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2) Immediately upon cell lysis, the pH stability curves of metabolically labeled native [ 32 P]prothymosin ␣ or a [ 32 P]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 ␣'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 ␣ decreased 8-fold when lysates were treated with borohydride; 2) the reagent caused 4 -8 glutamic acid residues/molecule to vanish; 3) using [ 3 H]NaBH 4 , label was introduced into proline, a product derived from reductive cleavage of phosphoglutamate; and 4) [ 3 H]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 ␣ is energy-rich by virtue of stoichiometric amounts of glutamyl phosphate.Prothymosin ␣ is a highly unusual protein with an unfortunate name. The protein is neither a precursor for processed polypeptides nor specifically associated with the thymus nor a member of a family with ␤ or ␥ homologues (1-3). Instead, it is probably the most acidic naturally occurring polypeptide in the eukaryotic world, with 54 carboxyl groups in 109 amino acids, resulting in an isoelectric point at or below pH 3.5 (1, 2, 4). The mRNA for prothymosin ␣ is distributed ubiquitously among mammalian nucleated cells and tissues (1). The protein possesses a potent nuclear localization signal and is present in amounts equivalent to those of histone H1 (5, 6). Because the amount of prothymosin ␣ mRNA (and presumably protein) found in a cell is directly proportional to cell growth, the protein is believed to play a role in cell proliferation (1). This idea was reinforced by the observation that synchronized human myeloma cells, in the presence of antisense oligodeoxyribonucleotides directed at prothymosin ␣ mRNA, were unable to divide while detectable amounts of the antisense oligonucleotides remained inside the cell (7). There are now many examples of a link between prothymosin ␣ and growth in systems as diverse as developing mouse embryos (8); normal, mitogenstimulated, and malignant lymphocytes (9, 10); and regenerating liver (10). There are also positive ...

Prothymosin ␣ is a small, highly acidic, abundant, nuclear, mammalian protein which is essential for cell growth. Our laboratory has recently shown that primate prothymosin ␣ contains stoichiometric amounts of phosphate on the glutamyl groups of the protein and that in vitro the phosphate undergoes rapid hydrolysis or transfer to a nearby serine residue. Here an assay for the presence of acyl phosphates in vivo has been developed by measuring stable phosphoserine and phosphothreonine in vitro. The assay was used to determine the half-life of the acyl phosphates on prothymosin ␣ in vivo by pulse-labeling HeLa cells with [ 32 P]orthophosphate and chasing using three different techniques: permeabilization with digitonin to allow extracellular ATP to equilibrate with the intracellular pool; electroporation in the presence of ATP to reduce the specific activity of Prothymosin ␣ is a small, highly acidic (1, 2), nuclear (3-6) protein found in virtually all mammalian tissues (7-11). Under conditions of rapid growth, a cultured cell accumulates upwards of 17 million molecules, a number roughly equivalent to that of histone cores (12). When cells are disrupted with detergents, the protein readily leaks out of the nucleus, suggesting that stable interactions with nucleosomes or with the nuclear matrix are not an inherent part of its activity (2, 3). Its precise function is unknown. Nevertheless, a role in cell proliferation has been proposed based on the following observations: prothymosin ␣ mRNA is plentiful only in rapidly dividing cells (13)(14)(15); the level of the protein declines 10-fold in cells forced to subsist in stationary phase (12); the amount of prothymosin ␣ is directly proportional to the proliferative activity of the tissue from which it is isolated (13); and the uptake of antisense oligodeoxyribonucleotides directed toward various locations in prothymosin ␣ mRNA prevents synchronized human myeloma cells from entering mitosis (16). Hence, a deficiency in prothymosin ␣ is associated with failure to complete the cell cycle.Prothymosin ␣ has several unusual features. The human protein, which is almost identical to that of all other mammals (17), has 109 amino acids, nearly 50% of which are acidic (1, 2). A potent nuclear localization signal consisting of five basic amino acids has been identified near the carboxyl terminus (3, 6), while a second cluster of five basic residues near the amino terminus has no unambiguous role (3,18). The absence of all aromatic residues renders the protein transparent at 280 nm; it also lacks methionine, cysteine, and histidine. Based on biophysical data, it is believed to have an unfolded structure (19), a conclusion consistent with the presence of only seven widely dispersed hydrophobic residues in the human protein. Prothymosin ␣ does not bind SDS and stains anomalously with silver stain (2). The protein and the peptides derived from it exhibit poor immunogenicity. However, due to yet another aberrant property, the ability to partition quantitatively into the aqueous phase ...

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Amiodarone is a drug used in the treatment of cardiac arrhythmias and is believed to have a persistent interaction with cellular membranes. This study sought to examine the structure and location of amiodarone in a membrane bilayer. Amiodarone has a high membrane partition coefficient on the order of 10(6). Small angle x-ray diffraction was used to determine the position of the iodine atoms of amiodarone in dipalmitoylphosphatidylcholine (DPPC) lipid bilayers under conditions of low temperature and hydration where the DPPC bilayer is in the gel state. The time-averaged position of the iodine atoms was determined to be approximately 6 A from the center (terminal methyl region) of the lipid bilayer. A dielectric constant of kappa = 2, which approximates that of the bilayer hydrocarbon core region, was used in calculating a minimum energy structure for membrane-bound amiodarone. This calculated structure when compared with the crystal structure of amiodarone demonstrated that amiodarone could assume a conformation in the bilayer significantly different from that in the crystal. The results reported here are an attempt to correlate the position of a membrane-active drug in a lipid bilayer with its time-averaged conformation. This type of analysis promises to be of great use in the design of drugs with greater potency and higher specificity.

Our study examines the effect of apoptosis on prothymosin alpha, an abundant, nuclear protein intimately involved with proliferation of all mammalian cells. When HeLa cells were treated with actinomycin D, with etoposide, or with staurosporine following synchronization with hydroxyurea, they underwent apoptosis based on several specific criteria, including fragmentation of DNA and activation of specific caspases. Similarly treated NIH3T3 cells arrested and displayed no indicators of apoptosis. In HeLa, but not in NIH3T3 cells, prothymosin alpha levels declined precipitously and a truncated version of the protein was formed. The following observations implicate caspase activity: (1) The truncated polypeptide arose only in the treated HeLa cell cultures. (2) The appearance of the truncated polypeptide coincided with the activation of caspase 3 and the cleavage of poly(ADP-ribose) polymerase, a known caspase substrate. (3) Carbobenzoxy-DEVD-fluoromethylketone, a cell-permeable caspase 3 inhibitor, blocked cleavage and degradation of prothymosin alpha. (4) The same inhibitor, when added to mixed extracts of apoptotic and normal cells, prevented cleavage of intact prothymosin alpha. (5) Recombinant caspase 3 and, to a much lesser extent, caspase 7 truncated purified prothymosin alpha. (6) In HeLa cells, cleavage occurred at three overlapping caspase 3-like sites with the consensus sequence D-X-X-D and released 10 to 14 residues from the carboxyl terminus, including the core nuclear localization signal. Two immediate consequences of the cleavage were observed: truncated prothymosin alpha was no longer confined to the nucleus and it was deficient in phosphate. These data suggest that the disabling of prothymosin alpha is a significant event in apoptosis. J. Cell. Physiol. 182:256-268, 2000. Published 2000 Wiley-Liss, Inc.

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