Blair J B, Cimbala M A, Foster J L, Morgan R A
J Biol Chem. 1976 Jun 25;251(12):3756-62.
A reversible interconversion of two kinetically distinct forms of hepatic pyruvate kinase regulated by glucagon and insulin is demonstrated in the perfused rat liver. The regulation does not involve the total enzyme content of the liver, but rather results in a modulation of the substrate dependence. The forms of pyruvate kinase in liver homogenates are distinguished by measurements of the ratio of the enzyme activity at a subsaturating concentration of P-enolpyruvate (1.3 mM) to the activity at a saturating concentration of this substrate (6.6 mM). A low ratio form of pyruvate kinase (ratio between 0.1 and 0.2) is obtained from livers perfused with 10(-7) M glucagon or 0.1 mM adenosine 3':5'-monophosphate (cyclic AMP). A high ratio form of the enzyme is obtained from livers perfused with no hormone (ratio = 0.35 to 0.45). The regulation of pyruvate kinase by glucagon and cyclic AMP occurs within 2 min following the hormone addition to the liver. Insulin (22 milliunits/ml) counteracts the inhibition of pyruvate kinase caused by 5 X 10(-11) M glucagon, but has only a slight influence on the enzyme properties in the absence of the hyperglycemic hormone. The low ratio form of pyruvate kinase obtained from livers perfused with glucagon or cyclic AMP is unstable in liver extracts and will revert to a high ratio form within 10 min at 37 degrees or within a few hours at 0 degrees. Pyruvate kinase is quantitatively precipitated from liver supernatants with 2.5 M ammonium sulfate. This precipitation stabilizes the enzyme and preserves the kinetically distinguishable forms. The kinetic properties of the two forms of rat hepatic pyruvate kinase are examined using ammonium sulfate precipitates from the perfused rat liver. At pH 7.5 the high ratio form of the enzyme has [S]0.5 = 1.6 +/- 0.2 mM P-enolpyruvate (n = 8). The low ratio form of enzyme from livers perfused with glucagon or cyclic AMP has [S]0.5 = 2.5 +/- 0.4 mM P-enolpyruvate (n = 8). The modification of pyruvate kinase induced by glucagon does not alter the dependence of the enzyme activity on ADP (Km is approximately 0.5 mM ADP for both forms of the enzyme). Both forms are allosterically modulated by fructose 1,6-bisphosphate, L-alanine, and ATP. The changes in the kinetic properties of hepatic pyruvate kinase which follow treating the perfused rat liver with glucagon or cyclic AMP are consistent with the changes observed in the enzyme properties upon phosphorylation in vitro by a clyclic AMP-stimulated protein kinase (Ljungström, O., Hjelmquist, G. and Engström, L. (1974) Biochim. Biophys. Acta 358, 289--298). However, other factors also influence the enzyme activity in a similar manner and it remains to be demonstrated that the regulation of hepatic pyruvate kinase by glucagon and cyclic AMP in vivo involes a phosphorylation.
在灌注的大鼠肝脏中，已证实肝丙酮酸激酶的两种动力学上不同的形式可通过胰高血糖素和胰岛素进行可逆的相互转化。这种调节并不涉及肝脏中酶的总量，而是导致底物依赖性的调节。通过测量在次饱和浓度的磷酸烯醇丙酮酸（1.3 mM）下的酶活性与在该底物饱和浓度（6.6 mM）下的活性之比，可区分肝脏匀浆中丙酮酸激酶的形式。从用10(-7) M胰高血糖素或0.1 mM腺苷3':5'-单磷酸（环磷酸腺苷）灌注的肝脏中可获得低比值形式的丙酮酸激酶（比值在0.1至0.2之间）。从未灌注激素的肝脏中可获得高比值形式的该酶（比值 = 0.35至0.45）。胰高血糖素和环磷酸腺苷对丙酮酸激酶的调节在激素添加到肝脏后2分钟内发生。胰岛素（22毫单位/毫升）可抵消5×10(-11) M胰高血糖素对丙酮酸激酶的抑制作用，但在无高血糖激素的情况下对酶的性质影响很小。从用胰高血糖素或环磷酸腺苷灌注的肝脏中获得的低比值形式的丙酮酸激酶在肝脏提取物中不稳定，在37℃下10分钟内或在0℃下数小时内会恢复为高比值形式。丙酮酸激酶可通过2.5 M硫酸铵从肝脏上清液中定量沉淀。这种沉淀可使酶稳定并保留动力学上可区分的形式。使用从灌注的大鼠肝脏中获得的硫酸铵沉淀物来研究大鼠肝丙酮酸激酶两种形式的动力学性质。在pH 7.5时，该酶的高比值形式的[S]0.5 = 1.6±0.2 mM磷酸烯醇丙酮酸（n = 8）。从用胰高血糖素或环磷酸腺苷灌注的肝脏中获得的低比值形式的酶的[S]0.5 = 2.5±0.4 mM磷酸烯醇丙酮酸（n = 8）。胰高血糖素诱导的丙酮酸激酶修饰不会改变酶活性对ADP的依赖性（两种形式的酶的Km约为0.5 mM ADP）。两种形式均受到1,6-二磷酸果糖、L-丙氨酸和ATP的变构调节。在用胰高血糖素或环磷酸腺苷处理灌注的大鼠肝脏后，肝丙酮酸激酶动力学性质的变化与在体外由环磷酸腺苷刺激的蛋白激酶磷酸化后观察到的酶性质变化一致（Ljungström, O., Hjelmquist, G.和Engström, L.（1974）Biochim. Biophys. Acta 358, 289 - 298）。然而，其他因素也以类似方式影响酶活性，并且仍有待证明胰高血糖素和环磷酸腺苷在体内对肝丙酮酸激酶的调节涉及磷酸化作用。
