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Purification and characterization of bovine brain glucocerebrosidase 牛脑糖脑苷酶的纯化与特性研究
Biochemical medicine
Pub Date : 1985-04-01 Epub Date: 2004-04-15 DOI: 10.1016/0006-2944(85)90028-6
P.U.M. Reddy, G.J. Murray, J.A. Barranger

Glucocerebrosidase was isolated from bovine brain by cholate extraction, ammonium sulfate fractionation, acid precipitation at pH 5.35, and hydrophobic chromatography. The purification is about 2400-fold with a specific activity of about 286,000 nmole/hr/mg protein. Molecular weight as determined by chromatography on Bio-Gel P-200 was 138,000. On SDS-polyacrylamide gel electrophoresis the enzyme protein resolved into two bands with apparent molecular weights of 63,000 and 56,000. These bands are cross-reactive to monospecific polyclonal antibody to homogeneous human placental glucocerebrosidase. The enzyme was found to be a complex glycoprotein based on its lectin binding specificity. Brain enzyme was found to be similar to placental glucocerebrosidase in its pH optima, heat stability at 52°C, and substrate affinity. Enzyme kinetics were measured in the presence of conduritol-β-epoxide, an irreversible inhibitor, and gluconolactone, and competitive inhibitor.

采用胆酸盐萃取、硫酸铵分馏、pH 5.35酸沉淀、疏水色谱等方法从牛脑中分离得到葡萄糖脑苷酶。纯化约2400倍，比活性约为286,000 nmol /hr/mg蛋白。Bio-Gel P-200色谱测定分子量为138,000。在sds -聚丙烯酰胺凝胶电泳上，酶蛋白分解成两个表观分子量为63,000和56,000的条带。这些条带与均质人胎盘糖脑苷酶单特异性多克隆抗体交叉反应。根据其凝集素结合特异性，发现该酶是一种复杂的糖蛋白。脑酶在pH值、52℃热稳定性和底物亲和力方面与胎盘糖脑苷酶相似。在不可逆抑制剂孔杜糖醇-β-环氧化物和竞争性抑制剂葡萄糖内酯存在的情况下，测定了酶动力学。
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引用次数: 2
Developmental aspects of mitochondrial creatine kinase in rat myocardial cell cultures 线粒体肌酸激酶在大鼠心肌细胞培养中的发育
Biochemical medicine
Pub Date : 1985-04-01 Epub Date: 2004-04-15 DOI: 10.1016/0006-2944(85)90050-X
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引用次数: 0
Anabolic response of some tissues to diabetes 一些组织对糖尿病的合成代谢反应
Biochemical medicine
Pub Date : 1985-04-01 Epub Date: 2004-04-15 DOI: 10.1016/0006-2944(85)90037-7
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引用次数: 0
Dr. Samuel P. Bessman. Samuel P. Bessman博士。
Biochemical medicine
Pub Date : 1985-04-01
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引用次数: 0
Dissociation of aortic tissue renin and the plasma levels of active and inactive renin in the one- and two-kidney goldblatt hypertensive rat 单肾和双肾高血压大鼠主动脉组织肾素解离及血浆活性和非活性肾素水平
Biochemical medicine
Pub Date : 1985-04-01 Epub Date: 2004-04-15 DOI: 10.1016/0006-2944(85)90036-5
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引用次数: 0
The regulation of mitochondrial-bound hexokinases in the liver 肝脏中线粒体结合己糖激酶的调节
Biochemical medicine
Pub Date : 1985-04-01 Epub Date: 2004-04-15 DOI: 10.1016/0006-2944(85)90031-6
U. Weiler, I. Riesinger, G. Knoll, D. Brdiczka

A functional coupling between bound hexokinase and the inner mitochondrial compartment has been shown. It is based structurally on the binding of hexokinase to a pore protein which is present in zones of contact between the two boundary membranes. The latter was observed by electron microscopic localization of antiporin and hexokinase at the mitochondrial surface.

The four isoenzymes present in liver differ considerably in their activity after binding to the mitochondrial surface. This was found by binding studies using the four isoenzymes isolated from the supernatant. Isoenzyme IV did not bind at all. Isoenzymes I–III did bind and became activated: I, 5.9-fold; II, 39-fold; and III, 1.3-fold. These results suggest that the in vivo activity of hexokinase in the mitochondrial fraction is much larger than so far observed.

Furthermore the binding of isoenzymes was differently affected by metabolites. Glucose 6-phosphate exclusively desorbed isoenzyme I from the mitochondrial membrane whereas free fatty acids predominantly liberated isoenzymes II and III. A reciprocal change of the levels of free fatty acids and glucose 6-phosphate in livers of starved rats therefore, can explain why exclusively mitochondrial-bound isoenzymes II and III decreased 10-fold while at the same time isoenzyme I increased.

结合己糖激酶和线粒体内室之间的功能偶联已被证实。它在结构上是基于己糖激酶与存在于两个边界膜之间的接触区中的孔蛋白的结合。后者通过电镜观察到抗孔蛋白和己糖激酶在线粒体表面的定位。肝脏中存在的四种同工酶在与线粒体表面结合后的活性差异很大。这是通过使用从上清中分离的四种同工酶的结合研究发现的。同工酶IV完全不结合。同工酶I - iii确实结合并被激活:I, 5.9倍;第二,39倍;III, 1.3倍。这些结果表明，线粒体部分的体内己糖激酶活性比目前观察到的要大得多。此外，同工酶的结合受到代谢物的不同影响。葡萄糖- 6-磷酸仅从线粒体膜上解吸同工酶I，而游离脂肪酸主要释放同工酶II和III。因此，饥饿大鼠肝脏中游离脂肪酸和葡萄糖6-磷酸水平的相互变化可以解释为什么线粒体结合的同工酶II和III减少了10倍，而同工酶I增加了。
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引用次数: 52
Lactic acid formation in human tumor cells 人肿瘤细胞中乳酸的形成
Biochemical medicine
Pub Date : 1985-04-01 Epub Date: 2004-04-15 DOI: 10.1016/0006-2944(85)90042-0
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引用次数: 0
Maternal, fetal, and newborn tissue PO2 in sheep measured with galvanic oxygen electrodes 用氧电电极测定绵羊的母体、胎儿和新生儿组织PO2
Biochemical medicine
Pub Date : 1985-04-01 Epub Date: 2004-04-15 DOI: 10.1016/0006-2944(85)90026-2
M.E. Towell , J. Johnson , I. Lysak , D.F. Sayler , S.P. Bessman

Tissue PO2 was measured with galvanic oxygen electrodes chronically implanted in maternal and fetal tissues of two pregnant ewes. Labor commenced after 3 days in one ewe and after 11 days in the other. Tissue PO2 was measured in both newborn lambs. Fetal tissue PO2 during labor was less than 3 mm Hg but rose to values similar to those found in adult tissues in the newborn period. Function of the electrode and response of the tissue to change in oxygenation was challenged by administration of 100% oxygen to the pregnant ewe or newborn lamb, and by administration of intravenous epinephrine to the ewe. These tests suggested that all electrodes were functioning satisfactorily in vivo and that tissue PO2 was responding as anticipated. Recalibration of electrodes after removal from the tissues confirmed that their response in vitro was similar to that obtained before implantation.

用长期植入母羊和胎羊组织的电氧电极测量组织PO2。一只母羊3天后开始分娩，另一只母羊11天后开始分娩。测定了两只新生羔羊的组织PO2。分娩时胎儿组织PO2低于3毫米汞柱，但在新生儿期间上升到与成人组织相似的值。对怀孕母羊或新生羔羊给予100%的氧气，对母羊静脉注射肾上腺素，对电极的功能和组织对氧合变化的反应进行了挑战。这些测试表明，所有电极在体内的功能令人满意，组织PO2的反应如预期的那样。从组织中取出电极后重新校准，证实其在体外的反应与植入前相似。
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引用次数: 5
The role of stress hormones in the development of hyperglycemia 应激激素在高血糖发生中的作用
Biochemical medicine
Pub Date : 1985-04-01 Epub Date: 2004-04-15 DOI: 10.1016/0006-2944(85)90052-3
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引用次数: 0
The effect of insulin on high-energy nucleotides, glycolytic intermediates, and the Krebs cycle in isolated rat hepatocytes 胰岛素对离体大鼠肝细胞高能核苷酸、糖酵解中间体和Krebs循环的影响
Biochemical medicine
Pub Date : 1985-04-01 Epub Date: 2004-04-15 DOI: 10.1016/0006-2944(85)90053-5
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引用次数: 0
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