Kato K, Shinohara H, Kurobe N, Inaguma Y, Shimizu K, Ohshima K
Department of Biochemistry, Institute for Developmental Research, Aichi, Japan.
Biochim Biophys Acta. 1991 May 24;1074(1):201-8. doi: 10.1016/0304-4165(91)90062-l.
In order to determine the quantitative distribution of alpha B crystallin (alpha B) in non-lenticular tissues, we have established a sensitive immunoassay system for specific measurement of alpha B. Antisera were raised in rabbits by injecting alpha B purified from bovine lenses, or C-terminal decapeptide (KPAVTAAPKK) of alpha B (alpha Bpep). The antibodies to alpha B and alpha Bpep were purified by the use of alpha B-coupled Sepharose column. The F(ab')2 fragments of antibody IgG to alpha B were immobilized on polystyrene balls and the Fab' fragments of antibody IgG to alpha Bpep were labeled with beta-D-galactosidase from Escherichia coli. The sandwich-type enzyme immunoassay consisted of the above two antibodies was sensitive, and the minimum detection limit of the assay was 10 pg alpha B without any crossreactivity with alpha A. By using the assay method, it is revealed that the alpha B was distributed in most of the tissues examined. Among the non-lenticular tissues, alpha B was present at high levels in the heart and striated muscles, especially in the soleus muscle, and kidney. High levels of alpha B in the muscle tissues were also seen in various animals. Developmental increases of alpha B in rat muscle tissues and kidney were observed from 16 days of gestational age to 1 or 5 weeks of postnatal age. In contrast, the alpha B in the brain kept a low level during the same period. After 5 weeks of age, alpha B concentrations in the brain increased sharply, reaching the adult levels at 9 weeks of age. Immunohistochemical staining with anti-alpha Bpep revealed that alpha B was positive not only in glial cells, in the central nervous tissues, but also in some neurons of spinal cord, brainstem, hippocampus, and olfactory bulb. Spermatocytes in the testis were also immunopositive for alpha B.
为了确定αB晶状体蛋白（αB）在非晶状体组织中的定量分布，我们建立了一种灵敏的免疫分析系统来特异性测量αB。通过注射从牛晶状体中纯化的αB或αB的C末端十肽（KPAVTAAPKK）（αBpep），在兔体内产生抗血清。使用αB偶联的琼脂糖柱纯化针对αB和αBpep的抗体。将抗αB抗体IgG的F(ab')2片段固定在聚苯乙烯球上，并用来自大肠杆菌的β-D-半乳糖苷酶标记抗αBpep抗体IgG的Fab'片段。由上述两种抗体组成的夹心型酶免疫分析灵敏，该分析的最低检测限为10 pg αB，且与αA无任何交叉反应。通过使用该分析方法，发现αB分布于大多数检测的组织中。在非晶状体组织中，αB在心脏和横纹肌中含量较高，尤其是比目鱼肌和肾脏。在各种动物的肌肉组织中也观察到高水平的αB。从胎龄16天到出生后1或5周，观察到大鼠肌肉组织和肾脏中αB的发育性增加。相比之下，同期大脑中的αB保持低水平。5周龄后，大脑中αB浓度急剧增加，在9周龄时达到成年水平。用抗αBpep进行免疫组织化学染色显示，αB不仅在中枢神经组织的神经胶质细胞中呈阳性，在脊髓、脑干、海马体和嗅球的一些神经元中也呈阳性。睾丸中的精母细胞对αB也呈免疫阳性。
