Sueko Hayashi

Effect of periodate oxidation on the structure and properties of glucose oxidase.

In order to elucidate the molecular structure of glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) and the roles of its carbohydrate moiety, chemical, physiochemical and immunological experiments were performed with enzyme samples before and after periodate oxidation. Hydrodynamic parameters indicated that the native enzyme was a globular protein with values of 1.21 for the frictional ratio and 43 A for the Stokes radius. The enzyme contained about 12% carbohydrate by weight, of which the main component was mannose. The periodate treatment decreased the carbohydrate content to about 40% of its original value. Slight modifications were detected in the absorbance spectrum and the content of arginyl residue. However, no significant alteration was brought about by this treatment in the catalytic parameters, immunological reactivities of the gross structure, not in the secondary and quaternary structures of the protein moity. Thermal denaturation temperature (about 72.5 degrees C) and the enthalpy of denaturation (about 450 kcal/mol) were common to the native and the periodate-oxodozed enzymes. The native was found to be quite resistant to sodium dodecyl sulfate and fairly stable to urea and heating. The periodate-oxidized enzyme was also stable to heat treatment, but it showed a diminished stability when denaturing agents were present. Kinetic analyses of the thermal inactivation processes showed that the entropy of activation was greatly decreased by the denaturing agents, especially in the case of the periodate-oxidized enzyme. It is concluded that the carbohydrate moiety of the enzyme plays a role in increasing the stability of the protein moiety, but does not directly participate in the catalytic activity, the immunological reactivity, or in maintaining the conformation of the enzyme protein.

Multiple forms of glucose oxidase with different carbohydrate compositions.

A glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) sample purified from Aspergillus niger by a previously reported method was subjected to isoelectric focusing and gel electrophoresis, and found to be composed of at least six component enzymes. The isoelectric points of the component enzymes ranged from pH 3.9 to 4.3. Analyses of the enzymes indicated that they all possess an identical protein moiety, since the amino acid compositions, the C-terminal sequences, the catalytic parameters, the quantitative and qualitative immunological properties and the electrophoretic patterns of the peptide fragments, obtained by the CNBr-cleavage, were practically the same. On the other hand, the carbohydrate contents of the isolated component enzymes were found to be different, and these differences were associated in the main with a particular peptide fragment. We suggest that the multiplicity of the enzyme is due to variation in the carbohydrate and their structures, rather than in the protein moiety.

Comparison of fungal glucose oxidases. Chemical, physicochemical and immunological studies.

Glucose oxidases (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) from two fungal genera (Aspergillus and Penicillium) were studied chemically, physicochemically and immunologically to elucidate the similarities and dissimilarities between these enzymes. Investigation of circular dichroism spectra revealed that these enzymes proteins possess essentially identical conformations. However, differences found in thermal inactivation parameters, catalytic parameters and quantitative immunological reactivities indicate that these enzymes must have some minor but distinct variations in their structures. Interestingly, it was observed that the Penicillium enzyme cross-reacted with the antiserum against the Aspergillus enzyme with an association constant of two orders of magnitude lower than that of the Aspergillus enzyme, and that the precipitin one of the Penicillium enzyme fused together with that of the Aspergillus enzyme in the immunodouble diffusion test. These results lead to the conclusion that these enzymes are closely related but not completely identical, and suggest that they might have evolved from a common ancestral precursor.

Corynebacterium sarcosine oxidase: A unique enzyme having covalently-bound and noncovalently-bound flavins

Abstract A sarcosine oxidase (sarcosine: oxygen oxidoreductase (demethylating), EC, 1.5.3.1) was purified to homogeneity from Corynebacterium sp. U-96 by the use of ionic exchange chromatographies and gel filtrations. The enzyme contained two mol FAD per mol enzyme (one covalently-bound and one noncovalently-bound; mol. wt., 174,000). The “semiapoenzyme”, which contains the covalently-bound FAD alone, was prepared by the acid-ammonium sulfate treatment. The semiapoenzyme had practically no activity for sarcosine oxidation, but retained intact back-bone structure judging from the circular dichroic spectrum in the far ultraviolet region. On the contrary, the circular dichroic spectrum of the semiapoenzyme in the visible region (a large negative band around 443 nm) was quite distinct from that of the holoenzyme (positive bands at 387, 456 and 489 nm).

Subcellular distribution of hepatic allantoinase varies among fishes

Abstract 1. 1. Intracellular and intraperoxisomal localization of hepatic allantoinase was examined by sucrose density gradient centrifugation with thirteen different fishes. 2. 2. On the basis of the localization of allantoinase, fishes were found to be classified into two groups. 3. 3. In group 1, allantoinase was located both in the peroxisomal soluble matrix and in the cytosol. In group 2, allantoinase was located only in the cytosol.

Steady-state kinetics and spectral properties of Corynebacterium sarcosine oxidase

The overall reaction kinetics of Corynebacterium sarcosine oxidase were investigated and the reaction was shown to follow a ping-pong, bi-bi mechanism with two substrates, sarcosine and molecular oxygen. Sarcosine analogs, such as acetate, propionate and methoxyacetate, were competitive inhibitors of the reaction. Acetate caused characteristic alterations in optical and circular dichroic spectra, indicating that the microenvironment of the substrate-binding region of the enzyme increased in hydrophobicity on binding with the substrate analog. The dissociation constants of the analogs calculated from the spectral changes were in agreement with the kinetic inhibition constants. Inorganic metallic ions were also inhibitory. Of interest was the finding that the inhibition by Hg2+ was proportional to the square of its concentration, which suggests that at least two sulfhydryl groups are related to the catalytic activity of the enzyme.

Response of hepatic alanine:glyoxylate aminotransferase 1 to hormone differs among mammalia

The subcellular distribution and substrate specificity of hepatic alanine:glyoxylate aminotransferase 1 have been reported to differ among mammalia. In the present study, the response of this enzyme to hormone (glucagon) was found to differ among mammalia.

Alanine:Glyoxylate aminotransferase 1 is present in the peroxisomes of guinea pig kidney

The subcellular distribution of alanine: glyoxylate aminotransferase 1 in guinea pig and rabbit kidneys was examined by centrifugation in a sucrose density gradient. The enzyme was located in the peroxisomes of guinea pig kidney and cross-reactive with the antibody against rat liver alanine: glyoxylate aminotransferase 1. This is the first report on the presence of the enzyme in the peroxisomes of mammalian kidney. The enzyme was found to be located in the mitochondria but not in the peroxisomes in rabbit kidney.

Heparinoid activities of depolymerized chondroitin polysulfates

Forty-one samples of sulfated chondroitin 6-sulfate (ChPS), differing in both the degree of sulfation and the number average molecular weight (5.65%–15.57% sulfur, Mn 600–73,800), were examined for their heparinoid activity in relation to the chemical properties of ChPS. Their anticoagulant activity was low and the highest activity was only 9.4 units/mg for the sample with 13.77% sulfur and Mn 27,100. On the other hand, their lipemia-clearing activity was very potent and comparable to that of heparin. The activity increased in proportion to the degree of sulfation (sulfur > 13.61%) and the optimum Mn range for the activity was 25,000–40,000. The nature of the clearing activity induced by injection of ChPS was also investigated and compared with that of heparin and dextran sulfate.

17. The role of NF-κB on osteoclast differentiation and tooth development

ス と p50 欠損 マ ウ ス を交配 し,野生型, aly , p50 − / 一,お よ び aly /p50 − / 一 マ ウ ス を作出 した.各 々 の マ ウ ス の ・軟 X 線撮影,骨密度測定お よ び組織学的検討を行 な っ た.・胸腺 およ び脾細胞 の T,B 細胞 の 分化を FACS で 解析 した.・脾 細胞を RANKL で刺激し , 破骨細胞を誘導し た . aly /p50−/一マ ウ ス は , 他の マ ウ ス と比較 して , ・著し い 成長障害 と 骨硬化症 を 呈 し た が,骨組織 に破骨細胞 は 存在 し た.歯 は 萌出 し た が,エ ナ メル 質形成不全 が 認 め られ た.・B 細胞 の 分化 が 抑制 され た.・RANKL 刺激 に よ る 破骨細胞形成 は 完全 に 抑制 され た.破骨細胞分化 に は p52 の 前駆体 p100 の プ ロ セ シ ン グ で は な く, p100 が 存在す る こ と が 重 . 要で あ る可能性が 示唆 され た. The role of NF一κ B on osteoclast differentiation and tooth development Eijiro Jimi, Wataru Masuda and Sueko Hayashi (Division of M61ecular Biochemistry, Kyushu Dental College) The transcription factor nuclear factor 一κ B (NF 一κB)participates in the expression of a wide variety of gerles that are involved in the regulation of immune and inflammatory responses , proliferation, tumorgenesis and survival . Gene targeting of p50 and p52 subunits of NF 一κ B have shown that NF一κ Bhas a critical role in osteoclast differentiation . However . the molecular mechanism by which NF .rcB