Purification of lysosomal phospholipase A and demonstration of proteins that inhibit phospholipase A in a lysosomal fraction from rat kidney cortex.
Purification of lysosomal phospholipase A and demonstration of proteins that inhibit phospholipase A in a lysosomal fraction from rat kidney cortex.
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溶酶体磷脂酶 A 的纯化以及大鼠肾皮质溶酶体部分中抑制磷脂酶 A 的蛋白质的演示。
DOI：
10.1021/bi00369a017
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Giordano,JR
中科院分区：
文献类型：
--
作者：
Hostetler,KY;Gardner,MF;Giordano,JR
材料和方法化学品。1，2-二[1 - 14 C]油酰磷脂酰胆碱，1，2-二[1- 14 C]棕榈酰磷脂酰胆碱，2-[1- 14 C]油酰-f这项工作得到了NIH赠款AM 32159和GM 24979以及退伍军人管理局医学中心研究服务处（拉霍亚，CA）的部分支持。在退伍军人管理局医疗中心与作者通信。在这项工作的过程中，他是退伍军人管理局的临床研究员。磷脂酰胆碱和[1 - 14 C]棕榈酰溶血磷脂酰胆碱得自阿默舍姆，阿灵顿海茨，IL。以下购自Sigma Chemical Co，St. Louis，MO：兔丙种球蛋白、Sephadex G-150、氟磷酸二异丙酯、二油酰磷脂酰胆碱、二棕榈酰磷脂酰胆碱、三（羟甲基氨基）甲烷、甲基α-甘露糖苷、EDTA、对溴苯甲酰甲基溴和1-棕榈酰溶血磷脂酰胆碱。Bio-Gel HTP（羟基磷灰石）和考马斯亮蓝蛋白测定试剂盒购自BioRad，里士满，CA。Superose 12 HPLC柱、PBE 94交换剂和Poly-buffer 74购自Pharmacia Fine Chemicals，皮斯卡特维，NJ。溶酶体磷脂酶A的分离和增溶。将18 - 30只Fischer 344品系大鼠禁食过夜，并通过颈部骨折处死，取出肾脏并置于由0.25 M蔗糖、5 mM Tris（pH 7.4）和2 mM EDTA组成的冰缓冲液A中。切除组织，称重，并在冰冷的缓冲液中冲洗。将重18-40 g的肾皮质切成小块，并如前所述制备缓冲液A中的5%匀浆（Hostetler & Hall，1982）。使匀浆通过四层粗棉布，并以160 g离心6 min。将沉淀物重悬于缓冲液A中，并以160 g离心6 min;重复洗涤三次，并将相应的上清液与原始的核后上清液合并。弃去核沉淀，合并上清液，以20000 g离心20 min。将沉淀加入70-80 mL含有10 mM磷酸钠缓冲液（pH 7.2）和50 mM NaCl的缓冲液中，并进行5次冷冻循环，然后解冻。将所得悬浮液以20000 g X 60 min离心以沉淀膜状物质，将其弃去。用0.25体积的冷甘油稀释含有可溶性蛋白的上清液，并如所述进一步纯化该材料。测定蛋白
Materials and Methods Chemicals. l, 2-Di [l-14C] oleoylphosphatidylcholine, 1, 2-di [1-14C] palmitoylphosphatidylcholine, 2-[1-14C] oleoyl-f This work was supported in part by NIH Grants AM 32159 and GM 24979 and by the Research Service of the Veterans Administration Medical Center, La Jolla, CA.* Address correspondence to this author at the Veterans Administration Medical Center. During the course of this work he was a Clinical Investigator of the Veterans Administration. phosphatidylcholine, and [l-14C] palmitoyllysophosphatidylcholine were obtained from Amersham, Arlington Heights, IL. The following were purchased from Sigma Chemical Co, St. Louis, MO: rabbit gamma globulin, Sephadex G-150, diisopropyl flurophosphate, dioleoylphosphatidylcholine, di-palmitoylphosphatidylcholine, tris (hydroxymethylamino)-methane, methyl a-mannoside, EDTA, p-bromophenacyl bromide, and 1-palmitoyllysophosphatidylcholine. Bio-Gel HTP (hydroxyapatite) and the Coomassie brilliant blue protein assay kit, were purchased from BioRad, Richmond, CA. Superose 12 HPLC column, PBE 94 exchanger, and Poly-buffer 74 were obtained from Pharmacia Fine Chemicals, Piscataway, NJ. Isolation and Solubilization of Lysosomal Phospholipase A. Eighteen to thirty rats of the Fischer 344 strain were fasted overnight and killed by cervical fracture, and the kidneys were removed and placed in iced buffer A consisting of 0.25 M sucrose, 5 mM Tris (pH 7.4), and 2 mM EDTA. The tissue was excised, weighted, and rinsed in ice-cold buffer. Kidney cortex weighing 18-40 g was cut into small pieces, and a 5% homogenate inbuffer A was prepared as previously described (Hostetler & Hall, 1982). The homogenate was passed through four layers of cheesecloth and centrifuged at 160g for 6 min. The pellet was resuspended in buffer A and recen-trifuged at 160g for 6 min; this washing was repeated three times, and the respective supernatants were combined with the original postnuclear supernatant. The nuclear pellet was discarded, and the combined supernatants were centrifuged 20000g for 20 min. The pellet was taken up 70-80 mL of buffer containing 10 mM sodium phosphate buffer, pH 7.2, and 50 mM NaCl and subjected to five cycles of freezing followed by thawing. The resulting suspension was centrifuged at 20000g X 60 min to sediment membranous material, which was discarded. The supernatant containing the soluble proteins was diluted with 0.25 volumes of cold glycerol, and this ma-terial was purified further as noted. Protein was measured
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影响因子：
7.3
作者：
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影响因子：
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作者：
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DOI：
10.1016/0005-2760(80)90151-4
发表时间：
1980
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Matsuzawa,Y;Hostetler,KY
通讯作者：
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DOI：
--
发表时间：
1982
期刊：
The Journal of pharmacology and experimental therapeutics
影响因子：
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作者：
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通讯作者：
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海胆组蛋白 mRNA 末端位于假定调控序列下游的基因区域
影响因子：
64.8
作者：
C. Hentschel;J. Irminger;P. Bucher;M. Birnstiel
通讯作者：
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