IDENTIFICATION OF THE MAJOR REGULATORY PHOSPHORYLATION SITE IN SUCROSE-PHOSPHATE SYNTHASE

被引:72
作者
MCMICHAEL, RW
KLEIN, RR
SALVUCCI, ME
HUBER, SC
机构
[1] N CAROLINA STATE UNIV, USDA ARS, RALEIGH, NC 27695 USA
[2] UNIV KENTUCKY, USDA ARS, LEXINGTON, KY 40546 USA
[3] UNIV KENTUCKY, DEPT AGRON, LEXINGTON, KY 40546 USA
[4] N CAROLINA STATE UNIV, DEPT CROP SCI, RALEIGH, NC 27695 USA
关键词
D O I
10.1006/abbi.1993.1586
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 [γ-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. © 1993 Academic Press, Inc.
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页码:248 / 252
页数:5
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