thymosin and sodium-borohydride

Topics: Adenosine Triphosphatases; Amino Acids; Bacterial Proteins; Biomarkers; Borohydrides; Carbon Radioisotopes; Collagen; Glutamate-Ammonia Ligase; Glutamic Acid; Membrane Proteins; Methyl-Accepting Chemotaxis Proteins; Myelin Proteolipid Protein; Oxidation-Reduction; Peptide Synthases; Phosphates; Phosphorylation; Phosphotransferases; Phosphotransferases (Phosphomutases); Protein Precursors; Pyrrolidonecarboxylic Acid; Thiolester Hydrolases; Thymosin; Ubiquitin Thiolesterase

Human and monkey prothymosin alpha contain activated carbonyl groups on glutamic acid residues. Three lines of evidence indicate the existence of unusual phosphates. 1) Prothymosin alpha continued to be metabolically labeled with [32P]orthophosphoric acid despite a mutation at Ser1, the sole site of phosphate in purified bovine prothymosin alpha (Sburlati, A. R., De La Rosa, A., Batey, D. W., Kurys, G. L., Manrow, R. E., Pannell, L. K., Martin, B. M., Sheeley, D. M., and Berger, S. L. (1993) Biochemistry 32, 4587-4596). 2) Immediately upon cell lysis, the pH stability curves of metabolically labeled native [32P]prothymosin alpha or a [32P]histidine-tagged variant resembled the pH stability curve of acetyl phosphate. 3) After a brief incubation at pH 7, these curves changed from a pattern diagnostic for an acyl phosphate to that characteristic of a serine or threonine phosphate, an observation consistent with transfer of phosphate in vitro. Our data indicate that most of prothymosin alpha's phosphates are subject instantaneously to hydrolysis, based on the observation that greater than 90% of the phosphate initially found at pH 7 disappeared at the extremes of pH. Rapid loss of phosphate was not affected by the presence of phosphatase inhibitors including 50 mM sodium fluoride, 1 mM okadaic acid, and 0.5 mM calyculin A. The amount of phosphate missing could not be ascertained, but the trifling amount recovered on Ser or Thr depended heavily on conditions favoring the transient survival of labile phosphate. Further analysis using COS cells lysed in the presence of sodium borohydride showed that: 1) phosphate recovered on prothymosin alpha decreased 8-fold when lysates were treated with borohydride; 2) the reagent caused 4-8 glutamic acid residues/molecule to vanish; 3) using [3H]NaBH4, label was introduced into proline, a product derived from reductive cleavage of phosphoglutamate; and 4) [3H]proline was localized almost exclusively to a peptide with pronounced homology to the histone binding site of nucleoplasmin, a chromatin remodeling protein found in Xenopus laevis. Our data demonstrate that prothymosin alpha is energy-rich by virtue of stoichiometric amounts of glutamyl phosphate.

Topics: Amino Acid Sequence; Animals; Borohydrides; Cattle; Cloning, Molecular; COS Cells; Glutamic Acid; HeLa Cells; Humans; Hydrogen-Ion Concentration; Molecular Sequence Data; Phosphorylation; Protein Precursors; Thymosin