Kantaro Suzuki

Enzyme Patterns of Glycolysis in Rabbit Gingiva and Effects of pH on the Patterns

The specific activities of individual enzymes associated with glycolysis in rabbit gingiva were systematically evaluated, and the enzyme patterns at pH 7.4 were established on the glycolytic and gluconeogenic pathways. As a result, it was suggested that there was no capability for gluconeogenesis. The effects of alkaline pH values on the enzyme patterns appeared to lead to the overall stimulation of glycolysis.

Characterization of a 28-kDa collagenous protein extracted with EDTA from adult rabbit alveolar bone

Bone proteins in mandibular alveolar bone from young adult rabbits (3-month-old) were extracted with 4 M guanidine hydrochloride (GuHCl), followed by 0.5M ethylenediaminetetraacetic acid (EDTA). The proteins in the EDTA extract were fractionated on Sepharose CL-6B in the presence of 4 M GuHCl, followed by HPLC using hydroxyapatite and then ‘Mono Q’ resin in the presence of 7 M urea, and a 28 kDa protein was isolated. The purified 28-kDa protein showed intense staining with silver following SDS-PAGE separation under reducing conditions. When the protein was digested with bacterial collagenase, a 19-kDa fragment was produced. However, the protein was not susceptible to cyanogen bromide. On SDS-PAGE under non-reducing conditions, the protein migrated as two bands; a new band at about 85-kDa, and the original 28-kDa band. The amino acid composition of the protein was similar to that of α1-pN-propeptide of type I procollagen from other tissues. Moreover, the characteristics of the purified 28-kDa protein were similar to those of a 28-kDa protein synthesized by rabbit alveolar bone-derived cellsin vitro.

Properties of malate dehydrogenase isozymes in rabbit dental pulp.

Delbruck et al. (Biochem Z 331:273, 1959) first demonstrated a multiplicity of malate dehydrogenases (MDH:EC 1.1.1.37) in tissues. Thorne et al. (Biochim Biophys Acta 42:175, 1962) isolated and characterized mitochondria and supernatant MDH (M-MDH and S-MDH) from pig or beef heart. However, the isozymes of MDH have not been reported in dental pulp. In this annotation, it is shown that MDH in rabbit dental pulp can be separated into three anionic isozymes and one cationic isozyme by electrophoresis. The enzymatic properties of each isozyme are described. Dental pulp (wet weight 500 mg) from molars of albino rabbits (weight approx. 2 kg) was homogenized in 5 ml of 0.25 M sucrose with a Teflon homogenizer. The mitochondria and supernatant fractions were obtained by the method of Hogeboom et al. (J Biol Chem 172:619, 1948). Each fraction was applied on starch-gel § (1 x 1 x 2 5 cm) previously equilibrated with 0.1 M veronal buffer, pH 8.6. After 16 hours of electrophoresis (conditions: 8 V/cm, 4 mA at 40C), the gel was cut into blocks (1 x 1 x 0.5 cm). Then each block was extracted with 5 in! of 0.15 M KCl during two hours of occasional stirring. The incubation assay mixture for MDH contained 20 pmole phosphate buffer, pH 7.6; 0.4 ,umole oxalacetate; and 0.08,mole sodium NADH, in 0.4 ml. The reaction was carried out at 370C for 30 min and terminated by the addition of 0.4 ml of 10% perchloric acid. After 15 min from the termination, 2.0 ml of 10 N NaOH was added to be reincubated for one h at 370C. The fluorescence originating from the NAD formed was determined by a fluorescence spectrophotometert

Characteristics of Human Alveolar Bone-Derived Cells. III. Alteration of Phosphatase Activities during Bone Nodule Formation.

ヒトの歯槽骨片を細菌性コラゲナーゼ処理した後に, 骨片から増殖してくる細胞群 (E-AB) のin vitroでの石灰化物形成, ならびにアルカリホスファターゼおよび酸性ホスファターゼ活性を, 42日間にわたって経日的に調べた。細胞は, 培養3日目から対数増殖期に入り, 10日目から14日目には定常期に達する傾向を示したが, 14日目以降も42日目まで次第に細胞数は増加した。noduleの形成は, 培養14日目頃に始まり, 28日目にはアリザリン赤にわずかに染色されることが肉眼でも観察され, 石灰化の開始時期であることを示した。培養42日目には, 形成されたすべてのnoduleがアリザリン赤に濃染し, 石灰化したことを示した。細胞のアルカリホスファターゼ活性はnoduleの形成が開始する培養14日目に最も高くなり, 石灰化開始期と思われる28日目にはその50%まで低下していた。酸性ホスファターゼ活性値は, アルカリホスファターゼ活性値に比べて約1/10程度の低い値であり, 培養日数の増加とともに若干の減少傾向を示した。

Characteristics of Human Alveolar Bone Derived Cells. II. Determination of Protein Synthesis by 14C-Proline Labeling.

ヒトの歯槽骨由来の骨芽細胞様細胞の特性を調べるために, 細菌性コラゲナーゼで処理した歯槽骨片から増殖してくる細胞群 (E-AB) と無処理の骨片から増殖してくる細胞群 (N-AB) とに分けて培養し, 両細胞群のタンパク質合成を14C-prolineの取込みで比較検討した。両細胞群の培養上清に分泌するタンパク質に占めるコラーゲン性タンパク質の割合は, E-ABで12.5%, N-ABで10.4%と異なり, その結果放射能 (14C) の取込量はE-ABの方がN-ABよりわずかに高い値を示したが, 両細胞群の全タンパク質合成能には差異を認めなかった。両細胞群の分泌タンパク質をコラゲナーゼあるいは臭化シアンで処理したものをSDS-ポリアクリルアミドゲル電気泳動で分離して比較すると, 両細胞群が合成する各タンパク質の量的比率には相違はあるが, 種類は類似していた。また, 両細胞群ともにI型コラーゲンの他に少量のIII型コラーゲンを分泌し, 型別合成比率もほぼ同程度であった。索