一磷酸腺苷（AMP）促进肌动球蛋白解离的作用机制研究

Tenderness is an important indicator of meat quality. The applicant’s previous study showed actomyosin dissociation could reduce the shrinkage of myofibrils and improve meat tenderness. The subsequent study indicated adenosine 5'-monophosphate (AMP) could remarkably promote actomyosin dissociation. However, much remains unknown about how AMP promotes actomyosin dissociation, what biochemical changes involved and how to enhance the tenderizing effect of AMP. In the present project, the characteristics of the interaction between AMP and actomyosin will be studied, and the specific protein (actin or myosin) in actomyosin interacting with AMP will be identified with the aid of fluorescence spectrum and nuclear magnetic resonance (NMR). The regulation of the protein-AMP interaction will be set up by optimizing of conditions for the interaction between AMP the specific protein; Under the optimized conditions, effect of AMP competitive occupying, induction of electric repulsion and changes in protein conformation will be evaluated by atomic force microscope (ATM), Zeta potential and circular dichroism spectra (CD), and the relationship between all these impacts and actomyosin dissociation will be established. Muscles will be treated with composites consisted of AMP and isomers, and the relationship of actomyosin dissociation with these supplements, surface charge and protein conformation will be evaluated. The tenderizing effect of AMP and its characteristics will be validated. The result of this project will contribute to the understanding of meat tenderization mechanism and regulation approach, and also provide the basis for the development of new tenderizers.

嫩度是肉品品质的重要指标。申请者前期研究显示，肌动球蛋白解离可以缓解加热中肌原纤维收缩，从而改善嫩度；进一步研究发现，一磷酸腺苷（AMP）能够显著促进肌动球蛋白解离；但AMP如何促进解离、涉及哪些变化以及怎样提高其作用效果，均有待阐明。本项目从AMP与肌动球蛋白的相互作用特征入手，运用荧光光谱和核磁共振法，确定与AMP作用的位置蛋白（肌动或肌球蛋白）；筛选AMP与位置蛋白相互作用的最佳条件，建立AMP与位置蛋白结合程度调控方法；在最佳条件下，运用原子力显微镜、Zeta电位和圆二色谱法，分析AMP的竞争性占位、诱发蛋白表面电荷斥力和构象变化效应，明确相关效应与肌动球蛋白解离的关系；最后通过AMP分子部位单体和结构相似物处理肌肉，分析肌动球蛋白解离与添加物、表面电荷和蛋白构象相关性，验证AMP的作用特征和效应。项目成果不仅为肉品嫩度形成机理及调控研究提供新思路，也为新型嫩化剂研发奠定理论基础。

本项目以肌动球蛋白为研究对象，探究一磷酸腺苷（AMP）与肌动球蛋白的分子间相互作用特性，筛查AMP与肌动球蛋白相互作用影响因素，进一步分析AMP通过分子间相互作用促进肌动球蛋白解离效应，最后在肉品环境中验证AMP促进肌动球蛋白解离的作用机制。结果表明，AMP与肌动球蛋白主要通过疏水作用形成的结合位点，使肌动球蛋白二级结构发生变化，氨基酸残基周围极性降低，肌球蛋白尾部发生解螺旋，进而发生肌动球蛋白解离；pH值、时间、缓冲溶液及离子强度均能影响 AMP-肌动球蛋白体系稳定性，当pH在6.0~7.0时AMP-肌动球蛋白体系的稳定性较好，在5.0~5.5时稳定性较差，而Cl-使二者结合能力增加；AMP腌制及其与超声波联合处理均可显著增加肉品的小片化指数（MFI）、显著减小肉品蒸煮损失和剪切力，使肌原纤维的蛋白热敏性、组织学结构和表面微观结构发生明显变化，同时游离态肌动蛋白含量显著增加，肌动球蛋白表面微观结构发生明显改变。研究成果为深度挖掘肉品嫩度形成机制奠定了理论基础，也为系统分析肉品组分之间的相互作用提供了技术支撑。

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