Structural and Enzymatic Studies of the T4 DNA Replication System

Vol. 264, No. 21, Issue of July 25, pp. 12709-12716,1989 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural and EnzymaticStudies of the T4 DNA Replication System I. PHYSICAL CHARACTERIZATION OF THE POLYMERASE ACCESSORY PROTEIN COMPLEX* (Received for publication, November 28, 1988)

Thale C. JarvisS, Leland S. Paul$, and Peter H. von Hippelll From the Instituteof Molecular Biology and Department of Chemistry, Universityof Oregon, Eugene, Oregon97403

In this study, we have investigated the structural and physical properties of the bacteriophage T4 DNA polymerase accessory proteins. We find that T4 gene 4 4 and 6 2 proteins associate to form a tight, highly homogeneous complex, containing four gene 44 protein subunits and one gene 6 2 protein subunit. The molecular mass of the complex is 163,700daltons. Sedimentation results suggest that the complex is quite asymmetric, witha prolate ellipsoid axial ratioof about 5:1. This protein complex is known to carry a DNA-dependent ATPase activity; we show by photoaffinity labeling that the ATP-binding sites reside in the gene 4 4 protein subunits of the complex. Equilibrium sedimentation and chemical cross-linking studies indicate that the T4 gene 46 protein self-associates to form a trimer in solution. This trimer species also appears to be quite asymmetric, showing an axial ratiofor a prolate ellipsoid of about 6:1, assuming normal hydration.

The bacteriophage T4 gene 43, 44, 62, 45, and 32 proteins, known to be required for DNA replication i n uiuo, can be formed into a reconstituted complex that is able to carry out leading strand DNA synthesis i n vitro (1,Z). The “polymerase accessory proteins,” encoded by genes 44, 62, and 45, show a DNA-dependent ATPase activity (3, 4), and in the presence of ATP these proteinsgreatly stimulate the enzymatic activity of the polymerase (5-7). Mechanistic studies of functional replication complexes require a structural understanding of the component proteins. Thus, we have undertaken aphysical characterization of the T4 gene 44, 62, and 45 proteins. In this paper we present structural and physical evidence for the association states and subunitcompositions of the T4 polymerase accessory proteins. In the accompanying paper (8), we examine functional aspects of the complex as manifested by its DNA-dependent ATPase activity. Through a combination of physical and enzymatic characterization, we can begin to elucidate the molecular mechanisms whereby the

accessory proteins interact with the DNA template and the other proteins within the replication complex. T4 gene 44 and 62 proteins associate to form a tight complex, dissociable only under denaturing conditions (3, 9, 10). Both of the genes have been cloned (11, 12), and the monomer molecular masses are known to be 35,584 and 21,347 daltons, respectively. Estimates in the literature for the size and composition of the complex vary (9, 11, 13). Thus, in order to understand the mechanism we must first define the stoichiometry of the complex. We have approached this by molecular weight determination, using velocity sedimentation, dynamic laser light scattering, and equilibrium sedimentation techniques. In addition, we have determined subunit composition directly by using reverse-phase HPLC’ to separate the gene 44 and 62 protein subunits from the complex. The gene 45 protein has also been cloned (14) and has a monomer molecular mass of 24,710 daltons. In order to understand how this protein interacts with the gene 44/62 protein complex, it is important to determine the association state of gene 45 protein both in solution and in association with the gene 44/62 protein complex. We have used equilibrium sedimentation and chemical cross-linking to determine the molecular weight of the associated species of gene 45 protein. MATERIALS ANDMETHODS

Preparation of Proteins and Nucleic Acids”T4gene 45 protein was preparedaccording to the method of Morris et al. (10)with the following modification. After the norleucine-Sepharose chromatography step, the proteinwas dialyzed into 50 mM NaC1, 1 mM EDTA, 10% glycerol, 1 mM /3-mercaptoethanol, and 20 mM Tris-HC1, pH 8.1, and runover single-strandedDNA-cellulose in series with Bio-Rex-70 (Bio-Rad), followed by a second DEAE-cellulose column (Whatman DE52). Under these conditions, the gene 45 protein passes through the first two columns and binds to DE52. It was then batch eluted from DE52 with a similar buffercontaining 400 mM NaC1. T 4 gene 44/62 protein was also purified according to Morris et al. (101, except that the protein was eluted from the hydroxylapatite column with a linear salt gradient (13). In addition, the final pool was dialyzed into I buffer (as defined by Morris et al. (lo)), loaded * This work was supported in part by United States Public Health onto single-stranded DNA-cellulose,and elutedwith a linear gradient Service (USPHS) Research Grants GM-15792 and GM-29158 (to P. from I buffer to I buffer plus 100 mM NaC1. The gene 44/62 protein H. v. H.) and by a grant from the Lucille P. Markey Charitable Trust. complex eluted a t about 40 mM NaCl. T4 gene 32 protein was purified according to Bittner et al. (15), The costs of publication of this article were defrayed in part by the payment of page charges. Thisarticlemusttherefore be hereby using T4 phage amN134, amBL292, amE219 (33-, 55-, 58-61-). The marked “advertisement” in accordance with 18 U.S.C. Section 1734 buffers for norleucine-Sepharose chromatography contained 0.05 M solely to indicate this fact. NaCl, instead of the 0.5 M prescribed by Bittner et al. (15). An $. Predoctoral trainee supportedby USPHS Institutional Research additional chromatography step,DEAE-Sephacel, was performed acService Award GM-07759. Submitted to the Graduate School of the cording to Alberts and Frey (16). T4 gene 43 protein was purified according to Morris et al. (10). University of Oregon in partial fulfillment of the requirements for the Ph.D. degree in Chemistry. Present adress: Dept. of Molecular, Escherichia coli rho protein andNusA protein were gifts from JohanCellular, and Developmental Biology, University of Colorado, Boulder, CO 80309. ’ The abbreviations used are:HPLC, high performance liquid chro§ Predoctoral trainee supportedby USPHS Institutional Research matography; DMS, dimethylsuberimidate; HEPES, 4-(2-hydroxyService Award GM-07759. Present address:Abbott Laboratories, ethyl)-l-piperazineethanesulfonicacid; SDS-PAGE, sodium dodecyl Abbott Park, IL 60064. sulfate-polyacrylamide gel electrophoresis; NsATP, 8-azidoadenosine 7 American Cancer Society Research Professor of Chemistry. 5”triphosphate.

12709

12710

Physical Propertiesof the Accessory Protein Complex

nes Geiselmann and Stanley C. Gill, respectively (this laboratory). All proteinpreparations were judged >98% pure based on SDSPAGE and were shown to he free of contaminating endonuclease by incubation with supercoiled pBR322. Poly(dA) was purchased from Pharmacia LKB Biotechnology Inc. Partial Specific Volume and Extinction Coefficient-Amino acid sequence information (11, 12, 14) allows us to calculate theoretical values for various physical properties of the accessory proteins, such as themolar extinction coefficient and the partialspecific volume. In general, the implicit assumption in these calculationsis that the physical characteristics of the individual amino acid residues will remain essentially the same in the native, folded protein as they are free in solution.While there are clearly examples of proteins for which this is not the case, the assumption proves surprisingly valid for the majority of proteins, and thus such calculations can provide valuable estimates of the physical properties of proteins of known sequence. The theoretical partial specific volume, 6, of gene 45 protein is 0.743 g/ml. This is based on known partial specific volumes of individual amino acid residues (17). Gene 44 and 62 proteins have calculated values of B of 0.739 and 0.746 g/ml, respectively. Theoreticalmolar extinction coefficients for theproteins were calculated based on the number of tryptophans and tyrosines they contain, and using the molar extinction coefficients determined by Edelhoch (18)for these residues cz80 = N~,5690 M~,,1280. (Procedures for carrying out such calculations and their limits of error, based on an extensiveanalysis of abasis set of experimentally determined values for a variety of proteins, have been described by Gill and von Hippel.)' N and M represent the numbersof tryptophan and tyrosine residues/protein monomer, respectively. The molar extinction coefficients are 1.91 X lo4, 2.29 X lo4, and3.17 X 10' I/molcm for gene 45, 44, and 62 proteins respectively. Velocity Sedimentation-Sedimentationcoefficients for gene 45 and 44/62 proteins were determined by velocity sedimentation in a Beckman model E Ultracentrifuge. A 12-mm double sector cell with quartz windows was filled with 0.9 ml of sample and a slightly greater amount of reference buffer. The buffer for both proteins consisted of 200 mM KCI, 25 mM Tris-HCI, pH 7.4, and 0.4 mM dithiothreitol. The sedimentation boundary was monitored using a UV scanner set a t a wavelength of 280 nm, and the boundaries were analyzed as described by Van Holde and Weischet (19).The initial gene 45 protein concentration was 0.38 mg/ml, and the rotor speed was 36,000 rpm. Gene 44/62 protein was sedimented at 34,000 rpm, with an initial concentration of 0.40 mg/ml. Light Scattering-Thediffusioncoefficientfor the gene 44/62 protein complex was determined by dynamic laser light scattering using the 514.4-nm line of a n argon ion laser (Spectra Physicsmodel 5). An intracavity etalon was used to ensure single frequency output. Scattered light was detected by a photomultiplier a t scattering angles of 60", go", 120", and 135". Translational diffusion coefficients were obtained using a Langley-Fordmodel 1096 autocorrelator. The analysis was performed as described by Bloomfield and Lim (20). The sample buffer contained 2.5% (w/v) glycerol, 25 mM Tris-OAc, pH 7.5, 60 mM KOAc, and 0.5 mM dithiothreitol and protein at 1.1mg/ ml. Sedimentation Equilibrium-Equilibrium ultracentrifugation was performed in a Beckmanmodel E Ultracentrifuge, using the meniscus depletion method (21). The sample buffer for both gene 45 and 44/ 62 proteins contained 100 mM KCl, 25 mM Tris-HCI, pH7.4, and 0.4 mM dithiothreitol. 0.13 ml of sample and 0.14 ml of reference buffer were placed in a double-sector cell with sapphire windows and interference window holders, and the cell was placed in an AN-D rotor. Interference optics were used, with 0.75-mm slits. Interference fringe photos were taken on Kodak Spectrograde 11-G photographic plates, and thefringes were analyzed on a Nikon model 6C Profile Projector. In general, after centrifugation for 40 to 48 h, the fringe patterns showed no furtherchanges with time,and we judged that equilibrium had been attained. In all cases, the meniscus depletion condition was clearly achieved, as judged by the levelness of the fringes in the upper half of the cell image. Reuerse-phase HPLC-Reverse-phase HPLCseparation of the gene 44 and 62 proteins was performed on an Aquapore Butyl (C4) column(330 X 4.6 mm, 7 pm), usingBeckman llOA Pumps, 421 Controller, 163 Variable Wavelength Detector, and a Spectra Physics SP4270 Integrator. The mobile phases contained 0.05% (v/v) trifluoroacetic acid (Applied Biosystems) and 0.05% (v/v) triethylamine (Fluka). Thegene 44/62 protein was diluted from storage buffer (10)

+

S. C. Gill and P. H. von Hippel, submitted for publication.

5-fold in0.1% trifluoroacetic acid, and a total of 50 pg of protein were loaded onto the column a t 15% (v/v) acetonitrile (UV grade, Brand). The subunits were eluted from the column over a 30-min period, using a linear 15-62% acetonitrile gradient with a flow rate of 0.35 ml/min. Chemical Cross-linking-The chemicalcross-linking of gene 45 protein was done with DMS (Pierce Chemical Co).The proteinswere dialyzed into 40 mM HEPES, pH 8.1, 100 mM KCI, 0.2 mM dithiothreitol, and10% (w/v) glycerol. The DMSwas freshly prepared each time by dissolving in 40 mM HEPES, pH 8.1, and adjusting the pH to approximately 8.5 with NaOH. Reactions contained protein a t 0.51.0 mg/ml and DMS at5 mg/ml. Reactions were performed a t room temperature and quenched by addition of ethanolamine to a final concentration of 0.6 M. The products were analyzed by SDS-PAGE as described by Laemmli (22) and silver-stained by the method of Morrisey (23). Azido-ATP PhotoaffinityLabeling-Photoaffinity labeling wasperformedusing theATPanalog 8-azidoadenosine5"triphosphate (NaATP). [ T - ~ ' P ] N ~ A Twas P obtained from Du Pont-New England Nuclear, and unlabeled NSATP was purchased from Schwarz-Mann. NsATP-containing solutionswere irradiated with an ultraviolet lamp with a 302-nm emission peak (Spectronics model BLE-1T158). Samples were mixed in a microtiter dish (Falcon) in volumes of approximately 20 pl and irradiated for 4 min a t a distance of about 3 cm. The labeled products were analyzed by SDS-PAGE, with the addition of 6 M urea to thegel and samplebuffer. RESULTS

Determination of the Molecular Weight and Subunit Composition of the Gene 44/62 Protein Complex As noted above, the productsof T4 genes 44 and 62 associate to form a tight complex. Subunit exchange studies (9) have failed todetectany dissociation and reassociation of the complex under native conditions, indicating that complex, the once formed, is quite stable. Although the molecular weights of the monomers are accurately known from the DNA sequence, the precise molecular weightof the totalcomplex has only been estimated. Various stoichiometries have been reported for the subunits of this complex, ranging from a ratio of four gene 44 to two 62 subunits (9) based on Coomassie staining of the proteins eluted from denaturing gels, to a 5:l ratio (13), obtained by densitometry of a Coomassie-stained gel. Most recently, Spicer et al. (11) have reported a 3.6 (k 0.6): 1 ratio, based on quantitation of the size of the peaks obtained at each step of protein sequencing. This is probably the best estimate currentlyavailable, but leaves some uncerhence as to tainty as to the exact subunit stoichiometry, and the molecular weight of the complex. T o firmly establish the composition of this multiprotein complex, we have re-examined the issue of subunit stoichiometry and determined the molecular weight of the complex by two different methods. Molecular Weight Determination Using theSvedberg Equation-When a macromolecular experiences acentrifugal field, the rateat which it sedimentswill be directly proportional to its reduced mass and inversely proportional to its frictional coefficient. The frictional coefficient is influenced by factors such as shape and hydration and these same frictional forces will modulate the rate at which the macromolecule diffuses. The Svedberg equation (for a derivation, see Ref. 24): M=

RTS D ( l - 6p)

(1)

allows the calculation of molecularweight using both the sedimentation and diffusion coefficients. Here M equals the molecular weight of the solute,R is the gas constant, T is the temperature, S is thesedimentation coefficient, D isthe diffusion coefficient, d is the partial specific volume, and p is the solute density. The frictional factors cancel, and we can

Physical Properties

Accessory of the Protein Complex

obtain a molecular weight that is independent of shape and hydration. The molecular weight of the gene 44/62 protein complex was determined by the sedimentation and diffusion method. A sedimentation coefficient of 7.1 f 0.2 S was obtained by velocity sedimentationinananalyticalultracentrifuge. A diffusioncoefficient of 3.9 X cm2/s was determined by dynamic laser light scattering. The sedimentation and diffu20 “C inwater. The datawere sion constants are corrected to not obtained over a wide concentration range and thus are not corrected to infinite dilution. The samples are already quite dilute, however, and as notedby Van Holde (25), the S and S o (sedimentation coefficient extrapolatedtoinfinite dilution) valuesfor most globular proteins differ by onlyabout 0.5% a t protein concentrationsof 1 mg/ml. In addition, several lines of evidence support the homogeneity of the species, including laser light scattering andsedimentation equilibrium. Therefore, we feel justified incombining the sedimentation anddiffusion data using theSvedburg equation, to give a molecular mass of 170,000 daltons for the complex. This relies ontheoretical valuesfor thepartial specific volumes of the proteins, based on amino acid sequence (see “Materials and method^").^ The sedimentation constant of 7.1 S for the gene 44/62 protein complex is in good agreement with that determined by Barry and Alberts (9) using sucrose density gradient sedimentation. Consistentlylow values of the “quality parameter”, p / y 2 , for the light-scattering data show that the autocorrelation function fits well to a single exponential decay, providing a sensitive indication of sample homogeneity (20). Determination of Molecular Weight by Equilibrium Sedimentation-The molecular weight of the gene 44/62 protein complex was further studied by equilibrium sedimentation, a technique capable of high precision. At sufficiently low rotor speeds, the centrifugal force that results in transport of the macromolecule bysedimentation will be balanced by diffusion transport in the opposite direction.An equilibrium condition is established, generating a concentration gradient throughout the cell. A homogeneous species, in an ideal two component system (i.e. soluteandsolvent) will show a concentration distribution described by: d In c - M ( 1 dr

“

2RT

12711

TABLE I Determination of accessory protein molecular masses by equilibrium ultracentrifugation The molecular weights of the gene 44/62 protein complex and the gene 45 protein were determined by equilibrium ultracentrifugation as described (see“Materials and Methods”). The initial concentration of gene 44/62 protein was 0.48 mg/ml for both runs, and the initial concentration of gene 45 protein was 0.69 mg/ml for the 24,000 and 28.000 m m runs, and 0.46 m d m l for the 20,000 m m run.

(2)

Molecular mass

14,400 15,000

163,000 177,000

76,600 20,000 24,000 28.000

78,700

daltons

Gene 44/62 protein 45 Gene

protein 75.900

i

-5F , 50.2

50.4

50.6

50.8

51.0

51.2

51.4

51.0

51.2

51.4

r* -2 t

50.2

Up)w2

mm

50.4

50.6

50.8

r2

FIG. 1. Fringe displacement data from sedimentation equi-

where c is the concentration of the solute, r is the distance librium. The naturallogarithm of the fringe displacement (in cm)is from the centerof rotation, and w is the angularvelocity (for plotted uersus ?, with r equal to the distance from the center of rotation (also in cm). Since the fringe displacement is proportional a review see Schachman (24)). to the solute concentration, the slope of the plot is proportional to The molecularweightvaluesfor the gene 44/62 protein the apparent molecular weight of the protein. A, gene 44/62 protein complex, obtained by equilibrium sedimentation at two difsedimented a t 14,000 rpm. B, gene 45 protein sedimented a t 20,000 ferent rotor speeds, are shown in Table I. The results are in rpm. excellent agreement with those obtained by the sedimentation and diffusion method (see above). Calculationof the molecu- of error in themolecular weight determination. Experimental lar weights relies on theoretical valuesfor the partial specific error, such as uncertainties in rotor speed, temperature, and known amino acid quantitation of the interference fringe patterns, can be estivolumes of the proteins, obtained from the sequences. Since the experimentally determined partial spe- mated and brings the total uncertainty in molecular weight cific volume of most proteins falls within 2% of the value to a maximum of about +lo%. predicted from sequence: thisassumptionis expected to The In c uersus r2 plots for equilibrium sedimentation excontribute an uncertainty of up to &6% in the final calculated periments run at 14,000 rpm are shown in Fig. 1A. Since the molecular weight, thus constituting the largest single source fringe displacement is proportional to solute concentration, The calculated partial specific volumes of the gene 44 and 62 proteins are close enough in value so that thechoice of stoichiometry (3:1, 4:1, 5 1 , etc.) for the complex makes little differencein the calculated molecularmass. A complex of four gene 44 protein subunits and one gene 62 subunit should have a partial specific volume of 0.740 g/ml. S. C. Gill, personal communication.

the slope of such a plotisproportionaltotheapparent molecular weight. The straightnessof the line is indicative of the homogeneity of the complex. As noted by Van Holde (25), In c uersus r 2 curves can be relatively insensitive to heterogeneity in some cases because the upward curvature produced by heterogeneity can be compensated by a downward curvature due tononideality, making the line appear straight. The

12712

Physical Properties

of the Accessory Protein Complex

TABLE I1 laser light-scattering experiments, however, are quite sensitive to heterogeneity and argue strongly in favor of a high Subunit stoichiometryof the gene 44/62 protein complex determined by HPLC separation of the subunits degree of homogeneity for the gene 44/62 protein complex. In Native gene 44/62 protein complex was loaded onto the column view of these results, the data of Fig. 1A can be most simply interpreted as representing ahomogeneous complex behaving and the gene 44 and 62 protein subunits were separated by reversephase HPLC. The effluent was monitored a t both 220 and 280 nm. in a relatively ideal fashion. It should also be noted that the Integration of the absorbance peaks gives the relative areas correYphantis (21) meniscus depletion method used here, although sponding to the gene 44 and 62 proteins. quite sensitive to low molecular weight contaminants, can Wavelength of detection entirely miss very high molecular weight species. Again, be220 nm 280 nm cause the light scattering technqiue is very sensitive to high 14.4 23.1 molecular contaminants, the results give us confidence that % Gene 62 protein peak (relative area) gene 44/62 protein in solution exists as a single species of molecular mass 170 f 20 kDa. Subunit ratio 3.71 4.5:l Determination of Subunit Ratios by Reverse-phase HPLC(G44P:G62P) (3.3 to 6.6:l) (2.8 to 5.01)” The molecular mass studies described above have sufficient The errors are estimates based on the following considerations. error to make it difficult to distinguish,for example, a complex The standard deviation of the integrated peak areas from four chrocontaining four gene 44 subunits and one gene 62 subunit matograms (for each wavelength) gives values of 14.4 k 2.8 at 220 (molecular mass 164 kDa) from one containing four gene 44 nm and 23.1 k 2.6 at 280 nm for the relative area of the gene 62 subunits andtwo gene 62 subunits (molecular mass 185 kDa). protein peak. This corresponds to 3.7 k 0.6 and 4.5 -+ 0.5, respectively, Therefore, we have also investigated the stoichiometry of the for the subunit ratios. These relatively small errors demonstrate the reproducibility of the data. There is, however, an uncertainty inherent subunits by using reverse-phase HPLC to separate and quan-in detection when monitoring the steep absorbance shoulder a t 220 titate theindividual protein components of the complex. The nm, increasing the total error in that measurement. The interpretagene 44 and 62 proteins separate on a C4 column with a tion of the peak area determined at 280 nm relies on theoretical gradient of increasing acetonitrile concentration.The elution extinction coefficients (see “Materials and Methods”). This method profiles are shown in Fig. 2. At least 90% separation of the of molar extinction coefficient calculation has been applied to prosubunits is achieved under these conditions. Gel electropho- teins of known sequence and shown to yield an average standard deviation of less than k5% from the extinction coefficients of the resis of fractions collected from this column identify the proteins as determined by a variety of other methods.’ Therefore, we species that elutes first to be gene 62 protein; the larger peak have incorporated that uncertainty in estimating the final error in with the longer retention timecorresponds to gene 44 protein. the 280-nm measurement. The elution of the proteins from the column was monitored by a UV detector at two different wavelengths. The dominant making it difficult to distinguish, for example, a 4:l from a chromophore at 220 nm is expected to be the peptide bond. 5:l complex. This accounts for the asymmetric errors shown Therefore, the relative areas at 220 nm have been corrected in Table 11. Although the errors are too large to allow us by the molecular masses of the gene 44 and 62 protein mon- uniquely to specify the subunit stoichiometry, this technique omers (35,584 and 21,347 daltons, respectively) to give the is particularly good at distinguishing between complexes with calculated subunit stoichiometry. At 280 nm, the absorbance low subunit ratios; the difference between a 2:l and a 3:l is principally due to tryptophan andtyrosine residues. Thus, complex is quite large. Thus, we can feel confident in ruling the theoretical extinction coefficients for the gene 44 and 62 out the2:1 gene 44 to 62 protein ratio originally suggested by proteins at thiswavelength were used to convert the relative Barry and Alberts (9). The Gene 44 and 62 Proteins Form a 4:l Complex-The areas into subunit stoichiometries. The resulting stoichiometries are shown in Table 11. Note that higher subunit ratios molecular weight and subunit stoichiometry data described show incrementally smaller changes in therelative peak areas, above for the gene 44/62 protein complex are summarized in Table 111. Several hypothetical subunit compositions are listed along with predicted molecular weights that span the range of compositions previously reported in the literature. The results of the HPLC experiments (determining subunit stoichiometry of the complex), and the sedimentation experiments (yielding complex molecular weight), are scored in terms of agreement with the proposed subunit composition. While the molecular weight determination could not unambiguously rule out the 3:2 and 4:2 stoichiometries, the HPLC data is clearly inconsistent with such low subunit ratios. Only the 4:l ratio is corroborated by both the sedimentation and HPLC data. Therefore, the combined evidence indicates that T4 gene 44 and 62 proteins form a tight, homogeneous species consisting of four gene 44 subunits, and one gene 62 protein subunit, with a total molecular mass of 163,700 d a l t ~ n s . ~ 20

25 Retention Time (min)

20

25 Retention Time bin)

FIG. 2. Chromatograms of HPLC separation of gene 44 and 62 proteins. The elution profile of the reverse-phase HPLC separation of gene 44 and 62 protein is shown, with the detection wavelength set at 220 nm ( A ) and 280 nm ( B ) . Fractions were collected and analyzed by SDS-PAGE, identifying the faster elutingspecies to be gene 62 protein and the slower to be gene 44 protein (data not shown).

A molecular mass for this protein complex wasreported by Barry et al. (26), using sucrose density gradient sedimentation and gel filtration. They obtained a sedimentation constant of 7.1 and a calculated molecular mass of 164,000 daltons, in excellent agreement with our current study. The stoichiometry of the complex was a t the time, however, incorrectly thought to be 2:l (44-62 subunits). Therefore Barry et al. (26) concluded that theactual molecular mass of the complex must be 176,000 daltons (i.e. a 4:2 subunit stoichiometry), based on apparent molecular masses of 34 and 20 kDa for gene 44 and 62 proteins, respectively.

s,

220,600


12713

SedimentationEquilibrium Yields a Trimer Molecular Weight for the Associated Species of Gene 45 Protein-The results of the sedimentationequilibrium experiments on gene 45 protein are displayed in Table I. As was the case for gene 44/62 protein, the In c versus ? plot (shown in Fig. 1B) is quite linear. This indicates that the protein species is probably homogeneous, judging by the fact that themolecular weights obtained a t three different rotor speeds and two different initial protein concentrations are quite consistent. Any heterogeneity or nonideality of the solute shouldbe revealed by comparison of runs made at different speeds and concentrations. The averagemolecular mass of the gene 45 protein from the three runs is 77,000 daltons, which is very close to the predicted trimer molecular mass of 74,100 daltons. Therefore, we conclude that thegene 45 protein exists primarily as a trimer in solution. Chemical Cross-linking Shows Associated Species-The asSubunit composition sociation state of gene 45 protein was additionally probed by (G44P:G62P) Molecular mass HPLC Sedimentation chemical cross-linking, using a bifunctional reagent (DMS) daltons that reacts with primary amines. Such diimidoesters have 3:2 150,400 been used extensively as structural probesof multimeric pro4:2 185,000 teins (29). Itmust be notedthat anegative resultfrom 5:2 chemical cross-linking (ie. no cross-linkedproducts observed) 128,100 3: 1 is inconclusive because the spatial positionsof lysine residues 163,700 4: 1 on adjacent subunitsof an oligomer may not be favorable for 199,300 5: 1 cross-link formation. The formation of discrete higher molecular weight species, however, provides reasonably good eviShape Factors Indicate an Asymmetric Complex-We can dence forthe existenceof multimeric proteinspecies, presummake some predictions about the shape of the gene 44/62 ing one can establish that the reaction conditions unlikely are protein complex on the basisof the sedimentationcoefficient. to favor collisional cross-linking (ie. cross-linking between A frictional coefficient forthe complex can be calculated using initially free protein monomers, rather than within pre-existthe relationship: ing oligomers). Samples of gene 45 protein were treated with DMS and the M ( l - Up) s= cross-linking reaction was quenched at various times by the Nf addition of excess ethanolamine. The productsof such a time We can also calculate the frictional coefficient expected for course, separated on a denaturing gel, are shown in Fig. 3A. a n anhydrous sphere of the same size. Using this approach Clearly, distinct higher molecular weight bands can be seen we obtain a maximumvalue of 1.45 for the Perrin shape after only a few minutes of cross-linking. Duplicate samples factor, F (for a general discussion see Ref. 27). If we assume were analyzed on a 7.5% gel, and the mobilities compared that the hydrationof the gene 44/62 protein complex falls in with thoseof protein standards to obtain the apparent molecthe range of 0.3-0.4 g of water/g of protein, as is typicalfor ular weights of the products (not shown). The approximate most proteins, then the shape factor, F, is about 1.25. This molecular masses were 89, 76, 55, and 44 kDa, respectively, corresponds toa prolate ellipsoid with an axial ratioof about for bands A, B, C, and D. These values can only be used as 5:l. This agrees with the shape factor reportedby Barry and rough indicators of the size of the cross-linked products since Alberts (9) for this complex, based onsucrose density gradient DMS cross-linked proteins frequently show anomalous misedimentation and gel filtration. Thus, it appears that the gration on denaturing gels, presumably as a consequence of complex either has a very asymmetric shape or a n unusually the branched chain structures that are formed by the interhigh degree of hydration. Since such a degree of hydration is subunit cross-links. very unlikely, a relatively asymmetric complex seems the most Clearly, species larger than dimers of gene 45 protein are plausible explanation of this shape factor. beingcovalently linked by the DMS. Formation of crosslinked species stops abruptly at an apparent molecular mass Gene 45 Protein Associates to Form a Trimer of about 89 kDa, providing a rough estimate of the uppersize Gene 45 protein has a monomer molecular mass, based on limit for the gene 45 protein oligomer. The absence of yet the DNAsequence, of 24.7 kDa (14). Previousliterature higher molecular weight species cannot conclusively rule out reports had suggested that the protein associates to form a larger species. However, as a positive control to determine dimer in solution (10, 28) corresponding to a calculated mo- that the cross-linking reagentis active, we have cross-linked large as lecular mass of 49 kDa. Gene 45 protein elutes with thevoid E. coli rhoprotein,and shown thatproductsas volume on a Bio-Gel P-60 column,'j for which the exclusion hexamers are formed. This protein associates.to form a hexlimit is approximately60 kDa. This could be consistent with amer under these buffer condition^,^ and has been shown by a n asymmetric dimermodel but is more likely to be indicative Finger and Richardson (30) to form a ladder of cross-linked of higher states of gene 45 protein association. Wehave products, from monomer to hexamer, when subjected to DMS. undertaken a more rigorous analysis of the physical state of It is also important to establish that the formation of crossgene 45 protein in order to ascertain whether the protein linked products reflects covalent linkage of subunits within exists principally as a dimer, or as some higher associated the same oligomer, rather than collisional cross-linking. The species. TABLE I11

Summary of evidence on the molecular weight of the native gene 44/62 protein complex Severalhypothetical subunit ratios of the gene 44/62 protein complex are listed, along with predicted molecular weights. The list focuses on those ratios most consistent with the current data aswell as estimates in the literature of the molecular weight of the native complex. The results from the HPLC separation of thesubunits (Table 11), which yields subunit stoichiometries, are compared with those from the sedimentation and light scattering experiments, which provides molecular weight information. For each type of experiment, (+) indicates that the results were consistent with the subunit composition in question, (+) indicates that the composition is unlikely based on the experiment, and (-) indicates that the results of the experiment clearly rule out the subunitcomposition. Thus, it canbe seen that a complex consisting of four gene 44 protein subunits and one gene 62 protein subunit, having a molecular mass of 163,700 daltons, is the only one consistent with both the HPLC and the sedimentation data.

J. Hockensmith, unpublished results.

J. Geiselmann, T. Yager, and P. H. von Hippel, manuscript in preparation.

12714

Physical Properties of the Accessory Protein Complex A.

f

I

1 2 3 4 5

It

M -w-,

4-D

+

Uncross-linked 94% monornel

0 2 51040 minutes

B. Gel Band

A:

Postulated Structure

& &

FIG.4. Velocity sedimentation of gene 45 protein. Apparent S , values for gene 45 protein are plotted as a function oft%,for w = 0.125,0.250,0.375,0.500,0.625,0.750, and 0.875 (see text). The least squares fitted lines for each value of w converge on an uncorrected S value of 4.2 f 0.1.This is corrected to 3.9 S for 20 “C.in water.

can postulate structures that might give rise to such bands on a gel, based on the trimer model for the gene 45 protein B: (diagrams in Fig. 3 B ) . The uncross-linked monomer gradually disappears with time. Within 2 min, a significant amount of band C is seen, which probably results from a single crossc: link between two adjacent subunits to form a dimer. Bands A and D form on a slightly slower time scale, appearing within 5 min, and might each result from the formationof two crossD links. In the case of peak A, these cross-links could form a covalently linkedtrimer, while the two cross-links in the peak FIG.3. The association state of gene 45 protein revealed by a doubly chemical cross-linking. Panel A shows the products of a time D species could join the same subunits, forming course for DMS cross-linkingof gene 45 protein, separated ona 10% linked dimer species with altered mobility from that of the SDS-polyacrylamide gel. Lane I shows uncross-linked gene 45 protein peak C form. Finally at long times (10 and 40 min) we see 10,and that the amount of peak A is depleted in favor of the more monomer. Lanes 2-5 show cross-linked products after 2, 5, 40 min, respectively. The band appearing just below the gene 45 rapidly migrating peakB, which might result from formation protein monomer (in the DMS cross-linked lanes) is probably the of a third cross-link. B, hyporesult of cross-link formation within the monomer subunit. The schematiccross-linking diagrams of Fig. 3B are merely thetical structures that might yield the cross-linked products labeled peaks A-D in panel A. The hatch narks indicate DMS molecules intended to represent subunitcross-linking connectivity and not tosuggest that theoverall trimer is quitecompact. In fact covalently linking to two different subunits. (see following section) the actual trimer is quite asymmetric, protein concentrations used in this study are low enough ( 4 and perhaps one simple way to visualize this in the context of Fig. 3B is to consider the cross-linking species as repremg/ml) to make the probability of collisional cross-linking very low (29).In addition,we have testedE. coli NusA protein, senting end-onviews of quite asymmetric (cylindrical?) subwhich has been shown to exist as a monomer in solution? units. Shape Information from theSedimentationCoefficientUnder identical reaction conditions,NusA showsno tendency to form collisional cross-links. Thus, we can feel fairly confi- Velocity sedimentation of gene 45 protein yields a sedimendent that the presence of higher molecular weight cross-linked tation coefficient, S20,w, of 3.9 -t 0.1 S. Fig. 4 shows the species of gene 45 protein accurately reflects the association apparent (uncorrected) S values plotted uersus tlh for seven state of the species pre-existing in the solution?Although it values of w = C(r)/Cp,the ratioof the concentrationof protein is difficult to obtain accurate molecularweights of cross- in the ultracentrifuge cell at radius r to the plateau concenlinked species, the approximatemolecular weightof the high- tration. This analysis, described by Van Holde and Weischet est molecular weight band we observed falls between that (19),results ina “fan plot”for ahomogeneous sample, because expected fora trimer and that expected for atetramer, clearly at infinite time, the apparentS, values converge to the same of gene 45 demonstrating associated species larger than a dimer. Thus, limit, S. Therefore, it appears that the trimer protein exists as a fairly homogeneous species.’O If we assume the cross-linking results corroborate the molecularweight determination by equilibrium sedimentation, which showsthe lo It should be noted that if the subunits of the trimer associate associated species of gene 45 protein tobe a trimer. The cross-linking time course presented in Fig. 3A shows and dissociate on a rapid time scale, the resulting sedimentation boundary could appear to behomogeneous. In thiscase, the sedimenthe appearance of a t least four major cross-linkedspecies. We tation constant obtainedwould be somewhat lower than thatfor the

68 &

* S. C. Gill and P. H. von Hippel, manuscript

in preparation. or efficiency was seen on No change in the cross-linking pattern association state of the addition of DNA or ATP, indicating that the protein isprobably not affected by binding of either of these potential cofactors (data not shown).

true trimerspecies, being an intermediate value between, for example, monomers and trimers. Since no indication of lower molecular weight species was seen in equilibrium sedimentation, however, we have no reason to believe that any significant fractionof the gene 45 protein exists as monomersor dimers under the solution conditions we have studied.


A. Coumassie-stainedgel 1

2

3

4

12715

B. Autoradiograph 5

6

1

2

3

4

5

6

FIG. 5. Photoaffinity labeling of T4 DNA replication proteins with azido-ATP. Potential ATP-binding sites on the five-protein replication system were investigated by photoaffinity labeling. Each reaction contained 4 pM NsATP in a buffer consistingof 10 mM HEPES, pH 7.5,50 mM NaC1,6 mM MgC12,and 1 mM 8-mercaptoethanol. The Coomassie-stained gel of the labeled proteins is shown in panel A. Lune 1 contains gene 44/62 protein; lune 2 contains gene 45 protein; lune 3 contains gene 32 protein (single-stranded DNA binding protein); lane 4 contains gene 43 protein (polymerase); lane 5 contains a mixture of all five proteins; lane 6 contains the gene 44/62 protein complex without Mg2'. Panel B shows the autoradiograph of the same gel.

work of Staros (34), suggesting that the lifetimes of reactive intermediates may be on a many millisecond to second time scale.However, under proper reaction conditions photoaffinity labeling can nonetheless be made quite specific. Gene 44 Protein Reacts Specifically with Azido-ATP-Samples of each of the five T4 replication proteins (gene 44, 62, 32,45, and 43 proteins),alone and in combination, were irradiated with 300 nm light in the presence of [y-32P]N3ATP. The results are shown in Fig. 5. The autoradiogram shows that gene 44 protein reacts specifically with NSATP. Since the gene 44/62 protein complex is the only one among the group for which ATPase activity has been demonstrated, it is notsurprising that one of itssubunits binds ATP. This The ATP-bindingSite Resides in the Gene 44 Subunit specific labeling is notably absent when no Mg+ is present. The T4polymerase accessory proteins have been shown to Since the substratefor ATP hydrolysis is Mg-ATP, this lends have an ATPase activity that is stimulated by DNA (for credence to theidea that N3ATP is binding to the true ATPdetails and references, see (8)).Piperno et a1. (3), examined binding site ina mannersimilar to thenormal substrate. Gene the identity of the ATPase protein by treating gene 44/62 62 protein shows virtually no labeling. Thus, it appears that protein and gene 45 protein in turn with 6-mercaptopurine the ATPhydrolysis activity resides in the gene 44subunits of the complex. This agrees with the results of Lin et al.," ribonucleoside 5'-triphosphate, which reacts at the ATPbinding site of various ATPases and inhibits ATPaseactivity showing that gene 44 protein purified from the cloned T4 gene has a low level DNA-dependent ATPase activity, while (31). Gene 45 protein treated in this manner is able to stimgene 62 protein (also purified from a clone) does not. ulate the ATPase activity of normal gene 44/62protein, while The only other protein to show specific photoaffinity labelmodified gene 44/62 protein shows no ATPase activity in the ing under theseconditions is the polymerase (gene 43 protein). presence of normal gene 45 protein. Thus, thefunctional site Although it lacks any detectable ATPase activity, the polymfor ATP hydrolysis appeared to reside in the gene 44/62 erase clearly must contain a deoxynucleoside triphosphateprotein complex. binding site, and this site probably has considerable affinity We have further investigated the locations of potential for N3ATP. None of the other proteins show any significant ATP-binding sites on the replication proteins by covalently labeling by the photoaffinity probe. In particular, the lack of labeling the proteins with anATP photoaffinity analog, labeling of gene 45 protein supportsthe conclusion of Piperno NATP. Aromatic azido compounds such as N3ATP form et al. (3), that the gene 44/62 protein complex is responsible highly reactive nitrenes when exposed to ultraviolet light. for the ATPase activity of the accessory proteins. Photoactivation is possible outside the normal absorption Evidence That Photoaffinity Labeling Occursat the Normal range of most biological macromolecules (i.e. above 300 nm). ATP-binding Site-In order to demonstrate unequivocally Thus, absorption of the incident light responsible for pho- that the observed labeling of gene 44 protein occurs specifitoactivation is likely to be minimal and photochemical reac- cally at (or near) the normal ATP-binding site,several criteria tion of the macromolecules themselves to be slight. Covalent should be met. The most important criterion is that ATP can bond formation between the affinity probe and a macromol- compete for the binding site, and reduce the amount of ecule is likely if the probe is actually bound at themoment of photoactivation. Ideally, unbound affinity probe will react "Lin, T. C., Rush, J., McKim, I., and Konigsberg, W. (1985) with solvent, although earlier estimates of solution lifetimes Abstracts from the 1985 Evergreen International T4 Meeting, August, on the order of milliseconds (33) have been challenged by the 1985, Olympia, WA.

that the hydration is between 0.3 and 0.4 g water/g protein, then the Perrin shape factor for gene 45 protein would be about 1.32, corresponding to anaxial ratio of 6 1 for a prolate ellipsoid. Thus, it appears that the trimer gene 45 protein may also be quite asymmetric in shape. Since the literature reports identifying gene 45 protein as a dimer were presumably based on sucrose density gradient sedimentation (28), this may explain the discrepancy between our results and those previously reported in the literature. Molecular weight determinations based only on sedimentation transport will tend to err on the small side when the complex in question exhibits a high degree of asymmetry.

12716

Physical Propertiesof the Accessory Protein Complex TABLE IV

binding sites reside in the gene 44 protein subunits. Thus, the complex has the potential capacity to bind up to four ATP molecules at once. Covalently hound N3ATP was separated from unbound material The gene 45 protein is seen to exist primarily as a trimer using spin columns (32). Radioactive incorporation was determined in dilutesolution, contrary to previous reports inthe literature by scintillation counting inaqueous mixture. Each reaction contained (10, 28). Since the association constant between the gene 44/ gene 44/62 protein a t 1.1 PM complexes, and 4 pM N3ATP (3.3 pCi/ ml), in a buffer consisting of 10 mM HEPES, pH 7.5,50 mM NaC1,6 62 protein complex and gene 45 protein is relatively weak (8) mM MgCl,, and 1 mM 0-mercaptoethanol. The poly(dA) concentra- it has not been possible to isolate the complete accessory tion was 60 U M and the ATP concentrationwas 1 mM. protein complex. Therefore, the stoichiometry of binding of gene 45 protein trimers to gene 44/62 protein complexes can Reaction components cpm" only be surmised from enzymatic studies. This will be disG44/62P 157 cussed in greater detail in the accompanying paper. In addiG44/62P irradiation) (no 23 tion, the interaction of the accessory protein complex with G44/62P + ATP 49 primer-template junction DNA (the functional site for repliG44I62P + Dolv(dA) 153 cation) will be discussed. Counts per min of 32Pcovalently incorporated into the gene 44/ Covalent incorporation of azido-ATP by the gene 44/62 protein complex

62 protein complex.

labeling. We have addressed this by photoaffinity labeling the gene 44/62 protein complex in the presence and abesence of ATP (Table IV). The amount of N3ATP covalently incorporated upon photolysis is reduced by inclusion of ATP in the reaction mixture. This incorporation is not decreased when adenosine is added (not shown) suggesting that the effect is specific and not due to an artifact such as absorption of the photoactivating light by the added nucleotide. Inclusion of DNA in the reaction mix also does notinhibit covalent incorporation of N3ATP, so it is unlikely that a DNA-binding site is being labeled. These resultssuggest that theazido-ATP analog is binding in the normal ATP-binding site." DISCUSSION

The T4 polymerase accessory proteins are anessential part of a fully functional DNA replication complex. We have shown that the gene 44 and 62 proteins form a complex consisting of four gene 44 protein subunits and one gene 62 protein subunit, Under our buffer conditions, the complex appears to be quite homogeneous. The anomalous sedimentation properties of the complex are indicative of an asymmetric ( i e . non-spherical) configuration. Although the current study does not address the number of ATP molecules bound/ complex, it is clear from photoaffinity labeling that theATP-

'' The argument for the specificity of the labeling is strengthened when certain additional criteria are met. First, when the level of analog is raised, the amount covalently incorporated should reach saturation and thereafter remain relatively constant. In addition, the analog should be usable as an enzyme substrate and show values of K , and VmaX similar to thoseof ATP. N3ATP has been shown tomeet all of these criteria for several types of ATPases, but not in other cases, possibly due to the fact that the bulky azido group tends to force the molecule into the cis-conformation rather than the characteristic trans-conformation (35). In the gene 44/62 protein labeling studies, we have beenunable to observe saturation of the photoaffinity labeling a t concentrations up to1 mM N3ATP. N3ATP is also hydrolyzed very poorly by the gene 44/62 protein complex, even in the presence of gene 45 protein and DNA. Although the K,,, for ATP of gene 44/62 protein alone is not known, the K , in the presence of gene 45 proteinand DNA isquite high (about 0.2mM). In this concentration range, nonspecific labeling by N3ATP of proteins that do notnormally bind ATP is prevalent(L. Paul, unpublished results). If the K,,, for N,ATP is similar to thatof ATP, then saturation may not be observed due to nonspecific labeling. Although labeling may be specific at low concentration levels, nonspecific labeling is prevalent at theconcentrations expected for saturation.

Acknowledgments-We are very grateful to Johannes Geiselmann for extensive assistance with the dynamic laser light-scattering experiments and to StanleyC. Gill for useful discussions of the calculation of molar extinction coefficients and partial specific volumes from amino acid sequence information. REFERENCES 1. Nossal, N. G., and Peterlin, B. M. (1979) J. Biol. Chem. 254,6032-6036 2. Sinha, N. K., Morris, C. F., and Alberts, B. M. (1980) J. Biol. Chem. 2 5 6 , 4290-4303 3. Piperno, J. R., Kallen, R. G., and Alberts, B. M. (1978) J. Biol. Chem. 2 5 3 , 5180-5185 4. Mace, D. C., and Alberts, B. M. (1984) J. Mol. Biol. 177,279-293 5. Newport, J. W., Kowalczykowski, S. C., Lonberg, N., Paul, L. S . , and von Hippel, P. H. (1980) in Mechanistic Studtes of DNA Repllcatzon and Genetic Recombznatzon ICN-UCLA Symp.Mol. Cell. Bwl. (Alberts, B. M., ed) Vol. 19, pp. 485-505, Academic Press, New York 6. Piperno, J. R., and Alberts, B. M. (1978) J. Bml. Chem. 253,5174-5179 7. Mace, D. C., and Alberts, B. M. (1984) J. Mol. Biol. 177,313-327 8. Jarvis, T. C., Hockensmith, J. W., Paul, L. S., and von Hippel, P. H. (1989) J. Biol. Chem. 264,12717-12729 9. Barry, J., and Alberts, B. M. (1972) Proc. Natl. Acad. Sci. U. S. A. 69,2717-2721 10. Morris, C. F., Hama-Inaba, H., Mace, D., Sinha, N. K., and Alherts, B. M. (1979) J. Biol. Chem. 254,6787-6796 11. Spicer, E. K., Nossal, N. G., and Williams, K. R. (1984) J. Biol. Chem. 2 5 9 , 15425-15432 12. Spicer, E. K., and Konigsherg, W. H. (1983) in Bacteriop e T4 (Mathews, C. K., Kutter, E. M., Mosig, G., and Berget, P. B., e s) pp. 291-301, American Society for Microbiology, Washington, DC 13. Nossal, N. G. (1979) J. Biol. Chem. 254,6026-6031 14. Spicer E. K., Noble, J. A,, Nossal, N. G., Konigsberg, W. H., and Williams, K. R. (1982) J. Biol. Chem. 257,8972-8979 15. Bittner, M., Burke, R. L., and Alberts, B. M. (1979) J. Biol. Chem. 2 5 4 , 9565-9572 16. Alberts B. and Frey L. (1970) Nature 227,1313-1318 17. Cohn, E. J.: and Edsdll, J. T. (1943) in Proteins, Amino Acids and Peptides, p. 370, Academic Press, New York 18. Eglhoch, H. (1967) Biochemistry 6,1948-1954 19. Van Holde, K. E., and Weischet, W. 0.(1978) Biopolymers 1 7 , 1387-1403 20. Bloomfield, V. A,, and Lim, T.K. (1978) Methods Enzyml. 48,415-494 21. Yphantis, D. A. (1964) Biochemistry 3,297-317 22. Laemmli, U. K. (1970) Nature 227,680-685 23. Morrissey, J. H. (1981) Anal. Biochem. 117,307-310 24. Schachman, H.K. (1959) Ultracentrifugation in Biochemistry, Academic Press, New York 25. Van Holde, K. E. (1975) in The Protei? (Neurath, H., and Hill, R. L., eds) 3rd Ed., Vol. 1,pp. 225-291, Academlc Press, New York 26. Barry J. Hama-Inaba, H., Moran, L., and Alberts, B. (1973) in DNA Sy&&is in Vitro (Wells, R. D., and Inman, R. B., e&) pp. 195-214, University Park Press, Baltimore 27. Cantor, C. R., and Schimmel, P. R. (1980) Biophysical Chemistry, Part 11, W. H. Freeman and Co., San Francisco 28. Alberts, B. M., Morris, C. F., Mace, D., Sinha, N., Bittner, M., and M o m , L. (1975) in DNA Synthesis and Its Regulation ICN-UCLA Symp. Mol. Cell. Biol. (Goulian, M., and Hanawalt, P., eds) Vol. 3, pp. 241-269, w. A. Benjamin, Inc., Menlo Park 29. Davies, G. E., and Stark, G. R. (1970) Proc. Natl. Acad. Sei. U. S. A. 66,651-656 30. Finger, L. R., and Richardson, J. P. (1982) J. Mol. Biol. 156,203-219 31. Patzelt-Wenczler, R., Pauls, H., Erdmann, E., and Schoner,W. (1975) Eur. J. Biochem. 53,301-311 32. Czarnecki, J., Geahlen, R., and Haley, B. (1979) Methods Enzymol. 5 6 , 642-653 33. Neal, M. W., and Florini, J. R. (1973) Anal. Biochem. 55,328-330 34. Staros, J. V. (1980) Trends Biochem. Sci. 5,320-322 35. Haley, B. E., and Hoffman, J. F. (1974) Pmc. Natl. Acad. sci. U. S. A. 71,3367-3371

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