Poduslo J F, Curran G L
Molecular Neurobiology Laboratory, Department of Neurology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 559051, USA.
J Neurochem. 1996 Aug;67(2):734-41. doi: 10.1046/j.1471-4159.1996.67020734.x.
Our previous studies have demonstrated that modification of superoxide dismutase (SOD) with the naturally occurring polyamines--putrescine (PUT), spermidine, and spermine--dramatically increases the permeability-coefficient surface area (PS) product at the blood-brain barrier and blood-nerve barrier after parenteral administration. Because of this increased permeability, the efficient delivery of polyamine-modified SOD (pSOD) across these barriers may enhance its therapeutic usefulness in treating ischemic neuronal degeneration, neurodegenerative disease, or even aging as an important antioxidant therapeutic strategy. Because PUT-SOD had the highest PS values, SOD was modified in the present experiments by activating carboxylic acid groups to the reactive ester with water-soluble carbodiimide and then reacted with PUT as the nucleophilic reagent. Preservation of SOD enzyme activity while maximizing the permeability was accomplished by adjusting the ionization of the protein carboxylic acid with pH. Both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing analyses demonstrated graded conversion of SOD to its polyamine-modified derivative when performed at different pH. Although modification at pH 4.7 resulted in only 6.6% retained SOD activity and the highest PS value (43.35 +/- 3.81 x 10(-6) ml/g/s for the hippocampus), modification at pH 5.7 resulted in 50.1 % retained activity with a PS value of 24.48 1.30 x 10(-6) ml/g/s for nerve endoneurium and 21.95 +/- 1.62 x 10(-6) ml/g/s for hippocampus. This contrasts with a PS of 1.8-3.2 x 10(-6) ml/g/s for native SOD in nerve and various brain regions. Reaction conditions are therefore defined that titrate enzyme activity of PUT-SOD with PS changes in the intact animal after intravenous administration. These studies will allow an evaluation of the therapeutic usefulness of pSOD in animal models of neuronal degeneration.
我们之前的研究表明，用天然存在的多胺——腐胺（PUT）、亚精胺和精胺——修饰超氧化物歧化酶（SOD），在肠胃外给药后可显著增加血脑屏障和血神经屏障处的通透系数表面积（PS）乘积。由于通透性增加，多胺修饰的SOD（pSOD）有效穿过这些屏障可能会增强其在治疗缺血性神经元变性、神经退行性疾病甚至衰老方面作为一种重要抗氧化治疗策略的治疗效用。由于PUT-SOD具有最高的PS值，在本实验中，通过用水溶性碳二亚胺将羧酸基团活化成活性酯，然后与作为亲核试剂的PUT反应来修饰SOD。通过用pH调节蛋白质羧酸的电离，在使通透性最大化的同时保留SOD酶活性。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和等电聚焦分析均表明，在不同pH下进行实验时，SOD可逐步转化为其多胺修饰衍生物。尽管在pH 4.7进行修饰时，仅保留了6.6%的SOD活性且PS值最高（海马体为43.35±3.81×10⁻⁶ ml/g/s），但在pH 5.7进行修饰时，保留了50.1%的活性，神经内膜的PS值为24.48±1.30×10⁻⁶ ml/g/s，海马体的PS值为21.95±1.62×10⁻⁶ ml/g/s。这与天然SOD在神经和各个脑区的PS值1.8 - 3.2×10⁻⁶ ml/g/s形成对比。因此确定了反应条件，可在静脉给药后完整动物体内随着PS变化滴定PUT-SOD的酶活性。这些研究将有助于评估pSOD在神经元变性动物模型中的治疗效用。
