Domain structure analysis of elongation factor-3 from Saccharomyces cerevisiae by limited proteolysis and differential scanning calorimetry.

Elongation-factor-3 (EF-3) is an essential factor of the fungal protein synthesis machinery. In this communication the structure of EF-3 from Saccharomyces cerevisiae is characterized by differential scanning calorimetry (DSC), ultracentrifugation, and limited tryptic digestion. DSC shows a major transition at a relatively low temperature of 39 degrees C, and a minor transition at 58 degrees C. Ultracentrifugation shows that EF-3 is a monomer; thus, these transitions could not reflect the unfolding or dissociation of a multimeric structure. EF-3 forms small aggregates, however, when incubated at room temperature for an extended period of time. Limited proteolysis of EF-3 with trypsin produced the first cleavage at the N-side of Gln775, generating a 90-kDa N-terminal fragment and a 33-kDa C-terminal fragment. The N-terminal fragment slowly undergoes further digestion generating two major bands, one at approximately 75 kDa and the other at approximately 55 kDa. The latter was unusually resistant to further tryptic digestion. The 33-kDa C-terminal fragment was highly sensitive to tryptic digestion. A 30-min tryptic digest showed that the N-terminal 60% of EF-3 was relatively inaccessible to trypsin, whereas the C-terminal 40% was readily digested. These results suggest a tight structure of the N-terminus, which may give rise to the 58 degrees C transition, and a loose structure of the C-terminus, giving rise to the 39 degrees C transition. Three potentially functional domains of the protein were relatively resistant to proteolysis: the supposed S5-homologous domain (Lys102-Ile368), the N-terminal ATP-binding cassette (Gly463-Lys622), and the aminoacyl-tRNA-synthase homologous domain (Glu820-Gly865). Both the basal and ribosome-stimulated ATPase activities were inactivated by trypsin, but the ribosome-stimulated activity was inactivated faster.     

Characterization of structural domains of the human epidermal growth factor receptor obtained by partial proteolysis

Partial cleavage with trypsin has been used to study the structure of the epidermal growth factor (EGF) receptor purified from human carcinoma cells. Following affinity labeling of the receptor with 125I-EGF or the ATP analogue 5'-p-fluorosulfonyl benzoyl[14C]adenosine, metabolic labeling with [35S]methionine, [3H]glucosamine, or [32P]orthophosphate, or in vitro autophosphorylation with [gamma-32P]ATP, tryptic cleavage defines the following three regions of the 180-kDa receptor protein: 1) a 125-kDa trypsin-resistant domain which contains sites of glycosylation, EGF binding, and an EGF-specific threonine phosphorylation site; 2) an adjacent 40-kDa fragment which contains serine and threonine phosphorylation sites and is further cleaved to a 30-kDa trypsin-resistant domain; and 3) a terminal 15-kDa portion of the receptor that contains the sites of tyrosine phosphorylation and is degraded to small fragments in the presence of trypsin. Both the 125- and 40-kDa regions of the EGF receptor appear to be required for receptor-associated protein kinase activity since separation of these regions by tryptic cleavage abolishes this activity, and both regions are specifically labeled with an ATP affinity analogue, suggesting that both are involved in ATP binding. Additional 63- and 48-kDa phosphorylated fragments are generated upon trypsin treatment of EGF receptor from EGF-treated cells. The potential usefulness of partial tryptic cleavage in studying the EGF receptor and the possible biological function of the 30-kDa trypsin-resistant fragment of the receptor are discussed.     

Identification of the sites in the eukaryotic elongation factor 1 alpha involved in the binding of elongation factor 1 beta and aminoacyl-tRNA.

In this article we report the identification of the sites which are involved in the binding of the GDP-exchange factor EF-1 beta and aminoacyl tRNA to the alpha-subunit of the eukaryotic elongation factor 1 (EF-1) from Artemia. For this purpose the polypeptide chain of EF-1 alpha, having 461 amino acid residues, was proteolytically cleaved into large fragments by distinct proteases. Under well defined conditions, a mixture of two large fragments, free from intact EF-1 alpha and with molecular masses of 37 kDa and 43 kDa, was obtained. The 37-kDa and 43-kDa fragments comprise the residues 129-461 and 69-461, respectively. However, in aqueous solution and under non-denaturing conditions, the mixture still contained a short amino-terminal peptide, encompassing the residues 1-36, that remained tightly bound. The ability of the mixture of the 37+43-kDa fragments, including this amino-terminal peptide 1-36, to bind GDP or to facilitate aminoacyl tRNA binding to salt-washed ribosomes was severely reduced, compared to intact EF-1 alpha. However, both of these complexes were able to bind to the GDP-exchange-stimulating subunit EF-1 beta. A 30-kDa fragment, comprising the residues 1-287, was generated after treatment of the protein with endoproteinase Glu-C. This fragment contained the complete guanine nucleotide binding pocket. Although it was able to bind GDP and to transport aminoacyl tRNA to the ribosome, no affinity towards EF-1 beta was observed. We propose that the guanine-nucleotide-exchange stimulation by EF-1 beta is induced through binding of this factor to the carboxy-terminal part of EF-1 alpha. As a result, a decreased susceptibility towards trypsin of the guanine-nucleotide-binding pocket of EF-1 alpha, especially in the region of its presumed effector loop is induced.     

Photoaffinity labelling of gizzard myosin with 3'-O-(4-benzoyl)-benzoic-adenosine 5'-triphosphate

The reaction of a photoaffinity analog, 3'-O-(4-benzoyl)-benzoic-adenosine 5'-triphosphate (BZ2ATP) with gizzard myosin is described. The incorporation of BZ2ATP into myosin is both specific and stoichiometric. About 2.2 mol BZ2ATP are incorporated/mol myosin resulting in the significant loss of EDTA(K+) ATPase activity. The Mg2+ and actin-activated ATPase activities are slightly inhibited. Addition of ATP (millimolar) during the photolysis reaction significantly inhibits incorporation of BZ2ATP into myosin. Our data show that the label is mainly incorporated into the heavy chain of myosin with some label in the 20-kDa light chain. Limited proteolysis of radioactively labeled myosin subfragment 1 with trypsin reveals the presence of radioactivity mainly in the 50-kDa fragment and some in the 29-kDa and 25-kDa fragments. However, our data on the ATP-sensitive incorporation of BZ2ATP into the tryptic fragments suggest that the 50-kDa peptide, not the 29-kDa peptide, may be located at or around the active site.     

Stepwise phosphorylation of the NH2-terminal region of the simian virus 40 large T antigen

The simian virus 40 (SV40) large T antigen was immunoprecipitated from extracts of infected monkey cells and cleaved with trypsin under conditions of mild proteolysis. The digestion generated fragments from the NH2-terminal region of T antigen which were released from the immunoprecipitates. Pulse-chase experiments showed that most of the newly made T antigen (form A) generated an NH2-terminal fragment of 17 kDa in size, whereas most of the T antigen that had aged in the cell (form C) generated a fragment of 20 kDa. An intermediate form of T antigen (form B), which generated an 18.5- kDa NH2-terminal fragment, was produced in part from form A and was converted to form C during the chase. Phosphate-labeling experiments showed that form C was the species of T antigen that incorporated the most 32P radioactivity at the NH2-terminal region, although some label was also incorporated into forms A and B. In vitro dephosphorylation of gel-purified 18.5- and 20-kDa fragments labeled with [35S]methionine increased the electrophoretic mobility of the fragments to that of 17 kDa. This signified that phosphorylation of the NH2-terminal fragments was directly responsible for their aberrant behavior in acrylamide gels. Although peptide maps of the methionine-labeled tryptic peptides of the 17-, 18.5-, and 20-kDa fragments were very similar to one another, maps of the 32P-labeled tryptic Pronase E peptides of these fragments contained qualitative and quantitative differences. Analysis of the labeled phosphoamino acids of various peptides from these fragments indicated that the 20-kDa fragment was highly phosphorylated at Ser 123 and Thr 124, whereas the 17- and 18.5-kDa fragments were mostly unphosphorylated at these sites. These experiments indicated that T antigen is phosphorylated at the NH2-terminal region in a specific stepwise process and, therefore, that this post-translational modification of T antigen is tightly regulated.     

Localization of the sites of ADP-ribosylation and GTP binding in the eukaryotic elongation factor EF-2

Tryptic cleavage of EF-2, molecular mass 93 kDa, produced an 82-kDa polypeptide and a 10-kDa fragment, which was further degraded. By a slower reaction the 82-kDa polypeptide was gradually split into a 48-kDa and a 34-kDa fragment. Similarly, treatment with chymotrypsin resulted in the formation of an 82-kDa polypeptide and a small fragment. In contrast to the tryptic 82-kDa polypeptide the corresponding chymotryptic cleavage product was relatively resistant to further attack. The degradation of the 82-kDa polypeptide with either trypsin or chymotrypsin was facilitated by the presence of guanosine nucleotides, indicating a conformational shift in native EF-2 upon nucleotide binding. No effect was observed in the presence of ATP, indicating that the effect was specific for guanosine nucleotides. After affinity labelling of native EF-2 with oxidized [3H]GTP and subsequent trypsin treatment the radioactivity was recovered in the 48-kDa polypeptide showing that the GTP-binding site was located within this part of the factor. Correspondingly, tryptic degradation of EF-2 labelled with [14C]NAD+ in the presence of diphtheria toxin showed that the site of ADP-ribosylation was within the 34-kDa polypeptide. By cleavage with the tryptophan-specific reagent N-chlorosuccinimide the site of ADP-ribosylation could be located at a distance of 40-60 kDa from the GTP-binding site and about 4-11 kDa from the nearest terminus.     

Long range allosteric control of cytoplasmic dynein ATPase activity by the stalk and C-terminal domains

The dynein motor domain consists of a ring of six AAA domains with a protruding microtubule-binding stalk and a C-terminal domain of unknown function. To understand how conformational information is communicated within this complex structure, we produced a series of recombinant and proteolytic rat motor domain fragments, which we analyzed enzymatically. A recombinant 210-kDa half-motor domain fragment surprisingly exhibited a 6-fold higher steady state ATPase activity than a 380-kDa complete motor domain fragment. The increased ATPase activity was associated with a complete loss of sensitivity to inhibition by vanadate and an approximately 100-fold increase in the rate of ADP release. The time course of product release was discovered to be biphasic, and each phase was stimulated approximately 1000-fold by microtubule binding to the 380-kDa motor domain. Both the half-motor and full motor domain fragments were remarkably resistant to tryptic proteolysis, exhibiting either two or three major cleavage sites. Cleavage near the C terminus of the 380-kDa motor domain released a 32-kDa fragment and abolished sensitivity to vanadate. Cleavage at this site was insensitive to ATP or 5'-adenylyl-beta,gamma-imidodiphosphate but was blocked by ADP-AlF3 or ADP-vanadate. Based on these data, we proposed a model for long range allosteric control of product release at AAA1 and AAA3 through the microtubule-binding stalk and the C-terminal domain, the latter of which may interact with AAA1 to close the motor domain ring in a cross-bridge cycle-dependent manner.     

Substructure and higher structure of chicken smooth muscle alpha-actinin molecule

Substructure of chicken gizzard smooth muscle alpha-actinin molecule was deduced by domainal mapping of the proteolytic fragments with alpha-chymotrypsin. There were three chymotryptic cleavage sites (Sites I, II, and III, from the amino terminus). Cleavage at Site I generated two fragments, i.e. an NH2-terminal 36-kDa fragment and a COOH-terminal 70-kDa fragment. The 70-kDa fragment generated either a 55-kDa fragment by cleavage at Site II or a 65-kDa fragment by cleavage at Site III. Purified NH2-terminal 36-kDa fragment bound to F-actin, whereas the 55-kDa fragment formed a dimeric molecule. Circular dichroism and electron microscopic experiments demonstrated that the alpha-helical content of the 55-kDa fragment was 14% higher than that of native gizzard alpha-actinin, coinciding with the apparently rod-shaped configuration of this fragment. A 110-kDa product was generated from two 55-kDa fragments in a cross-linking study with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Two cross-linkable sites in the 55-kDa, A- and B-site, were shown to be involved in this reaction. Further, it was demonstrated by using N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide labeling and immunoblotting analyses that the A-site on one 55-kDa fragment was cross-linked to the B-site on the other. These results suggest that smooth muscle alpha-actinin formed an antiparallel dimeric molecule in which the 55-kDa fragments connected the two actin-binding domains composed of the 36-kDa fragments.     

Affinity labelling with MgATP analogues reveals coexisting Na+ and K+ forms of the alpha-subunits of Na+/K+-ATPase.

To test the hypothesis that Na+/K+-ATPase works as an (alpha beta)2-diprotomer with interacting catalytic alpha-subunits, tryptic digestion of pig kidney enzyme, that had been inactivated with substitution-inert MgATP complex analogues, was performed. This led to the demonstration of coexisting C-terminal Na+-like 80-kDa as well as K+-like 60-kDa peptides and N-terminal 40-kDa peptides of the alpha-subunit. To localize the ATP binding sites on tryptic peptides, studies with radioactive MgATP complex analogues were performed: Co(NH3)4-8-N3-ATP specifically modified the E2ATP (low affinity) binding site of Na+/K+-ATPase with an inactivation rate constant (k2) of 12 x 10-3.min-1 at 37 degrees C and a dissociation constant (Kd) of 207 +/- 28 microm. Tryptic digestion of the [gamma32P]Co(NH3)4-8-N3-ATP-inactivated and photolabelled alpha-subunit (Mr = 100 kDa) led, in the absence of univalent cations, to a K+-like C-terminal 60-kDa fragment which was labelled in addition to an unlabelled Na+-like C-terminal 80-kDa fragment. Tryptic digestion of [alpha32P]-or [gamma32P]Cr(H2O)4ATP - bound to the E1ATP (high affinity) site - led to the labelling of a Na+-like 80-kDa fragment besides the immediate formation of an unlabelled K+-like N-terminal 40-kDa fragment and a C-terminal 60-kDa fragment. Because a labelled Na+-like 80-kDa fragment cannot result from an unlabelled K+-like 60-kDa fragment, and because unlabelled alpha-subunits did not show any catalytic activity, the findings are consistent with a situation in which Na+- and K+-like conformations are stabilized by tight binding of substitution-inert MgATP complex analogues to the E1ATP and E2ATP sites. Hence, all data are consistent with the hypothesis that ATP binding induces coexisting Na+ and K+ conformations within an (alphabeta)2-diprotomeric Na+/K+-ATPase.     

Adipocyte insulin receptor. Generation of a cryptic domain of the alpha-subunit during internalization of hormone-receptor complexes.

The dynamics of the internalization of photoaffinity-labelled insulin-receptor complexes was investigated in isolated rat adipocytes by using tryptic proteolysis to probe both the orientation and cellular location of the labelled complexes. In cells that were labelled at 16 degrees C and not prewarmed, 150 micrograms of trypsin/ml rapidly degraded the labelled 125 kDa insulin-receptor subunit into a major proteolytic fragment of 70 kDa and minor amounts of 90- and 50-kDa fragments. With milder trypsin treatment conditions (100 micrograms of trypsin/ml, 15 s at 37 degrees C), the 90 kDa peptide (different from the 90 kDa beta-subunit of the insulin receptor) appeared as a major intermediate proteolytic product, but this species was rapidly and completely converted into the 70- and 50-kDa fragments with continued exposure to trypsin, such that it did not accumulate to appreciable amounts in cells that were not prewarmed before trypsin exposure. By contrast, trypsin treatment of cells prewarmed to 37 degrees C for various times showed that: first, a proportion of the labelled 125 kDa receptors was internalized (became trypsin-insensitive); secondly, the 90 kDa tryptic peptide was formed in large amounts, with proportionate decreases occurring in the amounts of the 70- and 50-kDa tryptic peptides. The increased accumulation of the 90 kDa tryptic peptide from cells preincubated at 37 degrees C, but not at 16 degrees C, indicated that trypsin cleavage sites within the 90 kDa segment of the insulin-receptor alpha-subunit that were exposed at 16 degrees C were made inaccessible by incubation at 37 degrees C, a finding that is consistent with generation of a cryptic domain of the receptor subunit. The tryptic generation of the 90 kDa peptide at 37 degrees C was rapid, becoming half-maximal in 4.4 +/- 0.6 min and maximal in 15-20 min, preceded the intracellular accumulation of labelled receptors (half-maximal in 12.6 +/- 0.7 min and maximal in 30-40 min), was highly correlated with receptor internalization, and was not observed in cultured IM-9 lymphocytes, a cell line in which photolabelled insulin receptors are primarily lost by shedding into the incubation media. These results show that, in adipocytes incubated at 37 degrees C, rapid masking of a previously (at 16 degrees C) accessible domain of the insulin-receptor alpha-subunit occurs and that this dynamic process happens at an early stage in the internalization of insulin-receptor complexes.     

The limited tryptic cleavage of chymotryptic S-1: an approach to the characterization of the actin site in myosin heads.

Limited tryptic proteolysis of S-1 (A₁+A₂) or S-1 (A₁) and S-1 (A₂) converts the heavy chain into 3 fragments of Mr = 27K-50K-20K. As a result the actin-stimulated ATPase activity of the fragmented heads is lost. When the digestion is performed using the complex F-actin-S-1, this ATPase activity is completely preserved and the heavy chain is split into only 2 fragments of Mr = 27K–70K. The specific protection by F-actin of the -COOH terminal region of the heavy chain at the joint 50K-20K against tryptic cleavage and loss of activity suggests that this part of the head can be involved in actin binding site and/or Mg²⁺ ATP hydrolysis by the acto-S-1 complex.     

Domain structure of rat liver carbamoyl phosphate synthetase I

Independently folded structural domains of rat liver carbamoyl phosphate synthetase I have been identified by partial proteolytic cleavage under nondenaturing conditions. The pattern of fragments produced was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal sequences of the fragments were determined by automated Edman degradation. Comparison of these fragment sequences with the sequence of the intact protein allowed alignment of the fragments. The hydrolysis of carbamoyl phosphate synthetase I (Mr 160,000) by either trypsin or elastase proceeded in two stages, with two alternative routes of degradation for elastase. The alignment of the final tryptic fragments from the NH2 terminus to the COOH terminus was: Mr 87,000 fragment-Mr 62,000 fragment-group of small peptides. The alignment of the final elastase fragments was: Mr 37,000 fragment-Mr 108,000 fragment-group of small peptides. The rates of cleavage were affected by the presence of the substrate ATP or the positive allosteric effector N-acetylglutamate; the preferred route of elastase cleavage was also affected. In addition to providing a map of the carbamoyl phosphate synthetase I domains and preliminary information on the interaction of substrates with these domains, the present studies provide further support for the proposal that domains serve as units of protein evolution since the 37-kDa fragment encompasses the region of the rat liver synthetase that is homologous to the 40-kDa subunit of the Escherichia coli synthetase.     

Conformational transitions in the myosin head induced by temperature, nucleotide and actin. Studies on subfragment-1 of myosins from rabbit and frog fast skeletal muscle with a limited proteolysis method

Tryptic digestion patterns reveal a close similarity of the substructure of frog subfragment-1 (S1) to that established for rabbit S1. The 97-kDa heavy chain of chymotryptic S1 of frog myosin is preferentially cleaved into three fragments with apparent molecular masses of 29 kDa, 49 kDa and 20 kDa. These fragments correspond to the 27-kDa, 50-kDa and 20-kDa fragments of rabbit S1, respectively; this is indicated by the sequence of their appearance during digestion, by the suppression by actin of the generation of the 49-kDa and 20-kDa peptides, and by a nucleotide-promoted cleavage of the 29-kDa peptide to a 24-kDa fragment and the 49-kDa peptide to a 44-kDa fragment, analogous to the nucleotide-promoted cleavage of the 27-kDa and 50-kDa fragments of rabbit S1 to the 22-kDa and 45-kDa peptides. The same changes in the digestion patterns as those produced by the presence of nucleotide (ATP or its beta,gamma-imido analog AdoP P[NH]P) at 25 degrees C were observed when the digestion was carried out at 0 degrees C in the absence of nucleotide. The low-temperature-induced changes were particularly well seen in the preparations from frog myosin. The presence of ATP or AdoP P[NH]P at 0 degrees C enhanced, whereas the complex formation with actin prevented, the low-temperature-induced changes. The results are consistent with there being two fundamental conformational states of the myosin head in an equilibrium that is dependent on the temperature, the nucleotide bound at the active site, and the presence or absence of actin.     

Binding sites for monoclonal antibodies and for mRNPs on SV40 large T-antigen determined with a cleavage map

Immune complexes of simian virus 40 large T-antigen with monoclonal papovavirus protein antibodies PAb 416, PAb 402, or PAb 423 were bound to protein-A-Sepharose and then cleaved into discrete fragments by limited tryptic proteolysis. PAb 402 protected a specific cleavage site, located approximately within amino acid residues 450-500, from tryptic proteolysis; PAb 423 protected another site within residues 675-699. As shown by immunoblotting, 125I-labeled PAb 416 was bound to a 17-kDa N-terminal fragment of large T-antigen (amino acid residues 1-130), and PAb 423 was bound to several overlapping fragments derived from the C terminus of large T-antigen. These monoclonal antibodies were then used as accessibility probes to study the interaction of mRNPs with cytoplasmic large T-antigen. Whereas small T-antigen and nuclear large T-antigen were fully immunoreactive, cytoplasmic large T-antigen reacted poorly with PAb 402 or polyclonal antibodies unless the mRNP moiety was removed by treatment with EDTA/RNase A. In contrast, mRNP/T-antigen complexes were fully immunoreactive with PAb 416 or PAb 423 and did not require treatment with EDTA/RNase A. The results suggest that the binding site of PAb 402 is blocked due to the interaction with mRNPs whereas the N-terminal binding site of PAb 416 and the C-terminal binding site of PAb 423 remain accessible to antibodies.     

DNA and nucleotide-induced conformational changes in the Escherichia coli Rep and helicase II (UvrD) proteins

The domain structures of the Escherichia coli Rep and Helicase II proteins and their ligand-dependent conformational changes have been examined by monitoring the sensitivity of these helicases to proteolysis by trypsin and chymotrypsin. Limited treatment of unliganded Rep protein (73 kDa) with trypsin results in cleavage at a single site in its carboxyl-terminal region, producing a 68-kDa polypeptide which is stabilized in the presence of ATP, ADP, or adenosine 5'-O-thiotriphosphate) (ATP gamma S). The purified 68-kDa Rep tryptic polypeptide retains single-stranded (ss) DNA binding, DNA unwinding (helicase), and full ATPase activities. When bound to ssDNA, the Rep protein can be cleaved by trypsin at an additional site in its carboxyl-terminal region, producing a 58-kDa polypeptide that also retains ssDNA binding and ATPase activities. This 58-kDa Rep tryptic polypeptide can also be produced by further tryptic treatment of the 68-kDa Rep tryptic polypeptide when the latter is bound to ssDNA. This 58-kDa polypeptide displays a lower affinity for ssDNA indicating that the 10-kDa carboxyl-terminal peptide facilitates Rep protein binding to ssDNA. The 58-kDa Rep tryptic polypeptide is also stabilized in the presence of nucleotides. Based on these and previous studies that showed that the 68-kDa Rep tryptic polypeptide cannot support DNA replication in a system that is dependent upon the phi X174 cis-A protein (Arai, N. & Kornberg, A. (1981) J. Biol. Chem. 256, 5294-5298), we conclude that the carboxyl-terminal end (approximately 5 kDa) of the Rep protein is not required for its helicase or ATPase activities. However, we suggest that this region of the Rep protein is important for its interactions with the phi X174 cis-A protein. Limited treatment of unliganded Helicase II protein (82 kDa) with chymotrypsin results in cleavage after Tyr254, producing a 29-kDa amino-terminal polypeptide and a 53-kDa carboxyl-terminal polypeptide, which remain associated under nondenaturing conditions. This chymotrypsin cleavage reduces the ssDNA binding activity and eliminates the ssDNA-dependent ATPase and helicase activities of the Helicase II protein. The binding of ATP, ADP, ATP gamma S, and/or DNA to Helicase II protein results in protection of this site (Tyr254) from cleavage by chymotrypsin. Limited treatment of Helicase II protein with trypsin results in cleavage near its carboxyl-terminal end producing two polypeptides with apparent Mr = 72,000, in a manner similar to that observed with the Rep protein; these polypeptides are also stabilized by binding ATP or single-stranded DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

A variant of arrestin-1 binds rod outer segment membranes in a light-independent manner

A 50-kDa-polypeptide band peripherally bound to retinal rod outer segment (ROS) membranes was purified by anion-exchange chromatography. When the 50-kDa protein was compared with purified arrestin-1, it was observed that: (1) both proteins comigrated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and were recognized by either anti-50-kDa protein polyclonal antibodies or anti-arrestin-1 monoclonal antibodies; (2) protein fragments and peptide fingerprint maps obtained following limited and complete proteolysis with specific proteases were very similar for both molecules; and (3) several chromatographically-purified tryptic peptides from the 50-kDa protein possessed the same amino acid composition as tryptic peptides deduced from the reported arrestin-1 primary structure. Consequently, arrestin-1 and the purified 50-kDa protein must correspond to variants of the same molecule. However, in contrast to arrestin-1 that associated to the ROS membranes only in the presence of light and ATP, the 50-kDa protein interacted with the ROS membranes in a light-independent manner, either in the presence or absence of ATP. These results clearly established that phosphorylated and illuminated rhodopsin is not the membrane anchor for this variant of arrestin-1.     

Controlled tryptic digestion of prostaglandin H synthase. Characterization of protein fragments and enhanced rate of proteolysis of oxidatively inactivated enzyme

Treatment of prostaglandin H (PGH) synthase (70 kDa) with trypsin generates fragments of 33 and 38 kDa. Each of the fragments was purified by reverse-phase high performance liquid chromatography (HPLC) using acetonitrile/water/trifluoroacetic acid gradients. Amino acid sequence analysis indicates that the 33-kDa protein contains the NH2 terminus of PGH synthase. Neither the 33- nor 38-kDa fragment isolated by HPLC exhibits any PGH synthase activity; however, cleavage of intact enzyme to 33- and 38-kDa fragments to the extent of 90% only reduces cyclooxygenase activity by 40%. This implies that the cleaved proteins or a complex formed between them retains the conformation necessary for enzyme activity. Extensive attempts to resolve active fragments from each other or from intact enzyme were unsuccessful; intact enzyme and digestion fragments cochromatograph under all conditions employed. Treatment of PGH synthase with [3H]acetylsalicylic acid followed by trypsin digestion introduces [3H]acetyl moieties into the intact protein and the 38-kDa fragment (0.8-0.9 acetyl group/subunit). Nearly complete conversion of PGH synthase to 33- and 38-kDa fragments by exposure to high concentrations of trypsin prior to [3H]acetylsalicylic acid treatment results in labeling of the 38-kDa fragment, but not the 33-kDa fragment. The present findings are consistent with the presence of a membrane-binding domain (33 kDa) and an active site domain (38 kDa) in the 70-kDa subunit of PGH synthase. They also suggest that, following cleavage, the 38-kDa fragment retains the structural features responsible for the cyclooxygenase activity and selective aspirin labeling of PGH synthase. PGH synthase undergoes self-catalyzed inactivation by oxidants generated during its catalytic turnover. When PGH synthase, inactivated by treatment with arachidonic acid or hydrogen peroxide, was treated with trypsin it was cleaved two to three times faster than unoxidized enzyme. Addition of heme to oxidized PGH synthase did not reconstitute cyclooxygenase activity or resistance to trypsin cleavage. Spectrophotometric studies demonstrated that oxidatively inactivated enzyme did not bind heme. This implies that oxidation of protein residues as well as the heme prosthetic group is an important determinant of proteolytic sensitivity. Oxidative modification may mark PGH synthase for proteolytic cleavage and turnover.     

The interaction and photolabeling of myosin subfragment 1 with 3'(2')-O-(4-benzoyl)benzoyladenosine 5'-triphosphate

The photoprobe 3'(2')-O-(4-benzoyl)benzoyladenosine 5'-triphosphate (Bz2ATP) was used to characterize the nucleotide-binding site of myosin subfragment 1 (SF1). Improved synthesis and purification of Bz2ATP are reported. 1H NMR and ultraviolet spectroscopy show that Bz2ATP is a 60:40 mixture of the 3'(2')-ribose isomers and that the epsilon M261 is 41,000 M-1 cm-1. Bz2ATP is hydrolyzed by SF1 comparably to ATP in the presence of actin or K+, NH4+, or Mg2+ ions; and the product, Bz2ADP, has a single binding site on SF1 (K'a = 3.0 X 10(5) M-1). [3H]Bz2ATP was photoincorporated into SF1 with concomitant loss of K+-EDTA-ATPase activity. Analysis of photolabeled SF1 showed that the three major tryptic peptides (23, 50, and 20 kDa) of the heavy chain fragment and the alkali light chains were labeled. The presence of ATP during irradiation protected only the 50-kDa peptide, indicating that the other peptides were nonspecifically labeled. If Bz2ATP was first trapped on SF1 by cross-linking the reactive thiols, SH1 and SH2, with p-phenylenedimaleimide, only the 50-kDa tryptic peptide was labeled. These results confirm and extend previous observations that [3H]Bz2ATP trapped on SF1 by cobalt(III) phenanthroline photolabeled the same 50-kDa peptide (Mahmood, R., and Yount, R.G. (1984) J. Biol. Chem. 259, 12956-12959). Thus, the 50-kDa peptide is labeled with or without thiol cross-linking, indicating that the relative position of SH1 and SH2 does not affect the labeling pattern.     

Limited proteolysis as a structural probe of the soluble alpha-glycerophosphate oxidase from Streptococcus sp

As reported previously [Parsonage, D., Luba, J., Mallett, T. C., and Claiborne, A. (1998) J. Biol. Chem. 273, 23812-23822], the flavoprotein alpha-glycerophosphate oxidases (GlpOs) from a number of enterococcal and streptococcal sources contain a conserved 50-52 residue insert that is completely absent in the homologous alpha-glycerophosphate dehydrogenases. On limited proteolysis with trypsin, the GlpO from Streptococcus sp. (m = 67.6 kDa) is readily converted to two major fragments corresponding to masses of approximately 40 and 23 kDa. The combined application of sequence and mass spectrometric analyses demonstrates that the 40-kDa fragment represents the N-terminus of intact GlpO (Met1-Lys368; 40.5 kDa), while the 23-kDa band represents a C-terminal fragment (Ala405-Lys607; 22.9 kDa). Hence, limited proteolysis in effect excises most of the GlpO insert (Ser355-Lys404), indicating that this represents a flexible region on the protein surface. The active-site and other spectroscopic properties of the enzyme, including both flavin and tryptophan fluorescence spectra, titration behavior with both dithionite and sulfite, and preferential binding of the anionic form of the oxidized flavin, were largely unaffected by proteolysis. Enzyme-monitored turnover analyses of the intact and nicked streptococcal GlpOs (at [GlpO] approximately 10 microM) demonstrate that the single major catalytic defect in the nicked enzyme corresponds to a 20-fold increase in K(m)(Glp); the basis for this altered kinetic behavior is derived from an 8-fold decrease in the second-order rate constant for reduction of the nicked enzyme, as measured in anaerobic stopped-flow experiments. These results indicate that the flexible surface region represented by elements of the GlpO insert plays an important role in mediating efficient flavin reduction.     

The effect of actin and phosphorylation on the tryptic cleavage pattern of Acanthamoeba myosin IA

The Mg2+-ATPase activity of Acanthamoeba myosin IA is activated by F-actin only when the myosin heavy chain is phosphorylated at a single residue. In order to gain insight into the conformational changes that may be responsible for the effects of F-actin and phosphorylation on myosin I ATPase, we have studied their effects on the proteolysis of the myosin IA heavy chain by trypsin. Trypsin initially cleaves the unphosphorylated, 140-kDa heavy chain of Acanthamoeba myosin IA at sites 38 and 112 kDa from its NH2 terminus and secondarily at sites 64 and 91 kDa from the NH2 terminus. F-actin has no effect on tryptic cleavage at the 91- and 112-kDa sites, but does protect the 38-kDa site and the 64-kDa site. Phosphorylation (which occurs very near the 38-kDa site) has no detectable effect on the tryptic cleavage pattern in the absence of F-actin or on F-actin protection of the 64-kDa site, but significantly enhances F-actin protection of the 38-kDa site. Protection of the 64-kDa site is probably due to direct steric blocking because F-actin binds to this region of the heavy chain. The protection of the 38-kDa site by F-actin may be the result of conformational changes in this region of the heavy chain induced by F-actin binding near the 64-kDa site and by phosphorylation. The conformational changes in the heavy chain of myosin IA that are detected by alterations in its susceptibility to proteolysis are likely to be related to the conformational changes that are involved in the phosphorylation-regulated actin-activated Mg2+-ATPase activities of Acanthamoeba myosins IA and IB.