CN1037154A - 肾生长促进剂及其生产方法 - Google Patents
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Abstract
本发明涉及一种肾生长促进剂,它基于促黄体激
素的一种异构体具有促进肾生长的作用这样一个发
现。
以前从未了解到促黄体激素或其异构体具有促
进肾生长的作用。
本发明预期患有肾细胞数量减少或肾功能减弱
的肾脏可通过服用所述促黄体激素异构体而得以活
化。
Description
本发明涉及肾生长促进剂及其生产方法。
更具体地说,本发明涉及以具有促进肾生长活性的促黄体激素异构体作为活性成分的肾生长促进剂及其生产方法。
本发明是基于这样一个发现,即促黄体激素的一种异构体具有促进肾生长的作用。因此预计服用本发明的肾生长促进剂能增加肾细胞的数目或增强肾功能,从而活化患有肾细胞减少或肾功能减弱的肾脏,这大体上属于医药领域。
以前从未了解到促黄体激素或其异构体具有促进肾生长的作用。
本发明人注意到切除垂体后的小鼠肾脏重量明显降低这样一个事实,认为垂体中可能有某种控制肾生长的因子。因此他们试图找到一种存在于垂体中的肾生长因子。结果从垂体中分离出一种促进肾生长的激素样成分,并将其命名为RGF(或促肾素(renotropin))(参见Nippon Rinsho,44(1),separate issue,84-88(1986年1月))。
图1显示了实例1中由粗制肾生长因子的等电聚焦得到的各组分的蛋白含量。
但是,发现如此得到的RGF是一种粗产物,其活性不稳定并含有具有拮抗作用的各种污染物。
为获得单一成分的RGF,本发明人进一步进行了研究。结果意外地发现,根据等电点的不同对上面分离出的粗制RGF进行分级分离和纯化,可得到活性成分的单一条带。
分析这个单一条带并测定其一级结构,令人惊奇地揭示出,它是促黄体激素的一种异构体。
已知促黄体激素并不是单一化合物,而是由几种异构体构成的糖蛋白。这是由它的微结构多态性造成的。
据报道,羊促黄体激素的α亚基具有不均一的氨基末端顺序,β亚基具有不均一的羧基末端顺序(D.N.Ward,Int.J.Peptide Protein Res.,27,70-78(1986))。又据报道,人促黄体激素在进行柱电聚焦时分出7个峰(参见M.Yano,J.Jap.Tocol.Gynecol.,37(5),703-712(1985))。
当然,以前从未了解到促黄体激素及其各种异构体会具有促进肾生长的作用。
本发明人随后所作的一些研究揭示出,有一种促黄体激素异构体的亚基α3与另一亚基β3结合,这个异构体可达到最高的促进肾生长活性。
基于这些发现完成了本发明。本发明涉及一种以促黄体激素异构体作为活性成分的肾生长促进剂。所述促黄体激素异构体最好由亚基α3与另一亚基β3结合而构成。
本发明还提供一种生产具有促进肾生长活性的促黄体激素异构体的方法,它包括根据等电点的不同对粗制肾生长因子进行分级分离和纯化。
这里所述的根据等电点不同进行分级分离和纯化处理,是指这样一种处理方法,即,使两性电解质如蛋白质按每种物质的固有等电点而彼此分级分离,然后分别收集。这可以利用例如层析聚焦或电聚焦来进行。
用作本发明起始原料的粗制肾生长因子,可以是从哺乳动物如小鼠、羊或猪的尿、血液或器官得到的促黄体激素粗提液。此外,也可以用从通过遗传工程人工制备的培养系统中得到的粗产物。也可以用从合成化学反应系统得到的物质。无论怎样,都是用一种具有促进肾生长作用的促黄体激素异构体与不具有这种作用的异构体的混合物作起始原料。
例如,用下列方法从器官中得到粗制肾生长因子。将硫酸铵水溶液加到垂体中,所得混合物匀浆后离心。纯化上清液并冰冻干燥。然后将冰冻干燥物溶解并用一系列处理方法进行纯化而得到粗制肾生长因子(RGF)。处理方法有SP Sephadex层析、Sephadex G100层析、CM纤维素层析、伴刀豆球蛋白A层析和Sephadex G100层析。
根据等电点不同对如此得到的粗制肾生长因子(RGF)进行分级分离和纯化,从而得到单一条带的具有促进肾生长活性的物质。
可以将如此得到的单一条带的具有促进肾生长活性的物质分成组成亚基α和β。当然,将该物质分成这两个亚基后,肾生长促进活性将消失。
分离操作可以利用例如反相液相层析来进行。在分离图谱中,取决于氨基酸链的长度,亚基α和β各显示出若干个峰。已证实这些峰相应于通过切割亚基α和β而得到的几个较短的氨基酸链,这几条链先前已有报道。
尽管亚基α3与另一亚基β3结合的物质略有一些促黄体激素的活性,但它不同于促黄体激素的其它异构体,即它具有显著的肾生长促进活性。
亚基α3和β3是承受最少切割的亚基。在亚基彼此重新结合的各种亚基结合中,具有亚基α3和β3的结合具有最高的促进肾生长活性。
下面将给出本发明的实施例。
实例1
将10mM苯甲磺酰氯加到约350克猪垂体和1升0.15M硫酸铵水溶液中。所得混合物用Waring捣碎器匀浆两分钟,同时加以冷却。将pH值调至4.0,然后将匀浆后的混合物在冷却下放置足够长的时间。然后将其离心并收集上清液。再用0.5M偏磷酸水溶液将pH值调至3.0,然后离心所得溶液并收集上清液。将上清液的pH值调至6.5至7.0,向其中加入硫酸铵至达到50%饱和。然后再将混合物在冷却下放置足够长的时间,然后离心。收集沉淀物并悬浮于50-100毫升0.2M磷酸氢二钾溶液中。将悬浮液移至透析管中,在冷却下充分透析。然后取出内容物,再将其pH值调至8.5。将所得物质冰冻干燥。
将所得的冻干粉溶于0.01M磷酸氢二钠溶液中,加到用上述缓冲液平衡的SP-Sephadex(Pharmacia公司出品)柱上。收集用0.1M磷酸氢二钠洗脱出的各组分并进行冰冻干燥。
将冻干粉溶于少量0.05M碳酸氢铵溶液中,利用Sephadex G-100(Pharmacia公司出品)柱通过分子筛进行分级分离,并采用同样的缓冲液系统。收集分子量约为40,000的组分并进行冰冻干燥。
将所得干粉溶于少量10mM Tris-HCl(pH7.5)和0.3M氯化钠的溶液中,然后利用伴刀豆球蛋白A-Sepharose(Pharmacia公司出品)柱进行分级分离,并采用同样的缓冲液系统。收集用0.2M甲基甘露糖苷洗脱出的各组分,冷却下充分透析后进行冰冻干燥,即得约25毫克粗制肾生长因子(RGF)。
接着,利用LKB等电聚焦柱(LKB Produkter AB,Bromma,Sweden出品;容量:100毫升),根据等电点不同对上述粗产物进行分级分离和纯化。所用的载体两性电解质为安福灵pH3.5-10(LKB出品)、安福灵pH9-11(LKB出品)和安福灵pH7-9(LKB出品)。制备出梨醇密度梯度(5-50%),将上述粗产物溶于2毫升重溶液中并置于柱中央(重梯度溶液:山梨醇27克、蒸馏水34.9毫升、安福灵(pH9-11)2.1毫升;轻梯度溶液:山梨醇2.7克、蒸馏水52.3毫升、安福灵(pH9-11)0.3毫升、安福灵(pH3.5-10)0.2毫升、安福灵(pH7-9)0.2毫升)。用1M氢氧化钠溶液和0.01M乙酸溶液作电极液。在4℃下施加800-1500伏的电流24小时。通电后,用部分收集器以1.5毫升为一份收集物料。然后在4℃下立即测定每个组分的pH值(COM-11:Denki Kagaku Kogyo K.K.出品,CE 105 C型电极;标准缓冲液:Wako Pure Chemicals Co.,Inc.出品)。同时测定每个组分的蛋白含量。图1显示了层析图谱。
结果,从约1.5毫克粗制肾生长因子中分别得到104微克、281微克、415微克和103微克pI大于10.32的组分、pI10.32-10.15的组分、pI10.15-9.89的组分和pI小于9.89的组分。
将各组分在冷却下充分透析,从中除去安福灵和山梨醇,然后冰冻干燥得到干粉。
在上面所用的相同条件下重复等电聚焦,将粗制肾生长因子纯化若干次。然后将pI范围相同的组分合并在一起。用pI为7.97的马心肌球蛋白(Ⅲ型:Sigma化学公司出品)作标准蛋白。
肾生长促进活性的评价:
确定上面所得的各组分中哪个组分可促进肾生长。
借助对肾细胞DNA合成作用促进的测定来测定促肾素的肾生长促进作用。
取雄性CD大鼠(得自Nippon Charles River Co.,同一天出生,平均体重:100克)进行垂体切除和阉割,然后饲养9天。确认手术进行得是否得当,然后将大鼠分成若干试验组和一个对对照组,每组至少由5只动物组成。
由上述等电聚焦分离出的各组分各取50微克溶于150微升含0.1%BSA的50mM硼酸缓冲液(pH7.5)中,对每只大鼠进行皮下注射。8小时后去头处死动物并放血,然后取出右肾和左肾。去掉被膜后,每个肾沿皮质髓质轴(corticomedullary axis)切成两片。于37℃下将这些肾片置于2毫升Krebs-Ringer碳酸氢盐缓冲液(pH7.5)中保温2小时。该缓冲液预先通以95%氧和5%二氧化碳的混合气并而后加入2微居/毫升1,2-甲基3H-胸腺嘧啶(New England Nuclear Corp.出品)。保温结束后,用4mM胸腺嘧啶溶液冲洗肾片并于-20℃下储存。
向储存的肾片中加入4毫升蒸馏水,所得混合物用超分散混合器(Yamato Kagaku K.K.出品)匀浆。然后按A.Fleck和H.M.Munro建议的方法(参见Biochim.Biophys.Acta,55,571(1962))提取其中所含的DNA。按J.R.W.Wannemacher、J.W.L.Banks和W.H.Wunner报道的方法(Anal.Biochem.,11,320(1965))水解所提取的DNA,然后用牛胸腺DNA(I型,Sigma化学公司出品)作标准DNA进行测定。同时用常规方法测定0.5毫升所提取DNA的放射性。
利用放射性/总DNA含量来测定肾细胞的DNA合成促进活性,每组实验都以百分比表示,而将对照组的百分比定义为100%。结果表明,pI大于10.32组分的DNA合成促进活性为109%,pI10.32-10.15组分为130%(图1中的粗箭头),蛋白含量最高的pI10.15-9.89组分没有DNA合成促进作用(98%),pI小于9.89组分为115%(图1中的细箭头)。就是说,pI10.32-10.15组分和pI小于9.89(9.89-9.32)组分含有肾生长促进成分。此外,pI大于10.32组分也含有肾生长促进成分,但其活性很小。
同时测定同一批粗制肾生长因子的DNA合成促进作用。所观察到的活性为112%。
肾生长促进成分在体内对肾生长的影响:
取雄性CD-1小鼠(得自Nippon Charles River Co.,同一天出生,平均体重:17克)进行垂体切除和阉割,然后饲养9天。确认手术进行得是否得当,然后将小鼠分成一个试验组和一个对照组,每组都由10只动物组成。将前面由等电聚焦得到的40微克pI10.32-10.15组分溶于150微升含0.1%BSA溶液的50mM硼酸缓冲液(pH7.5)中。用同样方法处理另外几份40微克pI10.32-10.15组分。用所得溶液以150微升/天的剂量对每只动物连续皮下注射5天。于第6天去头处死动物并放血,然后取出右肾和左肾。去掉被膜后,将这两个肾冰冻干燥。计算出干肾重与大鼠终体重之比,考察实验组和对照组肾重之差。结果表明,试验组肾重增加110%,说明所用的组分具有肾生长促进作用。肾生长促进成分的亚基α和β的分离和再结合:
取利用等电聚焦分离的pI10.32-10.15组分进行高效液相层析并收集所分离的亚基。将收集物溶于少量0.1%三氟乙酸(TFA)水溶液中,然后用反相柱(Baker Bond宽孔丁基C4:Baker Research Products出品)以线性梯度(10%-50%)进行分离。用含0.1%TFA的2-丙醇/乙腈(7∶3)作溶剂。
所得到的亚基α和β各为三个主要峰,分别以洗脱顺序命名为α1、α2、α3和β1、β2、β3。用3毫克样品进行分离得到230微克α1、450微克α2、350微克α3、410微克β1570微克β2和60微克β3。氨基酸组成分析等结果发现,α3和β3为分解程度最低的促黄体激素异构体。
将等摩尔量的α1和β1、α2和β2、α3和β3混合在一起。于37℃下,将所得的每个混合物保持在少量1%碳酸铵水溶液中,试图以此使α亚基与β亚基结合。接着用TSK凝胶G3000 SW柱(Toyo Soda Mfg.Co.,Ltd.出品)使混合物脱盐,然后冰冻干燥。将这些样品的活性与建立在上述DNA合成促进作用基础上的另一个样品的活性作比较。结果表明,α3/β3的结合具有最高活性,即与对照组之差约为α1/β1结合的2.5倍。
实例2
将100克羊垂体粉末(Waitaki NZ Refrigerating Ltd.出品)悬浮于400毫升冷水中,然后向其中加入1.5升0.15M硫酸铵溶液。向其中加入10mM苯甲磺酰氯,所得混合物用Polytron在冷却下匀浆。将pH值调至4.0,然后在冷却下将匀浆后的混合物放置足够长的时间,然后离心。收集上清液并用与实例1所述相同的方法进行处理。然后用SP-Sephadex柱、Sephadex G-100柱和伴刀豆球蛋白A-Sepharose柱进行处理,从而得到粗制肾生长因子。
如实例1所述,再用等电聚焦分级分离5毫克粗制肾生长因子。结果得到一个pI大于9.85的组分、一个pI9.85-9.60的组分、一个pI9.60-9.10的组分、一个pI9.10-8.60的组分、一个pI8.60-7.30的组分及一个pI小于7.30的组分。如实例1所述进行同样的生物测定,结果证实pI大于9.85的组分含有肾生长促进成分。
实例3
离心750毫升人垂体提取液(KabiVitrun AB出品)。收集所得沉淀物并悬浮于70毫升0.2M磷酸氢二钾水溶液中。所得悬浮液于60℃加热3分钟,然后在冷却下透析两天。取出内容物,向其中加入0.13M硫酸铵。将pH值调至4.0后,再用0.5M偏磷酸溶液将混合液的pH值调至3.0。然后将混合物在冷却下放置,离心。收集上清液,在冷却下充分透析后冰冻干燥。
接着,用与实例1所述相同的方法用SP-Sephadex柱和Sephadex G-100柱进行处理,从而得到促黄体激素的粗提液。
用伴刀豆球蛋白A-Sephadex柱处理这个促黄体激素粗提液,得到粗制肾生长因子。
所得粗制肾生长因子利用等电聚焦进行分级分离。得到的每个组分如实例1所述进行同样的生物测定。结果证实pI9.05-9.90的组分含有肾生长促进成分。
Claims (2)
1、一种肾生长促进剂,它包含促黄体激素或其异构体作为活性成分。
2、一种生产具有肾生长促进活性的促黄体激素异构体的方法,该方法包括根据等电点的不同对粗制肾生长因子进行分级分离和纯化。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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JP62095277A JPS63264530A (ja) | 1987-04-20 | 1987-04-20 | 腎成長促進剤及びその製造法 |
PCJP88/00385 | 1988-04-19 | ||
PCT/JP1988/000385 WO1988008307A1 (en) | 1987-04-20 | 1988-04-19 | Kidney growth accelerator and process for its preparation |
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Publication Number | Publication Date |
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CN1037154A true CN1037154A (zh) | 1989-11-15 |
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CN88107233A Pending CN1037154A (zh) | 1987-04-20 | 1988-10-18 | 肾生长促进剂及其生产方法 |
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EP (1) | EP0373218B1 (zh) |
JP (1) | JPS63264530A (zh) |
KR (1) | KR890015750A (zh) |
CN (1) | CN1037154A (zh) |
DE (1) | DE3881396T2 (zh) |
WO (1) | WO1988008307A1 (zh) |
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EP0348517A4 (en) * | 1987-12-03 | 1991-04-03 | The Calpis Food Industry Co., Ltd. | Kidney growth accelerator |
JPH0995455A (ja) * | 1995-09-29 | 1997-04-08 | Sumitomo Pharmaceut Co Ltd | 腎機能改善剤 |
Family Cites Families (1)
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JPS5743696A (en) * | 1980-08-27 | 1982-03-11 | Hayashibara Biochem Lab Inc | Preparation of human luteinizing hormone |
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1987
- 1987-04-20 JP JP62095277A patent/JPS63264530A/ja active Granted
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1988
- 1988-04-19 EP EP88903384A patent/EP0373218B1/en not_active Expired - Lifetime
- 1988-04-19 DE DE88903384T patent/DE3881396T2/de not_active Expired - Lifetime
- 1988-04-19 WO PCT/JP1988/000385 patent/WO1988008307A1/ja active IP Right Grant
- 1988-10-18 CN CN88107233A patent/CN1037154A/zh active Pending
- 1988-10-19 KR KR1019880013617A patent/KR890015750A/ko not_active Application Discontinuation
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Publication number | Publication date |
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JPH0478618B2 (zh) | 1992-12-11 |
EP0373218B1 (en) | 1993-05-26 |
JPS63264530A (ja) | 1988-11-01 |
WO1988008307A1 (en) | 1988-11-03 |
DE3881396D1 (de) | 1993-07-01 |
EP0373218A1 (en) | 1990-06-20 |
KR890015750A (ko) | 1989-11-25 |
DE3881396T2 (de) | 1993-12-16 |
EP0373218A4 (en) | 1990-04-10 |
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