Crepin K M, De Cloedt M, Vertommen D, Foret D, Michel A, Rider M H, Rousseau G G, Hue L
Hormone and Metabolic Research Unit, Louvain University Medical School, Brussels, Belgium.
J Biol Chem. 1992 Oct 25;267(30):21698-704.
The rat cDNA for the muscle-type (M) isozyme of 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) contains two putative translation initiation sites. To determine whether the M isozyme expressed in rat skeletal muscle corresponds to the short (PFK2M-sf) or the long (PFK2M-lf) isoform, we have expressed them in Escherichia coli. A third construction was also expressed in which the second ATG codon was deleted (PFK2M-lf delta ATG) to ensure that initiation started at the first ATG. The properties of these recombinant proteins were compared with those of the PFK-2/FBPase-2 present in rat skeletal muscle and liver. The recombinant proteins displayed PFK-2 and FBPase-2 activities and the M(r) values of the subunits measured by SDS-polyacrylamide gel electrophoresis were compatible with the calculated ones. The purified recombinant lf form contained not only the expected lf band (54,500 M(r)) but also the sf band (52,000 M(r)), indicating that the expression system could synthesize the long and the short isoforms from the same mRNA. The kinetic properties of the recombinant sf form were not different from those of the rat muscle enzyme. By contrast, lf delta ATG PFK-2 displayed a higher Km for its substrates and a lower Vmax. Immunoblotting with an antibody directed against the long isoform revealed a 54,500 M(r) band both in the lf and the lf delta ATG recombinant, but no band in rat skeletal muscle extracts. In these extracts, one band of 52,000 and a minor one of 54,500 M(r) were detected by an anti PFK-2/FBPase-2 antibody. The 54,500 M(r) band was recognized by an antibody directed against the L isozyme, suggesting that a small amount of the latter is expressed in skeletal muscle. Thus, the M isozyme differs from the L isozyme by replacement of the first 32 amino acids of the L isozyme by an unrelated nonapeptide.
6-磷酸果糖-2-激酶（PFK-2）/果糖-2,6-二磷酸酶（FBPase-2）肌肉型（M）同工酶的大鼠cDNA含有两个假定的翻译起始位点。为了确定在大鼠骨骼肌中表达的M同工酶是对应于短（PFK2M-sf）还是长（PFK2M-lf）同工型，我们在大肠杆菌中进行了表达。还构建并表达了第三种结构，其中第二个ATG密码子被删除（PFK2M-lf delta ATG），以确保起始于第一个ATG。将这些重组蛋白的特性与大鼠骨骼肌和肝脏中存在的PFK-2/FBPase-2的特性进行了比较。重组蛋白表现出PFK-2和FBPase-2活性，通过SDS-聚丙烯酰胺凝胶电泳测得的亚基M（r）值与计算值相符。纯化的重组lf形式不仅含有预期的lf条带（54,500 M（r）），还含有sf条带（52,000 M（r）），这表明表达系统可以从同一mRNA合成长同工型和短同工型。重组sf形式的动力学特性与大鼠肌肉酶的动力学特性没有差异。相比之下，lf delta ATG PFK-2对其底物显示出更高的Km和更低的Vmax。用针对长同工型的抗体进行免疫印迹分析，在lf和lf delta ATG重组体中均显示出54,500 M（r）的条带，但在大鼠骨骼肌提取物中未检测到条带。在这些提取物中，抗PFK-2/FBPase-2抗体检测到一条52,000的条带和一条次要的54,500 M（r）条带。54,500 M（r）条带被针对L同工酶的抗体识别，表明在骨骼肌中表达了少量的后者。因此，M同工酶与L同工酶的不同之处在于，L同工酶的前32个氨基酸被一个不相关的九肽所取代。
