McMichael R W, Klein R R, Salvucci M E, Huber S C
U.S. Department of Agriculture, North Carolina State University, Raleigh 27695-7631.
Arch Biochem Biophys. 1993 Dec;307(2):248-52. doi: 10.1006/abbi.1993.1586.
Sucrose-phosphate synthase (SPS; EC 2.4.1.14) is regulated in part by reversible protein phosphorylation. When dephospho-SPS is partially purified from illuminated spinach leaves and incubated with [gamma-32P]ATP the enzyme is phosphorylated by a copurifying protein kinase. In this report, 32P-phosphopeptides from tryptic digests of in vitro phosphorylated SPS were purified by metal-ion affinity chromatography and reversed-phase high-performance liquid chromatography. Three distinct 32P-phosphopeptides were resolved. Edman sequencing of the major phosphopeptide (which contained > 80% of the total 32P) identified the amino acid sequence as Ile-Ser-Ser(P)-Val-Glu-Met-Met-Asp-Asn-Trp-Ala-Asn-Thr-Phe-Lys. This sequence corresponds to residues 156 to 170 of the deduced amino acid sequence of spinach SPS [Klein, R. R., Crafts-Brandner, S. J., and Salvucci, M. E. (1993) Planta 190, 498-510, and Sonnewald, U., Quick, W. P., MacRae, E., Krause, K.-P., and Stitt, M. (1993) Planta 189, 174-181]. Identification of the phosphoseryl residue was accomplished by manual Edman sequencing. The two other phosphopeptides, which each contained less than 10% of the total 32P, were not sequenced. An Escherichia coli expressed, 26-kDa fragment of SPS which contains the major phosphorylation site was a substrate for the protein kinase which copurifies with SPS. Two-dimensional peptide mapping analysis of this fragment showed the major phosphopeptide was present but not the other site(s), suggesting that other peptides are derived from a site other than Ser158. These results provide additional indirect evidence for the presence of multiple phosphorylation sites in SPS.
蔗糖磷酸合酶（SPS；EC 2.4.1.14）部分受可逆蛋白质磷酸化调节。当从光照下的菠菜叶片中部分纯化去磷酸化的SPS，并与[γ-32P]ATP一起孵育时，该酶会被一种共纯化的蛋白激酶磷酸化。在本报告中，通过金属离子亲和色谱和反相高效液相色谱法对体外磷酸化的SPS胰蛋白酶消化产物中的32P-磷酸肽进行了纯化。分离出了三种不同的32P-磷酸肽。对主要磷酸肽（其包含总32P的80%以上）进行的埃德曼测序确定其氨基酸序列为Ile-Ser-Ser(P)-Val-Glu-Met-Met-Asp-Asn-Trp-Ala-Asn-Thr-Phe-Lys。该序列对应于菠菜SPS推导氨基酸序列的第156至170位残基[克莱因，R.R.，克拉夫茨-布兰德纳，S.J.，和萨尔武奇，M.E.（1993年）《植物》190，498 - 510，以及松内瓦尔德，U.，奎克，W.P.，麦克雷，E.，克劳斯，K.-P.，和斯蒂特，M.（1993年）《植物》189，174 - 181]。通过手动埃德曼测序确定了磷酸丝氨酸残基。另外两个磷酸肽，每个都包含不到总32P的10%，未进行测序。大肠杆菌表达的包含主要磷酸化位点的26 kDa SPS片段是与SPS共纯化的蛋白激酶的底物。对该片段的二维肽图谱分析表明存在主要磷酸肽，但不存在其他位点，这表明其他肽来自Ser158以外的位点。这些结果为SPS中存在多个磷酸化位点提供了额外的间接证据。
