200
`
`PROTEIN ACY LA TIONS/DEACYLA TIO NS
`
`[11)
`
`UT Assay Procedure. The mixture of 5 µ,l of UT sample is allowed to
`incubate for a few minutes at 26°, then 5 µ,I of E. coli PnA (0 .4 unit/ml) is
`added to initiate the uridylylation reaction. After a suitable incubation
`period, 70 µ,I of the deadenyJylation reaction mixture preequilibrated at
`26° is added to determine the amount of P1m formed. After 5 min of
`incubation, I ml of the y-glutamyltransferase mixture is added, and after
`another 4 min, the reaction is stopped by adding 1 ml of the stop mixture.
`The resulting solution is centrifuged, and its absorbance at 540 nm is
`recorded. The control tube is prepared with the UT sample by a similar
`procedure as the sample tube with the exception that the UT assay mix(cid:173)
`ture is substituted by the UT assay blank mixture.
`The procedures described above can be used to measure Puo and UT
`activity in S. typhimurium, 43 P. putida, 44 and K . aero genes. 45 This is
`possible because P1m from those organisms stimulates the deadenylyla(cid:173)
`tion reaction catalyzed by E.coli ATd and because E. coli PnA is uridylyl(cid:173)
`ated by UT activities from various organisms.
`
`[11] Determination and Occurrence of
`Tyrosine O-Sulf ate in Proteins
`By WIELAND 8. HUTTNER
`
`The term ' ' protein sulfa ti on' ' describes the modification of proteins by
`covalent attachment of sulfate. One can distinguish between two principal
`types of protein sulfation. The first type is the covalent linkage of sulfate
`to amino acid residues of proteins, i.e., the primary modification of the
`polypeptide chain itself. Only one amino acid, tyrosine, has so far been
`shown to undergo this modification. The second type is the covalent
`linkage of sulfate to carbohydrate moieties of glycoproteins and pro(cid:173)
`teoglycans. In structural terms this type of sulfation is a secondary modifi(cid:173)
`cation of the polypeptide chain, the primary one being protein glycosyla(cid:173)
`tion. Both sulfated tyrosine and sulfated carbohydrate residues can
`apparently occur in the same protein.
`The sulfate linked to tyrosine is present as an 0 4-sulfate ester. Such a
`tyrosine 0 4-sulfate residue was first identified in 1954 in bovine fibrino(cid:173)
`peptide B. 1 In the 28 years following this discovery, the presence of
`
`1 F. R . Bettelheim, J . Am. Chem. S oc. 76, 2838 (1954).
`
`METHODS IN ENZYMOLOG Y. VO L. 107
`
`Copyright © 1984 by Acade mic Press. Inc.
`All righ1s of reproduc1io n in any form reserved.
`
`I ISBN ~ 12- '82000'<
`
`-200-
`
`
`
`
`MAIA Exhibit 1022
`MAIA V. BRACCO
`IPR PETITION
`
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`

`(11]
`
`TYROSINE SULFATION OF PROTEINS
`
`201
`
`tyrosine sulfate, initiaJly believed to be restricted to fibrinogens and fi(cid:173)
`brins, 2 was detected in a few biological peptides, such as gastrin II,3
`phyllokinin,4 caerulein,5 cholecystokinin,6 and Leu-enkephalin,7 as well
`as in the small polypeptide hirudin.8 Except for these peptides (and their
`precursors), however, the occurrence of tyrosine sulfate in proteins re(cid:173)
`mained virtually unnoticed until recently. In fact, when proteins were
`found to be sulfated, this was, with a single exception,9 generally taken to
`be indicative of the presence of sulfated carbohydrate residues.
`In a more recent study , 10 the possible widespread occurrence of tyro(cid:173)
`sine sulfate in proteins was investigated. Tyrosine sulfate was detected in
`proteins from a wide variety of vertebrate tissues and cell cultures in
`many molecular weight ranges. Since this recognition of tyrosine sulfation
`as a widespread modification of proteins, at least 10 specific tyrosine(cid:173)
`sulfated proteins have been newly identified and partially characterized.
`These are (1) the four major sulfated proteins of a rat pheochromocytoma
`cell line (PC12), designated as pl 13, p105 , p86, and p84, which were also
`found to be phosphorylated on serine11 ; (2) the major soluble sulfated
`protein of the rat brain, designated as pl 20, which was found to be devel(cid:173)
`opmentally regulated 12 ; (3) three prominent sulfated proteins of the chick
`retina, designated as pl 10, p102, and p93, which were found to move by
`fast axonal transport down the optic nerve12, (4) an acidic secretory pro(cid:173)
`tein of the bovine anterior pituitary, 13 originally described by Rosa and
`Zanini 14
`; (5) immunoglobulin G of some hybridoma cell lines 15 ; and
`(6) vitellogenin of Drosophila. 15
`
`2 F. R. Jevons, Biochem. J. 89,62 1 (1963).
`3 H. Gregory, P. M. Hardy , D. S. Jones , G. W. Kenner, and R. C. Sheppard, Nature
`(L ondon) 204, 931 (1964).
`4 A. Anastasi, G. Bertaccini. and V. Erspamer, Br. J. Pharmacol. Chemother. 21, 479
`(1966).
`5 A. Anastasi, V. Erspamer. and R. Endean, Arch. Biochem. Biophys. 125, 57 (1968).
`6 V. Mutt and J.E. Jorpes, Eur. J. Biochem. 6, 156 (1968).
`7 C. D. Unsworth , J. Hughes, and J. S. Morley, Nature (London) 29S, 5 19 ( 1982).
`8 T. E. Petersen. H . R. Roberts, L. Sollrup-Jensen, S . Magnusson, and D. Bagdy, in
`"Protides of Biological Fluids " (H. Peeters, ed.), Vol. 23, p. 145. Pergamon. Oxford,
`1976.
`9 G. Scheele, D. Bartelt, and.W. Bieger, Gastroenterolol?Y 80,461 (198 1).
`10 W. B. Huttner, Nature (London) 299, 273 (1982).
`11 R. W. H. Lee and W. 8. Huttner, J . Biol. Chem. 258, 11 326 ( 1983); R. W. H. Lee.
`A . Hille, and W. B. Huttner, unpublished observations.
`12 S. B. Por and W. 8 . H uttner, manuscripts in preparation.
`13 P. Rosa. G. Fumagalli, A. Zanini. and W. B. Huttner, manuscript submitted for publi(cid:173)
`cation.
`14 P. Rosa and A. Zanini , Mot. Cell. Endocrinol. 24, 181 ( 198 1).
`'5 P. A. Baeuerle and W. 8. Huttner. manuscripts submitted for publication.
`
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`202
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`PROTEIN ACYLATIONS/ DEACYLATIONS
`
`[11)
`
`The role of tyrosine sulfation in the function of these proteins is the
`subject of current investigations. A possible common denominator may
`be the observation that aH polypeptides so far known to contain tyrosine
`sulfate either are secretory proteins or show properties consistent with
`them being secretory proteins. It is therefore possible that tyrosine sulfa(cid:173)
`tion is involved in the processing, sorting, or functioning of some secre(cid:173)
`tory proteins. In the case of the known proteins, tyrosine sulfation may
`occur during their passage through the Golgi complex; it appears to be
`only slowly reversible or even irreversible. 11
`It is, however, too early to draw general conclusions about the subcel(cid:173)
`lular compartmentation and the degree of reversibility of tyrosine sulfa(cid:173)
`tion. The proteins mentioned above are only some of the tyrosine-sulfated
`proteins existing in the respective cell systems, and only a few celI sys(cid:173)
`tems have so far been studied. Clearly, many more tyrosine-sulfated pro(cid:173)
`teins are yet to be discovered, and many more known proteins need to be
`tested for the presence of tyrosine sulfate. Hopefully, by learning more
`about tyrosine-sulfated proteins, we will achieve an understanding of the
`biological role(s) of this modification. In the following sections, some of
`the current procedures used to detect tyrosine-sulfated proteins and to
`study this modification are described. 16
`
`Chemical Synthesis and Properties of Tyrosine Sulfate
`
`Synthesis. L-Tyrosine O 4-sulfate is synthesized according to the
`method of Reitz et al. 17 by the reaction of L-tyrosine with concentrated
`sulfuric acid at low temperature. After the reaction, the sulfuric acid is
`neutralized and precipitated by addition of barium hydroxide. The tyro(cid:173)
`sine sulfate is freed from unreacted tyrosine and Ba2+ by passage through
`a cation exchanger. The final product may contain variable amounts of
`tyrosine 3'-sulfonate, which can be distinguished from tyrosine sulfate by
`its slightly different electrophoretic mobility at pH 3.5, its distinct ultravi(cid:173)
`olet absorption spectrum, and its stability to acid at elevated temperature.
`The appearance of tyrosine 3 '-sulfonate can be minimized (less than 1 % of
`the tyrosine sulfate) by limiting the reaction time of tyrosine with sulfuric
`acid to 15 min and by keeping the reaction temperature low (between
`
`16 For recent overviews of sulfation , the reader is referred to two excellent books: G . J .
`Mulder, " Sulfation of Drugs and Related Compounds," CRC Press, Boca Raton, Florida.
`1981 ; G. J. Mulder, J. Caldwell, G. M. J. Van Kempen , and R. J. Vonk, " Sulfate Metabo(cid:173)
`lism and Sulfate Conjugation," Taylor & Francis, London, 1982.
`17 H. C. Reitz, R. E . Ferrel, H. Fraenkel-Conrat, and H . S. Olcott, J. A m. Chem. Soc. 68,
`!024 (1946).
`
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`TYROSINE SULFATION OF PROTEINS
`
`203
`
`-25 and -10°). Tyrosine O4-[35S]sulfate is synthesized by the same proto(cid:173)
`col, using [35S]H2SO4.
`Properties. One of the most remarkable properties of tyrosine sulfate
`is the lability of the ester bond in acid and its stability in alkali. More than
`95% of the ester is hydrolyzed after 5 min in 1 M HCI at 100°, making it
`impossible to detect tyrosine sulfate after acid hydrolysis of proteins. The
`acid lability of the ester presumably explains why tyrosine sulfate is not
`observed when peptides known to contain this modified amino acid are
`sequenced by chemical methods. Fortunately, more than 90% of the ester
`remains after 24 hr in 0.2 M Ba(OH)2 at 110°, making alkaline hydrolysis
`the method of choice to detect tyrosine sulfate in proteins. Tyrosine sul(cid:173)
`fate (Ba2+, K ~, or Na+ salt) is stable in neutral aqueous solution at 4° for
`weeks, and standard solutions can be kept frozen at -20° for at least a
`year. The ultraviolet absorption spectrum of tyrosine sulfate is different
`from that of tyrosine, showing a peak at 260.5 nm with a molar extinction
`coefficient of 283 (pH 7 .0). 18
`
`Detection of Tyrosine Sulfate in Proteins
`
`This chapter focuses on the detection of tyrosine-sulfated proteins and
`on the determination of tyrosine sulfate by methods that are based on
`labeling proteins with 35SO4 • This approach has several advantages over
`the chemical determination of tyrosine sulfate in unlabeled proteins. It
`can be much more easily used to search for tyrosine-sulfated proteins and
`is considerably more sensitive and therefore requires less protein material
`for analysis. An immunological approach to detect tyrosine-sulfated pro(cid:173)
`teins has been developed in our laboratory .18 Antisera were raised against
`a synthetic antigen containing a large number of tyrosine sulfate residues,
`and antibodies were purified from the antisera by affinity chromatogra(cid:173)
`phy. These antibodies appeared to recognize tyrosine sulfate-containing
`proteins in "Western" blots and in solid-phase radioimmunoassay. The
`general usefulness of these antibodies to screen for, immunoprecipitate,
`or purify tyrosine-sulfated proteins is currently being tested.
`The standard method to detect tyrosine sulfate in proteins involves
`four main steps described in Sections 1-4 below: ( I) in vivo labeling of
`tissues in situ or of tissue explants, tissue slices, and ce1ls in culture with
`inorganic [35S]sulfate; (2) separation of proteins by polyacrylamide gel
`electrophoresis (PAGE); (3) elution from gels and hydrolysis of individual
`35SOrlabeled proteins; (4) separation of tyrosine [35S]sulfate by thin-layer
`electrophoresis. Major modifications of and additions to the standard
`
`18 P. A. Baeuerle, Diploma Thesis , University of Konstanz, 1983.
`
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`204
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`PROTEIN ACYLA TIONS/DEACYLA TIO NS
`
`[11]
`
`steps are described in separate paragraphs at the end of each section. A
`general scheme of the various procedures is shown in Fig. I.
`
`1. Labeling with 35 SO4
`When whole animals are labeled with 35S04 , one should bear in mind
`that the isotope becomes distributed throughout the body and that its
`specific activity is reduced by the endogenous unlabeled sulfate. Thus, in
`order to achieve sufficient 35S04 incorporation into proteins of the tissue
`of interest, relatively large quantities of 35S04 of high specific activity
`(> 900 Ci/mmol) should be used. For example, a single intraperitoneal
`injection of 20 mCi of 35S04 into a 100-g rat was sufficient for the detection
`of sulfated proteins in various tissues and in the plasma 18 hr after the
`injection by SOS-PAGE and fluorography of the gels for 30 hr. 10 More
`efficient labeling is found if the 35S04 is administered to the tissue of
`
`[35S}SULFATE LABELING OF TISSUES OR CELLS
`
`Tissue / cell
`homogeno te or
`subtroclion
`
`15S] PAJ'S labeling
`
`[
`
`OE NATURATION OF PROIEINS IN SOS OR UREA
`
`Jmmunoprec,p,I olion
`Acetone prec1pilol1an
`
`SOS - PAGE OR 20 - PAGE
`
`ARG OR F6
`
`ALKALINE HYDROLYSIS OR EXIENSIVE PRONASE DIGESTION
`
`10 OR 20 ! HIN - LAYER ELECTROPHORESIS . ARG OR FG
`
`Fro. I. Schematic outline of the sequence of procedures used to detect tyrosine sulfate in
`proteins. Thick arrows indicate the sequence of the standard method; thin arrows indicate
`modifications and additions. ARG, autoradiography; FG, fluorograph y.
`
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`
`TYROSINE SULFATION OF PROTEINS
`
`205
`
`interest directly. For example, sulfated proteins could easily be detected
`in the chick retina or the rat brain 1 hr after injection of 2 mCi of 35SO4 into
`the chick eye or the third ventricle of the rat brain, respectively, by SDS(cid:173)
`PAGE and fluorography of the gels for 15 hr.12
`Mammalian tissues are not capable of reducing 35SO4 for the synthesis
`of [35S]methionine and L35S]cysteine. Nevertheless, one encounters
`[35S]methionine and [35S]cysteine incorporation into proteins after label(cid:173)
`ing whole animals with 35SO4 . This is presumably due to the synthesis of
`[35S]methionine and [35S]cysteine by bacteria present in the gastrointesti(cid:173)
`nal tract of the animals and can be prevented by using germfree animals.
`Although this phenomenon makes the interpretation of fluorograms of
`SDS-polyacrylamide gels less straightforward, it poses no problem for
`the identification of tyrosine [35S]sulfate in protein hydrolysates, using
`thin-layer electrophoresis (see Section 4).
`When tissue ex plants and cells are labeled in culture with 35SO4 , effi(cid:173)
`cient 35SO4 incorporation into proteins is achieved by adding carrier-free
`35SO4 to sulfate-free medium. Many culture systems use medium supple(cid:173)
`mented with some sort of serum, and the presence of low concentrations
`of unlabeled sulfate (up to about 10-4 M) , as is the case when sulfate-free
`medium supplemented with undialyzed serum is used , still allows satis(cid:173)
`factory radioactive sulfate incorporation into proteins. In this case, the
`reduction in the specific activity of 35SO4 is presumably compensated to
`some extent by the increase in cellular sulfate uptake. The advantage
`of using the isotope at high specific activity should be balanced against
`the potential disadvantage that may result from the use of dialyzed serum
`and from starving cells of sulfate. It is therefore recommended to as(cid:173)
`certain that the use of sulfate-free medium and (when serum is required
`for the culture) the use of dialyzed serum during 35SO4 labeling does not
`reduce the viability of the cells under investigation. In particular, the
`capacity for protein synthesis should not be impaired, since protein syn(cid:173)
`thesis appears to be required for suifate incorporation into some pro(cid:173)
`teins .11 In our experience, the use of carrier-free sulfate (about 0.5 mCi/ml
`final concentration) in sulfate-free medium supplemented with a reduced
`concentration (1-5%) of undialyzed serum has resulted in very efficient
`incorporation of radioactive sulfate into PCl 2 cell proteins after labeling
`periods of up to 18 hr.
`Since incorporation of 35SO4 into proteins may occur at both tyrosine
`residues and carbohydrate residues, it can be informative to pe1form
`35SO4 labeling in the absence and in the presence of inhibitors of N(cid:173)
`glycosylation, e.g. , tunicamycin. For example, in the slime mold Dictyo(cid:173)
`stelium discoideum, protein sulfation was virtually abolished by tunicamy(cid:173)
`cin, and analysis of sulfated proteins for the presence of tyrosine sulfate
`
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`

`206
`
`PROTEIN ACY LA TIONS/ DEACYLA TIO NS
`
`[11]
`
`gave essentially negative results. 19 In PC12 cells, the four proteins desig(cid:173)
`nated pl13, pI05, p86, and p84, known to contain most of the incorpo(cid:173)
`rated sulfate as tyrosine sulfate, 11 were labeled similarly with 35SO4 in the
`absence and in the presence of tunicamycin.
`Labeling with 3'-Phosphoadenosine 5'-Phospho[35SJsu[fate (PAPS).
`The study of tyrosine sulfation of proteins has been extended to cell-free
`systems. ll Using the radiolabeled "activated sulfate" [35S]PAPS as sul(cid:173)
`fate donor, transfer of 35SO4 to tyrosine residues of endogenous protein
`acceptors, catalyzed by an endogenous tyrosylprotein sulfotransferase,
`can be observed in cell lysates and some subcellular fractions. At present,
`the labeling efficiency of proteins in cell lysates is less than that obtained
`in intact cells, for several reasons. (1) The specific activity of commercially
`available [35S]PAPS is relatively low (~2 Ci/mmol), compared with that
`of inorganic [35S]sulfate (>900 Ci/mmol). (2) The proportion of the pro(cid:173)
`tein of interest that is in the unsulfated form, and thus a substrate for
`labeling, may be small at any given time point including that of cell Jysis,
`whereas in intact ceJls ongoing protein synthesis continuously supplies
`unsulfated substrate protein. There can be little doubt, however, that, as
`the components of cell-free tyrosine sulfation of proteins are elucidated,
`sulfation of defined proteins by tyrosylprotein sulfotransferase will be(cid:173)
`come more efficient, and the results obtained will increasingly contribute
`to our understanding of the role of tyrosine sulfation.
`
`5SO4-Labeled Proteins
`2. Separation of 3
`After labeling of tissues or cell cultures with 35SO4 , reactions are
`terminated and proteins are solubilized for separation on polyacrylamide
`gels. For these purposes, the use of SDS together with boiling at neutral
`pH appears to be the most suitable method, since it fulfills all the follow(cid:173)
`ing requirements.
`
`1. Rapid inactivation of enzymes. Although at present little is known
`about the possible regulation of the sulfation of specific proteins by extra(cid:173)
`cellular and intracellular signals, only the preservation of the in vivo state
`of protein suJfation by rapid enzyme inactivation may allow the observa(cid:173)
`tion of such regulatory phenomena.
`2. Avoidance of low pH. The tyrosine sulfate ester is labile in acidic
`conditions at elevated temperatures. Although the ester bond may be
`stable in acidic conditions in the cold, it appears safer to avoid low pH
`
`19 J. Stadler, G. Gerisch, G. Bauer, C. Suchanek, and W. 8 . Huttner, EMBO J. 2, 1137
`(1983).
`
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`TYROSINE SULFATION OF PROTEINS
`
`207
`
`whenever possible. It is for this reason that we avoid the use of trichlo(cid:173)
`roacetic acid for terminating sulfation reactions.
`3. Complete solubilization of proteins. Proteins solubilized in SDS
`can be subjected to phenol extraction, immunoprecipitation, SDS-PAGE
`and, after acetone precipitation, two-dimensional PAGE (see Fig. I).
`
`An SOS-containing, neutral solution that we have found to be suitable
`for most purposes is the sample buffer according to Laemmli, 20 referred to
`as "stop solution." Cells attached to culture dishes and cell pellets are
`directly dissolved in stop solution [3% (w/v) SDS, 10% (w/v) glycerol,
`3.3% (v/v) 2-mercaptoethanol, a trace of bromophenol blue, and 62.5 mM
`Tris-HCl, pH 6.8), whereas cells in suspension are mixed with 0.5 volume
`of three times concentrated stop solution, followed in both cases by im(cid:173)
`mediate boiling of the samples for 3-5 min. Tissues are rapidly frozen in
`liquid nitrogen, crushed into a fine powder under liquid nitrogen, and then
`dissolved in stop solution followed by boiling.
`After 35S04 labeling, tissues and cells may be subjected to subcellular
`fractionation, and the subcellular fractions of interest can then be dis(cid:173)
`solved in stop solution. It should be borne in mind, however, that rapid
`regulatory phenomena in the sulfation of proteins, should they exist,
`might be lost during the fractionation process.
`In an attempt specifically to remove sulfated carbohydrate residues
`from proteins aner 35S04 labeling and at the same time preserve tyrosine
`sulfate residues, we have subjected PC12 cell proteins, known to contain
`tyrosine sulfate, 11 to treatment with anhydrous hydrogen fluoride accord(cid:173)
`ing to Mort and Lamport. 21 In our experience, however, all the sulfate
`was removed from these proteins by this treatment. We have not used
`enzymatic deglycosylation to distinguish between the presence of sulfated
`carbohydrate residues and tyrosine sulfate residues, but this may be. use(cid:173)
`ful in some cases.
`If specific proteins arc to be subjected to immunoprecipitation prior to
`electrophoresis, cells and tissues can be dissolved in a neutral buffer
`without 2-mercaptoethanol containing 1-3% (w/v) SOS followed by boil(cid:173)
`ing. The SOS is then diluted by addition of the nonionic detergent Nonidet
`P-40, and immunoprecipitation is pe1formed according to standard proce(cid:173)
`dures. 22 Alternatively, cells can be dissolved in RIPA buffer and subjected
`to immunoprecipitation as described.23 Immunoprecipitates can be dis-
`
`20 U. K. Laemmli , Nature (London) 227, 680 (1970).
`21 A. J. Mort and D. T . A. Lamport, Anal. Biochem. 82, 289 (1977).
`22 S. E. Goelz, E . J. Nestler, B. Chehrazi , and P Greengard , Proc. Natl. Acad. Sci. U.S .A.
`78, 2130 (1981).
`23 8. M. Sefton, K. Beemon, and T. Hunter, .I. Virol. 28,957 (1978).
`
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`

`208
`
`PROTEIN ACYLATIONS/DEACYLA TIONS
`
`[ 11]
`
`solved in stop solution (for SOS-PAGE) or in O'Farrell lysis buffer24 (for
`two-dimensional PAGE).
`Cells and tissues dissolved in stop solution can be subjected to any of
`the following protocols.
`
`1. A phenol extraction protocol as described in Procedure I, designed
`to separate the proteins from sulfated glycosaminoglycans, foJiowed by
`PAGE (see below). This procedure is similar to that introduced by Hunter
`and Sefton25 for the study of tyrosine phosphorylation of proteins, and
`used in that case to separate proteins from nucleic acids and phospho]i(cid:173)
`pids. The effect of phenol extraction is illustrated in Fig. 2.
`2. SDS-PAGE according to Laemmli. 20
`3. Two-dimensional PAGE using either isoelectric focusing or non(cid:173)
`equilibrium pH gradient electrophoresis in the first dimension, as de(cid:173)
`scribed by O'Farrell. 24 For two-dimensional PAGE, samples in stop solu(cid:173)
`tion are mixed with 5 volumes of acetone, kept at -20° until precipitation
`occurs, and centrifuged. The pellets are washed in 80% (v/v) acetone,
`dried, and dissolved in O'Farrell lysis buffer containing 5% (w/v) Nonidet
`P-40. Alternatively, cells attached to culture dishes or cell pellets can als~
`be directly dissolved into lysis buffer. In our experience, both types of
`sample preparation give rise to similar separations upon two-dimensional
`PAGE.
`
`Procedure I. Phenol Extraction of Sulfated Proteins
`1. Dissolve sample, e.g. , 35S04-labeled cells in culture, in stop solu(cid:173)
`tion (0.5-5 mg of protein per mi1liliter of stop solution). Boil immediately
`for 3-5 min.
`2. Unless otherwise indicated, the following steps are performed at
`room temperature. Prepare phenol solution: dissolve phenol in an equal
`amount (w/v) of HEN buffer (50 mM HEPES-NaOH, pH 7.4; 5 mM
`EDTA; 100 mM NaCl), mix vigorously for 5 min, let stand or centrifuge
`until phases are separated. The lower phase is HEN buffer-saturated
`phenol (referred to as phenol solution), the upper phase is phenol-satu(cid:173)
`rated HEN buffer (referred to as HEN solution).
`3. Mix the sample with an equal volume of phenol solution, vortex
`vigorously for at least 30 sec, centrifuge for IO min at 15,000 rpm in a
`Sorvall SS34 rotor.
`
`24 P.H. O'Farrell, J. Biol. Chem. 250, 4007 (1975); P. Z. O'Farrell, H. M. Goodman, and P.
`H. O'Farrcll, Cell 12, 1133 (1977).
`25 T. Hunter and B. M. Sefton, Proc. Natl. Acad. Sci. U.S.A. 77, 13 11 ( 1980).
`
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`

`[11]
`
`TYROSINE SULFATION OF PROTEINS
`
`209
`
`-
`
`+
`
`FIG. 2. Autoradiograms showing the effect of the phenol extraction protocol described in
`Procedure I. PC 12 cells were labeled with 35SO4 and subjected to SOS-PAGE, without (-)
`or with ( +) prior phenol extraction. Arrows indicate major tyrosine-sulfated proteins. 11
`Some of the proteins can be better seen after phenol extraction.
`
`4. Collect the aqueous (upper) phase. If flocculent material at the
`interface is present, avoid disturbing it. Keep phenol (lower) phase plus
`interface.
`5. Mix the aqueous phase with an equal volume of phenol solution~
`vortex and centrifuge as before. Discard the aqueous phase; keep the
`phenol phase plus interface.
`6. Pool the phenol phases plus interfaces from first and second extrac(cid:173)
`tion ; reextract 1-3 times with equal volume of HEN solution. Discard
`aqueous phases each time. The efficiency of extraction can be monitored
`by spotting aliquots of the aqueous phases on filter paper and observing
`the decline in radioactivity with a portable ,B-radiation monitor or by
`liquid scintillation counting.
`
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`

`210
`
`PROTEIN ACYLATIONS/ DEACYLATIONS
`
`(11]
`
`7. Add five volumes of cold ( - 20°) ethanol to final phenol phase plus
`intetface. If necessary, transfer to larger centrifuge tube. Mix well and
`keep at - 20° until precipitation has occurred (2 hr or longer). Centrifuge
`for IO min at 10,000 g in a Sorvall SS34 rotor. Discard the supernatant.
`Wash the precipitate once with chloroform-methanol (2: 1) and collect it
`by centrifugation as above. Allow the precipitate to dry.
`8. Dissolve the precipitate in stop solution (for SOS-PAGE), in lysis
`buffer (for two-dimensional PAGE), or in 0.2 M Ba(OHh (for alkaline
`hydrolysis and determination of tyrosine sulfate, see Procedure Ill).
`
`After electrophoresis, gels are fixed and (if desired) stained and de(cid:173)
`stained by conventional procedures, using acetic acid rather than trichlo(cid:173)
`roacetic acid. Fixed gels can also be subjected to an acid treatment that is
`described in a subsection at the end of this section. For the detection of
`35SOdabeled proteins, gels are prepared for fluorography, dried, and
`fluorographed at -70°. We use exclusively the sodium salicylate method26
`for fluorography, primarily because the salicylate is water-soluble and can
`therefore easily be removed from gels after fluorography. This renders the
`proteins present in the gel suitable for further biochemical analysis, e.g.,
`peptide mapping by limited proteolysis, tryptic fingerprinting, tyrosine
`sulfate analysis, (see below). If the labeling of proteins is sufficiently
`intense, the salicy)ate treatment of gels can be omitted, and the dried gels
`are subjected to autoradiography at room temperature. The X-ray film
`used in our laboratory for autoradiography and fluorography is Kodak
`XAR-5.
`An alternative, rapid procedure to obtain an autoradiogram after
`SDS-PAGE or two-dimensional PAGE is to transfer the proteins from
`the gel onto nitrocellulose filter paper using the " Western" blotting tech(cid:173)
`nique. After the transfer, the nitrocellulose fiJter paper is either air-dried
`and autoradiographed or is dipped in 20% PPO in toluene, dried, and
`fluorographed at - 70°. This procedure has two advantages.
`
`1. The time needed for autoradiography after transfer to nitrocellulose
`filter paper is shorter than with dried polyacrylamide gels.
`2. The sulfated material that is often found as a diffuse smear in the
`high molecular weight regions of SDS-polyacrylamide gels (presumably
`sulfated proteoglycans or glycosaminoglycans) does not appear to trans(cid:173)
`fer very well from the gel to the nitrocellulose filter paper. Thus, autora(cid:173)
`diograms obtained after transfer of 35SO4-]abeled proteins to nitrocellu(cid:173)
`lose filter paper tend to be "cleaner" than those of the corresponding gels
`
`26 J. P. Chamberlain, Anal. Biochem. 98, 132 (1979).
`
`-210-
`
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`
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`

`

`[11]
`
`TYROSINE SULFATION OF PROTEINS
`
`21 I
`
`and can be compared to autoradiograms obtained after SDS-P AGE of
`phenol-extracted samples (Fig. 2).
`35S04-labeled proteins separated in polyacrylamide gels can be used
`for hydrolysis (see Section 3 below) and tyrosine sulfate analysis (Section
`4). If desired (see Fig. 1), individual sulfated protein bands can first be
`subjected to limited proteolysis in SDS and peptide mapping in SDS
`polyacrylamide gels,27 or to extensive proteolysis and two-dimensional
`fingerprinting by thin-layer electrophoresis/chromatography. In these
`cases, the resulting peptide fragments can be used for hydrolysis and
`tyrosine sulfate analysis.
`HCl Treatment of Proteins in Polyacrylamide Gels. The details of the
`method are described in Procedure II, below. The rationale of this treat(cid:173)
`ment is to screen, among the variety of sulfated proteins present in a
`sample, for those likely to contain tyrosine sulfate. Since the tyrosine
`sulfate ester is remarkably acid-labile and appears to be hydrolyzed faster
`than most carbohydrate sulfate esters, a short acid treatment will lead to a
`preferential loss of sulfate from tyrosine residues. When such an acid
`treatment is performed on 35S04-labeled proteins fixed in polyacrylamide
`gels, the resulting autoradiographic pattern may show some labeled bands
`that disappear quite specifically after the acid treatment (i.e., their reduc(cid:173)
`tion is greater than the small overall reduction in labeled bands). These
`specifically acid-sensitive bands may contain a large proportion of their
`sulfate label as tyrosine sulfate. In the examples illustrated in Fig. 3, there
`appears to be a good correlation between the acid sensitivity of bands and
`their tyrosine sulfate content.
`It may, however, be too early to assume generally that a correlation
`between these two parameters will be found in every case because one
`cannot rule out false-positive and false-negative results. Possible explana(cid:173)
`tions for false-positive acid se1isitivity of bands incJude the following. (1)
`Sulfated residues other than tyrosine sulfate, e.g., certain carbohydrate
`sulfate esters, may exist that are as acid-sensitive as tyrosine sulfate
`ester. (2) The acid treatment may lead to hydrolysis of some peptide
`bonds. This may result in the formation of peptide fragments small
`enough to remain no longer fixed in the gel but to diffuse out. If sulfated
`residues other than tyrosine sulfate, e.g., sulfated carbohydrates, were
`located in such peptide fragments , an acid treatment-induced loss of 35S04
`label would be observed without the actual hydrolysis of a tyrosine sulfate
`ester. False-negative acid sensitivity, i.e., the apparent lack of acid sensi-
`
`27 D. W. Cleveland, S. G. Fischer, M. W. Kirschner, and U. K. Laemmli , J. Biol. Chem.
`252, 1102 (1977).
`
`-211-
`
`
`
`
`

`

`212
`
`PROTEIN ACYLA TIONS/DEACYLATIONS
`
`[11]
`
`A
`
`B
`
`C
`
`--
`
`F10. 3. Autoradiograms showing the effect of the HCI treatment described in Procedure
`II on proteins separated by SOS-PAGE: (A) proteins of rat pheochromocytoma cells
`(PC12), labeled with [35S]methionine; (B) same, labeled with [.l5S)sulfate; (C) proteins of
`Dictyostelium discoideum, labeled with (35S)sulfate. Compared with the control (- ), the HCl
`treatment ( +) does not lead to a loss of protein from the gel (A). The HCI treatment
`markedly reduces the 11S04 label of PC 12 cell proteins known to contain tyrosine sulfate 11
`(arrows) (B), but only slightly the 35S04 label of D. discoideum proteins known to contain
`sulfate on carbohydrate residues 19 (C).
`
`tivity of bands despite the presence of tyrosine sulfate, may also be ob(cid:173)
`served. For example, if a glycoprotein contained 10 sulfated carbohydrate
`residues and I tyrosine sulfate residue, the acid treatment-induced spe(cid:173)
`cific Joss of 35SO4 label of about 10% would not readily be noticed upon
`comparative autoradiography. Despite these considerations, the acid
`treatment of gels is a useful and simple tool in searching for tyrosine(cid:173)
`sulfated proteins, particularly when this method is combined with the
`
`-212-
`
`
`
`
`

`

`[11]
`
`TYROSINE SULFATlON OF PROTEINS
`
`213
`
`procedure to determine tyrosine sulfate in individual proteins obtained
`from polyacrylamide gels (Section