Schwägele F, Haschke C, Krauss G, Honikel K O
Institut für Chemie und Physik, Bundesanstalt für Fleischforschung, Kulmbach, Germany.
Z Lebensm Unters Forsch. 1996 Jul;203(1):14-20. doi: 10.1007/BF01267763.
A fast breakdown of glycogen is observed in muscles of stress-susceptible pigs leading to pale, soft and exudative (PSE) meat. We report a comparative study of pyruvate kinase from muscles of normal and PSE-prone pigs. Compared with the enzyme from normal muscle, pyruvate kinase isolated from PSE muscle shows a five times lower Michaelis constant, Km, for phosphoenol pyruvate and a more than ten times higher Kcat/Km value. The pH dependency of the enzymatic activity is shifted to more acidic values for pyruvate kinase from PSE muscles. According to isoelectric focusing, pyruvate kinase from PSE muscle consists of three isoforms, while only two isoforms are detectable in pyruvate kinase preparations from normal pigs. The various isoforms were isolated by preparative isoelectric focusing and their steady-state properties were compared. Isoform 3, which is found only in PSE muscle, shows a 10-fold higher specific activity, a 30-fold lower Km value and a 100-fold increased kcat/Km value for phosphoenol pyruvate as compared to isoform 1. The presence of isoform 3 in PSE muscle appears to be responsible for the high activity of this enzyme under the more acidic conditions prevailing in PSE muscle. In vitro phosphorylation and dephosphorylation experiments using total enzyme and purified isoenzyme 1 suggest that isoforms 2 and 3 arise from isoform 1 by phosphorylation. Thus protein phosphorylation seems to be responsible for the shift in activity of pyruvate kinase, a key enzyme of glycolysis, under the acidic conditions of PSE muscles.
在应激敏感型猪的肌肉中观察到糖原快速分解，导致肉色苍白、质地松软且有渗出液（PSE肉）。我们报告了一项对正常猪和易患PSE猪肌肉中丙酮酸激酶的比较研究。与正常肌肉中的酶相比，从PSE肌肉中分离出的丙酮酸激酶对磷酸烯醇丙酮酸的米氏常数Km低五倍，催化常数与米氏常数的比值Kcat/Km高十多倍。PSE肌肉中丙酮酸激酶的酶活性对pH的依赖性向更酸性的值偏移。根据等电聚焦分析，PSE肌肉中的丙酮酸激酶由三种同工型组成，而正常猪的丙酮酸激酶制剂中只能检测到两种同工型。通过制备性等电聚焦分离出各种同工型，并比较它们的稳态性质。仅在PSE肌肉中发现的同工型3，与同工型1相比，对磷酸烯醇丙酮酸的比活性高10倍，Km值低30倍，Kcat/Km值增加100倍。PSE肌肉中同工型3的存在似乎是该酶在PSE肌肉中更酸性条件下具有高活性的原因。使用全酶和纯化的同工酶1进行的体外磷酸化和去磷酸化实验表明，同工型2和3是由同工型1通过磷酸化产生的。因此，蛋白质磷酸化似乎是糖酵解关键酶丙酮酸激酶在PSE肌肉酸性条件下活性变化的原因。
