroism and ultraviolet spectra of pchE shifted in proportion to the concentration of capric acid added, suggesting a conformational change in the pchE molecule. The present study indicated that FA-induced inhibition of pchE is based on the irreversible interaction or binding of pchE with FA.

抄録全体を表示

Pseudocholinesterase (PchE EC 3.1. 1.8) inhibition by capric acid did not occur when the substrate (benzoylcholine) concentration was less than 50mM, but did occur, dose-dependently at higher substrate concentrations, double-reciprocal plots for the fatty acid (FA)-inhibited enzyme reaction of pchE showed that the inhibition was kinetically uncompetitive. It occurred at above room temperature (26-27°C), whereas pchE activity was stimulated at 4°C. The pchE activity was restored in proportion to the increasing concentration (0-1 mM) of human serum albumin (HSA) when it was added to the pchE solution before the capric acid solution, but not when HSA was added after capric acid. This result suggests that for effective restoration of the esterase activity, HSA must react with pchE or capric acid before the interaction of pchE with capric acid.

抄録全体を表示

Guanase from human liver was purified by two methods.
The first was a procedure involving ammonium sulfate fraction, TEAE column chromatography, hydroxyapatite column chromatography, affinity chromatography on CM Affi-gel blue and gel filtration on Sephacryl S-200. Buffers containing 0.1mM/L of dithiothreitol were used in all steps. Dithiothreitol was added to cut the S-S complex of guanase if it was present. Guanase was purified 1505-fold with a 17% yield in this method. Homogeneity was established by SDS-polyacrylamide gel electrophoresis.
The second method was employed to purify guanase without using dithiothreitol. It involved four repetitions of ammonium sulfate fractionation together with TEAE chromatography and gel filtration. Guanase in the seccnd method was resolved into two fractions, guanase 1 and 2 on the first TEAE chromatography.
The molecular weight of the enzyme purified with dithiothreitol according to SDS-polyacrylamide gel electrophoresis and gel filtration was estimated to be 55000 and 64000, respectively. When dithiothreitol was not used, the molecular weight of guanase 1 was 64000 and that of guanase 2 was 110000 according to gel filtration. In SDS-polyacrylamide gel electrophoresis, the molecular weights of both guanase 1 and 2 were 55000. This shows that the enzyme is composed of a subunit with a molecular weight of 55000. These results indicate that guanase 2 consists of polymeric form.
As guanase from crude extract of human liver showed the same bands of guanase 1 and 2 on agarose gel, polymeric forms of guanase are considered toThe present in human liver.

抄録全体を表示

The interaction of human hepatic and intestinal alkaline phosphatase (EC 3.1. 3.1) with 17 amino acids or its derivatives was studied.
Of these. homogentisate was a potent inhibitor for both alkaline phosphatases. The pH profile for the enzymes with and without homogentisate was similar, but the enzyme·homogentisate complex showed a marked heat-lability at higher temperature.
According to the results of S/v against S plots, the mechanism of homogentisate inhibition for the intestinal enzyme was uncompetitive type and the Ki value was found to be 1mM.
To investigate the binding sites of homogentisate, bismuth and L-phenylanine on the intestinal enzyme molecule, the effect of homogentisate on the enzyme activity with and without L-phenylalanine or bismuth was studied. From the results of kinetic analysis, the L-phenylalanine and homogentisate binding sites is apparently independent of each other, while the sites for bismuth and homogentisate binding are presumed to be proximal to one another.

抄録全体を表示

Direct radioimmunoassay for the determination of estradiol 3-sulfate 17-glucuronide in serum using highly specific antiserum without prior deconjugation has been developed. Antiserum was raised in the rabbit by immunization with antigen in which the steroid hapten is coupled to a carrier protein through the C-6 position. The proposed method employing this specific antiserum only needs a pretreatment on a Sep-pak C₁₈ cartridge. A simple and reliable assay procedure for the determination of estradiol 3-sulfate 17-glucuronide was established and applied to peripheral blood during pregnancy.

抄録全体を表示

Guanase from human liver was purified, without adding dithiothreitol, into two enzymes, guanase 1 and 2. Studies of these enzymes yielded the following characteristics of guanase.
1. Guanase 1 and 2 showed different substrate (guanine) concentration-activity relationships. Guanase 1 activity displayed typical Michaelis-Menten kinetics, while guanase 2 showed a sigmoid dependence on guanine concentration. Guanase 2 may accelerate catabolism of guanine when the concentration of guanine becomes higher than a fixed value. The K_m value of guanase 1 for guanine was 1.78×10^-5. Hill plots showed that Hill's n factor for guanase 2 was 1.6 but 1.0 for guanase 1. Therefore, guanase 2 seems to be an allosteric enzyme.
2. 5-Aminoimidazole-4-carboxamide was a competitive inhibitor of guanase, with a K_i of 1.55×10^-6. In the presence of 5-aminoimidazole-4-carboxamide, guanase 2 showed typical Michaelis-Menten kinetics. Variation of the concentration of 5-aminoimidazole-4-carboxamide may regulate guanase activity and efficiently accelerate or suppress the catabolism of guanine.
3. Guanase I was inactivated by 10 min treatment of p-chloromercuribenzoic acid at 37°C while approximately 50% of the activity in guanase 2 remained under the same conditions. About 30 to 40% of the guanase 2 activity remained when it was treated with p-chloromercuribenzoic acid up to 30min. Guanase 1 is considered to be inactivated by attack of p-chloromercuribenzoic acid by SH at its active site. In guanase 2, SH in the active site does not seem to be affected by p-chloromercuribenzoic acid because of the difference in its stereo-construction from guanase 1.
4. Al little difference between guanase 1 and 2 was observed in optimum pH and heat stability.
These results indicate that guanase 1 and 2 have different molecular structures.

抄録全体を表示

Sera of five patients with Lactate dehydrogenase (LDH)-Immunoglobulin A (IgA) Kappa (K) complexes and two patients with LDH-Immunoglobulin G (IgG) Lambda (L) complexes were analized. IgA K separated from the LDH-IgA complexes reassociate with the LDH-2 and LDH-3 isoenzymes.
IgG L obtained from LDH-IgG complexes reassociated with the LDH-4 and LDH-5 isoenzymes to form LDH-IgG complexes. The results of our experiments suggest that IgA K has antigenic properties against the H₂M trimer molecule which is common to LDH-2 and LDH-3 and that IgG L has antigenic properties against the M₃ trimer common to LDH-4 and LDH-5.

抄録全体を表示

Two groups of uremic peptides having 5 to 17 amino acids residues, one of which is composed of 20 peptides in total and the other of 16 peptides in total, were isolated respectively from two groups of hydrophobic middle molecule fractions in the hemofiltrates of two dialysis patients by the ion exchange separation into acidic, neutral and basic portions, followed by the adsorption of middle molecule substances on non-polar copolymer, and hydrophobic fractionation by HPLC. Uremic peptides were obtained universally from each hydrophobic fraction. These peptides are abundunt in glutamic acid, aspartic acid, threonine or serine and especially in glycine, while scarce in aromatic amino acids. A common characteristic of these peptides is their amino acid proportion with abundunt residues of hydrophilic amino acids and glycine. A significant difference of the hydrophobicity value against one glycine residue was observed between uremic and biologically active peptides. It seems that these uremic peptides do not conjugate with any plasma components and permeate into body cell. Therefore, it seems reasonable to assume that these peptides play an important role in patients with uremia.

抄録全体を表示

We have designed a new chelating method for the determination of L-ascorbic acid (AsA) in plasma. For the blank, AsA is oxidized with L-ascorbate oxidase before deproteinization with trichloroacetic acid (TCA). During the reaction, iron (Fe³⁺) is reduced to Fe²⁺ with AsA, which can be continuously monitored by chelating Fe²⁺ and 2-nitroso-5-(N-propyl-N-sulfopropylamino)phenol. We determined the optimal concentrations of substrate and reactants and developed a method, to eliminate proteins in the plasma sample by treatment with TCA. Results from this method correlate well with the 2, 4, 6-tris-(2-pyridyl)-s-triazine method for determination. We established a reference interval of 10-450μM; and at optimal conditions within this range, the variance of this method was less than 1.50%.

抄録全体を表示

A simple modified spectrophotometric method for determination of arginine aminopeptidase activity in the submaxillary gland of mice was studied. The present method is based on cleaving of the substrate α-N-benzoyle-DL- arginine-ρ-nitroanilide to ρ-nitroaniline which is liberated on enzymatic hydrolysis. Catalization by the enzyme activity is determined by measuring diazotization and coupling converted to a purple azo dye. The intensity of color is measured spectrophotometrically at 546nm. The sensitivity of this method is in accordance with the requirements. Further, it may be useful to determine the activity of arginine aminopeptidase of the submaxillary gland in mice by this method combined with the zymogram method.

抄録全体を表示