Analysis of actin and tropomyosin in hearts of cardiac mutant axolotls by two-dimensional gel electrophoresis, western blots, and immunofluorescent microscopy.
Analysis of actin and tropomyosin in hearts of cardiac mutant axolotls by two-dimensional gel electrophoresis, western blots, and immunofluorescent microscopy.
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通过二维凝胶电泳、蛋白质印迹和免疫荧光显微镜分析心脏突变蝾螈心脏中的肌动蛋白和原肌球蛋白。
DOI：
10.1002/jmor.1052010102
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发表时间：
1989
影响因子：
1.5
通讯作者：
Lemanski,LF
中科院分区：
文献类型：
--
作者：
Starr,CM;Diaz,JG;Lemanski,LF
当墨西哥钝口螈纯合时，隐性突变基因会导致胚胎心脏功能衰竭。这种失败显然是由于前内胚层的异常诱导影响导致正常肌节肌原纤维形成的缺失。进行生化和免疫荧光研究来评估正常和突变心脏中的收缩蛋白肌动蛋白和原肌球蛋白。对于免疫荧光研究，将心脏组织固定在高碘酸盐-赖氨酸-多聚甲醛中，冷冻切片，并通过间接方法用针对高度纯化的鸡心肌动蛋白和原肌球蛋白产生的单特异性多克隆抗体进行免疫染色。在正常心脏中，抗肌动蛋白和抗原肌球蛋白都会对肌原纤维 I 带进行强烈染色。在突变心脏中，抗肌动蛋白抗体的染色强度与正常相似，尽管没有观察到肌节模式。与正常心脏相比，突变心脏中抗原肌球蛋白抗体对原肌球蛋白的染色强度显着降低。生化研究用于评估正常和突变心脏组织中肌动蛋白和原肌球蛋白的抗体特异性、抗原变异性以及相对蛋白浓度。组织匀浆进行二维电泳，二维平板凝胶要么用考马斯蓝银染色，要么转印到硝酸纤维素上，然后用抗体对蛋白质进行染色。心脏蛋白的染色凝胶和免疫印迹表明，正常和突变心脏中肌动蛋白亚型的数量是相同的。然而，这些方法证明突变组织中原肌球蛋白的量显着减少。这证实了早期的研究表明，基于免疫学测定，突变心脏中原肌球蛋白的含量减少。因此，基因突变心脏中正常肌原纤维生成的失败似乎不是由于肌动蛋白异构体组成的变化引起的，而可能与原肌球蛋白的缺乏有关。
When homozygous, recessive mutant genecinAmbystoma mexicanumresults in a failure of embryonic heart function. This failure is apparently due to abnormal inductive influences from the anterior endoderm resulting in an absence of normal sarcomeric myofibril formation. Biochemical and immunofluorescent studies were undertaken to evaluate the contractile proteins actin and tropomyosin in normal and mutant hearts. For the immunofluorescent studies, cardiac tissues were fixed in periodate‐lysine‐paraformaldehyde, frozen sectioned, and immunostained by an indirect method with monospecific polyclonal antibodies produced against highly purified chicken heart actin and tropomyosin. In normal hearts, both antiactin and antitropomyosin stained the myofibrillar I‐bands intensely. In mutant hearts, intensity of staining with antiactin antibody was similar to normal, although sarcomeric patterns were not observed. Staining intensity for tropomyosin with antitropomyosin antibody was significantly reduced in mutant hearts when compared to normal. Biochemical studies were used to evaluate antibody specificity, antigenic variability, and relative protein concentrations of actin and tropomyosin in normal and mutant cardiac tissues. Tissue homogenates were electrophoresed in two dimensions, and second‐dimension slab gels were either Coomassie Blue silver‐stained or transblotted onto nitrocellulose and the proteins stained with antibodies. Stained gels and immunoblots of cardiac proteins reveal that the amounts of actin isoforms are identical in normal and mutant hearts. However, these methods demonstrate a significantly reduced amount of tropomyosin in mutant tissue. This confirms earlier studies suggesting reduced amounts of tropomyosin in mutant hearts based upon immunological assays. Thus, failure of normal myofibrillogenesis in genecmutant hearts does not appear to result from a change in actin isoform composition but may be related to a deficiency in tropomyosin.