Carbohydrate metabolism by Pseudomonas fluorescens. III. Purification and properties of a 6-phosphogluconate dehydrase.
Carbohydrate metabolism by Pseudomonas fluorescens. III. Purification and properties of a 6-phosphogluconate dehydrase.
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荧光假单胞菌的碳水化合物代谢。
DOI：
10.1016/s0021-9258(18)98206-2
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发表时间：
1955
期刊：
影响因子：
--
通讯作者：
W. Wood
中科院分区：
文献类型：
--
作者：
R. Kovachevich;W. Wood
方法细菌学-P。 jluorescens，菌株 A3。 12，在室温下在先前描述的矿物培养基中生长（5）。一个或多个装有 14 升培养基的细口大瓶分别接种 1 升在 1% 酵母提取物、1% 胰蛋白胨、0.5% KzHP04 和 0.5% 葡萄糖中生长 16 小时的培养物。通过以 1725 rpm 搅拌并迫使每分钟 1 至 2 体积的空气通过培养物的装置实现剧烈通气。 7小时后，在Sharples离心机中收获细胞，洗涤并在真空中或用丙酮干燥。 2至2.5克。获得每升培养基的细胞干重。 通过改进 Robison 和 King (7) 的程序，由结晶葡萄糖-6-磷酸钡七水合物 (6) 制备化学 6-磷酸葡萄糖酸盐，其中溴氧化在不存在钡离子的情况下进行。 1-U4-6-磷酸葡萄糖酸由 BL Horecker 博士制备 (8)。谷胱甘肽钠 (NaGSH) 购自 Schwarz Laboratories, Inc.。最终产物（推测为 KDPG）是通过 6-PG 脱水酶的作用从 6-PG 制备的，并通过醇沉淀从反应混合物中以单钡盐形式回收。结晶 Nas-2-酮-3-脱氧-6-磷酸葡萄糖酸酯。 2H20 由 M. Doudoroff 博士捐赠。三磷酸吡啶核苷酸，纯度为 80%，购自 Sigma Chemical Company。 DL-甘油醛-3-磷酸(G-3-P)由得自Concord Laboratories的间甘油醛-1-溴化-3-磷酸(二聚体)通过用氢氧化钠中和来制备。测定丙酮酸通过直接萃取法或Friedemann 和Haugen 的双萃取法测定(9)。 Sibley 和 Lehninger (lo) 在醛缩酶测定中没有采用肼和碱预处理的情况下，3-磷酸甘油醛不会显色。 KDPG形成不溶于极性溶剂的2,4-二硝基苯腙。在碱存在下，未提取的 2, 4-二硝基苯腙产生的颜色在不到 5 分钟内褪色为零。因此中间体磷酸酯和3-磷酸甘油醛不会干扰丙酮酸的测定。还在乳酸脱氢酶和还原二磷酸吡啶核苷酸(DPNH)存在下根据340rn/*的光密度降低来估计丙酮酸。 6-磷酸葡萄糖酸通过 Horecker 和 Smyrniotis (11) 的方法用纯化的酵母 6-PG 脱氢酶进行分光光度测定。 KDPG n-as det，通过硫酸高铈降解 (12) 或通过 KDPG 醛缩酶 (13) 转化为丙酮酸来确定，然后进行分光光度或量热丙酮酸测定。碳 14 浓度测定为 BaCY403，如所述
MethodsBacteriological-P. jluorescens, strain A3. 12, was grown at room temperature in a mineral medium described previously (5). One or more carboys cont’aining 14 liters of medium were each inoculated with 1 liter of a 16 hour culture grown in 1 per cent yeast extract, 1 per cent tryptone, 0.5 per cent KzHP04, and 0.5 per cent glucose. Vigorous aeration was attained by an arrangement which stirred at 1725 rpm and forced 1 to 2 volumes of air per minute through the culture. After 7 hours the cells were harvested in a Sharples centrifuge, washed, and dried either in vacua or with acetone. 2 to 2.5 gm. dry weight of cells per liter of medium were obtained.Chemical-6-Phosphogluconate was prepared from crystalline barium glucose-6-phosphat> e heptahydrate (6) by a modification of the procedure of Robison and King (7) in which the bromine oxidation was carried out in the absence of barium ions. 1-U4-6-Phosphogluconate was prepared by Dr. BL Horecker (8). Sodium glutathione (NaGSH) was obtained from t, he Schwarz Laboratories, Inc. The end-product, presumed to be KDPG, was prepared from 6-PG by the action of 6-PG dehydrase and recovered from t’he reaction mixture as the monobarium salt by alcohol precipitat’ion. Crystalline Nas-2-keto-3-deoxy-6-phosphogluconate. 2H20 was donated by Dr. M. Doudoroff. Triphosphopyridine nucleotide, 80 per cent pure, was obtained from the Sigma Chemical Company. DL-Glyceraldehyde-3-phosphate(G-3-P) was prepared from m-glyceraldehyde-1-bromide-3-phosphoric acid (dimeric) obtained from the Concord Laboratories by neutralization with sodium hydroxide. DeterminationsPyruvate was determined either by the direct or by the double extraction methods of Friedemann and Haugen (9). In the absence of both hydrazine and the alkali pretreatment which are employed in aldolase determinations by Sibley and Lehninger (lo), glyceraldehyde-3-phosphate did not give a color. KDPG formed a 2, 4-dinitrophenylhydrazone which was not soluble in polar solvents. In the presence of alkali the color produced by the unextracted 2, 4-dinitrophenylhydrazone faded to nil in less than 5 minutes. Thus the intermediate phosphate ester and glyceraldehyde-3-phosphate did not int’erfere with the pyruvate determinations. Pyruvate also was estimated in the presence of lactic dehydrogenase and reduced diphosphopyridine nucleotide (DPNH) from the decrease in optical density at 340 rn/*. 6-Phosphogluconate was determined spectrophotometrically by the method of Horecker and Smyrniotis (11) with purified yeast 6-PG dehydrogenase. KDPG n-as det, ermined by ceric sulfate degradation (12) or by conversion to pyruvate by KDPG aldolase (13), followed by spectrophotometric or calorimetric pyruvate determinations. Carbon14 concentrations were determined as BaCY403, as described