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The tRNA tertiary core region is important for both tRNA stability and activity in the translation elongation cycle. Here we report the effects of mutating each of two highly conserved base pairs in the tertiary core of Phe-tRNAPhe, 18-55 and 19-56, on rate and equilibrium constants for specific steps of this cycle, beginning with formation of aminoacyl-tRNA·EF-Tu·GTP ternary complexs and culminating with translocation of A-site-bound peptidyl-tRNA into the P-site. We find that codon-dependent binding of aminoacyl-tRNA to the A/T-site and proofreading of near-cognate tRNA are sensitive to perturbation of either base pair; formation of the ternary complex and accommodation from the A/T to the A-site are sensitive to 18-55 perturbation only, and translocation of peptidyl-tRNA from the A- to P-site is insensitive to perturbation of either. These results underline the importance of the core region in promoting the efficiency and accuracy of translation, and they likely reflect different requirements for structural integrity of the core during specific steps of the elongation cycle.

The pattern of yeast pyrophosphatase (Y-PPase) inhibition by fluoride suggests that it replaces active site Mg(2+)-bound nucleophilic water, for which two different locations were proposed previously. To localize the bound fluoride, we investigate here the effects of mutating Tyr(93) and five dicarboxylic amino acid residues forming two metal binding sites in Y-PPase on its inhibition by fluoride and its five catalytic functions (steady-state PP(i) hydrolysis and synthesis, formation of enzyme-bound PP(i) at equilibrium, phosphate-water oxygen exchange, and Mg(2+) binding). D117E substitution had the largest effect on fluoride binding and made the P-O bond cleavage step rate-limiting in the catalytic cycle, consistent with the mechanism in which the nucleophile is coordinated by two metal ions and Asp(117). The effects of the mutations on PP(i) hydrolysis (as characterized by the catalytic constant and the net rate constant for P-O bond cleavage) were in general larger than on PP(i) synthesis (as characterized by the net rate constant for PP(i) release from active site). The effects of fluoride on the Y-PPase variants confirmed that PPase catalysis involves two enzyme.PP(i) intermediates, which bind fluoride with greatly different rates (Baykov, A. A., Fabrichniy, I. P., Pohjanjoki, P., Zyryanov, A. B., and Lahti, R. (2000) Biochemistry 39, 11939-11947). A mechanism for the structural changes underlying the interconversion of the enzyme.PP(i) intermediates is proposed.

Serpins form enzymatically inactive covalent complexes (designated E*I*) with their target proteinases, corresponding most likely to the acyl enzyme that resembles the normal intermediate in substrate turnover. Formation of E*I* involves large changes in the conformation of the reactive center loop (residues P17 to P9′) and of the serpin molecule in general. The “hinge” region of the reactive center loop, including residues P10–P14, shows facile movement in and out of β-sheet A, and this movement appears to be crucial in determining whether E*I* is formed (the inhibitor pathway) or whether I is rapidly hydrolyzed to I* (the substrate pathway). Here, we report stopped-flow and rapid quench studies investigating the pH dependence of the conversion of the α1-antichymotrypsin·α-chymotrypsin encounter complex, E·I, to E*I*. These studies utilize fluorescent derivatives of cysteine variants of α1-antichymotrypsin at the P11 and P13 residues. Our results demonstrate three identifiable intermediates, EIa, EIb, and EIc, between E·I and E*I* and permit informed speculation regarding the nature of these intermediates. Partitioning between inhibitor and substrate pathways occurs late in the process of E*I* formation, most likely from a species occurring between EIc and E*I*.

Serpins, serine proteinase inhibitors, form enzymatically inactive, 1:1 complexes (denoted E*I*) with their target proteinases, that only slowly release I*, in which the P1-P1′ linkage is cleaved. Recently we presented evidence that the serpin antichymotrypsin (ACT, I) reacts with the serine proteinase chymotrypsin (Chtr, E) to form an E*I* complex via a three-step mechanism, E + I ⇌ E ·I ⇌ EI′ ⇌ E*I* in which EI′, which retains the P1-P1′ linkage, is formed in a partly or largely rate-determining step, depending on temperature (O’Malley, K. H, Nair, S. A., Rubin, H., and Cooperman, B. S. (1997) J. Biol. Chem. 272, 5354–5359). Here we extend these studies through the introduction of a new assay for the formation of the postcomplex fragment, corresponding to ACT residues 359 (the P1′ residue) to 398 (the C terminus), coupled with rapid quench flow kinetic analysis. We show that the E·I encounter complex of wild type-rACT and Chtr forms both E*I* and postcomplex fragment with the same rate constant, so that both species arise fromEI′ conversion to E*I*. These results support our earlier conclusion that the P1-P1′ linkage is preserved inEI′ and imply that E*I* corresponds to a covalent adduct of E and I, either acyl enzyme or the tetrahedral intermediate formed by water attack on acyl enzyme. Furthermore, we show that the A347R (P12) variant of rACT, which is a substrate rather than an inhibitor of Chtr, has a rate constant for postcomplex fragment formation from the E·I complex very similar to that observed for WT-rACT, implying that EI′ is the common intermediate from which partitioning to inhibitor and substrate pathways occurs. These results are used to elaborate a proposed scheme for ACT interaction with Chtr that is considered in the light of relevant results from studies of other serpin-serine proteinase pairs.

Escherichia coli ribosomal protein L23 was derivatized with [3H]2,4-dinitrofluorobenzene both at the N terminus and at internal lysines. Dinitrophenyl-L23 (DNP-L23) was taken up into 50 S subunits from a reconstitution mixture containing rRNA and total 50 S protein depleted in L23. Unmodified L23 competed with DNP-L23 for uptake, indicating that each protein form bound in an identical or similar position within the subunit. Modified L23, incorporated at a level of 0.7 or 0.4 DNP groups per 50 S, was localized by electron microscopy of subunits complexed with antibodies to dinitrophenol. Antibodies were seen at two major sites with almost equal frequency. One site is beside the central protuberance, in a region previously identified as the peptidyltransferase center. The second location is at the base of the subunit, in the area of the exit site from which the growing peptide leaves the ribosome. Models derived from image reconstruction show hollows or canyons in the subunit and a tunnel that links the transferase and exit sites. Our results indicate that L23 is at the subunit interior, with separate elements of the protein at the subunit surface at or near both ends of this tunnel.

Serine proteinase inhibitors (serpins) form enzymatically inactive, 1:1 complexes (denoted E*I*) with their target proteinases that release free enzyme and cleaved inhibitor only very slowly. The mechanism of E*I* formation is incompletely understood and continues to be a source of controversy. Kinetic evidence exists that formation of E*I* proceeds via a Michaelis complex (E.I) and so involves at least two steps. In this paper, we determine the rate of E*I* formation from alpha-chymotrypsin and alpha1-antichymotrypsin using two approaches: first, by stopped-flow spectrofluorometric monitoring of the fluorescent change resulting from reaction of alpha-chymotrypsin with a fluorescent derivative of alpha1-antichymotrypsin (derivatized at position P7 of the reactive center loop); and second, by a rapid mixing/quench approach and SDS-polyacrylamide gel electrophoresis analysis. In some cases, serpins are both substrates and inhibitors of the same enzyme. Our results indicate the presence of an intermediate between E.I and E*I* and suggest that the partitioning step between inhibitor and substrate pathways precedes P1-P1' cleavage.

Inhibition of human chymase by alpha 1-antichymotrypsin produces 3.5 mol of degraded inhibitor for every mol of chymase inhibited, resulting in a stoichiometry of inhibition (SI) of 4.5. In the present study, the substrate versus inhibitor properties of this reaction were examined further using wild type and mutant recombinant antichymotrypsins (rACT). Titration of chymase hydrolytic activity with rACT-L358 (wild type) and reactive site (P1) variants of ACT, L358W, L358M, and L358F revealed that the SI was sensitive to P1 residue replacements. SI values increased in the order of Trp

The interactions of bovine pancreatic chymotrypsin (Chtr) and recombinant alpha 1-antichymotrypsin (rACT) and rACT variants were studied by kinetic and gel electrophoretic analyses, leading to the formulation of a general kinetic scheme that accounts for all known results concerning this serpin-protease pair, as well as for results obtained with other such pairs. Incubation of rACT and Chtr leads rapidly to the formation of an inhibited complex, Chtr.rACT*, that is stable toward sodium dodecyl sulfate denaturation and boiling. The extent of release of active Chtr from this complex increases markedly as ionic strength, mu, is raised. The kinetic scheme quantitatively accounts for this effect on the basis of a partitioning of Chtr.rACT* between dissociation of the complex to yield active enzyme and cleaved rACT, and Chtr-catalyzed conversion of the complex to a form that is much more resistant to release of active enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of reaction mixtures of rACT and Chtr are consistent with the scheme. Also consistent are the results of experiments measuring the effects of 1) Chtr.rACT* concentration, 2) uncomplexed Chtr, and 3) added alpha 2-macroglobulin on active Chtr release from Chtr.rACT*. Proteolysis of Chtr.rACT* to give a resistant complex is also catalyzed by human neutrophil elastase, a process with potential physiological relevance. Comparison of the rates of Chtr dissociation from the complexes formed with rACT and with rACT variants mutated at the P1 site suggests that such rates are more sensitive to P1 substitution at low mu than at high mu. Several equivalents of the L358R-rACT variant are required for full inhibition of Chtr. This observation is also quantitatively accounted for by the proposed kinetic scheme, on the basis of another partitioning step between L358R-rACT acting as a substrate or as an inhibitor toward Chtr.

This is the third paper in a series (Olah, T. V., Olson, H. M., Glitz, D. G., and Cooperman, B. S. (1988) J. Biol. Chem. 263, 4795-4800; Olson, H. M., Olah, T., Cooperman, B. S., and Glitz, D. G. (1988) J. Biol. Chem. 263, 4801-4806) describing the use of 2,4-dinitrophenyl (DNP) derivatives of Escherichia coli 30 S ribosomal proteins to locate the positions of these proteins within the 30 S subunit by immune electron microscopy. In it we describe the derivatization of proteins S6, S13, S16, and S18 with [3H]2,4-dinitrofluorobenzene, identify the nature of the derivatized amino acids within each protein, and demonstrate that each DNP protein, denoted DNP-Sx, can be taken up into a reconstituted 30 S subunit when added to a reconstitution mixture containing 16 S rRNA and total 30 S protein depleted in Sx. We further demonstrate that each DNP-Sx binds within the 30 S subunit in a position identical or similar to that of the unmodified Sx protein, as judged by its meeting one or more of the following three criteria: (i) unmodified Sx competes with the uptake of DNP-Sx into 30 S subunits; (ii) DNP-Sx restores functional activity to those single protein omission reconstitution particles lacking full activity; (iii) DNP-Sx induces the uptake of proteins into 30 S subunits that depend on the presence of Sx. The fourth paper in this series (Montesano-Roditis, L., McWilliams, R., Glitz, D. G., Olah, T. V., Perrault, A. R., and Cooperman, B. S. (1993) J. Biol. Chem. 268, 18701-18709), which follows this one, describes the localization of the DNP-Sx proteins within the 30 S subunit by immune electron microscopy.

Purified Escherichia coli ribosomal proteins S6, S13, S16, and S18 were dinitrophenylated at their amino termini and/or at one or more internal lysine residues. Each dinitrophenyl protein was then separately incorporated into reconstituted small ribosomal subunits. Modified proteins were localized on the 30 S subunit surface by electron microscopy of reconstituted subunits complexed with antibodies to dinitrophenol (DNP). DNP protein S13 was placed on the subunit head above the platform and on the surface that faces the large subunit. DNP-S18 was localized to the subunit platform below the tip and in a region associated with binding to 50 S subunits. DNP proteins S6 and S16 were both localized near the junction of the subunit body and platform; DNP-S6 was available to antibody in 70 S ribosomes and was placed on the cytoplasm-facing side of the subunit in an area that overlaps the platform and body of the particle. DNP-S16 in 70 S ribosomes was not bound by antibody. It was localized to the 30 S body near its junction with the platform and on the surface facing the 50 S particle. The results complement and clarify data obtained using other approaches.

The ability of neutrophils to generate free radicals is a crucial component of host defense (Babior, B. M. (1978) N. Engl. J. Med. 298, 659-668, 721-725. Neutrophil oxidants, however, can cause significant host tissue destruction (Weiss, S. J. (1989) N. Engl. J. Med. 320, 365-376), and the regulation of free radical production is not well understood. We have previously shown that recombinant antichymotrypsin (rACT), a serine protease inhibitor, inhibits superoxide production in intact neutrophils (Kilpatrick, L., Johnson, J. L., Nickbarg, E. B., Wang, Z., Clifford, T. F., Banach, M., Cooperman, B. S., Douglas, S. D., and Rubin, H. (1991) J. Immunol. 146, 2388-2393). Using a cell-free NADPH oxidase preparation, we now demonstrate that rACT alone has no effect on superoxide production and that antichymotrypsin-chymotrypsin (rACT.CT) complexes are required to inhibit superoxide, suggesting that neutrophil chymotrypsin-like proteases produce conformational changes in ACT, allowing it to become active in regulating superoxide production. Additionally, we have identified NADPH oxidase itself as the target for rACT.CT and have demonstrated that rACT.CT interferes specifically with activation of the NADPH oxidase without changing the Km for NADPH or the rate constant describing the rate-limiting step in activation. These observations suggest an important role for antichymotrypsin in the regulation of NADPH-oxidase activation, which is a prerequisite for neutrophil superoxide production, and predict possible therapeutic uses for rACT in conditions where unregulated neutrophil-free radical production has been implicated in the mechanism of tissue destruction.

Human alpha 1-antichymotrypsin has been cloned, sequenced and expressed in Escherichia coli and recombinant protein as well as point-specific mutants have been purified and characterized. The corrected gene-deduced amino acid sequence has 45% overall identity with alpha 1-protease inhibitor, which is higher than the 42% previously reported (Chandra, T., Stackhouse, R., Kidd, V. J., Robson, J. H., and Woo, S. L. C. (1983) Biochemistry 22, 5055-5060). Recombinant antichymotrypsin (rACT) is similar to natural antichymotrypsin with respect to the specificity of its interactions with proteases. Its second-order rate constant for association with bovine chymotrypsin is 6-8 x 10(5) M-1 s-1, which is identical to that of the serum-derived inhibitor. Site-specific mutagenesis has been used to produce two variants of rACT in which the P1 position has been changed from leucine to either methionine (L358M-rACT) or arginine (L358R-rACT). L358M-rACT has a specificity of inhibitory activity toward serine proteases closely similar to that of native rACT. By contrast, the specificity of L358R-rACT is quite different from that of native rACT, most notably in efficiently inhibiting trypsin and human thrombin while showing a decreased ability to inhibit chymotrypsin.

Ribosomes from Escherichia coli strain Q13 have been photoaffinity labeled with [3H]puromycin in the presence of tetracycline. Puromycin-modified 50 S subunits appear to be identical with untreated subunits in electron micrographs and are precipitated by antibodies to the N6,N6'dimethyladenosine moiety of puromycin. Electron micrographs of subunit-antibody complexes show ribosomal subunits to which an individual antibody molecule is bound and pairs of subunits linked by an IgG molecule. Two regions of puromycin binding have been identified. The primary area, seen in 76% of the ribosome monomer complexes and 93% of the antibody-linked dimers, is beside (or on) the small central protuberance and on the side opposite the L7/L12 arm. A secondary area, maximally distant from the central protuberance, is seen in 22% of the monomeric complexes but only 7% of the antibody-linked dimers. In conjunction with our earlier localization of puromycin binding on the 30 S subunit (Olson, H. M., Grant, P. G., Glitz, D. G., and Cooperman, B. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 890-894), we now define a puromycin-binding neighborhood of the 70 S ribosome. In addition to providing evidence for the localization of the peptidyl transferase center within the 50 S subunit, our results contribute to the formulation of a model for tRNA binding to both 30 S subunits and 70 S ribosomes.

The ability of plasma proteinase inhibitors to inactivate human chymase, a chymotrypsin-like proteinase stored within mast cell secretory granules, was investigated. Incubation with plasma resulted in over 80% inhibition of chymase hydrolytic activity for small substrates, suggesting that inhibitors other than alpha 2-macroglobulin were primarily responsible for chymase inactivation. Depletion of specific inhibitors from plasma by immunoadsorption using antisera against individual inhibitors established that alpha 1-antichymotrypsin (alpha 1-AC) and alpha 1-proteinase inhibitor (alpha 1-PI) were responsible for the inactivation. Characterization of the reaction between chymase and each inhibitor demonstrated in both cases the presence of two concurrent reactions proceeding at fixed relative rates. One reaction, which led to inhibitor inactivation, was about 3.5 and 4.0-fold faster than the other, which led to chymase inactivation. This was demonstrated in linear titrations of proteinase activity which exhibited endpoint stoichiometries of 4.5 (alpha 1-AC) and 5.0 (alpha 1-PI) instead of unity, and SDS gels of reaction products which exhibited a banding pattern indicative of both an SDS-stable proteinase-inhibitor complex and two lower Mr inhibitor degradation products which appear to have formed by hydrolysis within the reactive loop of each inhibitor. At inhibitor concentrations approaching those in plasma where inhibitor to chymase concentration ratios were in far excess of 4.5 and 5.0, the rate of chymase inactivation by both serpin inhibitors appeared to follow pseudo-first order kinetics. The "apparent" second order rate constants of inactivation determined from these data were about 3000-fold lower than the rate constants reported for human neutrophil cathepsin G and elastase with alpha 1-AC and alpha 1-PI, respectively. This suggests that chymase would be inhibited about 650-fold more slowly than these proteinases when released into plasma. These studies demonstrate that although chymase is inactivated by serpin inhibitors of plasma, both inhibitors are better substrates for the proteinase than they are inhibitors. This finding along with the slow rates of inactivation indicates that regulation of human chymase activity may not be a primary function of plasma.

Escherichia coli small ribosomal subunits have been reconstituted from RNA and high performance liquid chromatography-purified proteins including protein S19 that had been modified at its amino-terminal proline residue with 1-fluoro-2,4-dinitrobenzene. As detailed in the accompanying paper (Olah, T. V., Olson, H. M., Glitz, D. G., and Cooperman, B. S. (1988) J. Biol. Chem. 263, 4795-4800), dinitrophenyl (DNP)-S19 was efficiently incorporated into the site ordinarily occupied by S19. Antibodies to DNP bound effectively to the reconstituted subunits and did not cause dissociation of the modified protein from the subunit. Electron microscopy of the immune complexes was used to localize the modified protein on the subunit surface. More than 95% of the antibody binding sites seen were consistent with a single location of protein S19 on the upper portion or head of the subunit, on the surface that faces the 50 S particle in a 70 S ribosome, and in an area relatively distant from the subunit platform. The S19 site is close to the region in which 30 S subunits are photoaffinity labeled with puromycin. Protein S19 is thus near protein S14 in the small subunit and in proximity to the peptidyl transferase center of the 70 S ribosome.

Small (30 S) ribosomal subunits from Escherichia coli strain TPR 201 were photoaffinity-labeled with [3H]puromycin in the presence of chloramphenicol under conditions in which more than 1 mol of antibiotic was incorporated per mol of ribosomes. The subunits were than washed with 3 M NH4Cl to yield core particles and a split protein fraction; the split proteins were further fractionated with ammonium sulfate. Subunits were then reconstituted using one fraction (core, split proteins, or ammonium sulfate supernatant) from photoaffinity-modified subunits and other components from unmodified (control) subunits. The distribution of [3H]puromycin in ribosomal proteins was monitored by one-dimensional polyacrylamide gel electrophoresis, and the sites of puromycin binding were visualized by immunoelectron microscopy. Two areas of puromycin binding were identified. A high affinity puromycin site, found on the upper third of the subunit and distant from the platform, is identical to the primary site previously identified (Olson, H. M., Grant, P. G., Glitz, D. G., and Cooperman, B. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 890-894). Binding at this site is maximal in subunits reconstituted with high levels of puromycin-modified protein S14, and is decreased when unmodified S14 is incorporated. Because the percentage of antibody binding at the primary site always exceeds the percentage of puromycin label in protein S14, the primary site must include components other than S14. A secondary puromycin site of lower affinity is found on the subunit platform. This site is enriched in subunits reconstituted from puromycin-modified core particles and may include protein S7. Our results demonstrate the feasibility of localizing specifically modified components in reconstituted ribosomal subunits.

In this first of two consecutive papers, the main objective of which is to present a new approach to the systematic localization of individual proteins located in the Escherichia coli ribosome by immunoelectron microscopy, we describe the derivatization of several purified 30 S proteins (S12, S21, S14, S19, S18, S17) with 2,4-[3,5-3H]dinitrofluorobenzene at pH 7.4 and 8.4 and the uptake of each dinitrophenylated protein in place of the corresponding unmodified protein into totally reconstituted 30 S subunits. Reverse-phase high performance liquid chromatography is used to purify the proteins, to separate and characterize the products of 2,4-[3,5-3H]dinitrofluorobenzene modification, and to analyze the protein composition of the reconstituted subunits. The extent of dinitrophenyl (DNP) modification is estimated by both radioactivity and integrated peak areas, using dual wavelength monitoring at 214 and 360 nm. DNP derivatives of each of the six proteins are efficiently incorporated into reconstituting 30 S subunits. Incorporation of any of the six DNP-modified proteins does not interfere with binding of Phe-tRNA(Phe) in a poly(U)-dependent manner. This result, as well as data showing that unmodified protein competes with DNP-protein for uptake during reconstitution, provide evidence that each DNP-protein occupies the same position in 30 S subunit as does unmodified protein. In general, for a given protein, unmodified and/or less modified forms are incorporated in preference to more modified forms. Modification of protein S19 at pH 7.4 proceeds with selective formation of one derivative in high yield. Reverse-phase high performance liquid chromatography analysis of acid hydrolysates of a purified sample of this derivative, as well as of peptides derived from it by digestion with Staphylococcus aureus protease, show the N-terminal proline to be the predominant site of DNP-derivatization.

Previous work (Nicholson, A. W., Hall, C. C., Strycharz, W. A., and Cooperman, B. S. (1982) Biochemistry 21, 3797-3808) showed that [3H]p-azidopuromycin photoaffinity labeled 70 S Escherichia coli ribosomes and that photoincorporation into 50 S subunit proteins was in the order L23 greater than L18/22 greater than L15. In the present work we report on immunoelectron microscopic studies of the complexes formed by p-azidopuromycin-modified 50 S subunits with antibodies to the N6,N6-dimethyladenosine moiety of the antibiotic. The p-azidopuromycin-modified 50 S subunits appear to be identical to unmodified control subunits in electron micrographs. Complexes of modified subunits with antibodies to the N6,N6-dimethyladenosine moiety of p-azidopuromycin were visualized in micrographs. Individual subunits with a single bound antibody (monomeric complexes) and pairs of subunits cross-linked by a single antibody (dimeric complexes) were separately evaluated and showed similar results. Two regions of p-azidopuromycin photoincorporation were identified. The primary site, seen in about 75% of the complexes, is between the central protuberance and small projection, on the side away from the L7/L12 arm, in a region thought to contain the peptidyltransferase center. The secondary site, of unknown significance, is at the base of the subunit maximally distant from the arm. These placements are essentially identical to those we observed in analyses of puromycin photoincorporation (Olson, H. M., Grant, P. G., Cooperman, B. S., and Glitz, D. G. (1982) J. Biol. Chem. 257, 2649-2656) and quantitatively similar to evaluations of monomeric puromycin-50 S subunit complexes. The data support the placement of proteins L23, L18/22, and L15 at or near the peptidyltransferase center at the primary site and suggest, in addition, that the secondary site includes a genuine area of puromycin affinity.

Evidence based upon a variety of experimental approaches is in accord with the conclusion that native yeast inorganic pyrophosphatase (EC 3.6.1.1, pyrophosphate phosphohydrolase) comprises two identical subunits approximately 32,000 in molecular weight with threonine and valine the NH2- and COOH-terminal residues, respectively. Gel filtration of reduced and carboxymethylated pyrophosphatase on a column of 6% agarose in 6 m guanidine hydrochloride revealed a single protein component of molecular weight 33,000; a value of 35,500 was obtained after exhaustive succinylation of the alkylated enzyme. The molecular weight thus derived for S-carboxymethylated pyrophosphatase was in close agreement with the values calculated from polyacrylamide gel electrophoresis in sodium dodecyl sulfate and amino acid compositional data which were, respectively, 33,000 and 30,250. Threonine was identified as the sole NH2-terminal residue by the dimethylaminonaphthalene-5-sulfonyl chloride end group procedure. Hydrolysis of alkylated pyrophosphatase by carboxypeptidase A was attended by the rapid release of COOH-terminal valine in quantities stoichiometric with the amount of monomeric protein present. Peptide maps of trypsin digests from S-carboxymethylated enzyme revealed a total of 30 peptides, five of which gave a positive test for arginine and three of which stained positively for tryptophan. These results are in accord with what would be expected on the basis of the lysine, arginine, and tryptophan content of the enzyme subunit. Finally, the partial sequence of NH2-terminal amino acid residues presented below was elucidated by Edman degradation with the aid of automated sequencing equipment. [see PDF for sequence] Yields of the phenylthiohydantoin amino acid derivatives obtained after each cycle of the degradative procedure were consistent with the quantity of monomeric protein subjected to analysis.