Abstract The purification procedure for isolating sn-glycerol-3-phosphate dehydrogenase (EC 1. 1. 1. 8) from Saccharomyces cerevisiaewas improved by the introduction of an ion-exchange step. Enzyme yields were doubled and the specific activity was increased as compared to the original procedure. A new value of 42, 000 was obtained for the molecular weight by several denaturing methods. By native gel chromatography the molecular weight appears to be 31, 000 as reported earlier. Michaelis constants were found to be 0. 37mM with dihydroxyacetone phosphate as the variable substrate and 0. 018mM for NADH as the variable substrate.
Prothymosin α is a small, highly acidic, abundant, nuclear, mammalian protein which is essential for cell growth. Our laboratory has recently shown that primate prothymosin α 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 α in vivo by pulse-labeling HeLa cells with [32P]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 [32P]ATP by expansion of the pool; and incubation with inorganic phosphate. Regardless of the method, the phosphate turned over with a half-life of 75–90 min. The ability of cells to phosphorylate old prothymosin α molecules was established by demonstrating equivalent labeling of the protein with [32P]orthophosphate in the presence and absence of cycloheximide. The half-life of the acyl phosphates was also studied in resting and growing NIH3T3 cells, with measured values of 30–35 and 70 min, respectively. Our data suggest that the "activity" of prothymosin α involves the turnover of its acyl phosphates and that it participates in a function common to all nucleated mammalian cells regardless of whether they are quiescent or undergoing rapid proliferation. This is the first measurement of the stability of protein-bound acyl phosphates in vivo. Prothymosin α is a small, highly acidic, abundant, nuclear, mammalian protein which is essential for cell growth. Our laboratory has recently shown that primate prothymosin α 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 α in vivo by pulse-labeling HeLa cells with [32P]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 [32P]ATP by expansion of the pool; and incubation with inorganic phosphate. Regardless of the method, the phosphate turned over with a half-life of 75–90 min. The ability of cells to phosphorylate old prothymosin α molecules was established by demonstrating equivalent labeling of the protein with [32P]orthophosphate in the presence and absence of cycloheximide. The half-life of the acyl phosphates was also studied in resting and growing NIH3T3 cells, with measured values of 30–35 and 70 min, respectively. Our data suggest that the "activity" of prothymosin α involves the turnover of its acyl phosphates and that it participates in a function common to all nucleated mammalian cells regardless of whether they are quiescent or undergoing rapid proliferation. This is the first measurement of the stability of protein-bound acyl phosphates in vivo. Prothymosin α is a small, highly acidic (1Haritos A.A. Blacher R. Stein S. Caldarella J. Horecker B.L. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 343-346Crossref PubMed Scopus (95) Google Scholar, 2Sburlati A.R. Manrow R.E. Berger S.L. Protein Expression Purif. 1990; 1: 184-190Crossref PubMed Scopus (27) Google Scholar), nuclear (3Manrow R.E. Sburlati A.R. Hanover J.A. Berger S.L. J. Biol. Chem. 1991; 266: 3916-3924Abstract Full Text PDF PubMed Google Scholar, 4Palvimo J. Linnala-Kankkunen A. FEBS Lett. 1990; 277: 257-260Crossref PubMed Scopus (15) Google Scholar, 5Watts J.D. Cary P.D. Crane-Robinson C. FEBS Lett. 1989; 245: 17-20Crossref PubMed Scopus (51) Google Scholar, 6Clinton M. Graeve L. El-Dorry H. Rodriguez-Boulan E. Horecker B.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6608-6612Crossref PubMed Scopus (64) Google Scholar) protein found in virtually all mammalian tissues (7Haritos A.A. Tsolas O. Horecker B.L. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1391-1393Crossref PubMed Scopus (120) Google Scholar, 8Economou M. Seferiadis K. Frangou-Lazaridis M. Horecker B.L. Tsolas O. FEBS Lett. 1988; 233: 342-346Crossref PubMed Scopus (27) Google Scholar, 9Clinton M. Frangou-Lazaridis M. Panneerselvam C. Horecker B.L. Arch. Biochem. Biophys. 1989; 269: 256-263Crossref PubMed Scopus (60) Google Scholar, 10Frillingos S. Frangou-Lazaridis M. Seferiadis K. Hulmes J.D. Pan Y.-C.E. Tsolas O. Mol. Cell. Biochem. 1991; 109: 85-94Google Scholar, 11Schmidt G. Werner D. Biochim. Biophys. Acta. 1991; 1088: 442-444Crossref PubMed Scopus (30) Google Scholar). Under conditions of rapid growth, a cultured cell accumulates upwards of 17 million molecules, a number roughly equivalent to that of histone cores (12Sburlati A.R. De La Rosa A. Batey D.W. Kurys G.L. Manrow R.E. Pannell L.K. Martin B.M. Sheeley D.M. Berger S.L. Biochemistry. 1993; 32: 4587-4596Crossref PubMed Scopus (34) Google Scholar). 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 (2Sburlati A.R. Manrow R.E. Berger S.L. Protein Expression Purif. 1990; 1: 184-190Crossref PubMed Scopus (27) Google Scholar, 3Manrow R.E. Sburlati A.R. Hanover J.A. Berger S.L. J. Biol. Chem. 1991; 266: 3916-3924Abstract Full Text PDF PubMed Google Scholar). Its precise function is unknown. Nevertheless, a role in cell proliferation has been proposed based on the following observations: prothymosin α mRNA is plentiful only in rapidly dividing cells (13Eschenfeldt W.H. Berger S.L. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9403-9407Crossref PubMed Google Scholar, 14Gomez-Marquez J. Segade F. Dosil M. Pichel J.G. Bustelo X. Freire M. J. Biol. Chem. 1989; 264: 8451-8454Abstract Full Text PDF PubMed Google Scholar, 15Bustelo X.R. Otero A. Gomez-Marquez J. Freire M. J. Biol. Chem. 1991; 266: 1443-1447Abstract Full Text PDF PubMed Google Scholar); the level of the protein declines 10-fold in cells forced to subsist in stationary phase (12Sburlati A.R. De La Rosa A. Batey D.W. Kurys G.L. Manrow R.E. Pannell L.K. Martin B.M. Sheeley D.M. Berger S.L. Biochemistry. 1993; 32: 4587-4596Crossref PubMed Scopus (34) Google Scholar); the amount of prothymosin α is directly proportional to the proliferative activity of the tissue from which it is isolated (13Eschenfeldt W.H. Berger S.L. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9403-9407Crossref PubMed Google Scholar); and the uptake of antisense oligodeoxyribonucleotides directed toward various locations in prothymosin α mRNA prevents synchronized human myeloma cells from entering mitosis (16Sburlati A.R. Manrow R.E. Berger S.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 253-257Crossref PubMed Scopus (149) Google Scholar). Hence, a deficiency in prothymosin α is associated with failure to complete the cell cycle. Prothymosin α has several unusual features. The human protein, which is almost identical to that of all other mammals (17Eschenfeldt W.H. Manrow R.E. Krug M.S. Berger S.L. J. Biol. Chem. 1989; 264: 7546-7555Abstract Full Text PDF PubMed Google Scholar), has 109 amino acids, nearly 50% of which are acidic (1Haritos A.A. Blacher R. Stein S. Caldarella J. Horecker B.L. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 343-346Crossref PubMed Scopus (95) Google Scholar, 2Sburlati A.R. Manrow R.E. Berger S.L. Protein Expression Purif. 1990; 1: 184-190Crossref PubMed Scopus (27) Google Scholar). A potent nuclear localization signal consisting of five basic amino acids has been identified near the carboxyl terminus (3Manrow R.E. Sburlati A.R. Hanover J.A. Berger S.L. J. Biol. Chem. 1991; 266: 3916-3924Abstract Full Text PDF PubMed Google Scholar, 6Clinton M. Graeve L. El-Dorry H. Rodriguez-Boulan E. Horecker B.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6608-6612Crossref PubMed Scopus (64) Google Scholar), while a second cluster of five basic residues near the amino terminus has no unambiguous role (3Manrow R.E. Sburlati A.R. Hanover J.A. Berger S.L. J. Biol. Chem. 1991; 266: 3916-3924Abstract Full Text PDF PubMed Google Scholar,18Kubota S. Adachi Y. Copeland T.D. Oroszlan S. Eur. J. Biochem. 1995; 233: 48-54Crossref PubMed Scopus (30) Google Scholar). 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 (19Gast K. Damaschun H. Eckert K. Schulze-Forster K. Maurer H.R. Müller-Frohne M. Zirwer D. Czarnecki J. Damaschun G. Biochemistry. 1995; 34: 13211-13218Crossref PubMed Scopus (189) Google Scholar), a conclusion consistent with the presence of only seven widely dispersed hydrophobic residues in the human protein. Prothymosin α does not bind SDS and stains anomalously with silver stain (2Sburlati A.R. Manrow R.E. Berger S.L. Protein Expression Purif. 1990; 1: 184-190Crossref PubMed Scopus (27) Google Scholar). 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 of a phenol extraction, prothymosin α can easily be obtained as a single band in a Coomassie Blue-stained gel. Recently, we have shown that prothymosin α contains phosphorylated glutamic acid residues (20Trumbore M.W. Wang R.-H. Enkemann S.A. Berger S.L. J. Biol. Chem. 1997; 272: 26394-26404Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Acyl phosphates occur in proteins when a glutamic or aspartic acid residue undergoes a posttranslational modification producing a mixed anhydride of a carboxylic acid with phosphoric acid. Such phosphates are highly unusual, energy-rich, and easily transferred to any convenient hydroxyl group. In the prokaryotic world, in yeast, and in plants, they are found as aspartyl phosphates on the response regulator proteins of two component systems (reviewed in Refs. 21Koshland Jr., D.E. Science. 1993; 262: 532Crossref PubMed Scopus (24) Google Scholar and 22Stock J.B. Stock A.M. Mottonen J.M. Nature. 1990; 344: 395-400Crossref PubMed Scopus (486) Google Scholar). These reactive acyl phosphates are acquired from phosphorylated histidine residues on the sensor components; they readily hydrolyze, with half-lives from 6 s to 3.5 min when measured in vitro (23Hess J.F. Oosawa K. Kaplan N. Simon M.I. Cell. 1988; 53: 79-87Abstract Full Text PDF PubMed Scopus (397) Google Scholar, 24Keener J. Kustu S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4976-4980Crossref PubMed Scopus (233) Google Scholar, 25Weiss V. Magasanik B. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8919-8923Crossref PubMed Scopus (186) Google Scholar). In higher eukaryotes, aspartyl phosphates occur as catalytic intermediates of P-type ATPases (reviewed in Ref. 26Pederson P.L. Carafoli E. Trends Biochem. Sci. 1987; 12: 146-150Abstract Full Text PDF Scopus (823) Google Scholar) with ATP as the physiological donor. Glutamyl phosphate has been identified on only one mammalian protein (prothymosin α), one amphibian protein (nucleoplasmin from Xenopus laevis), 1S. A. Enkemann, M. W. Trumbore, and S. L. Berger, manuscript in preparation. and one avian protein (bone collagen of the chicken (27Cohen-Solal L. Cohen-Solal M. Glimcher M.J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4327-4330Crossref PubMed Scopus (16) Google Scholar)). Little is known about the origins of glutamyl phosphates, and virtually nothing is understood about their contribution to cellular metabolism. To expand our grasp of the function of prothymosin α, we have determined the half-life of its glutamyl phosphates in vivo. To do so, we made use of the properties established by Trumboreet al. (20Trumbore M.W. Wang R.-H. Enkemann S.A. Berger S.L. J. Biol. Chem. 1997; 272: 26394-26404Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), who showed that these phosphates are unstable in cell lysates and undetectable, except for a small fraction that apparently transfer to serine 1 of the human protein (12Sburlati A.R. De La Rosa A. Batey D.W. Kurys G.L. Manrow R.E. Pannell L.K. Martin B.M. Sheeley D.M. Berger S.L. Biochemistry. 1993; 32: 4587-4596Crossref PubMed Scopus (34) Google Scholar) or to unspecified threonine residues located within the first 14 amino acids of the murine protein (28Barcia M.G. Castro J.M. Jullien C.D. González C.G. Freire M. FEBS Lett. 1992; 312: 152-156Crossref PubMed Scopus (15) Google Scholar). The degree of accumulation of the stable component depends on the conditions experienced by prothymosin α at the moment of cell lysis. Here we demonstrate that the fortuitous, albeit inefficient, migration of the labile glutamyl phosphate to positions of stability on serine or threonine on the same molecule can form the basis of a quantitative assay. When this technique was used together with three independent pulse-chase methods, we found that both new and old prothymosin α molecules were indistinguishable targets for phosphorylation on glutamic acid and that all acyl phosphates were rapidly lost in vivo with half-lives slightly in excess of 1 h. We believe that turnover of prothymosin α's phosphates might reflect a role that is continuously required by all cells, regardless of their metabolic state, and we present evidence for associating prothymosin α with processes required both for the maintenance of cells and for their growth. HeLa S3 cells, African green monkey kidney cells (COS-1), and NIH3T3 cells were grown in Dulbecco's modified Eagle's medium (DMEM) 2The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride-HCl; DEAE, diethylaminoethyl; MES, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PTMA, prothymosin α. from Life Technologies, Inc. (catalog number 15240-039) or Biofluids (catalog number 172; Rockville, MD). Human and monkey cells were cultured with 10% heat-inactivated fetal calf serum (Life Technologies, Inc. or HyClone (Logan, UT)), whereas the murine cells grew in 10% calf serum. All culture fluids contained 2 mm glutamine, 90 units/ml of penicillin, 90 μg/ml of streptomycin, and 0.22 μg/ml amphotericin B, and all cells were maintained in an environment of 5% CO2 at 37 °C. Quiescent NIH3T3 cells were obtained by incubating cells for 48 h in 0.25% calf serum. Cells were harvested from culture flasks by washing with Puck's saline and treating with 0.05% trypsin in Hanks' balanced salts containing 0.53 mm EDTA. A two-step approach was employed for relating 32P found in isolated prothymosin α on serine or threonine to the amount of glutamyl phosphate existing on the protein inside the cell. First, the relationship between the amount of 32P recovered stably on prothymosin α and the amount of prothymosin α was determined by transfecting 106 COS cells in 60-mm dishes with 0–3 μg of pRSV PTMA, a vector containing the prothymosin α gene (29Mol P.C. Wang R.-H. Batey D.W. Lee L.A. Dang C.V. Berger S.L. Mol. Cell. Biol. 1995; 15: 6999-7009Crossref PubMed Google Scholar), using the DEAE-dextran method (30Cullen B.R. Methods Enzymol. 1987; 152: 684-704Crossref PubMed Scopus (662) Google Scholar). These cells, expressing varying amounts of prothymosin α, were labeled 44 h post-transfection with 100 μCi/ml of carrier-free [32P]orthophosphoric acid for 4 h. Prothymosin α was isolated by means of a phenol extraction (2Sburlati A.R. Manrow R.E. Berger S.L. Protein Expression Purif. 1990; 1: 184-190Crossref PubMed Scopus (27) Google Scholar), further purified electrophoretically, and quantified both as a Coomassie Blue-stained band of protein and as a radioactive band on an autoradiograph (see below). Direct comparisons were carried out only with samples analyzed on the same gel and subjected to the same conditions of staining and autoradiography. The relationship between the amount of prothymosin α and the amount of glutamyl phosphate was determined by resuspending ∼4–10 × 107 COS cells in 2 ml of dimethyl sulfoxide containing 30 mm [3H]NaBH4 (NEN Life Science Products; specific activity, 1000 mCi/mmol) at a specific activity of 47 mCi/mmol (low specific activity method) and allowing the reaction to proceed for 10 min at room temperature (20Trumbore M.W. Wang R.-H. Enkemann S.A. Berger S.L. J. Biol. Chem. 1997; 272: 26394-26404Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 31Degani C. Boyer P.D. J. Biol. Chem. 1973; 248: 8222-8226Abstract Full Text PDF PubMed Google Scholar). Alternatively, COS cells were lysed in 1 ml of 5 mCi of [3H]NaBH4 (222 mCi/mmol) in dimethyl sulfoxide (high specific activity method), and reactions were carried out for 1–3 h. In both cases, the insoluble material was washed in dimethyl sulfoxide, centrifuged, and suspended in water. Prothymosin α was recovered from the soluble component with the aid of phenol (2Sburlati A.R. Manrow R.E. Berger S.L. Protein Expression Purif. 1990; 1: 184-190Crossref PubMed Scopus (27) Google Scholar), and purified using high pressure liquid chromatography (20Trumbore M.W. Wang R.-H. Enkemann S.A. Berger S.L. J. Biol. Chem. 1997; 272: 26394-26404Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). As a result of the borohydride reaction, tritium is incorporated into hydroxynorvaline, which is recovered as [3H]proline after acid hydrolysis, derivatization, and amino acid analysis (20Trumbore M.W. Wang R.-H. Enkemann S.A. Berger S.L. J. Biol. Chem. 1997; 272: 26394-26404Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 32Cohen-Solal L. Blouquit Y. Cohen-Solal M. Glimcher M.J. Anal. Biochem. 1985; 151: 82-87Crossref PubMed Scopus (5) Google Scholar). The reaction is specific, but even in well defined solutions yields are low (31Degani C. Boyer P.D. J. Biol. Chem. 1973; 248: 8222-8226Abstract Full Text PDF PubMed Google Scholar), and in crude solutions yields are quite poor (20Trumbore M.W. Wang R.-H. Enkemann S.A. Berger S.L. J. Biol. Chem. 1997; 272: 26394-26404Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Radioactivity incorporated into prothymosin α was measured in a Packard Tri-Carb model 1500 liquid scintillation analyzer in Hydrofluor (National Diagnostics), and protein was quantified from the amino acid analysis. The two sets of data, 32P and 3H found in measured quantities of prothymosin α, form the basis of the assay. Cell volume was ascertained using two techniques. In the first case, the volume occupied by 1.9 × 106 or 3.8 × 106HeLa cells in a total volume of 20 or 25 μl of medium was measured in a calibrated capillary tube. An average value of 1.65 μl was obtained. The second method is a dilution assay; 106 cells in quadruplicate were allowed to take up 1 or 2 μCi/ml [32P]orthophosphoric acid for 1.5 h. They were washed free of external radioactivity, resuspended in 1 ml of phosphate-buffered saline (PBS), and used for a determination of Cerenkov radiation to measure the amount of label imbibed. The cells were then removed from scintillation vials and recovered by centrifugation. The pellets from two samples were resuspended in 10 μl of PBS, while the pellets from the remaining two samples were lysed with 10 μl of 10 n NaOH. It should be understood that, at this point in the experiment, the total volume is 10 μl of PBS plus the volume of the cell or cell lysate and that the amount of radioactivity, but not its concentration, is known. To determine the concentration, aliquots of 2 and 4 μl of the cell suspension or lysate were subjected to scintillation counting. If the cells had contributed nothing to the volume, these samples would have contained 20 or 40%, respectively, of the initial radioactivity imbibed by the cells. However, because the intact cells or the lysed cells do occupy space, their volume can be determined by measuring the reduction in radioactivity caused by the dilution of the buffer or base by the cells or their contents. These measurements resulted in average values of 1.49 and 1.52 μl/106 cells for intact and lysed cells, respectively; they were essential for determining the degree of equilibration of ATP across a permeabilized plasma membrane (see below). The method of Lee et al. (33Lee J.J. Calzone F.J. Davidson E.H. Dev. Biol. 1992; 149: 415-431Crossref PubMed Scopus (18) Google Scholar), based on the work of Sasvári-Székely et al. (34Sasvári-Székely M. Vitez M. Staub M. Antoni F. Biochim. Biophys. Acta. 1975; 395: 221-228Crossref PubMed Scopus (17) Google Scholar), was used to determine the specific activity of the ATP pool of HeLa cells. Briefly, the technique makes use of the fact that the number of AMP residues equals the number of UMP residues when RNA is polymerized using poly(dA-dT) as the template. The ATP, which is synthesized in vivo by cells in the presence of [3H]adenosine, is supplied by the cell extract, whereas UTP of known specific activity is provided exogenously in amounts which are large relative to the amount of endogenous UTP in the same cell extract. Under these conditions, all of the ATP in the extract is used to generate polymer, with no free ATP remaining in the reaction mix. More specifically, an aliquot of 1% of a neutralized perchloric acid extract from 5 × 106cells labeled for 2 h with 50 μCi/ml [2,8-3H]adenosine (specific activity, 30.6 Ci/mmol; NEN Life Science Products) was used as a source of ATP for the polymerization of RNA in a 50-μl reaction containing 0.2 μg of poly(dA-dT) as template, 0.1 μCi of [U-14C]UTP (609 mCi/mmol, NEN Life Science Products) 0–5 nmol of nonradioactive UTP, and 3 units of RNA polymerase holoenzyme from Escherichia coli. The bacterial polymerase initiates synthesis of RNA at any location without the need for specific promoter sequences. Synthesis terminates when the limiting nucleotide, in this case ATP supplied by the extract, is exhausted. The specific activity of ATP given by the expression, (dpm in 3H/dpm in 14C) × specific activity of UTP, was 6.3 × 105 cpm/nmol. Thus, 106 cells with an average cell volume of 1.6 μl contained 1.4 nmol of ATP at a concentration of 0.88 mm. Conditions for the use of digitonin were selected after the evaluation of several parameters. Each dish of 5–6 × 106 HeLa S3 cells in a variation of a solution termed "cytomix" (35van den Hoff M.J.B. Moorman A.R.M. Lamers W.H. Nucleic Acids Res. 1992; 20: 2902Crossref PubMed Scopus (385) Google Scholar) (120 mmKCl, 150 mm CaCl2, 10 mm potassium phosphate at pH 7.6, 25 mm HEPES-KOH at pH 7.6, 2 mm EGTA, and 5 mm MgCl2) or complete DMEM was treated with 20–40 μg/ml digitonin (Sigma) at either 0 or 37 °C for periods ranging from 5 to 30 min. In some experiments, the cells were labeled with [3H]glutamic acid before initiating the permeabilization treatment. After removal of digitonin by gentle aspiration and washing with Puck's saline, the chase conditions were simulated by incubating the cells in either [γ-32P]ATP or [γ-32P]UTP in either cytomix or DMEM without serum at 37 °C for 0.5–4 h; here radioactive ATP or UTP served only as a marker for measuring the rate at which permeabilized cells could imbibe external substances. Optimal conditions appeared to be treatment with 5 ml of 20 μg/ml of digitonin for 10 min in cytomix on ice. Such conditions allowed labeled ATP, placed outside the cell in either DMEM or cytomix, to equilibrate with the interior in less than 1 h. For pulse-chase experiments, HeLa cells at 37 °C were labeled in 60-mm dishes with 100 μCi/ml of [32P]orthophosphoric acid (catalog number NEX-053, NEN Life Science Products) for 4 h in 2 ml of phosphate-free DMEM (Life Technologies, Inc.) containing the additions noted above. The cells were washed sequentially with warm Puck's saline followed by cytomix at 37 °C, permeabilized using the optimized conditions noted, and chased in 5 ml of DMEM (without serum and additives) containing 10 mm ATP and 10 mmMgCl2 at pH 7.0. Our methods were developed from published procedures (36Messing Eriksson A. Zetterqvist M.-A. Lundgren B. Andersson K. Beije B. DePierre J.W. Eur. J. Biochem. 1991; 198: 471-476Crossref PubMed Scopus (35) Google Scholar). Permeabilized cells were maintained at 37 °C in the incubator during the chase. A separate dish was used for each time point. At the stated time, the cells were harvested by aspirating the ATP-containing chase medium and by immersing them, still in the dish, in 1 ml of lysis buffer (10 mm Tris-HCl at pH 7.5, 5 mm EDTA, 12% sucrose, 1% Triton X-100, and 1 mm 4-(2-aminoethyl)-benzenesulfonyl fluoride-HCl (AEBSF, Sigma)). Cells and debris were scraped free with a cell lifter (Costar), transferred to an Eppendorf tube, cooled in ice, mixed vigorously with a vortex mixer, and centrifuged at 4 °C for 10 min at 15,000 rpm in a Tomy model MTX-150 centrifuge. The supernatant fluid was transferred to a 15-ml polypropylene tube, brought to 4 ml with cold filter-sterilized water, and extracted with phenol as described below. It is important to note that prothymosin α in its entirety leaks out of the nuclei when digitonin-treated cells, as well as electroporated or normal cells, are lysed with nonionic detergents (2Sburlati A.R. Manrow R.E. Berger S.L. Protein Expression Purif. 1990; 1: 184-190Crossref PubMed Scopus (27) Google Scholar) and that prothymosin α yields in all cases are virtually identical. HeLa S3 Cells were electroporated in the presence of [32P]ATP in the medium to determine the degree to which molecules outside the cell had equilibrated with the internal environment. An aliquot consisting of 1 ml of cells was placed in a disposable electroporation chamber with a 0.4-cm gap (catalog number 11601-028, Life Technologies, Inc.); the chamber was cooled in ice for 10 min and shocked with an electrical discharge of 875 V/cm and 330 microfarads in a Life Technologies, Inc. Cell-Porator at room temperature with the low resistance setting. The amount of a known concentration of [32P]ATP taken up by the cells, 70–80% of which survived, was measured and found to represent 70% equilibration. For pulse-chase experiments, HeLa S3 cells were labeled in 175 cm2 flasks under the conditions noted above, trypsinized, washed in Puck's saline, and resuspended at 5 × 106cells/ml in warm cytomix to which 20 mm ATP and 20 mm MgCl2 at pH 7.0 had been added. The chase was initiated by electroporating as noted above. These conditions resulted in a 14–15-fold instantaneous decrease in the specific activity of intracellular ATP. Following electroporation, the chamber was again cooled to 4 °C for 10 min and brought to room temperature for 10 min. Cells recovered from each chamber were washed thoroughly with Puck's saline, centrifuged free of the wash solution, resuspended in 5 ml of complete DMEM (DMEM containing serum and additives), seeded into a 60-mm dish, and maintained in an incubator as described earlier. The unphysiological concentration of ATP achieved inside the cells after electroporation did not significantly affect the viability of the cells for the duration of the chase. For the harvest, the cells in each dish were scraped free with a cell lifter, transferred to a centrifuge tube, cooled to 4 °C, washed with PBS, recovered as a pellet, and disrupted in 1 ml of cold lysis buffer. After removing the nuclei by centrifugation, the supernatant fluids were transferred to a clean polypropylene tube, diluted with water as detailed above for digitonin-treated cells, and subjected to a phenol extraction (see below). HeLa S3 cells were labeled in flasks in phosphate-free complete DMEM for 4 h as indicated for the electroporated cells. To initiate the chase, the cells were washed free of the labeling solution, trypsinized, washed again, resuspended in complete DMEM containing 40 mm sodium phosphate at pH 7.0, and seeded into dishes at a concentration of 6 × 106 cells/dish. At the end of the chase, the cells were recovered and lysed using the methods for electroporated cells. Supernatant fluids from lysed cells containing virtually all of the prothymosin α in the cell in 4 ml were made 0.5% in SDS and extracted at 65 °C with 2 ml of phenol saturated with 2 × ACE buffer (20 mm sodium acetate at pH 5.1, 100 mm NaCl, and 6 mm disodium EDTA). The aqueous phase was recovered by centrifugation and extracted twice with phenol using the same methods. The final aqueous phase was precipitated with 4 volumes of acetone in dry ice for 1 h or overnight at −20 °C, and the sample was recovered by centrifugation at 16,000 × g for 30 min, washed in 80% acetone, 20% 20 mm Tris-HCl at pH 7.5, and dissolved in 400 μl of 20 mm Tris-HCl at pH 7.5. RNA was destroyed by adding 40 μg of pancreatic ribonuclease A and incubating the sample at 37 °C for 20 min. Ribonuclease was removed by two additional phenol extractions. The sample was recovered by precipitation in acetone, and the pellet was dissolved in water and analyzed in an 18% polyacrylamide gel (catalog number EC6506, Novex). A general description of our methods for purification of the protein and for electrophoresis has appeared (2Sburlati A.R. Manrow R.E. Berger S.L. Protein Expression Purif. 1990; 1: 184-190Crossref PubMed Scopus (27) Google Scholar, 12Sburlati A.R. De La Rosa A. Batey D.W. Kurys G.L. Manrow R.E. Pannell L.K. Martin B.M. Sheeley D.M. Berger S.L. Biochemistry. 1993; 32: 4587-4596Crossref PubMed Scopus (34) Google Scholar). Prothymosin α was visualized by sta
Prothymosin alpha gene expression accompanies growth of all mammalian cells. The protein, which is abundant, exceedingly acidic, and localized to the nucleus, is further distinguished by the presence of clustered phosphorylated glutamic acid residues (Trumbore et al., 1997, J Biol Chem 272:26394-26404). These glutamyl phosphates are energy rich and unstable in vivo and in vitro (Wang et al., 1997, J Biol Chem 272:26405-26412). To understand the function of prothymosin alpha in greater detail, the turnover of its phosphates was examined in metabolically manipulated cells. Phosphate half-lives in growing, mock transfected, and vector-transfected COS cells were compared with the half-life in cells transfected with the prothymosin alpha gene to determine the fate of the predominantly ectopic phosphorylated protein. The values obtained--72-75 min in cells with normal levels of the protein, but 118 min in cells with surplus prothymosin alpha--led us to conclude that underutilized phosphates persist whereas functioning phosphates disperse. Cell-cycle-specific differences in the half-lives were observed in NIH3T3 cells: 72 min while cycling, 83 or 89 min during arrest in or progression through S phase, but 174 min during M-phase arrest. In the presence of actinomycin D, the value was about 145 min regardless of whether cells were quiescent or growing. In these experiments, reduced utilization of prothymosin alpha's glutamyl phosphates, signaled by an increase in their half-lives, accompanied the attenuation or abolition of transcription. Our data suggest that prothymosin alpha fuels an energy-requiring step in the production, processing, or export of RNA.
A rapid and sensitive method was developed for the preparative separation of laminin subunits. Laminin was extracted and purified from mouse EHS sarcoma. On SDS-PAGE, the reduced and carboxymethylated molecule separated into two components corresponding to molecular weights of about 400 KDa (subunit A) and 200 KDa (subunit B). These two subunits were preparatively separated using heparin-agarose affinity chromatography. The larger subunit quantitatively adhered to the affinity column while the smaller one did not adhere. Amino acid analyses of the separated subunits showed distinct differences. Subunit B was further resolved into two distinct polypeptides of 200 KDa, B1 and B2, by means of reverse-phase HPLC. Although the amino acid compositions of B1 and B2 were very similar, the peptide maps generated by digestion of the B1 and B2 chains with Staphylococcus aureus V8 protease or by cyanogen bromide showed B1 and B2 to differ from each other. Thus, at least three different polypeptide subunits are present in this laminin and probably arise from separate gene origins.These studies provide a basis for the subsequent localization and analysis of the specialized structural and functional domains of laminin.
This study evaluated a unique formulation of lidocaine 4% in an emollient aerosol foam microemulsion system to facilitate rapid delivery of the active ingredient and reduce pain associated with cosmetic dermatologic laser treatment.This was a noncontrolled, open-label, paired, comparison study.Private practice dermatology clinic.Ten patients undergoing various cosmetic laser treatments, 18 years of age or older and considered clinically appropriate for study participation.Primary endpoints were patient and clinician assessments of procedural pain intensity for the treated and untreated areas. Ratings were recorded on a visual analog scale ranging from "no pain at all" to "the most intense pain imaginable." Secondary study endpoints included clinician and patient subjective assessments of the lidocaine 4% foam.Mean patient and clinician ratings of pain were significantly lower for areas treated with the lidocaine 4% foam compared with pain ratings for untreated areas. No adverse events were reported. Clinician's mean ratings for ease of application and overall satisfaction were favorable.The results from this pilot, 10-patient, open-label study suggest that the lidocaine 4% foam may be acceptable to both patients and clinicians for the safe and effective reduction of pain associated with cosmetic dermatologic laser procedures. However, a blinded, placebo-controlled study of a larger population is needed to confirm these preliminary results.
