Farnesyl protein transferase: identification of K164 alpha and Y300 beta as catalytic residues by mutagenesis and kinetic studies.

Farnesyl protein transferase (FPT) is an alpha/beta heterodimeric zinc enzyme that catalyzes posttranslational farnesylation of many key cellular regulatory proteins, including oncogenic Ras. On the basis of the recently reported crystal structure of FPT complexed with a CVIM peptide and alpha-hydroxyfarnesylphosphonic acid, site-directed mutagenesis of the FPT active site was performed so key residues that are responsible for substrate binding and catalysis could be identified. Eight single mutants, including K164N alpha, Y166F alpha, Y166A alpha, Y200F alpha, H201A alpha, H248A beta, Y300F beta, and Y361F beta, and a double mutant, H248A beta/Y300F beta, were prepared. Steady-state kinetic analysis along with structural evidence indicated that residues Y200 alpha, H201 alpha, H248 beta, and Y361 beta are mainly involved in substrate binding. In addition, biochemical results confirm structural observations which show that residue Y166 alpha plays a key role in stabilizing the active site conformation of several FPT residues through cation-pi interactions. Two mutants, K164N alpha and Y300F beta, have moderately decreased catalytic constants (kcat). Pre-steady-state kinetic analysis of these mutants from rapid quench experiments showed that the chemical step rate constant was reduced by 41- and 30-fold, respectively. The product-releasing rate for each dropped approximately 10-fold. In pH-dependent kinetic studies, Y300F beta was observed to have both acidic and basic pKa values shifted 1 log unit from those of the wild-type enzyme, consistent with a possible role for Y300 beta as an acid-base catalyst. K164N alpha had a pKa shift from 6.0 to 5.3, which suggests it may function as a general acid. On the basis of these results along with structural evidence, a possible FPT reaction mechanism is proposed with both Y300 beta and K164 alpha playing key catalytic roles in enhancing the reactivity of the farnesyl diphosphate leaving group.

Tricyclic farnesyl protein transferase inhibitors: crystallographic and calorimetric studies of structure-activity relationships.

Crystallographic and thermodynamic studies of farnesyl protein transferase (FPT) complexed with novel tricyclic inhibitors provide insights into the observed SAR for this unique class of nonpeptidic FPT inhibitors. The crystallographic structures reveal a binding pattern conserved across the mono-, di-, and trihalogen series. In the complexes, the tricycle spans the FPT active site cavity and interacts with both protein atoms and the isoprenoid portion of bound farnesyl diphosphate. An amide carbonyl, common to the tricyclic compounds described here, participates in a water-mediated hydrogen bond to the protein backbone. Ten high-resolution crystal structures of inhibitors complexed with FPT are reported. Included are crystallographic data for FPT complexed with SCH 66336, a compound currently undergoing clinical trials as an anticancer agent (SCH 66336, 4-[2-[4-(3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo[5, 6]cyclohepta[1, 2-b]pyridin-11-yl)-1-piperidinyl]-2-oxoethyl]-1-piperidinecarbo xamide ). Thermodynamic binding parameters show favorable enthalpies of complex formation and small net entropic contributions as observed for 4-[2-[4-(3,10-dibromo-8-chloro-6,11-dihydro-11H-benzo[5, 6]cyclohepta[1, 2-b]pyridin-11-ylidene)-1-piperidinyl]-2-oxoethyl]pyridine N-oxide where DeltaH degrees bind = -12.5 kcal/mol and TDeltaS degrees bind = -1.5 kcal/mol.

A continuous spectrophotometric assay for the hepatitis C virus serine protease.

The hepatitis C virus (HCV) encodes a chymotrypsin-like serine protease responsible for the processing of HCV nonstructural proteins and which is a promising target for antiviral intervention. Its relatively low catalytic efficiency has made standard approaches to continuous assay development only modestly successful. In this report, four continuous spectrophotometric substrates suitable for both high-throughput screening and detailed kinetic analysis are described. One of these substrates, Ac-DTEDVVP(Nva)-O-4-phenylazophenyl ester, is hydrolyzed by HCV protease with a second-order rate constant (kcat/Km) of 80,000 +/- 10,000 M-1 s-1. Together with its negligible rate of nonenzymatic hydrolysis under assay conditions (0.01 h-1), analysis of as little as 2 nM protease can be completed in under 10 min.

High-level expression, purification, kinetic characterization and crystallization of protein farnesyltransferase beta-subunit C-terminal mutants.

Protein farnesyltransferase (FPT) is a 97 000 Da heterodimeric enzyme that catalyzes post-translational farnesylation of many cellular regulatory proteins including p21 Ras. To facilitate the construction of site-directed mutants, a novel translationally coupled, two-cistron Escherichia coli expression system for rat FPT has been developed. This expression system enabled yields of >5 mg of purified protein per liter of E.coli culture to be obtained. The E.coli-derived FPT demonstrated an activity comparable to that of protein isolated from other sources. The reported expression system was used to construct three beta-subunit C-terminal truncation mutants, Delta5, Delta10 and Delta14, which were designed to eliminate a lattice interaction between the beta-subunit C-terminus of one molecule and the active site of a symmetry-related molecule. Steady-state kinetic analyses of these mutants showed that deletion up to 14 residues at the C-terminus did not reduce the value of kcat; however, Km values for both peptide and FPP increased 2-3-fold. A new crystalline form of FPT was obtained for the Delta10 C-terminal mutant grown in the presence of the substrate analogs acetyl-Cys-Val-Ile-Met-COOH peptide and alpha-hydroxyfarnesylphosphonic acid. The crystals diffract to beyond 2.0 A resolution. The refined structure clearly shows that both substrate analogs adopt extended conformations within the FPT active site cavity.

Structural and biological stability of the human interleukin 10 homodimer.

Human interleukin 10 (huIL-10) is a cytokine that regulates the synthesis of type 1 helper T cell derived cytokines such as gamma-interferon, interleukin 2, and tumor necrosis factor alpha. The potential immunosuppressive activities of huIL-10 suggest that this protein may be clinically useful for treating autoimmune diseases. Due to the potential clinical value of this cytokine, physicochemical studies have been performed regarding its association state and biological/structural stability. These studies include performing size-exclusion chromatography, chemical cross-linking, equilibrium ultracentrifugation, and circular dichroism spectroscopy. The results indicate huIL-10 is predominantly a noncovalent homodimer at neutral pH and 4 degreesC for concentrations greater than 0.003 mg/mL (0.08 microM dimer). An apparent pKa value of approximately 4.8 was calculated for both the pH-dependent subunit dissociation and pH-induced loss in MC/9 biological activity. A temperature analysis revealed a linear relationship between the percent dimer and relative MC/9 activity, thus, these results and the pH-dependent activity results suggest that the huIL-10 dimer is the active species. The GndHCl-induced unfolding of rhuIL-10, monitored by far-UV circular dichroism, revealed a unique biphasic unfolding process which contained both a subunit dissociation process (<1.6 M GndHCl) as well as the unfolding of a highly alpha-helical monomer intermediate ([GndHCl]1/2 = 3.5 M). The monomer intermediates generated with 1.6 M GndHCl or pH 2.5 retained approximately 80% and 89% of the alpha-helical content of the native protein, respectively. Although a soluble and highly helical monomer state can be generated, the observed correlation between unfolding studies and biological activity suggests the dimer is the active species. These results are consistent with both the recent observation that the three-dimensional structure of rhuIL-10 is a 2-fold symmetric homodimer and that a complex between the extracellular domain of the recombinant human IL-10 receptor and IL-10 is consistent with two IL-10 homodimers and four receptors.

Crystal structure of farnesyl protein transferase complexed with a CaaX peptide and farnesyl diphosphate analogue.

The crystallographic structure of acetyl-Cys-Val-Ile-selenoMet-COOH and alpha-hydroxyfarnesylphosphonic acid (alphaHFP) complexed with rat farnesyl protein transferase (FPT) (space group P61, a = b = 174. 13 A, c = 69.71 A, alpha = beta = 90 degrees, gamma = 120 degrees, Rfactor = 21.8%, Rfree = 29.2%, 2.5 A resolution) is reported. In the ternary complex, the bound substrates are within van der Waals contact of each other and the FPT enzyme. alphaHFP binds in an extended conformation in the active-site cavity where positively charged side chains and solvent molecules interact with the phosphate moiety and aromatic side chains pack adjacent to the isoprenoid chain. The backbone of the bound CaaX peptide adopts an extended conformation, and the side chains interact with both FPT and alphaHFP. The cysteine sulfur of the bound peptide coordinates the active-site zinc. Overall, peptide binding and recognition appear to be dominated by side-chain interactions. Comparison of the structures of the ternary complex and unliganded FPT [Park, H., Boduluri, S., Moomaw, J., Casey, P., and Beese, L. (1997) Science 275, 1800-1804] shows that major rearrangements of several active site side chains occur upon substrate binding.

Probing the substrate specificity of hepatitis C virus NS3 serine protease by using synthetic peptides.

We probed the substrate specificity of a recombinant noncovalent complex of the full-length hepatitis C virus (HCV) NS3 serine protease and NS4A cofactor, using a series of small synthetic peptides derived from the three trans-cleavage sites of the HCV nonstructural protein sequence. We observed a distinct cleavage site preference exhibited by the enzyme complex. The values of the turnover number (k(cat)) for the most efficient NS4A/4B, 4B/5A, and 5A/5B peptide substrates were 1.6, 11, and 8 min(-1), respectively, and the values for the corresponding Michaelis-Menten constants (Km) were 280, 160, and 16 microM, providing catalytic efficiency values (k(cat)/Km) of 92, 1,130, and 8,300 M(-1) s(-1). An alanine-scanning study for an NS5A/5B substrate (P6P4') revealed that P1 Cys and P3 Val were critical. Finally, substitutions at the scissile P1 Cys residue by homocysteine (Hcy), S-methylcysteine (Mcy), Ala, S-ethylcysteine (Ecy), Thr, Met, D-Cys, Ser, and penicillamine (Pen) produced progressively less efficient substrates, revealing a stringent stereochemical requirement for a Cys residue at this position.

Thermodynamic and structural analysis of phosphotyrosine polypeptide binding to Grb2-SH2.

A thermodynamic analysis using isothermal titration calorimetry (ITC) has been performed to examine the binding interaction between the SH2 (Src homology 2) domain of growth factor receptor binding protein 2 (Grb2-SH2) and one of its phosphotyrosine (pY) polypeptide ligands. Interaction of the Shc-derived phosphotyrosine hexapeptide Ac-SpYVNVQ-NH2 with Grb2-SH2 was both enthalpically and entropically favorable (DeltaH = -7.55 kcal mol-1, -TDeltaS = -1.46 kcal mol-1 , DeltaG = -9.01 kcal mol-1, T = 20 degrees C). ITC experiments using five alanine-substituted peptides were performed to examine the role of each side chain in binding. The results were consistent with homology models of the Grb2-SH2-Shc hexapeptide complex which identified several possible hydrogen bonds between Grb2-SH2 and the phosphotyrosine and conserved asparagine(+2) side chains of the Shc hexapeptide. These studies also demonstrated that the hydrophobic valine(+1) side chain contributes significantly to the favorable entropic component of binding. The thermodynamic and structural data are consistent with a Grb2-SH2 recognition motif of pY-hydrophobic-N-X (where X is any amino acid residue). The measured heat capacity of binding (DeltaCp = -146 cal mol-1 K-1) was very similar to computed values using semiempirical estimates (DeltaCp = -106 to -193 cal mol-1 K-1) derived from apolar and polar accessible surface area values calculated from several homology models of the Grb2-SH2-Shc hexapeptide complex. The homology model which most closely reproduced the measured DeltaCp value is also the model which had the lowest RMS deviation from the subsequently determined crystal structure. Calculations based on the thermodynamic data and these semiempirical estimates indicated that the binding event involves burial of nearly comparable apolar (677 A2) and polar (609 A2) surface areas.

Characterization of Ha-ras, N-ras, Ki-Ras4A, and Ki-Ras4B as in vitro substrates for farnesyl protein transferase and geranylgeranyl protein transferase type I.

Ras proteins are small GTP-binding proteins which are critical for cell signaling and proliferation. Four Ras isoforms exist: Ha-Ras, N-Ras, Ki-Ras4A, and Ki-Ras4B. The carboxyl termini of all four isoforms are post-translationally modified by farnesyl protein transferase (FPT). Prenylation is required for oncogenic Ras to transform cells. Recently, it was reported that Ki-Ras4B is also an in vitro substrate for the related enzyme geranylgeranyl protein transferase-1 (GGPT-1) (James, G. L., Goldstein, J. L., and Brown, M. S. (1995) J. Biol. Chem. 270, 6221-6226). In the current studies, we compared the four isoforms of Ras as substrates for FPT and GGPT-1. The affinity of FPT for Ki-Ras4B (Km = 30 nM) is 10-20-fold higher than that for the other Ras isoforms. Consistent with this, when the different Ras isoforms are tested at equimolar concentrations, it requires 10-20-fold higher levels of CAAX-competitive compounds to inhibit Ki-Ras4B farnesylation. Additionally, we found that, as reported for Ki-Ras4B, N-Ras and Ki-Ras4A are also in vitro substrates for GGPT-1. Of the Ras isoforms, N-Ras is the highest affinity substrate for GGPT-1 and is similar in affinity to a standard GGPT-1 substrate terminating in leucine. However, the catalytic efficiencies of these geranylgeranylation reactions are between 15- and 140-fold lower than the corresponding farnesylation reactions, largely reflecting differences in affinity. Carboxyl-terminal peptides account for many of the properties of the Ras proteins. One interesting exception is that, unlike the full-length N-Ras protein, a carboxyl-terminal N-Ras peptide is not a GGPT-1 substrate, raising the possibility that upstream sequences in this protein may play a role in its recognition by GGPT-1. Studies with various carboxyl-terminal peptides from Ki-Ras4B suggest that both the carboxyl-terminal methionine and the upstream polylysine region are important determinants for geranylgeranylation. Furthermore, it was found that full-length Ki-Ras4B, but not other Ras isoforms, can be geranylgeranylated in vitro by FPT. These findings suggest that the different distribution of Ras isoforms and the ability of cells to alternatively process these proteins may explain in part the resistance of some cell lines to FPT inhibitors.

Purification and crystallization of a complex between human interferon gamma receptor (extracellular domain) and human interferon gamma.

X-ray diffraction quality crystals have been obtained from a complex between interferon gamma and the extracellular domain of its high-affinity cell surface receptor. The crystals were obtained from interferon gamma/interferon gamma receptor complexes purified by size exclusion chromatography. Diffraction quality crystals required analyzing these complex samples by isoelectric focusing gels to select purified complex fractions devoid of unbound interferon gamma. These studies used interferon gamma receptor engineered with an eight amino acid N-terminal deletion to eliminate heterogeneity generated due to proteolytic cleavage. In addition, the receptor was expressed in an E. coli secretion cell line which eliminated the need to refold the protein. Hexagonal crystals were grown from 1.6 M ammonium phosphate solutions and belong to a spacegroup of P6(5)22 with unit cell dimensions a = 145.9 A and c = 180.3 A. These crystals diffract to at least 2.9 A resolution when exposed to synchrotron radiation. SDS PAGE analysis of the crystals demonstrated that both interferon gamma and the receptor were present. Analysis of the x-ray diffraction data revealed that the crystals contain complexes with a stoichiometry of 2:1 receptor: ligand within the crystallographic asymmetric unit and consist of approximately 55% solvent.

Crystal structure of a complex between interferon-gamma and its soluble high-affinity receptor.

The crystal structure of interferon-gamma bound to the extracellular fragment of its high-affinity cell-surface receptor reveals the first view of a class-2 cytokine receptor-ligand complex. In the complex, one interferon-gamma homodimer binds two receptor molecules. Unlike the class-1 growth hormone receptor complex, the two interferon-gamma receptors do not interact with one another and are separated by 27 A. Upon receptor binding, the flexible AB loop of interferon-gamma undergoes a conformational change that includes the formation of a 3(10) helix.

Crystallization and preliminary X-ray investigation of recombinant human interleukin 10.

Crystals of recombinant human interleukin 10 have been grown from solutions of ammonium sulfate. The crystals are tetragonal, space group P4(1)2(1)2 or P4(3)2(1)2; the unit cell axes are a = 36.5 A and c = 221.9 A. There is the equivalent of one polypeptide chain in the asymmetric unit. The crystals are stable to X-rays and diffract to at least 2.5 A resolution.

A homology model of human interferon alpha-2.

An atomic coordinate five alpha-helix three-dimensional model is presented for human interferon alpha-2 (HuIFN alpha 2). The HuIFN alpha 2 structure was constructed from murine interferon beta (MuIFN beta) by homology modeling using the STEREO and IMPACT programs. The HuIFN alpha 2 model is consistent with its known biochemical and biophysical properties including epitope mapping. Lysine residues predicted to be buried in the model were primarily unreactive with succinimidyl-7-amino-4-methylcoumarin-3-acetic acid (AMCA-NHS), a lysine modification agent, as shown by mass spectrometric analysis of tryptic digests. N-terminal sequence analysis of polypeptides generated by limited digestion of HuIFN alpha 2 with endoproteinase Lys-C demonstrated rapid cleavage at K31, which is consistent with the presence of this residue in a loop in the proposed HuIFN alpha 2 model. Based on this model structure potential receptor binding sites are identified.

Disulfide bond assignments and secondary structure analysis of human and murine interleukin 10.

Interleukin 10 (IL-10), which was first discovered by its ability to inhibit the synthesis of various cytokines, most notably gamma interferon, from Th1 helper cells, displays pleiotropic immunoregulatory properties. Human and murine IL-10 have a high amino acid sequence identity (ca. 73%) which includes the conservation of all four cysteine residues in human IL-10 and the first four out of five cysteine residues for murine IL-10. Chemical analysis was used to determine that both recombinant human and recombinant murine IL-10 contain two disulfide bonds. The disulfide pairs for each were determined by mass spectrometric and reversed-phase HPLC analysis of trypsin-derived polypeptide fragments. The disulfide bond assignments for both species were similar in that the first cysteine residue in the sequence paired with the third and the second paired with the fourth. The fifth cysteine in murine IL-10 was determined by chemical modification to be unpaired. Far-UV circular dichroism analysis indicated that the secondary structure of recombinant human and murine IL-10 are composed of ca. 60% alpha-helix. Reduction of the disulfide bonds structurally destabilized the protein and led to a structure containing only 53% alpha-helix. The reduced protein displayed no in vitro biological activity in a mast cell proliferation assay. These studies indicate that IL-10 is a highly alpha-helical protein containing two disulfide bonds, either one or both of which are critical for its structure and function. In addition, these properties suggest that this interesting cytokine may belong to the alpha helical cytokine class of hematopoietic ligands.

Kinetics of oxygen binding and subunit assembly for the hemoglobin alpha subunit.

A thorough kinetic characterization of the O2-binding and self-association reactions of alpha-subunits of human hemoglobin A has been performed. All of the rate constants for a five step reaction model linking the monomer-dimer reaction to the O2-binding steps have been determined for the first time. Our analysis of the ligand binding reaction shows that both monomer and dimer have nearly identical intrinsic O2-association and dissociation rate constants and therefore identical affinities for oxygen. During this investigation we discovered a small absorbance difference between the oxy-monomer and oxy-dimer alpha-subunits. This difference spectrum enabled direct measurements of the alpha O2 self-association reaction. We find an association rate constant of, 2.0 10(5) M(-1)s-1, similar to that for other subunit assembly processes in the hemoglobin system. Our results also suggest that the deoxy-subunit assembly kinetics must be similar to that for the oxy-subunit. These kinetic results together with the equilibrium constants obtained for these solution conditions by Ackers and coworkers provides, for the first time, a complete kinetic and thermodynamic description of all the intrinsic ligand binding and association reactions for alpha-subunits.

Selective proteolytic cleavage of recombinant human interleukin 4. Evidence for a critical role of the C-terminus.

Human interleukin 4 is a 129 amino acid lymphokine secreted by activated T cells that exerts pleiotropic biological effects on B and T lymphocytes and other hematopoietic cells. Structure-function relations were studied by employing selective proteolytic cleavage of purified recombinant human interleukin 4 (rhuIL-4). Limited proteolysis with endoprotease Glu-C from Staphylococcus aureus (V8) produced two digestion products that were observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weight values of 19K (I) and 15K (II), respectively. These species were isolated by reversed-phase HPLC. Amino acid sequencing indicated that species II was an 84 amino acid core fragment extending from Gln-20 to Glu-103 and containing a hydrolyzed peptide bond at Glu-26. On the basis of known disulfide bond assignments, it was concluded that species II was stabilized by two disulfide bonds (Cys-24/Cys-65 and Cys-46/Cys-99). Analysis of its secondary structure by circular dichroism revealed a high content of alpha helix. Species I was the full-length rhuIL-4 with selective cleavage at Glu-26 and Glu-103. Both species I and II were inactive in an in vitro assay based on proliferation of peripheral blood lymphocyte blasts and lacked the ability to bind to teh rhuIL-4 receptor on Daudi cells. In order to elucidate further the role of the residues removed by S. aureus V8 protease, rabbit antisera were raised to synthetic peptides corresponding to residues 1-26 at the N-terminus and 104-129 at the C-terminus. Only antisera directed to the C-terminal peptide inhibited binding of 125I-rhuIL-4 to Daudi cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the conformation and stability of Escherichia coli derived recombinant human interleukin 4 by circular dichroism.

The conformation and stability of Escherichia coli derived recombinant human interleukin 4 (rhuIL-4) have been examined by circular dichroism (CD). Protein unfolding was detected by ellipticity changes at 222 nm with increasing concentrations of guanidine hydrochloride (GdnHCl). The unfolding midpoint ([GdnHCl]1/2) was 3.7 M, the free energy of unfolding, (delta GDH2O), was 5.9 kcal/mol and the dependence of delta GD on the GdnHCl concentration (m) was 1.6 (kcal/mol)/M. This unfolding was demonstrated to be reversible upon removal of the GdnHCl by dialysis. Analysis of the far-UV CD spectrum indicated the presence of a high percentage of alpha-helical structure (ca. 73%). A small change in ellipticity was noted over the pH range 1.9-9.6, suggesting that the protein undergoes a minor conformational change with an apparent pKa of 4.17. Virtually complete biological activity, measured in vitro in a T-cell proliferation assay, was recovered following exposure to extreme values of pH (i.e., pH 3 and 10). An analysis of the near-UV CD spectrum indicated that the single tryptophan residue at position 91 was unconstrained and most likely exposed to the solvent. Titration with 4,4'-dithiodipyridine and 2-nitrothiosulfobenzoate established that the six cysteine residues in rhuIL-4 were involved in intramolecular disulfide linkages. These data support that rhuIL-4 has a highly stable three-dimensional structure.

Xenopus transcription factor IIIA binds to the flanking regions of the 5 S RNA gene intragenic control region in a unique and highly ordered state.

The interaction of Xenopus transcription factor IIIA (TFIIIA) with the intragenic control region (ICR) of the 5 S RNA gene was studied by footprinting techniques under conditions which elicited a unique DNase I digestion pattern. Although a typical full footprint at the ICR was apparent at a 5 nM TFIIIA concentration, higher concentrations (greater than or equal to 50 nM) resulted in a decrease in the size of the footprint (on the 5' side of the ICR) concomitant with the appearance of an alternating protection pattern (APP) located both in the 3'- and 5'-flanking regions of the ICR with a periodicity of approximately 10 base pairs. This periodicity indicates that TFIIIA binding occurs on only one side of the DNA helix. The minimum size of the highly ordered and apparently cooperative APP effect was determined to be at least 250 base pairs in length and could be abolished through competition with nonspecific DNA. However, binding of TFIIIA to nonspecific DNA alone was not sufficient to generate the APP effect at any of the TFIIIA concentrations studied. Removal of the C-terminal domain of the protein by either tryptic or papain digestion resulted in the abolition of the APP effect at all concentrations studied, indicating the necessity of the protein-protein interactions for this effect. A nucleation site, most likely at or near the ICR, is proposed to exist through which TFIIIA specifically interacts and orients the binding of additional protein molecules in a cooperative and highly ordered manner to the flanking DNA sequences on either side of the ICR. The APP effect near the ICR may play a role in the initiation and stabilization of 5 S RNA gene transcription.

Purification of Xenopus transcription factor IIIA and 5 S RNA from 7 S ribonucleoprotein particle by ammonium sulfate precipitation.

A simple and efficient method for purifying Xenopus transcription factor IIIA from the 7 S particle has been developed by taking advantage of the differential solubilities of the protein factor and 5 S RNA in ammonium sulfate solution. Conditions under which ammonium sulfate dissociates the 7 S particle and selectively precipitates factor IIIA while the 5 S RNA moiety remains in the supernatant were found. The method simultaneously purifies, in a nondestructive manner, both factor IIIA and 5 S RNA in high yield. Purification proceeds through several ammonium sulfate precipitations of the 7 S particle. Factor IIIA obtained by this method contains no detectable RNA and is highly active as judged by DNase I footprinting and in vitro transcription of the 5 S RNA gene, as well as reconstitution with 5 S RNA to form the 7 S particle. The molar extinction coefficients of factor IIIA at 205 and 280 nm were determined from the ultraviolet absorption spectra measured with the purified protein.