Fishbein W N, Davis J I, Foellmer J W
Division of Biochemistry, Armed Forces Institute of Pathology, Washington, DC 20306.
J Exp Pathol. 1983;1(1):7-25.
Muscle biopsies from patients with myoadenylate deaminase deficiency (mADD) have been evaluated kinetically and immunologically to ascertain the origin of residual enzyme activity. Kinetic evaluation employed 5 mM AMPS/1 mM AMP ratios, which were 0.7-0.8 for the human muscle isozyme, but 0-0.25 for the isozyme(s) of all other human blood cells and tissues examined. Of 14 control biopsies, 13 showed a ratio greater than 0.60 (one gave 0.47) regardless of the enzyme specific activity, while all 14 mADD biopsies showed a ratio less than 0.24, suggesting that a fetal muscle isozyme and/or blood cell isozyme were responsible for the residual activity. Confirmation was provided by rabbit antisera to purified human muscle AMP deaminase. These antisera fully precipitate the isozyme from crude human muscle biopsy homogenates, regardless of fiber-type composition, and cross-react effectively with the muscle isozyme of Rhesus monkeys and thoroughbred horses, but are inactive toward the isozymes of all other human blood cells and tissues examined. Of 18 mADD homogenates tested, 14 showed less than 20% reactivity with the antisera, at levels that precipitated 10 x more enzyme in control specimens. The residual activity in most cases of mADD must therefore arise from some source other than normal AMP deaminase. To evaluate the possibility of a single common determinant, 9 mADD homogenates were tested for soluble immune complexes. Seven of the 9 then showed 20-42% reactivity, suggesting that part of their residual activity may be due to an isozyme sharing one antigenic determinant with the normal muscle isozyme. Competitive antigen binding was used to assess whether catalytically inactive AMP deaminase was present in mADD. The method was demonstrated effective in identifying spontaneously inactivated purified enzyme and alkaline-inactivated crude enzyme. Nevertheless, homogenates from 17 mADD cases failed to produce more than 14% activation, under conditions in which 63-99% activation was expected. Triton X-100 extracts of homogenate residues of 11 mADD cases were also tested, in a search for insoluble antigen; none produced significant competition. The evidence thus indicates that most cases of mADD are due to a complete gene block, with total absence of all normal muscle AMP deaminase protein.
对肌腺苷酸脱氨酶缺乏症（mADD）患者的肌肉活检样本进行了动力学和免疫学评估，以确定残余酶活性的来源。动力学评估采用5 mM AMPS/1 mM AMP比例，对于人肌肉同工酶，该比例为0.7 - 0.8，但对于所有其他检测的人血细胞和组织的同工酶，该比例为0 - 0.25。在14份对照活检样本中，13份显示该比例大于0.60（有一份为0.47），与酶的比活性无关，而所有14份mADD活检样本的该比例均小于0.24，这表明胎儿肌肉同工酶和/或血细胞同工酶是残余活性的原因。用兔抗纯化人肌肉AMP脱氨酶的抗血清提供了证实。这些抗血清能从粗制的人肌肉活检匀浆中完全沉淀出同工酶，与纤维类型组成无关，并且能与恒河猴和纯种马的肌肉同工酶有效交叉反应，但对所有其他检测的人血细胞和组织的同工酶无活性。在检测的18份mADD匀浆中，14份与抗血清的反应性低于20%，而在对照样本中，该抗血清沉淀的酶量要多10倍。因此，大多数mADD病例中的残余活性必定来自正常AMP脱氨酶以外的某种来源。为了评估单一共同决定簇的可能性，对9份mADD匀浆检测了可溶性免疫复合物。其中7份的反应性为20% - 42%，这表明其部分残余活性可能是由于一种同工酶与正常肌肉同工酶共享一个抗原决定簇。采用竞争性抗原结合来评估mADD中是否存在催化无活性的AMP脱氨酶。该方法被证明可有效鉴定自发失活的纯化酶和碱失活的粗酶。然而，在预期激活率为63% - 99%的条件下，17例mADD病例的匀浆未能产生超过14%的激活。还检测了11例mADD病例匀浆残余物的Triton X - 100提取物，以寻找不溶性抗原；均未产生明显的竞争。因此，证据表明大多数mADD病例是由于完全的基因阻断，导致所有正常肌肉AMP脱氨酶蛋白完全缺失。
