A peptidohydrolase from mammalian fibroblasts (bovine dental pulp).
A peptidohydrolase from mammalian fibroblasts (bovine dental pulp).
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来自哺乳动物成纤维细胞（牛牙髓）的肽水解酶。
DOI：
10.1021/bi00865a021
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发表时间：
1966
期刊：
影响因子：
2.9
通讯作者：
G. Kalnitsky
中科院分区：
文献类型：
--
作者：
C. Schwabe;G. Kalnitsky
材料与方法组织来源与制备.与爱荷华州锡达拉皮兹的Wilson Packing公司合作获得牛牙髓（100 kg湿重）。如前所述（施瓦贝和Kalnitsky，1965），将纸浆提取、冻干并降解成60目粉末。这种粉末在-20 ℃下不定期稳定，是一种方便的原料，称为纸浆粉。柱色谱材料。将DEAE-纤维素（Schleicher & Schiill）悬浮于0.1n NaOH中，搅拌30分钟，用蒸馏水洗涤除去碱，并悬浮于所需缓冲液中。在每个10分钟的沉降期后，通过四次倾析除去细颗粒。羟基磷灰石是按照Jenkins（1962）的方法制备的。磷酸钙沉淀的安排（施瓦贝，1965）已证明对我们有利。该装置允许自动控制两种试剂的相对添加速率以及从恒定体积的水中沉淀。Dowex 50-X2（Dow Chemical Co.）如Schroeder等人所述制备。将Bio-Gel（Calbiochem）悬浮于0.1N乙酸中，并在洗脱缓冲液中于4 ℃下溶胀过夜。用0.1 N乙酸填充二甲基二氯硅烷涂覆的柱。然后将柱的顶部安装有广口漏斗，并引入足够的Bio-Gel悬浮液以在流动下填充柱。这需要大约5-6小时。床层沉降后，将0.1 n乙酸从LKB微型流动双注射泵以6或12 ml/小时的速率泵送通过柱数小时，或在分子量实验中泵送2天。
Materials and Methods Tissue Source andPreparation. Bovine dental pulp (100 kg wet weight) was obtained in cooperation with the Wilson Packing Co. of Cedar Rapids, Iowa. The pulps were extracted, lyophilized, and degraded to a 60-mesh powder as previously described (Schwabe and Kalnitsky, 1965). This powder, which is stable for indefinite periods at—20 and constitutes a con-venient starting material, is referred to as pulp powder. Materials for Column Chromatography. DEAE-cellulose (Schleicher & Schiill) was suspended in 0.1 n NaOH, stirred for 30 min, washed free from alkali with distilled water, and suspended in the desired buffer. Fine particles were removed by four decantations after each 10-min settling period. Hydroxylapatite was prepared following the pro-cedure of Jenkins (1962). The arrangement for the pre-cipitation of calcium phosphate (Schwabe, 1965) has proved to be advantageous in our hands. The apparatus permits automatic control of the relative rates of addi-tion of the two reagents as well as precipitation from a constant volume of water. Dowex 50-X2 (Dow Chemical Co.) was prepared as described by Schroeder et al.(1962).Bio-Gel (Calbiochem) was suspended in 0.1 n acetic acid and left to swell overnight at 4 in the eluting buffer. A dimethyldichlorosilane-coated column was filled with 0.1 n acetic acid. The top of the column was then fitted witn a wide-mouth funnel and enough Bio-Gel suspension introduced to fill the column under flow. This took about 5-6 hr. After the bed had settled, 0.1 n acetic acid was pumped through the column at a rate of either 6 or 12 ml/hr from an LKB miniflow dual-syringe pump for several hours, or in the molecular weight experiment, for 2 days.