Isolation of Prolyl-tRNA Synthetase as a Free Form and as a Form ...

3 downloads 8339 Views 6MB Size Report
that appears to be the free enzyme, and another form which is associated with a ..... Analysis of the Domain Structure ofthe Free Form of Prolyl-. tRNA Synthetase ...
Vol. 267, No . 25, Issue of September

THEJOURNALOF BIOLOGICAL CHEMISTRY IC

1992 by The American Society for Biochemistry and Molecular Biology, Inc.

1i701-17709,1992 Printed in U.S A.

Isolation of Prolyl-tRNA Synthetaseas a Free Form and as a Form Associated with Glutamyl-tRNA Synthetase* (Received for publication, April 9, 1992)

Shu Mei Ting, PeterBognerS, and John David Dignamj From the Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43699-0008

Rat liver prolyl-tRNA synthetase was purified as a dimer of M , 60,000 subunits not associated with other aminoacyl-tRNA synthetases and as a form associated with glutamyl-tRNA synthetase. Proteolysis of the dimeric enzyme generated a less active form with M , 52,000 subunits and an inactive form with M, 40,000 subunits. A second species was isolated with polypeptides ofM, 60,000 and 150,000. This form dissociated during gel filtration chromatography being partially resolved into theM , 150,000 and 60,000 components; glutamyl-tRNA synthetasewasassociatedwith the larger polypeptide and prolyl-tRNA synthetase with the smaller component. Antibodies againstthe M, 60,000 polypeptide reacted with the M, 60,000 and 150,000 polypeptides. Gel filtration of extractsrevealed multiple forms of prolyl- and glutamyl-tRNA synthetase. Antibody against theM , 60,000 component detectedthe M , 60,000 and 150,000 polypeptides throughout the chromatogram; these forms could be partially separated by polyethylene glycol fractionation. The M , 150,000 and 60,000 polypeptides were detected by Western blot analysis of crude extracts prepared under several conditions. Antibody to prolyltRNA synthetase reacted with a M , 150,000 polypeptide of the aminoacyl-tRNA synthetase core complex identified previously as glutamyl-tRNA synthetase.

(designated the valyl-tRNA synthetase complex) contains valyl-tRNA synthetase, elongationfactor 1, and two additional polypeptides of unidentified function (14, 15).In spite of difficulties encountered in the isolation of the complexes due to proteolysis and the dissociation of some components during purification, there is little doubt that they are distinct entities with a defined structural organization (16). The reason theseenzymes exist incomplexes is unclear since individual, active components can be isolated from the complexes (17-19), and because some aminoacyl-tRNAsynthetases (alanyl-, glycyl-, histidyl-, seryl-, and threonyl-tRNA synthetases) are consistently isolated as individual components. It may be that all aminoacyl-tRNA synthetases are associated in a complex whose size and fragility precludes its isolation by standard biochemical techniques, but is essential for the efficiency or control of protein synthesis. Webegan examiningprolyl-tRNAsynthetasetounderstand how this enzyme is regulated in animal cells and undertook its purification as the first step in analysis. this While enzymatic activities have been assigned to most of the polypeptides in the complexes, the identityof the polypeptide with prolyl-tRNAsynthetasehasnot beenestablished, even though this enzymatic activity is detected at varying levels in preparations of the core complex (13). A recent report (20) indicates that this activity may be contained ona polypeptide of M , 180,000which also contains glutamyl-tRNA synthetase. In the process of purifying prolyl-tRNA synthetase we detected multiple forms of the enzyme, including a small form Aminoacyl-tRNA synthetases are a structurally and func- that appears tobe the free enzyme, and another form which tionally heterogeneous group of enzymes which perform akey is associatedwith a largerpolypeptide that appears to be role in protein synthesis (1-4) and perform other, somewhat glutamyl-tRNA synthetase. unanticipated functions inmacromolecular synthesis andmeEXPERIMENTAL PROCEDURES tabolism (5-8). While these enzymes are generally isolated as individual entities from bacteria and fungi, many exist in Materials-DEAE- and sulfopropyl-HPLC’ columns, Superose 12 higher eucaryotic organisms as multienzyme complexes with FPLC column, Sepharose CLGB, and Q-Sepharose were purchased up to 13 polypeptides. One type of complex (designated the from Pharmacia LKB Biotechnology, Inc. Supported nitrocellulose core complex) contains glutamyl-, glutaminyl-, aspartyl-, ly- was from Schleicher & Schuell. Opti-Vant was from TSI Center for syl-, arginyl-, methionyl-, leucyl-, and isoleucyl-tRNA synthe- Diagnostic Products. [32P]Pyrophosphate, tetrasodium, and [‘Hlglutamic acid were from Du Pont-New England Nuclear. [“C]Proline tases; activities have been assigned to many of the polypep- purchased from Amersham was further purified by ascending paper tides in this complex, whilethe functionsof some polypeptides chromatography on Whatman 3” as described for [“Clserine (21) have not been determined (9-13). A second type of complex except that the chromatogram was developed in ethanol, ammonia, * This work was supported by Grant DE09669 from the National

Institute of Dental Medicine; the recombinant DNA facility was supported by Harold A. and Helen McMaster. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adoertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Present address: Pannon College of Agriculture, CT-Biological Ctr., P. 0. Box 16, 7401 Paposvar, Hungary. To whom correspondence shouldbe addressed Dept. of Biochemistry and Molecular Biology, Medical College of Ohio, P. 0. Box 10008, Toledo, OH 43699-0008. Tel.: 419-381-4136; Fax: 419-3827395.

and water (201:4). PEG 6000,5-bromo-4-chloro-3-indolyl phosphate, nitro blue tetrazolium, and L-1-tosylamido-2-phenylethylchloromethylketone-treatedtrypsin were from Sigma. The trypsin was further purified by column chromatography on S-Sepharose(22). Electrophoresis supplies (acrylamide, bisacrylamide, and SDS) and goat anti-rabbit alkaline phosphatase conjugate were from Bio-Rad. Polyvinylidene difluoride membrane (Immobilon-P) was from Millipore. Buffers-Wash buffer contained 50 mM Tris-C1 (pH 7.5), 2 mM



The abbreviations used are: HPLC, high-performance liquid chromatography; SDS, sodium dodecyl sulfate; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PEG, polyethylene glycol; FPLC, fast-proteinliquid chromatography.

17701

17702

Synthetase

Prolyl-tRNA

EDTA, 0.5 mM D T T buffer A, 50 mM Tris-C1 (pH 7.5), 10%glycerol, 0.2 mM EDTA, 5 mM magnesium acetate, 0.5 mM DTT, 1 mM PMSF, 1 mM tosylargininemethylester, and 10 pg/ml soybean trypsin inhibitor; buffer B, KPO, (pH 7.5) at concentrations indicated, 20% glycerol, 0.2 mM EDTA, and 0.5 mM D T T buffer C, 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 10% glycerol, 0.2 mM EDTA, and 0.5 mM DTT; buffer D, 50 mM Tris-C1 (pH 7.5), 150 mM NaCl, 10%glycerol, 0.2 mM EDTA, and 0.5 mM DTT; 50% PEG 6000 was made up in 50 mM Tris-C1 (pH 7.5) and 2 mM EDTA. Enzyme Assays-Prolyl- and glutamyl-tRNA synthetase activities were determined as previously described (23). The rabbit tRNAPm and tRNA8'" were partially purified on benzoylated DEAE-cellulose (23, 24). One unit of activity corresponds to the incorporation of 1 nmol of amino acid in 1 min into acid-insoluble material. ATPpyrophosphate exchange activity was measured by using [32P]pyrophosphate as previously described (21) except that proline or glutamate was substituted for serine. Analytical Methods-Amino acid composition was determined on samples that had been hydrolyzed for 24 and 48 h in 6 N HC1. Cysteine and methionine were determined as cysteic acid and methionine sulfone, respectively (25), on samples oxidized with performic acid followed by acid hydrolysis in 6 N HCI. N-terminal sequence determinations were carried out by automated Edman degradation on an Applied Biosystems 470A gas-phase sequencer. Phenylthiohydantoin derivatives were identified by reversed-phase chromatography withan on-line HPLC (26). Protein was measured by a Coomassie Brilliant Blue G-250 dye binding assay (27) or by absorbance at 280 nm. Gel Electrophoresis-SDS-polyacrylamide gel electrophoresis in a Tris/glycine buffer (28) was used for monitoring the purity of the enzyme and for analyzing protease-digestion products; a phosphate buffer system (29) was used to determine thesubunit molecular weight. Gels were stained with Coomassie Brilliant Blue R250 or with silver nitrate (30). Samples were treated with SDS (1%)and mercaptoethanol (5%)and heated for 1 min at 100 "C prior to electrophoresis. Isoelectric Focusing-The isoelectric point was determined as previously described (31) except that a pH gradient of 6.0 to 9.0 rather than 4.5 to 7.0 was used. Determination of Stokes Radius and Sedimentation Constant-The Stokes radius was determined by HPLC gel filtration using ferritin (7.8 nm), catalase (5.2 nm), aldolase (4.6 nm), and bovine serum albumin (3.5 nm) as standard proteinsusing values taken from Ref. 32. Standard proteins at 0.5 mg/ml in 200 p1 were applied to a Superose 12 FPLC column (1.0 cm X 30.0 cm) equilibrated in buffer D. The column was eluted at 0.3 ml/min, and fractions of 0.24 ml were collected. Standard proteinswere run separately on the column under thesame conditionsas thesample. The sedimentation constant was determined by sucrose density gradient sedimentation as previously described (23, 33) using catalase, alcohol dehydrogenase, and hemoglobin as standards. Preparation of Antibody and Western Blot Analysis-Antibodies were raised against the purified enzyme as previously described (34) except that Opti-Vant was used as anadjuvant (250 Fg/rabbit). Each of four New Zealand White rabbits received 300 pg of enzyme in the first injection given intramuscularly at two sites and subdermally at five sites. Lower doses of 90-150 pg/rabbit were used for later booster injections given after 21, 42, and 69 days following the initial injections. The rabbitswere bled at day 83. The serum collected was stored a t -80 "C. The antibody was affinity-purified by adsorbing the antibody to the pure prolyl-tRNA synthetase that had been transferred to a nitrocellulose filter and eluting it from the nitrocellulose with 0.1 M glycine (pH 2.5) and 0.1 M NaCl (35).Purified antibody detected both the M , 60,000 prolyl- and M , 150,000 glutamyl-tRNA synthetases at 1:100, 1:500, 1:2500, and 1:5000 dilutions; both polypeptides were detected when either the prolyl- or glutamyl-tRNA synthetase was used for affinity purification. Western blot analysis was carried out as previously described (34) using alkaline phosphatase-conjugated goat anti-rabbit IgG as the secondary antibody; the secondary antibody was detected using nitro blue tetrazolium and 5-bromo-4chloro-3-indolyl phosphate (36). Purification of Prolyl-tRNA Synthetase-Thirty rats (175-200 g each) were decapitated, and their livers were removed and washed twice with 500 ml of cold wash buffer. Steps prior to sulfopropyl- and DEAE-HPLC steps were performed at 0 to 4 "C. Crude Extract Preparation and PEG Fractionation-Liver (350 g) was homogenized in buffer A (875 ml) for 60 s in a Waring Blendor at high speed. The homogenate was centrifuged for 45 min at 15,000 X g. The supernatantfraction (crude extract) was brought to 5%PEG

by adding 0.11 volume of 50% PEG 6000, stirring for 20 min, and centrifuging for 20 min at 15,000 X g. The supernatant fraction was brought to 10% PEG by adding 0.125 volume of 50% PEG, stirring, and centrifuging as described above. The precipitates from PEG fractionation were dissolved in a minimum volume of buffer B. Chromatography on Q-Sepharose-The 5-10% PEG fraction was applied to a column of Q-Sepharose (2.5 X 30 cm) equilibrated with buffer B. The column was washed with 400 ml of starting buffer, or until the eluentwas no longer red, then eluted with a 1000-ml linear gradient from 10 to 100 mM KPO, (pH 7.5) in buffer B at 90 ml/h. The enzyme eluted in the last 35% of the gradient at approximately 70 mM KPO,. The active fractions were combined and concentrated using an Amicon ultrafiltration cell employing a PM-10 membrane. When the gel filtration step on Sepharose CL6B was omitted the concentrated material was dialyzed against 50 volumes of buffer C for 12 h prior to chromatography on the sulfopropyl-HPLC column. Chromatography on Sephurose CUB-The concentrated material from Q-Sepharose was applied to a column of Sepharose CL6B (2.5 X 110 cm) equilibrated with buffer C. The flow rate was 18 ml/h, and fractions of 5 ml were collected. The fractions containing enzyme activity were pooled. In some preparations this stepwas omitted and the material from Q-Sepharose was applied to thesulfopropyl-HPLC column as indicated above. Samples of 15 ml of the crude extract, the 0-5% PEG fraction, and the 5-10% PEG fraction (see Figs. 6 and 7 in the text) were also analyzed on Sepharose CL6B under the same conditions. Chromatography on Sulfopropyl-HPLC Column-Chromatography on a Sulfopropyl-HPLCcolumn was performed at room temperature. The pooled fractions from the preceding step (either gel filtration on Sepharose CL6B or Q-Sepharose) were applied at 1.0 ml/min to a LKB Ultro Pac SP 5-PW column (7.5 X 75 mm) equilibrated with buffer C (25 mM KPO,). The column was washed for 10 min with starting buffer then eluted with a gradient of 25 to 100 mM KPO, (pH 7.5) in buffer C over 20 min followed by isocratic elution at 100 mM KPO, for 10 min. Fractions of 1.0 ml were collected and placed on ice. The enzyme eluted at approximately 100 mM KPO,. The active fractions (2 ml) were combined and dialyzed against buffer C at 0 to 4 "C. Chromatography on DEAE HPLC Column-Chromatography on DEAE was performed at room temperature. The dialyzed material was applied at 1.0 ml/min to a LKB Ultro Pac DEAE 5-PW column (7.5 X 75 mm) equilibrated with buffer C. The column was washed for 5 min with starting buffer, then eluted with a gradient of 25 to 100 mM KPO, (pH 7.5) in buffer C over 20 min. Fractions of 0.5 ml were collected and placed on ice. The enzyme eluted at approximately 85 mM KPO,. Active fractions were quick-frozen on dry ice and stored at -80 "C. Determination of Apparent K,,, and k,, Values-Apparent K , values were obtained from substrate saturation experiments. At least five substrate concentrations in duplicate were used over the following ranges: for rabbit liver tRNAPro,0.1-5 PM;for proline, 20-400 pM; for ATP, 0.1-5 mM. The experiments with ATP were done at a magnesium:ATP ratio of 1.2:l. The data were analyzed using double-reciprocal plots. Tryptic Digestion of Prolyl-tRNA Synthetase-To examine the effect of trypsin digestion on enzymatic activity (see Fig. 4), prolyltRNA synthetase (0.2 mg/ml in buffer B) was digested with trypsin at 1 or 5 pg/ml at 30 "C. Aliquots were removed at 0, 15,30, 60, and 120 min for SDS-polyacrylamide gel electrophoresis, and separate aliquots were treated with 10 mM PMSF and 0.1 mg/ml soybean trypsin inhibitor and assayed for enzyme activity immediately. To examine the effect of ligands on enzymaticactivity (see Fig. 5), prolyltRNA synthetase (0.2 mg/ml) was treated with trypsin at 1 gg/ml for 30 min or at 5 pg/ml for 60 min at 30 "C in the presence of proline (0.2 mM), ATP (5 mM), tRNAPro(9 p ~ )or, ATP (5 mM) and tRNAPro (9 p ~ together. ) At the end of each incubation, samples were treated with SDS andmercaptoethanol and electrophoresed on a 12.5%SDSpolyacrylamide gel. RESULTS

Purification of Prolyl-tRNA Synthetaseas a Free Form and as a Complex with Glutamyl-tRNA Synthetase-Table I summarizes the purification of the freeformof prolyl-tRNA synthetase (form I) and the prolyl-glutamyl-tRNA synthetase complex (form 11) from rat liver. The specific activity of the free enzyme was 1260 units/mg of protein, representing an

TAMEI Purification of prol.vl-tRNA synthrtasr forms I and I I from rat Iilvr 3 9 The purification given is from a single prep starting with 337 g of rat liverandperformed as descrihedunder “F:xperimental Procedures.“ Step Total activity Specific acti\*itv Recovery ~ ~ ~ _ _ _ _ ~ _ _ _ _ _ -~nmol min”

extract

nmol mrn” ma”

30

-

a14 5

43 “

9 7-

,p

45600

1.68

(100)

I’olvet hylene glycol 0 0 ; to 5% 5c;, to 1 0 5 10“;. t o 20%

moo 204oo 6000

1.95 2.86 0.84

3n 45 13

9260

8.83

20

Sulfopropyl-HP1.C form I form I1

2250 1247

44 1 461

4.93 2.73

DEAE-HPIX form I form I1~-~

1895 618 -

1260 6 1 :i

4.15 1.35

~

37

PI

Crude

Q-Sepharose pool the on 5 t o IOri, PEG fraction

A

L

r

4 5-

31-

B 0.3

1 .oo

1

1600 0 03

N

0.2

.

m

n

0

0.1 .

fraction

FIG. 1. Sulfopropyl-HPLC chromatography of prolyl-tRNA synthetase from Q-Sepharose.Active fractions from Q-Sepharose wereconcentratedanddialyzedagainsthufferCand suhjectetl to chromatography on a sulfopropyl-HPLC column,as descrihed under “ExperimentalProcedures.“ Svmhols: dottrd linr, A?,,; A, prolvltRNA synthetase activity, units/ml.

tamyl-tRNA synthetase activity (shown in Fig. 3 ) . Gel filtraincrease in specific activity of about 750-fold with a recovery tion on Sepharose CL6R after the (2-Sepharose step usually of4.15% fromthecrudeextract.Thespecificactivity of gave a broad peak of activity, hut in one experiment thisstep various preparations ranged up to 1600 units/mg of protein. partially resolved two peaksof prolyl-tHSA synthetase activProlyl-tRNA synthetase formI1 had less than half the specific ity which were purified separately through the sulthpropylactivity of the free form of the enzyme, which is consistent and DEAE-HPLC steps. The prolyl-t RNA sqnt hetase activity with the presence of an additional M, 150,000 polypeptide. eluting in the first peak from the gel filtration step in this T h e relatively low recovery of activityforthetwoforms experiment was subjected to the I>EAE-HI’I,C step and conresults in part from their separation from other complex forms tained the M, 60,000 polypeptide that correspondedto proiylduring fractionation with polyethylene glycol. Gel filtration a polypeptide of .%I, 150.000 whose tRNAsynthetaseand on Sepharose CLBB helped resolve form I (the free form of subsequent experiments inidentity was initially unknown: theenzyme)fromform I1 (a complexwith a M , 150,000 dicated that this pol?.peptide was glutamyl-tR S A synthetase. polypeptide) and other smaller contaminants and produceda gel filtration step in this preparaThe second peak from the purer preparation of enzyme (approximately 90% based on gel electrophoresis). Enzyme purified by the procedure which tion gave the free form when chromatographed through the sulfopropyl- and DEAE-HPIX steps. included this step was used in experiments analyzing the Physical and Catalytic I’roprrtirs of I’ro!yl-tltAVAS>~nthdnsr domain structure of the free enzyme (see Figs.3 and 4). If gel nf ratliver filtration on Sepharose CLBB was omitted, twowell resolved Form I-Thephysicalandcatalvticproperties prolyl-tRNA svnthetase are summarized in ’I’atde 11. T h e peaks of activity were detected at the sulfopropyl-HPLC step subunit molecular weight determined t ~ ySDS-p~)lyacrylamirie as shown in Fig. 1. With this approach, approximately onethird of the prolyl-tRNA synthetase activity was recovered ingel electrophoresis is 60.000. T h e oligomeric molerulnr weight of 133,000 was calculated from the Stokes radius (4.7 n m ) , the second peak. When the two peaks from the sulfopropylHPLC step were chromatographed separately on the DEAE- sedimentation constant (7.0 S),and partial specific volume (0.71ml/g,determinedfromtheaminoacidcomposition). HPLC column, the first peak gave a preparation that was highly purified and corresponded to the free enzyme (form I, The results indicate that prolyl-tRNA synthetase is n dimer shown in Fig. 2), while the second peak from the sulfopropyl- of similar or identical subunits. Theprotein has a rather high HPLC step eluted with a M, 150,000 polypeptide and gluof 2.09). indicative o f asymmetry. Its frictional ratio (f/f,,,,,,

Prolyl-tRNA Synthetase

17704

A

FIG.3. DEAE chromatography of form I1 prolyl-tRNA synthetase from sulfopropyl-HPLC chromatography. Fract.ions in the second activity peakshownin Fig. 1 werepooled,dialyzed against buffer C, and further purified by Chromatography on DEAE as descrihedunder"ISxperimentalProcedures." A , 10-pI aliquots of the indicated fractions were treated with SDS and subjectedtoSDS-gelelectrophoresisand stained with Coomassie Blue. R, symhols arethesame as in Fig. 2 except that glutamyl-tRNAsynthetaseactivity is represented by open triangles.

46

44

50

48

45-

-- 4 0.2c

-3 0

0.1 5

a3 hl

2 0

-2 0.10

I f -1

0.05

20 24 28 32 36

40

44

40

52

56

16

0

fraction Properties of the free form of rat lioerprolvl-tRNA svnthdasr -~ ~Property

Subunit molecular weight Stokes radius Sedimentation constant Part ial specific volume Oligomeric molecular weight

Method

SDS-polyacrylamide gel electrophoresis Gel filtration Sucrose gradient sedimentation Amino acid composition Calculated from Stokes radius, partial specific volume and sedimentation constant Calculated from the diffusion and sedimentation constant Isoelectric focusing

2.09 8.2

Determined from suhstrate Saturation experiments For the dimer

basicisoelectricpoint(8.2) is also reflectedin a relatively high value for lysine (9 mol%) observed in the amino acid composition (Table 111). T h e K,, values for proline, tRNA'"", and ATP were determined from substrate saturation experimentswheretheexistence of initialrateconditions was verified by examining the time course of the reaction at the extreme substrate concentrations. The k,.,,, values ranged from 2.0 to 4.0 s" for the dimer, depending on the preparation of the enzyme assayed. To determine the N-terminal sequenceof the enzyme and the tryptic fragments, the untreated enzyme and the appropriate tryptic digests were subjected to SDS-acrylamide gel

electrophoresis and transferredto a polwinvlidene difluoride membrane for sequence analysis. The undigested enzvme and t h e M , 52,000 and 40,000 fragments were transferred from an acrylamide gel to a pol-winylidene difluoride membrane and subjected to automated sequence analvsis. No phenvlthiohydantoin derivatives could be detected above background for the two tryptic fragments and the undigested enzvme, suggesting that the protein has a blocked S terminus and that trypsin may be removing fragments from the C terminus of the protein. An equivalent amount of mvoglohin was analyzed immediatelypriortothesequenceattemptsandgavethe expected sequence.

Prolyl-tRNA Synthetase TARLE I11 Amino acid compositionof prol-vl-tRNA s y n t h ~ t a s ~ Amino acid mol% acid

.4 4.3

4.4

1.3 1.0

acid

Aspartic Threonine" Serine" Glutamic Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Half-cystineh Methionine"

A Iow

con t rot 0

8.7

60

97-

66- .

...

IS

-.

30

high 60

15

30

60

. - -

43-

'? X

,31-

I 21-

1.8

" Determined by extrapolation to zero time. *Determined as cysteicacidandmethioninesulfoneafterperformic acid oxidat,ion.

B 1

.->z .c Y

V

0 Analysis of the Domain Structure of the Free Form of ProlylC tRNA Synthetaseandthe Effect of SubstratesonTrypsin 9) 0 Sensitivity-Prolyl-tRNA synthetase was digested with trypL sin under conditions where the enzyme retains activity is and Wa presumably native; two concentrations of the protease were employed to generate awide range of digestion. Aliquots were taken at times up to 60 min and assayed for pyrophosphate exchange and aminoacyl-tRNA synthetase activities, while 0 20 40 60 separate samples of the reaction mixtures were treated with SDS and mercaptoethanol and subjected to SDS-gel electrominutes phoresis. The results of this experiment are shown in Fig. 4. FIG. 4. T i m e c o u r s eof digestion of p r o l y l - t R N A s v n t h e t a w Tryptic digestion of the enzyme generated a M , 52,000 frag- w i t h t r y p s i n . Prolyl-t R S A synthr*tase (0.2 mg/ml in h l f e r I ! ) was ment at early times, which appeared tobe a precursor to M a , digestedwithtrypsin at 1 pg/ml ( l o w ) or .5 pg/ml (high). fnr the 40,000 fragment upon longer incubation. Decline in amino- indicated times at 20 *C. Parallel reaction mixtrrrrs larking t p p s i n acylationactivity by 60%accompaniedconversion of the were assayed to correct for enzyme inactivation: less than I O r ; of the was lost in the ahsence of trypsin. A t the inrliratetl times. protein to the M , 52,000 fragment. Pyrophosphate exchange activity samples of 10 pI were treated with 1 0 mxf I'lfSF followed I)? SI)S activity, however, was not significantly affected until the M , andmercaptoethanol,heated for 2 min,andelrrtrnphoresetlon a 52,000 fragment wasdigested to the M , 40,000 fragment. 12.5%SDS-polyacn.Iamidc pel which WRS stainedwith('oomacsie Digestion a t high concentrations of trypsin for 1 h resulted in Blue (shown in A ). Samples were assayed fnr pyrophosphate exchange and aminoacyl-tRNA synthetase artivities after dilution in hrrffer ( 2 0 loss of both pyrophosphate exchange and aminoacyl-tRNA synthetaseactivity. T o investigatethe possible effects of mM KPO,, 20rA glvrerol. 0.2 r n \ f Er)TA, and 0.5 msf I)'I"T, conraining 0.1 mg/ml soyhean tnpsin inhibitor (shtrwn in /{I. I'yrophosphnte substrates on trypsin sensitivity, proline, ATP, and tRNA exchange activity at low ( A , and high ( A )tppsin; aminoacyl-t RSA were included singly or in combination in the digestion reac- synthetase activity at low (0) and high 1 0tr>psin. ) tion as shown in Fig. 5. tRNA appeared to increase sensitivity to the protease, while both proline and ATP afforded some ity (Fig. 6). Western blot analvsis (Fig. 6.A) using antibody level of protection. Proline appears to inhibit the conversion raised against the free form of prolvl-tRNA synthetase deM , 40,000 fragment, while tected primarily a polypeptide of M , 150,000 in the peak of of the M , 52,000 polypeptide to the ATPappearstoinhibittheconversion of the undigested highest molecular weight and primarilv a polypeptide of M , species to theM , 52,000 form. This latter effect of ATP was 60,000 in the peak of lowest molecular weight. 1'ol.ypeptides most evident when prolinewas also present, perhapsbecause of both sizes were detected in the intermediate fractions. The proline's inhibitory effect on the conversion of the M , 52,000 fractions were alsoassaved for glrltamyl-t RYA synthetase species to the M , 40,000 form allowed the M , 52,000 species which also exhibitedheterogeneous behavior. The distrihr~tion to accumulate. No amino acid sequence could be obtained of the M ,60,000 and 150,000 polypeptides detected by \Vestfrom any of the species generated by partial tryptic digestion ern blot analysis followed the activities for prolvl- and gluor from the undigestedenzyme; this result may indicate that tamyl-tRNA synthetases, respectively. Fractional precipitathe N terminus is blocked and that smaller fragments are tion with 5% PEG effectively separates the high and low M, removed from the C terminus of the protein by trypsin. forms so that primarilv the larger species was detected when Distribution of High and LAW M , Forms of Prolyl-tRNA this fraction was examined by gel filtration (Fig. 7.4 ). ChroSynthetase-To examine the size distribution of prolyl-tRNA matographyof the5 to lorn PE(; fraction on the same column synthetase, crude extract and various partially purified frac- indicated that i t contained primarilv smallerforms of the tions were subjected togel filtration chromatography on Seph- enzyme (Fig. 7 R ) . With material obtained after Q-Sepharose arose CL6R and assayed for enzymatic activity. When the chromatography of the 5 to lor; PEG fraction, the pcnk of crude extractwas examined the elutionprofile of the column prolyl-tRNA synthetase activitv was further shifted to a rerevealed a t least three peaksof prolyl-tRNA synthetase activ- gion of lower molecular weight when examined by chromatog-

Prolyl-tRNA Synthetnw

17706 low 1

1

1

7

3

4

hiqh 5

6

7

8

1

2

3

4

5

6

7

8

66-

31

-

FIG. 5. Effect of substrates on the susceptibility of prolyl-tRNA synthetase to d i g e s t i o n w i t h t r y p s i n . l'rolyl-tRS:\ s . n t h r t n w (0.2 mg/ml in buffer I?) was incul)ated with 1 pg/ml llnnrs 1-8, low) or 6 pg/ml ( I r t n c ~/ - x , IliCh) tr>7)sin at :{!I ( ' f o r (X) min in t h r a l w n c r of ligands ( h e I ) ; in the presence of 1.0 mM proline (lane 2); 5 mM A T P (Ianc 3); 9 p~ tRSA"'" ( / o m , 4 ) : 1.1) nix! prnlinr : t n d 5 mxf ; \ T I ' (Inn(,5 ) ; 1.0 mM proline and 9 p~ tRNA'"" ( h n c fi); 5 mM A T P a n d 9 ph4 tRNA'"" (Innr 7 ) ; 1.0 mM prnlinr, 5 m\f ,\TI', nntl !I pxf t RSA""' (Ianc 8 ) . T h e lane labeled is the undigested enzyme. At the end of incuhations. samples were treated with SIIS. hoilrd l o r 2 min. and subjected to electrophoresis on 12.50; SDS-polyacrylamide gels which was stained with Conmassie Hluc.

tRNAsynthetase,some of thematerialfromthe DEAEHPLC column which contained 170th activities was sr~hjectetl t o gel filtrationonSuperose I:! asshown inFig. 8. T h e behavior of glutamyl- and prolyl-tRNA synthetases on this column indicated that the two enzymes were heterogeneous, withmost of the glutamyl- and some of theprolyl-tRNA synthetaseactivitiescoelutingearly in thechromatogram, while the remaining prolyl-t RNA synthetase activity eluted later. Analysis of the active fractions by SDS-gel electrophoresis (Fig. 8 A ) and on Western hlots (Fig.R H ) , indicated that the M, 60,000 pdypeptide eluted withprolyl-t RNA synt hetase activity and the M , 150,000 polypeptide was coincident with glutamyl-tRNAsynthetaseactivityandthattheantihody E3 2.5 hoth detected pol.ypeptides. hehavior The of' the t w n enzymes upon gel filtration is consistent with their dissnciating during 2.0 forms \wight molecular lower chromatographv to since nctiv- ity could he detected in hoth high a n d low molecr~larweight 0 species (Fig. 7C). The chromatographic behavior of the two a 1.5 n column DEAE the onenzymes (I.'$. 4 ) is also consistent with ul n the two enzymes dissociating from a complex since the ac1.0 tivities were not at a constant ratio throughout the peak. Analvsis of Immunolngicnl C'ross-rrnctir,ity I'rol>,l0.5 and immr~nolog~ h t n m $ - tapparent f < ! v AS'.vnthta.w-The ical similarity between prolyl- and glut;1myl-t R N A synthetases was examined further to ascertain whether the larger 0.0 20 76 32 38 44 50 56 62 68 74 80 form might precursor be a the to free form of prolyl-t R S A fraction synthetase through proteolysis. hypothesis I f this is correct one would expect the M , 150.000 pol>-peptide t o contain hoth FIG. 6. Distribution of high and low molecular weight forms enzymatic activities and that the ratio of the two polypeptides of prolyl-tRNA synthetase in a crude extract on Sepharose CL6R. Sample preparation and chromatographic conditions are de- detected by the antibody might vary depending on the extractionmethod.First,twodifferentpreparations o f the high scribrd under "Experimental Procedures." A , Western hlot analysis of IO-pl samples o f theindicatedfractionsprobedwithaffinitymolecular weight aminoacyl-t RNA synthetase complex (propurified antihody to prolyl-tRNA synthetase. The sample designated vided by M. 7'. Norcum, LTniversity of Xfississippi Xhdical m is 20 ng of lorm I1 prolyl-tRNA synthetase run as a marker. H , 0, Center) from rabbit reticulocytes were examined b y \!'cstern A, prolyl-tRNA synthetase; A, glutamyl-tRNA synthetase. A r blot analysis as shown in Fig. 9 A . Examination o f t he silverrows in H indicate the elution position of ferritin ( F ) and aldolase stained gel run on the same samples (shownin 913) indicated ( A ). that the M , 1:i0,000 pol?lpeptide comigrated with the largest raphy on Sepharose CL6R as shown in Fig. 7C. Prolyl-tRNA component of the complex,b u t no polypeptidesin the complex 0 to 5 and 5 t o 10% PEG corresponded to thesize of the M , ( I O . 0 0 0 polypeptide of synthetase activity in both the prolyl-tRNA synthetase. While the antihotly readilydetected fractions exhibited clear indicationsof heterogeneity. both polypeptides in form I1 and only the smaller polypeptide Association of Prolyl-andGlutamyl-tRNASynthetasesin form I, only the largerM , 15O.f)OO p o l p e p t ide was detected We observed that during the later purification steps one form of prolyl-tRNA synthetase could be isolated which contained in the two preparations of the rabhit reticrllocyte complex. Whileglutamyl-tRNAsynthetaseactivity was readily dea larger polypeptide associated with it and glutamyl-tRNA tected in these same two preparations n t the complex, w e synthetaseactivity(seeFig. 3). T o examinethepossihle association between glutamyl-tRNA synthetase and prolylcould detect no activity for prolyl-t HSA synthetase. A l l the

A4 03 6

6 46 44 05 65 24 8

68

72

m

hrltrrwn

Prolyl-tRNA Synthetase ‘00

4

A

-.

A

19 2 7

17

F

212 5

23

97-

-5

p?

:66-

d

X

e

=

L

iI

5.0

VI

n

43-

0

2.5

B

0.0

19

17

21

25

23

27

froc!ion

:

97-

p?

6.0

12

66-

4

=

“ _“

L

-

43-

C F

Q

?

0

-

10

-

E

fraction

0

L

Q,

.

0.

.+, .” C (I)

2

i

o

I

0

5 -

ie/;o

0

a N

0

~i

*/

VI

n

Q

/’

?

0

0 10

A

a.

,

15

20

25

*

0 30

!

35

Fraction

FIG.8. Gel filtration of prolyl-tRNA svntheta.sepeak

11 from sulfopropvl-HPIX on Suprrose 12. A “1(1-p1 sample lrorn the DEAE-HI’I,(: s t c y (see Fic. :!!{I cnntaininghothprolyl-and frqc::on glutamvl-t RSA svnthvtase nrtivitiw was applied to a rolrlrnn ( 1 . 0 X FIG.7. Distribution of high- and low-M, forms of prolyl30 c m ) of Superose 12 eclrlilihratetl with hllfcr TI. The rolumn was tRNA synthetase in fractions taken during purification on eluted at 0.2 rnl/min. and frartinns o f 200 P I were rollerted starting Sepharose C L B R . Samples of t h e 0 5 to Tir; ( A ) polyethylene glycol a t 6.8 minandassayedfnrprolyl(01andglrltamyl- ( 0 1t H S A fraction, the 5‘; to 1 0 5 polyethylene glycol fraction ( E ) and the Q- synthetase as shown in C. Aliqrtots of the indicated fractions were Sepharose pool (C) were chromatographed on Sepharose CLGH and . treated with SDS and applied to I O r ; S1)S-polyarrylamitle ~ c l s One assayed for prolyl-tRNA synt.hetase activity. Sample preparation and was stained with silver nitrate (11 I * and the other was srlhjectetl to chromatographic conditions are described in “Experiment,al ProceWestern hlot analysis using affinity-purified antihody ( H ). Ferritin dures.” Symhols are as inFig. 6. ( F )and aldolase ( A I were run separately as atandnrcl.; and elrlte)d at fractions 2 1 nnd 28. respert ively. 70

76

52

:E

14

:.j

‘6

62

68

74

flr)

Western blot analyses shown in these experiments were performed with antibody that had been affinity-purified using the low molecularweightform I prolyl-tRNAsynthetase. However, we also used the M , 150,000 polypeptide to affinity still purifytheantibodyand observed thattheantibody reacted with both the larger M , 150,000 and smallerM , 60,000 polypeptides and that both polypeptides were detected irrespective of the dilution of the antibody for adsorptionto antigenduringitspurification.Inaddition,theantibody detected both polypeptides in crude extracts prepared under the following conditions: 1) extractsmade employing the protocol used in purification of the enzyme and treating the resultingextract with SDS andmercaptoethanol; 2) after extracting the tissue directly in SDS sample buffer used for electrophoresis; 3) after extracting the tissue in 6 M guanidineHCI, followed by acetone precipitation and treatment with

SDS andmercaptoethanol.Theseresults suggest thatthe pol-ypeptide larger pol-ypeptide is not a precursor to the smaller through proteolysis during extract preparation and that the larger polypeptide does not contain prolvl-tRNA synthetase activity. DISCUSSION

Prolyl-tRNA synthetase can be isolated from liver as multiple forms, one of which is the free enzyme, not associated with othercomponents.While it is possible that this size heterogeneity reflects oligomeric heterogeneitv in cico, a more likely explanation is that prolvl-tRNA svnthetaseis part of a larger complex andthat it dissociates from this complex during extraction and isolation. Consistent with this hypothesis is our observation that prolyl-tKNA svnthetnse can he

17708

Prolyl-tRNA Synthetaw

observed for prolyl- and glutamyl-tRSA synthetase maygive information concerning the organization oft hese two enzymes in the complex, hut would not he physiologically significant. T h e weakassociation of prolyl-tRNAsynthetasewiththe aminoacyl-tRNA synthetase complex andits possihle identity as a protein with a suhunit molecular weight of M , 60,000 has been noted earlier hy another lahoratory ( 1 3 ) . T h e free form of prolyl-tRNA synthetase is a n ( 1 , dimer of similar or identical suhunits and in this respect is similar to a numher of aminoacyl-t RNA svnthetases from hot h procaryotic and eucaryotic sources. The association of prolyl- and glutamyl-tRNA svnthetases appears to he noncovalent , since t hese t wn activities could he partially resolved hy gel filtration. and the prolyl-t R 9 A synthetase activity that remained associated with the M , l.50,OOO polypeptide was accompanied hy a smaller polypeptide of the subunit, size of the free enzyme which reacted with antihody to the small form of the enzyme. The conclusion that prolyltRNA synthetase activity resides exclusively in the smaller M , 60,000 polypeptide would appear to he unequivocal were o f the antihody with the it not for the specific interaction largerpolypeptideandtherecentreportthatprolyl-tRNA synthetase and glutamyl-tRNA synthetase are containedin a hifunctional polypeptide in Drosophiln ( 2 0 ) .A possihle interpretation of the \Vestern hlotsis that the small formof prolyltRNAsynthetase is derivedfromthelargeformthrough proteolysis. If the smaller polypeptide arises through proteolysis one might expect its ratio to the large fnrm to depend on the method of tissue extraction or on the length of time prior to the addition of achaotropicagenttoanextract; 4 5however, several crude extracts stored for various times at 4 “C and crude extracts prepared in tissue disruptedin chaotropic solvents containing guanidine-HClo r S I X followed hy Western hlot analysis contained hot h pol>peptides: the relative intensity of the staining did not appear to depend on the method of extract preparation. In addition, if prolyl-tRNA FIG. 9. Westernblotanalysis of prolyl-tRNA synthetase o f the M , and rabbit reticulocyte aminoacyl-tRNA synthetase complex.synthetaseactivitywerecontainedwithinpart 150,000 polypeptide along with glutamyl-tRNA synthetase. A , samples were applied to a IOr;. acrylamide SDS gel and \Vestern hlot analysis using antihody to prolyl-tRNA synthetase. Lane I , 20 the two activities ought to he present in preparations that ng of prolvl-tRNA synthetase from DEAE-HPLC that contains hoth containonlythe M , 150.000 polypeptideandoughtto he prolyl- and glntamyl-tRNA synthetase activities: lnnc 2, 10 ng of the present at a constant ratio. However, two preparations of the free form of prolvl-tRNA synthetase; lnnrs 9 and 4 , two different preparations (100 and 75 ng, respectively) of rahhit reticulocyte aminoacvl-tRNAsvnthetasecomplexfromrahhitreticuloM , 150,000 polypeptide and glutamyl-tRNA aminoacyl-tRNA synthetase complex. R. samples were applied to a cytes in which the 10‘6 acrylamide gel and stained with silver nitrate. I m w I , 100 ng of synthetase activity were readily detected had negligihlelevels prolyl-tRNA synthetase from the DEAE-HPLC step containing hoth of prolyl-tRNAsvnthetaseactivity.an nhsen.:ltion that is prolyl- and glntamyl-tRNA synthetase activities: lnnr 2, 50 ng of the inconsistent with the M , 150.000 polypeptide having prolylfree form of prolyl-tRNA synthetase; lnnrs .? and 4. two preparations of rahhit reticulocyte aminoacyl-tRNA synthetase core complex (2.0 tRNA synthetase activity as suggested earlier ( ‘ L O ) . ‘This activity,however,wasreadilydetected in form I1 o f prolyland 1.5 pcg, respectively). tRNA synthetase which contained hoth the M , 60,000 and 150,000 polypeptides. I t is possihle that the M, 1.50.000 polyisolated associated with glutamyl-tRNA synthetase. An earlier study indicated that glutamyl-tRNA synthetase could he peptidehasprolvl-tRNAsynthetaseactivity,hutthatthis form of theenzyme is differentiallyinactivated or that it isolated associated with predominantly Iysyl-tRNA syntheexists in a latent state. W’hile none of our data supports this tase (37). The various sized formsof prolyl-tRNA synthetase prnlylcould be accounted for if glutamyl- and prolvl-tRNA synthe- hypothesis,theohservationthattheglutamyl-and he contained in a single polypeptases are associated with one another and the complex of tRNA synthetases appear to tidein Drosophila suggeststhatthisexplanationcould he these two components is in turn associated with some other components of the aminoacyl-tRNA synthetase complex. Par-correct. A third possihilitv that is consistent with our data is that there are two antigenically related formsof prnlyl-tRNA tial dissociation from the complex would result in some activsynthetase, one thatis part of the M , 1.50.000 pol>-peptide and ity associated with the complex, some existing as a prolylglutamyl-tRNAsynthetasecomplexandafraction of t h e a second that is a separate entity that is not the result o f a trivial explanation, such asprotenlyt ic digest ion o f the large activity existing as free prolyl-tRNA synthetase. A similar form. This latter explanation would alsn have t he requirement phenomenon has been observed with the arginyl- and Iysylin the lilrgfl pnlypeptRNA synthetases which can be isolated as an n,&’,!tetramer that the prolyl-t,RNA svnthetase activity tide exhibit differential stahility or activityrelative t o the containing hoth activities (38) or as part of the aminoacylI)rosophiln ( 2 0 ) . the tRNA synthetase complex.If this explanation for the multipleglutamyl-tRNAsvnthetaseactivity.In appears t o he enforms of prolyl-tRNA synthetase is correct, the heterogeneity hifunctionalgluprolvl-tRNAsynthetase

Prolyl-tRNA Synthetase coded by a 6.1-kb mRNA; however, there is an additional 3.4kb mRNA that appears to be related to 3' end of the 6.1-kb mRNA in sequence that encodes the putative prolyl-tRNA synthetase domain. Theobservation of two mRNAs may suggest the existenceof a smaller formof the protein containing only prolyl-tRNAsynthetase activity. It is likely that cDNAs mammals containa similar bifunctional protein since originally thoughtto encode glutaminyl-tRNAsynthetase (39) encode a protein that is strikingly similar to gluprolylthe tRNA synthetase in Drosophila (approximately 70% identity noted by the authors of Ref. 20). If the immunological cross-reactivity is takenat face value, it suggests that thelarge and smallpolypeptides in theprolylglutamyl-tRNA synthetase complex have common antigenic determinants;thissimilarity maybeindicative of similar structures and perhaps related primary sequences. A trivial explanation would be that theoriginal antigen (form I prolyltRNAsynthetase) was contaminatedwiththe larger M , 150,000 polypeptide. Several aspects of the methodology for preparation and analysis of the antisera suggest that this is not the case. First, the antigen used for immunization of the rabbits appeared to be free of the large polypeptide, but was further purifiedfrom a n SDS-acrylamide gel that readily separates thetwo polypeptides. Second, the antibody reacted with bothpolypeptides after it had been affinity-purified with either the larger or the smaller polypeptide. Third, four rabbits immunized with the antigen all showed reactivity with both polypeptides, indicating that the specificity of the antibody did not represent theidiosyncratic response of one animal. The results we haveobtained suggest that prolyl-tRNA synthetase and glutamyl-tRNA synthetase are noncovalently associated with one another and may share some similarity in primary structure. There are some interesting issues that remain to be resolved. First, what is the nature of the apparent antigenic similarity between the glutamyl- and prolyl-tRNA synthetases? Second, can the relative concentrations of prolyl- and glutamyl-tRNA synthetases vary depending on the growth conditions or the state of the cell or do they always exist in a defined stoichiometry? Third, are there two pools of prolyl-tRNA synthetase in vivo, one associated with glutamyl-tRNA synthetase and a second that exists independently, or is the existence of a free form merely a result of the isolation procedure? Fourth, how are prolyl- and glutamyltRNA synthetasesorganized in the aminoacyl-tRNA synthetase complex with respect to the other polypeptide components?

17709

Acknowledgments-We are indebted to Dr. Mona Trempe Norcum, Department of Biochemistry, University of Mississippi Medical Center, for providing samples of the aminoacyl-tRNA synthetase core complex from rabbit reticulocyte and for many productive discussions during the course of these studies. REFERENCES 1. Schimmel, P. (1987) Annu. Reu. Biochem. 5 6 , 125-158 2. Mirande, M. (1991) Prog. Nucleic Acid Res. 40,95-124 3. Yang, D. C. H., Garcia, J. V., Johnson, Y. D., and Wahab, S. (1988) Curr. Top. Cell. Regul. 26,325-335 4. Dang, C. V., and Dang, C. V. (1986) Bicchem. J. 239,249-255 5. Cherniack. A. D.. Garriea. G.. Kittle., J. D.., Akins. R. A,. and Lambowitz. A. M. (1990) Cell 62,-745-755 6. Miseta, A,, Woodley, C. L., Greenberg, J. R., and Slobin, L. I. (1991) J. Biol. Chem. 266,19158-19161 7. Huang, D.-D., and Wang, W. (1986) J. Biol. Chem. 2 6 1 , 13451-13455 8. Schon, A., Krupp, G., Gough, S. P., Berry-Lowe, S., Kannangara, C. G., and SOH, D. (1986) Nature 322,281-284 9. Kellermann, O., Tonetti, H., Brevet, A,, Mirande, M., Pailliez, J.-P., and Waller, J.-P. (1982) J. Biol. Chem. 2 5 7 , 11041-11048 10. Mirande, M., Kellermann, O., and Waller, J.-P. (1982)J. Biol. Chem. 2 5 7 , 11049-11055 11. Mirande, M., Cirakoglu, B., and Waller, J.-P. (1982) J. Biol. Chem. 2 5 7 , 11056-11063 12. Mirande, M., Le Corre, D., and Waller, J.-P. (1985) Eur. J. Biochem. 1 4 7

"-3R1-384 ""

13. Godar, D. E., Godar, D. E., Garcia, V., Jacobo, A,, Aebi, U., and Yang, D. C. H. (1988) Biochemistry 27,6921-6928 14. Motorin, Y., Wolfson, A., Orlovsky, A,, and Gladilin, K. (1988) FEBS Lett. 238. 262-264 15. Bec, G., Kejan, P., Zha, X. D., and Waller, J.-P. (1989) J . Biol. Chem. 2 6 4 , 21131-21137 16. Norcum, M. T. (1989) J. Biol. Chem. 2 6 4 , 15043-15051 17. Kellerman, O., Brevet, A,, Tonetti, H., and Waller, J.-P. (1978) Eur. J . Biochem. 8 8 , 205-210 18. Lazard, M., Mirande, M., and Waller, J.-P. (1985) Biochem. 24,5099-5106 19. Bec, G., and Waller, J.-P. (1989) J. Biol. Chem. 264,21138-21143 20. Cerini, C., Kerjan, P., Astier,M., Gratecos, D., Mirande, M., and Semeriva, M. (1991) EMBO J. 10,4267-4277 21. Viswanathan, S. V., and Dignam, J. D. (1988) J. Biol. Chem. 263,535-541 22. Beely, J. G., and Neurath, H. (1968) Biochemistry 7 , 1239-1251 23. Dignam, S. S., and Dignam, J. D. (1984) J . Biol. Chem. 259,4043-4048 24. Gillam, I. C., and Tenor, G. M. (1981) in Selected Methods in Enzymology: RNA and Protein Synthesis (Colwick, S. P., and Kaplan, N. O., eds) pp. 43-58, Academic Press, New York 25. Hirs, C. H. W. (1967) Methods Enzymol. 11,59-62 26. Hunkapillar, M. W.,and Hood, L. E. (1983) Methods Enzymol. 91,60-75 27. Bradford, M. M. (1976) Anal. Biochem. 7 2 , 248-254 28. Laemmli, U. K. (1970) Nature 227,680-685 29. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244,4406-4412 30. Wray, T. B., Wray, V. P., and Hancock, R. (1981) Anal. Biochem. 1 1 8 , 197-203 31. Dignam, J. D., Dignam, S. S., and Brumley, L. (1991) Eur. J. Biochem. 198,201-210 32. Tanford, C., Nozaki, Y., Reynolds, J. A,, and Makino, S. (1974) Biochemtstry 1 3 , 2369-2376 33. Siegel, L.M., and Monty, K. J. (1966) Biochim. Biophys. Acta 1 1 2 , 346362 34. Viswanathan, S., Dignam, S. S., and Dignam, J. D. (1988) Deu. Biol. 129, 350-357 35. Smith, D. E., and Fisher, P. A. (1984) J. Cell Biol. 9 9 , 20-28 36. Blake, M. S., Johnston, K. H., Russell-Jones, G. J., and Gotschlich, E. C. (1984) A d . Bwchem. 136,175-179 37. Deutscher, M. P. (1967) J. Biol. Chem. 2 4 2 , 1123-1131 38. Dang, C. V., Glinski, R. L., Gainey, P. C., and Hilderman, R. H. (1982) Biochemistry 21,1959-1966 39. Fett, R., and Knippers, R. (1991) J. Biol. Chem. 266,1448-1455 I~

~

~

~