大鼠肝脏D-双功能蛋白与胆汁酸代谢关系的研究
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摘要
目的
D-３-羟脂酰辅酶A脱水酶／D-３-羟脂酰辅酶A脱氢酶，简称D-双功能蛋白（D-bifunctional protein, DBP），存在于哺乳动物过氧化物酶体内，特异地催化D-3-羟脂酰辅酶A的脱水与脱氢，参与过氧化物酶体β-氧化，于1996年由姜玲玲等发现并命名。
胆汁酸合成与过氧化物酶体β-氧化途径有关。肝脏胆汁酸合成的经典途径是胆固醇在胆固醇7α-羟化酶(CYP7A)的作用下生成7α-羟胆固醇，再经多个酶促反应生成27碳的三羟（或二羟）胆甾烷酰基辅酶Ａ（THC-CoA或DHC-CoA），然后进入过氧化物酶体进行侧链的β-氧化。CYP7A是胆汁酸经典合成途径的限速酶，此酶的活性对胆固醇转化为胆汁酸的速度起制约作用。固醇侧链由过氧化物酶体β-氧化途径氧化。在DBP发现之前，一直认为是L-双功能蛋白参与了THC-CoA（或DHC-CoA）侧链的β-氧化。直到DBP发现之后，研究发现，①体外用纯化的DBP可将24-烯-THC-CoA转变成24-酮-THC-CoA；②病人血清中有大量的不成熟的27碳胆汁酸堆积是因为DBP缺陷；③DBP基因敲除小鼠的胆汁中也大量存在携带不饱和侧链的27碳胆汁酸。这些资料表明胆汁酸合成过程中过氧化物酶体内固醇侧链的β-氧化由DBP催化，DBP为胆汁酸合成所必需。
但是在机体处于生理状态下，DBP是否是胆汁酸合成的
调节靶点，即DBP的表达及活性是否随胆汁酸合成量的变化而变化，诱导DBP的表达，增加其酶活性是否会加速胆汁酸合成，还是一个有待研究的问题。
胆汁酸合成速度受底物胆固醇和产物胆汁酸的调节。增加食物中的胆固醇可激活胆固醇7α-羟化酶(CYP7A)，增加胆汁酸合成。胆酸或鹅脱氧胆酸可反馈抑制胆固醇7α-羟化酶(CYP7A)活性，抑制胆汁酸合成；胆汁酸螯合剂考来烯胺可通过干扰胆汁酸的肠肝循环，促进胆汁酸从肠道排出而增加胆汁酸的合成。
资料表明过氧化物酶体增殖剂邻苯二甲酸二异辛酯（DEHP）可使大鼠肝脏过氧化物酶体增值，DBP的mRNA表达增强，酶活性增加。
本研究从整体水平上一方面通过应用考来烯胺和高胆固醇食物促进胆汁酸合成，观察DBP的表达及活性的变化；另一方面应用过氧化物酶体增殖剂DEHP诱导DBP增加，观察DBP增加后胆汁酸合成量是否发生改变。以探讨在生理条件下，肝脏DBP是否是胆汁酸合成途径的调节靶点。
方法
1 动物分组及取材
将10周龄成年雄性Wistar大鼠40只，随机分为①对照组（Control组）：给普通大鼠饲料；②考来烯胺组（CHY组）：给含2%考来烯胺普通饲料；③诱导剂组（DEHP组）：给含2%DEHP普通饲料；④高胆固醇组（CH组）：给高胆固醇饲料（普通饲料含2%胆固醇和10%玉米油）。各组均自由饮食饮水，饲养2周。2周后，大鼠禁食不禁水一晚后于上午8点至11点之间处死。麻醉后，颈动脉放血处死大鼠，收集血
液,血清用于血清胆固醇测定。迅速取下肝脏，一部分肝脏组织立刻置于液氮中，然后-70℃保存，用于RNA提取；另取一部分肝脏立即用于过氧化物酶体提取，用于D-双功能蛋白活性测定。
2 肝脏过氧化物酶体粗提物的制备
称取5g肝脏，快速剪碎后，加预冷的20ml提取缓冲液（PH7.4Tris/HCl10mM, EDTA1mM，蔗糖0.25M，乙醇0.1%，PMSF 0.5mM，Benzamidin 1mM）匀浆。肝匀浆4℃3000g离心10min，上清置于（冰浴）中保存；沉淀加入预冷的20ml提取缓冲液，再次匀浆，然后4℃3000g离心10min，弃沉淀，取上清。合并两次上清，用提取缓冲液补足体积至50ml(肝重的10倍体积)。4℃，7000g，离心5min后，弃沉淀；取上清4℃11000g离心20min，弃上清，留沉淀。沉淀用15ml提取液（肝重的3倍）混悬，4℃11000g离心20min，弃上清，留沉淀。沉淀用1ml提取缓冲液悬浮，此样品即为过氧化酶体的粗提物。放置-20℃保存，用于D-双功能蛋白活性的测定。
3 血清胆固醇测定
用全自动生化分析仪，酶法测定。
4 DBP酶活性测定
以D-（-）-3-羟辛酰辅酶A作为底物，测定单位时间内产物3-酮辛酰辅酶A的生成。用303nm波长处吸光度的变化幅度反映酶活性高低。DBP酶活性用每毫克蛋白所含酶的毫单位数表示（mU/mg protein）。
5 肝脏总RNA的提取
用异硫氰酸胍一步法提取。
6 mRNA表达的定量
用β-actin作为内参照，利用RT-PCR法测定肝脏CYP7A mRNA、DBP mRNA及PPARα mRNA相对表达量。
结果
1 考来烯胺组（CHY）的变化
①与对照组相比，血清胆固醇无明显变化；②CYP7A mRNA相对表达量（0.872±0.0781）比对照组（0.473±0.0646）显著增高（p<0.01），是对照组的1.8倍；③DBP活性无明显变化；④DBP mRNA相对表达量无明显变化；⑤PPARα mRNA相对表达量无明显变化。表明大鼠应用考来烯胺后，血胆固醇无明显变化，胆汁酸合成加强，但DBP活性及mRNA表达无明显变化。
2 诱导剂组（DEHP）的变化
①与对照组相比，血清胆固醇无明显变化；②CYP7A mRNA相对表达量（0.768±0.0461）比对照组（0.473±0.0646）显著增高（p<0.01），是对照组的1.6倍；③DBP活性（69.636±11.103mU/mg pr）比对照组（8.811±2.528mU/mg pr）显著升高，是对照组的7.9倍（p<0.01）；④DBP mRNA相对表达量（1.047±0.0379）比对照组（0.721±0.063）显著增高（p<0.01），是对照组的1.5倍；⑤PPARαmRNA相对表达量（1.010?
Objective: D-3-Hydroxyacyl-CoA dehydratase /D-3- Hydroxyacyl-CoA dehydrogenase (D-bifunctional protein, DBP), located in mammalian peroxisomes and involved in peroxisomal beta-oxidation, was first found and named by Jiang LL et al in 1996,which catalyzes the dehydration and dehydrogenation of D-3-hydroxyacyl-CoA.
The synthesis of bile acids is associated with peroxisomal beta-oxidation. In the classical pathway for the disposal of cholesterol in mammals, Cholesterol 7α-hydroxylase(CYP7A) is the first and rate-limiting enzyme that catalyzes the conversion of cholesterol to bile acids. In peroxisomes，conversion of the enoyl-forms of intermediates tri- and dihydroxycholestanoyl-CoA (THC-CoA or DHC-CoA) in bile acid formation to oxo-forms via D-3-hydroxyacyl-CoA is catalyzed by DBP but not L-bifunctional protein. In DBP deficiency patients, the serum unmature 27-carbon-bile acids increased remarkably. The same phenomenon was observed in DBP knock-out mice. From these evidences ,we can draw a conclusion that DBP takes part in theβ-oxidation reactions of cholesterol side chains and it is a necessary enzyme for bile acid synthesis.
But, in vivo, whether D-bifunctional protein is a key enzyme in the regulation of bile acids synthesis, whether the mRNA expression and enzyme activity change with the amount of bile acids synthesis are unclear.
Recently, many works have been done on the factors which regulating the synthesis and secretion of bile acids. These may be the potential ways to prevent and treat the hypercholesterolemia and related diseases in the future. So the research on relationships between D-bifunctional protein and bile acid synthesis in vivo is a valuable work.
The target of our experiment is to find whether there are some relationships between mRNA expression and activity of DBP and the synthesis of bile acids, and to investigate whether D-bifunctional protein regulates the bile acid synthesis.
For this purpose, we design our experiment in two directions. One is to observe the changes of expression and activity of hepatic DBP of rats fed high cholesterol or cholestyramine which are used to induce bile acid synthesis. On the other hand, we want to know how the rat bile acids synthesis change after the quantity and activity of liver DBP was induced by Diethylhexyl phthalate(DEHP), one of the peroxisome proliferators.
Methods
1 Animals and diets
Forty male Wistar rats were divided randomly into four groups:①control group（Control）,fed common rodent diet②cholestyramine group（CHY）,fed 2% cholestyramine diet③
cholesterol group（CH）,fed 2% cholesterol plus 10%corn oil diet④inducer group（DEHP）,fed 2% DEHP diet. Rats were given free access to food and water for 2 wk. After overnight fasting, rats were killed by releasing blood from carotid artery. Blood were selected for serum total cholesterol assay. Liver samples were cut rapidly, and a portion of liver was throw into liquid Nitrogen and stored at -70℃ for the extraction of RNA. In paralleled, 5g of liver were quickly homogenized to isolate crude peroxisome for measurement of DBP activity.
2 Isolation of liver crude peroxisome
5 g of liver were quickly homogenized in 20ml of an ice-cold buffer(Tris-HCL 10mM,sucrose 250mM, EDTA 1mM,ethanol 0.1%, PMSF 0.5mM，Benzamidin 1mM, PH7.4).The homogenate was first centrifuged at 3000g (10min,4℃);the resulting supernatant kept in ice-water, the pellets were homogenized again in 20ml buffer .The supernatant of two times were put together adding volume to 50ml and then centrifuged at 7000g(5min,4℃).The resulting supernatant was then centrifuged at 11000g(20min,4℃). The pellets were resuspended in 15ml of the buffer. The centrifugation procedure was repeated and the resulting pellets resuspended in 1ml of the buffer. The peroxisomal preparation was stored at -20℃ until measurement of DBP activity.
3 Serum total cholesterol assay
Serum total cholesterol was measured by Auto-biochemical-analyst using enzymatic procedure.
4 DBP activity assay
The activity of D-3-hydoxyacyl-CoA d
D-３-羟脂酰辅酶A脱水酶／D-３-羟脂酰辅酶A脱氢酶，简称D-双功能蛋白（D-bifunctional protein, DBP），存在于哺乳动物过氧化物酶体内，特异地催化D-3-羟脂酰辅酶A的脱水与脱氢，参与过氧化物酶体β-氧化，于1996年由姜玲玲等发现并命名。
胆汁酸合成与过氧化物酶体β-氧化途径有关。肝脏胆汁酸合成的经典途径是胆固醇在胆固醇7α-羟化酶(CYP7A)的作用下生成7α-羟胆固醇，再经多个酶促反应生成27碳的三羟（或二羟）胆甾烷酰基辅酶Ａ（THC-CoA或DHC-CoA），然后进入过氧化物酶体进行侧链的β-氧化。CYP7A是胆汁酸经典合成途径的限速酶，此酶的活性对胆固醇转化为胆汁酸的速度起制约作用。固醇侧链由过氧化物酶体β-氧化途径氧化。在DBP发现之前，一直认为是L-双功能蛋白参与了THC-CoA（或DHC-CoA）侧链的β-氧化。直到DBP发现之后，研究发现，①体外用纯化的DBP可将24-烯-THC-CoA转变成24-酮-THC-CoA；②病人血清中有大量的不成熟的27碳胆汁酸堆积是因为DBP缺陷；③DBP基因敲除小鼠的胆汁中也大量存在携带不饱和侧链的27碳胆汁酸。这些资料表明胆汁酸合成过程中过氧化物酶体内固醇侧链的β-氧化由DBP催化，DBP为胆汁酸合成所必需。
但是在机体处于生理状态下，DBP是否是胆汁酸合成的
调节靶点，即DBP的表达及活性是否随胆汁酸合成量的变化而变化，诱导DBP的表达，增加其酶活性是否会加速胆汁酸合成，还是一个有待研究的问题。
胆汁酸合成速度受底物胆固醇和产物胆汁酸的调节。增加食物中的胆固醇可激活胆固醇7α-羟化酶(CYP7A)，增加胆汁酸合成。胆酸或鹅脱氧胆酸可反馈抑制胆固醇7α-羟化酶(CYP7A)活性，抑制胆汁酸合成；胆汁酸螯合剂考来烯胺可通过干扰胆汁酸的肠肝循环，促进胆汁酸从肠道排出而增加胆汁酸的合成。
资料表明过氧化物酶体增殖剂邻苯二甲酸二异辛酯（DEHP）可使大鼠肝脏过氧化物酶体增值，DBP的mRNA表达增强，酶活性增加。
本研究从整体水平上一方面通过应用考来烯胺和高胆固醇食物促进胆汁酸合成，观察DBP的表达及活性的变化；另一方面应用过氧化物酶体增殖剂DEHP诱导DBP增加，观察DBP增加后胆汁酸合成量是否发生改变。以探讨在生理条件下，肝脏DBP是否是胆汁酸合成途径的调节靶点。
方法
1 动物分组及取材
将10周龄成年雄性Wistar大鼠40只，随机分为①对照组（Control组）：给普通大鼠饲料；②考来烯胺组（CHY组）：给含2%考来烯胺普通饲料；③诱导剂组（DEHP组）：给含2%DEHP普通饲料；④高胆固醇组（CH组）：给高胆固醇饲料（普通饲料含2%胆固醇和10%玉米油）。各组均自由饮食饮水，饲养2周。2周后，大鼠禁食不禁水一晚后于上午8点至11点之间处死。麻醉后，颈动脉放血处死大鼠，收集血
液,血清用于血清胆固醇测定。迅速取下肝脏，一部分肝脏组织立刻置于液氮中，然后-70℃保存，用于RNA提取；另取一部分肝脏立即用于过氧化物酶体提取，用于D-双功能蛋白活性测定。
2 肝脏过氧化物酶体粗提物的制备
称取5g肝脏，快速剪碎后，加预冷的20ml提取缓冲液（PH7.4Tris/HCl10mM, EDTA1mM，蔗糖0.25M，乙醇0.1%，PMSF 0.5mM，Benzamidin 1mM）匀浆。肝匀浆4℃3000g离心10min，上清置于（冰浴）中保存；沉淀加入预冷的20ml提取缓冲液，再次匀浆，然后4℃3000g离心10min，弃沉淀，取上清。合并两次上清，用提取缓冲液补足体积至50ml(肝重的10倍体积)。4℃，7000g，离心5min后，弃沉淀；取上清4℃11000g离心20min，弃上清，留沉淀。沉淀用15ml提取液（肝重的3倍）混悬，4℃11000g离心20min，弃上清，留沉淀。沉淀用1ml提取缓冲液悬浮，此样品即为过氧化酶体的粗提物。放置-20℃保存，用于D-双功能蛋白活性的测定。
3 血清胆固醇测定
用全自动生化分析仪，酶法测定。
4 DBP酶活性测定
以D-（-）-3-羟辛酰辅酶A作为底物，测定单位时间内产物3-酮辛酰辅酶A的生成。用303nm波长处吸光度的变化幅度反映酶活性高低。DBP酶活性用每毫克蛋白所含酶的毫单位数表示（mU/mg protein）。
5 肝脏总RNA的提取
用异硫氰酸胍一步法提取。
6 mRNA表达的定量
用β-actin作为内参照，利用RT-PCR法测定肝脏CYP7A mRNA、DBP mRNA及PPARα mRNA相对表达量。
结果
1 考来烯胺组（CHY）的变化
①与对照组相比，血清胆固醇无明显变化；②CYP7A mRNA相对表达量（0.872±0.0781）比对照组（0.473±0.0646）显著增高（p<0.01），是对照组的1.8倍；③DBP活性无明显变化；④DBP mRNA相对表达量无明显变化；⑤PPARα mRNA相对表达量无明显变化。表明大鼠应用考来烯胺后，血胆固醇无明显变化，胆汁酸合成加强，但DBP活性及mRNA表达无明显变化。
2 诱导剂组（DEHP）的变化
①与对照组相比，血清胆固醇无明显变化；②CYP7A mRNA相对表达量（0.768±0.0461）比对照组（0.473±0.0646）显著增高（p<0.01），是对照组的1.6倍；③DBP活性（69.636±11.103mU/mg pr）比对照组（8.811±2.528mU/mg pr）显著升高，是对照组的7.9倍（p<0.01）；④DBP mRNA相对表达量（1.047±0.0379）比对照组（0.721±0.063）显著增高（p<0.01），是对照组的1.5倍；⑤PPARαmRNA相对表达量（1.010?
Objective: D-3-Hydroxyacyl-CoA dehydratase /D-3- Hydroxyacyl-CoA dehydrogenase (D-bifunctional protein, DBP), located in mammalian peroxisomes and involved in peroxisomal beta-oxidation, was first found and named by Jiang LL et al in 1996,which catalyzes the dehydration and dehydrogenation of D-3-hydroxyacyl-CoA.
The synthesis of bile acids is associated with peroxisomal beta-oxidation. In the classical pathway for the disposal of cholesterol in mammals, Cholesterol 7α-hydroxylase(CYP7A) is the first and rate-limiting enzyme that catalyzes the conversion of cholesterol to bile acids. In peroxisomes，conversion of the enoyl-forms of intermediates tri- and dihydroxycholestanoyl-CoA (THC-CoA or DHC-CoA) in bile acid formation to oxo-forms via D-3-hydroxyacyl-CoA is catalyzed by DBP but not L-bifunctional protein. In DBP deficiency patients, the serum unmature 27-carbon-bile acids increased remarkably. The same phenomenon was observed in DBP knock-out mice. From these evidences ,we can draw a conclusion that DBP takes part in theβ-oxidation reactions of cholesterol side chains and it is a necessary enzyme for bile acid synthesis.
But, in vivo, whether D-bifunctional protein is a key enzyme in the regulation of bile acids synthesis, whether the mRNA expression and enzyme activity change with the amount of bile acids synthesis are unclear.
Recently, many works have been done on the factors which regulating the synthesis and secretion of bile acids. These may be the potential ways to prevent and treat the hypercholesterolemia and related diseases in the future. So the research on relationships between D-bifunctional protein and bile acid synthesis in vivo is a valuable work.
The target of our experiment is to find whether there are some relationships between mRNA expression and activity of DBP and the synthesis of bile acids, and to investigate whether D-bifunctional protein regulates the bile acid synthesis.
For this purpose, we design our experiment in two directions. One is to observe the changes of expression and activity of hepatic DBP of rats fed high cholesterol or cholestyramine which are used to induce bile acid synthesis. On the other hand, we want to know how the rat bile acids synthesis change after the quantity and activity of liver DBP was induced by Diethylhexyl phthalate(DEHP), one of the peroxisome proliferators.
Methods
1 Animals and diets
Forty male Wistar rats were divided randomly into four groups:①control group（Control）,fed common rodent diet②cholestyramine group（CHY）,fed 2% cholestyramine diet③
cholesterol group（CH）,fed 2% cholesterol plus 10%corn oil diet④inducer group（DEHP）,fed 2% DEHP diet. Rats were given free access to food and water for 2 wk. After overnight fasting, rats were killed by releasing blood from carotid artery. Blood were selected for serum total cholesterol assay. Liver samples were cut rapidly, and a portion of liver was throw into liquid Nitrogen and stored at -70℃ for the extraction of RNA. In paralleled, 5g of liver were quickly homogenized to isolate crude peroxisome for measurement of DBP activity.
2 Isolation of liver crude peroxisome
5 g of liver were quickly homogenized in 20ml of an ice-cold buffer(Tris-HCL 10mM,sucrose 250mM, EDTA 1mM,ethanol 0.1%, PMSF 0.5mM，Benzamidin 1mM, PH7.4).The homogenate was first centrifuged at 3000g (10min,4℃);the resulting supernatant kept in ice-water, the pellets were homogenized again in 20ml buffer .The supernatant of two times were put together adding volume to 50ml and then centrifuged at 7000g(5min,4℃).The resulting supernatant was then centrifuged at 11000g(20min,4℃). The pellets were resuspended in 15ml of the buffer. The centrifugation procedure was repeated and the resulting pellets resuspended in 1ml of the buffer. The peroxisomal preparation was stored at -20℃ until measurement of DBP activity.
3 Serum total cholesterol assay
Serum total cholesterol was measured by Auto-biochemical-analyst using enzymatic procedure.
4 DBP activity assay
The activity of D-3-hydoxyacyl-CoA d
引文
Reddy JK, Mannaerts GP. Peroxisomai lipid metabolism. Annu Rev Nutr, 1994,14:343~370
Kunau WH, Dommes V, Schulz H. β-Oxidation of fatty acids in mitochondria, peroxisomes, and bacteria: A century of continued progress. Prog lipid Res, 1995,34:267~342
Jiang LL, Miyazawa S, Hashimoto T. Purification and properties of rat D-3-hydroxyacyl-CoA dehydratase: D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein. J Biochem (Tokyo), 1996,120:633~41
Jiang LL, Miyazawa S, Souri M, et al. Structure of D-3-Hydroxyacyl-CoA Dehydratase/D-3-Hydroxyacyl-CoA Dehydrogenase Bifunctional Protein. J Biochem, 1997,
121:364~369
Jiang LL, Kurosawa T, Sato M, et al. Physiological role of D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein. J Biochem (Tokyo), 1997,121:506~13
Osumi T, Hashimoto T. Peroxisomal beta-oxidation system of rat liver. Copurification of enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase. Biochem Biophys Res Commun, 1979,89:580~4
Verhoeven NM, Jakobs C. Human metabolism of phytanic acid and pristanic acid. Prog Lipid Res, 2001,40:453~66
Moller G, Leenders F, van Grunsven EG, et al. Characterization of the HSD17B4 gene: D-specific multifunctional protein 2/17beta-hydroxysteroid dehydrogenase IV. J Steroid Biochem Mol Biol, 1999,69:441~6
Leenders F, Prescher G, Dolez V, et al. Assignment of human 17 beta-hydroxysteroid dehydrogenase IV to chromosome 5q2 by fluorescence in situ hybridization. Genomics, 1996,37:403~4
Moller G, Luders J, Markus M, et al. Peroxisome targeting of porcine 17beta-hydroxysteroid dehydrogenase type IV /D-specific multifunctional protein 2 is mediated by its C-terminal tripeptide AKI. J Cell Biochem, 1999, 73:70~8
Adamski J, Normand T, Leenders F, et al. Molecular cloning of a novel widely expressed human 80 kDa 17
beta-hydroxysteroid dehydrogenase IV. Biochem J, 1995,311:437~43
Leenders F, Tesdorpf JG, Markus M, et al. Porcine 80-kDa protein reveals intrinsic 17 beta-hydroxysteroid dehydrogenase, fatty acyl-CoA-hydratase/dehydrogenase, and sterol transfer activities. J Biol Chem, 1996,271:5438~42
van Grunsven EG, van Berkel E, Ijlst L,et al. Peroxisomal D-hydroxyacyl-CoA dehydrogenase deficiency: resolution of the enzyme defect and its molecular basis in bifunctional protein deficiency. Proc Natl Acad Sci U S A, 1998,95:2128~33
de Launoit Y, Adamski J. Unique multifunctional HSD17B4 gene product: 17beta-hydroxysteroid dehydrogenase 4 and D-3-hydroxyacyl-coenzyme A dehydrogenase/hydratase involved in Zellweger syndrome. J Mol Endocrinol, 1999,22:227~40
Castagnetta LA, Carruba G, Traina A, et al. Expression of different 17beta-hydroxysteroid dehydrogenase types and their activities in human prostate cancer cells. Endocrinology, 1997,138:4876~82
Kurosawa T, Sato M, Jiang LL, et al. Stereospecific formation of (24R, 25R)-3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid catalyzed with a peroxisomal bifunctional D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase. Biol Pharm Bull, 1997,20:295~7
Suzuki Y, Jiang LL, Souri M, et al D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein deficiency: a newly identified peroxisomal disorder. Am J Hum Genet. 1997,61:1153~62
Une M, Konishi M, Suzuki Y, et al Bile acid profiles in a peroxisomal D-3-hydroxyacyl-CoA dehydratase /D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein deficiency. J Biochem (Tokyo), 1997,122:655~8
Baes M, Huyghe S, Carmeliet P, et al Inactivation of the peroxisomal multifunctional protein-2 in mice impedes the degradation of not only 2-methyl-branched fatty acids and bile acid intermediates but also of very long chain fatty acids. J Biol Chem, 2000,275:16329~36
Dieuaide-Noubhani M, Asselberghs S, Mannaerts GP, et al. Evidence that multifunctional protein 2, and not multifunctional protein 1, is involved in the peroxisomal beta-oxidation of pristanic acid. Biochem J, 1997,325:367-73
Clayton PT. Clinical consequences of defects in peroxisomal beta-oxidation. Biochem Soc Trans, 2001,29:298~305
Ferdinandusse S, Meissner T，Wanders RJ. Identification of the peroxisomal beta-oxidation enzymes involved in the degradation of leukotrienes. Biochem Biophys Res Commun, 2002,293:269~73
Su HM, Moser AB, Moser HW, et al. Peroxisomal straight-chain Acyl-CoA oxidase and D-bifunctional protein are essential for the retroconversion step in docosahexaenoic
acid synthesis. J Biol Chem, 200,276:38115~20
Itoh M, Suzuki Y，Akaboshi S. Developmental and pathological expression of peroxisomal enzymes: their relationship of D-bifunctional protein deficiency and Zellweger syndrome. Brain Res, 2000,858:40~7
Itoh M,Suzuki，Takashima S Y A novel peroxisomal enzyme, D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein: its expression in the developing human brain. Microsc Res Tech, 1999,45:383~8
Moller G, van Grunsven EG, Wanders RJ, et al. Molecular basis of D-bifunctional protein deficiency. Mol Cell Endocrinol, 2001,171:61~70
Gloerich J, Denis S, van Grunsven EG, et al. A novel HPLC-based method to diagnose peroxisomal D-bifunctional protein enoyl-CoA hydratase deficiency. J Lipid Res, 2003,44:640~4
Aoyama T, Peters JM, Iritani N, et al. Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha). J Biol Chem, 1998,273:5678~84
Caira F, Clemencet MC, Cherkaoui-Malki M, et al. Differential regulation by a peroxisome proliferator of the different multifunctional proteins in guinea pig: cDNA cloning of the guinea pig D-specific multifunctional protein 2. Biochem J, 1998,330:1361~8
Carstensen JF, Tesdorpf JG, Kaufmann M, et al.
Characterization of 17beta-hydroxysteroid dehydrogenase IV. J Endocrinol, 1996,150:3~12
Kunau WH, Dommes V, Schulz H. β-Oxidation of fatty acids in mitochondria, peroxisomes, and bacteria: A century of continued progress. Prog lipid Res, 1995,34:267~342
Jiang LL, Miyazawa S, Hashimoto T. Purification and properties of rat D-3-hydroxyacyl-CoA dehydratase: D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein. J Biochem (Tokyo), 1996,120:633~41
Jiang LL, Miyazawa S, Souri M, et al. Structure of D-3-Hydroxyacyl-CoA Dehydratase/D-3-Hydroxyacyl-CoA Dehydrogenase Bifunctional Protein. J Biochem, 1997,
121:364~369
Jiang LL, Kurosawa T, Sato M, et al. Physiological role of D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein. J Biochem (Tokyo), 1997,121:506~13
Osumi T, Hashimoto T. Peroxisomal beta-oxidation system of rat liver. Copurification of enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase. Biochem Biophys Res Commun, 1979,89:580~4
Verhoeven NM, Jakobs C. Human metabolism of phytanic acid and pristanic acid. Prog Lipid Res, 2001,40:453~66
Moller G, Leenders F, van Grunsven EG, et al. Characterization of the HSD17B4 gene: D-specific multifunctional protein 2/17beta-hydroxysteroid dehydrogenase IV. J Steroid Biochem Mol Biol, 1999,69:441~6
Leenders F, Prescher G, Dolez V, et al. Assignment of human 17 beta-hydroxysteroid dehydrogenase IV to chromosome 5q2 by fluorescence in situ hybridization. Genomics, 1996,37:403~4
Moller G, Luders J, Markus M, et al. Peroxisome targeting of porcine 17beta-hydroxysteroid dehydrogenase type IV /D-specific multifunctional protein 2 is mediated by its C-terminal tripeptide AKI. J Cell Biochem, 1999, 73:70~8
Adamski J, Normand T, Leenders F, et al. Molecular cloning of a novel widely expressed human 80 kDa 17
beta-hydroxysteroid dehydrogenase IV. Biochem J, 1995,311:437~43
Leenders F, Tesdorpf JG, Markus M, et al. Porcine 80-kDa protein reveals intrinsic 17 beta-hydroxysteroid dehydrogenase, fatty acyl-CoA-hydratase/dehydrogenase, and sterol transfer activities. J Biol Chem, 1996,271:5438~42
van Grunsven EG, van Berkel E, Ijlst L,et al. Peroxisomal D-hydroxyacyl-CoA dehydrogenase deficiency: resolution of the enzyme defect and its molecular basis in bifunctional protein deficiency. Proc Natl Acad Sci U S A, 1998,95:2128~33
de Launoit Y, Adamski J. Unique multifunctional HSD17B4 gene product: 17beta-hydroxysteroid dehydrogenase 4 and D-3-hydroxyacyl-coenzyme A dehydrogenase/hydratase involved in Zellweger syndrome. J Mol Endocrinol, 1999,22:227~40
Castagnetta LA, Carruba G, Traina A, et al. Expression of different 17beta-hydroxysteroid dehydrogenase types and their activities in human prostate cancer cells. Endocrinology, 1997,138:4876~82
Kurosawa T, Sato M, Jiang LL, et al. Stereospecific formation of (24R, 25R)-3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid catalyzed with a peroxisomal bifunctional D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase. Biol Pharm Bull, 1997,20:295~7
Suzuki Y, Jiang LL, Souri M, et al D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein deficiency: a newly identified peroxisomal disorder. Am J Hum Genet. 1997,61:1153~62
Une M, Konishi M, Suzuki Y, et al Bile acid profiles in a peroxisomal D-3-hydroxyacyl-CoA dehydratase /D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein deficiency. J Biochem (Tokyo), 1997,122:655~8
Baes M, Huyghe S, Carmeliet P, et al Inactivation of the peroxisomal multifunctional protein-2 in mice impedes the degradation of not only 2-methyl-branched fatty acids and bile acid intermediates but also of very long chain fatty acids. J Biol Chem, 2000,275:16329~36
Dieuaide-Noubhani M, Asselberghs S, Mannaerts GP, et al. Evidence that multifunctional protein 2, and not multifunctional protein 1, is involved in the peroxisomal beta-oxidation of pristanic acid. Biochem J, 1997,325:367-73
Clayton PT. Clinical consequences of defects in peroxisomal beta-oxidation. Biochem Soc Trans, 2001,29:298~305
Ferdinandusse S, Meissner T，Wanders RJ. Identification of the peroxisomal beta-oxidation enzymes involved in the degradation of leukotrienes. Biochem Biophys Res Commun, 2002,293:269~73
Su HM, Moser AB, Moser HW, et al. Peroxisomal straight-chain Acyl-CoA oxidase and D-bifunctional protein are essential for the retroconversion step in docosahexaenoic
acid synthesis. J Biol Chem, 200,276:38115~20
Itoh M, Suzuki Y，Akaboshi S. Developmental and pathological expression of peroxisomal enzymes: their relationship of D-bifunctional protein deficiency and Zellweger syndrome. Brain Res, 2000,858:40~7
Itoh M,Suzuki，Takashima S Y A novel peroxisomal enzyme, D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein: its expression in the developing human brain. Microsc Res Tech, 1999,45:383~8
Moller G, van Grunsven EG, Wanders RJ, et al. Molecular basis of D-bifunctional protein deficiency. Mol Cell Endocrinol, 2001,171:61~70
Gloerich J, Denis S, van Grunsven EG, et al. A novel HPLC-based method to diagnose peroxisomal D-bifunctional protein enoyl-CoA hydratase deficiency. J Lipid Res, 2003,44:640~4
Aoyama T, Peters JM, Iritani N, et al. Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha). J Biol Chem, 1998,273:5678~84
Caira F, Clemencet MC, Cherkaoui-Malki M, et al. Differential regulation by a peroxisome proliferator of the different multifunctional proteins in guinea pig: cDNA cloning of the guinea pig D-specific multifunctional protein 2. Biochem J, 1998,330:1361~8
Carstensen JF, Tesdorpf JG, Kaufmann M, et al.
Characterization of 17beta-hydroxysteroid dehydrogenase IV. J Endocrinol, 1996,150:3~12