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1.
Topographic labelling of pore-forming proteins from the outer membrane of Escherichia coli.
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The topography of three pore-forming proteins from the outer membrane of Escherichia coli has been explored by using two labelling techniques. Firstly, the distribution of nucleophilic residues has been investigated by selective chemical modification using arylglyoxals (for arginine residues), isothiocyanates (for lysine residues), carbodi-imides (for carboxy residues) and diazonium salts. Secondly, the membrane-embedded domains have been investigated by labelling with photoactivatable phospholipid analogues and a reagent that partitions into the membrane. Few nucleophilic groups are found to be freely accessible to pore-impermeant probes reacting in the aqueous medium. More groups are accessible to small, pore-permeant probes, suggesting that several groups of each sort are contained within the pore. In addition, there appear to be a number of arginine, lysine, carboxyl and many tyrosine residues that are rather inaccessible and that react only with small, hydrophobic probes, if at all. Amongst these more deeply buried residues there are four arginine residues and an as-yet-undetermined number of carboxy residues that appear to be essential to the structural integrity of the oligomeric molecule. 相似文献
2.
Adler M Bryant J Buckman B Islam I Larsen B Finster S Kent L May K Mohan R Yuan S Whitlow M 《Biochemistry》2005,44(26):9339-9347
This paper presents the crystal structure of porcine pancreatic carboxypeptidase B (pp-CpB) in complex with a variety of thiol-based inhibitors that were developed as antagonists of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). Recent studies have indicated that a selective inhibitor of TAFIa could enhance the efficacy of existing thrombolytic agents for the treatment of acute myocardial infarction, one of the most prevalent forms of heart attacks. Unfortunately, activated TAFIa rapidly degrades in solution and cannot be used for crystallographic studies. In contrast, porcine pancreatic CpB is stable at room temperature and is available from commercial sources. Both pancreatic CpB and TAFIa are zinc-based exopeptidases, and the proteins share a 47% sequence identity. The homology improves considerably in the active site where nearly all of the residues are conserved. The inhibitors used in this study were designed to mimic a C-terminal arginine residue, one of the natural substrates of TAFIa. The X-ray structures show that the thiol group chelates the active site zinc, the carboxylic acid forms a salt bridge to Arg145, and the guanidine group forms two hydrogen bonds to Asp255. A meta-substituted phenyl was introduced into our inhibitors to reduce conformational freedom. This modification vastly improved the selectivity of compounds against other exopeptidases that cleave basic residues. Comparisons between structures indicate that selectivity derives from the interaction between the guanidine group in the inhibitors and an acidic active site residue. The location of this acidic residue is not conserved in the various carboxypeptidases. 相似文献
3.
RXPA380 (Cbz-PhePsi[PO(2)CH]Pro-Trp-OH) was reported recently as the first highly selective inhibitor of the C-domain of somatic angiotensin-converting enzyme (ACE), able to differentiate the two active sites of somatic ACE by a selectivity factor of more than 3 orders of magnitude. The contribution of each RXPA380 residue toward this remarkable selectivity was evaluated by studying several analogues of RXPA380. This analysis revealed that both pseudo-proline and tryptophan residues in the P(1)' and P(2)' positions of RXPA380 play a critical role in the selectivity of this inhibitor for the C-domain. This selectivity is not due to a preference of the C-domain for inhibitors bearing pseudo-proline and tryptophan residues, but rather reflects the poor accommodation of these inhibitor residues by the N-domain. A model of RXPA380 in complex with the ACE C-domain, based on the crystal structure of germinal ACE, highlights residues that may contribute to RXPA380 selectivity. From this model, striking differences between the N- and C-domains of ACE are observed for residues defining the S(2)' pocket. Of the twelve residues that surround the tryptophan side chain of RXPA380 in the C-domain, five are different in the N-domain. These differences in the S(2)' composition between the N- and C-domains are suggested to contribute to RXPA380 selectivity. The structural insights provided by this study should enhance understanding of the factors controlling the selectivity of the two domains of somatic ACE and allow the design of new selective ACE inhibitors. 相似文献
4.
Methylglyoxal (MG) is an important glycating agent produced under physiological conditions. MG could react with DNA and proteins to generate advanced glycation end products. Human hemoglobin, the most abundant protein in blood cells, has not been systematically investigated as the target protein for methylglyoxal modification. Here we examined carefully, by using HPLC coupled with tandem mass spectrometry (LC-MS/MS), the covalent modifications of human hemoglobin induced by methylglyoxal. Our results revealed that hemoglobin could be modified by methylglyoxal, and the major form of modification was found to be the hydroimidazolone derivative of arginine residues. In addition, Arg-92 and Arg-141 in the alpha chain as well as Arg-40 and Arg-104 in the beta chain were modified, whereas two other arginine residues, that is, Arg-31 in the alpha chain and Arg-30 in the beta chain, were not modified. Semiquantitative measurement for adduct formation, together with the analysis of the X-ray structure of hemoglobin, showed that the extents of arginine modification were highly correlated with the solvent accessibilities of these residues. The facile formation of hydroimidazolone derivatives of arginine residues in hemoglobin by methylglyoxal at physiologically relevant concentrations suggested that this type of modification might occur in vivo. The unambiguous determination of the sites and extents of methylglyoxal modifications of arginines in hemoglobin provided a basis for understanding the implications of these modifications and for employing this type of hemoglobin modification as molecular biomarkers for clinical applications. 相似文献
5.
The arginine residue(s) necessary for tetrahydrofolate binding to sheep liver serine hydroxymethyltransferase were located by phenylglyoxal modification. The incorporation of [7-14C]phenylglyoxal indicated that 2 arginine residues were modified per subunit of the enzyme and the modification of these residues was prevented by tetrahydrofolate. In order to locate the sites of phenylglyoxal modification, the enzyme was reacted in the presence and absence of tetrahydrofolate using unlabeled and radioactive phenylglyoxal, respectively. The labeled phenylglyoxal-treated enzyme was digested with trypsin, and the radiolabeled peptides were purified by high-performance liquid chromatography on reversed-phase columns. Sequencing the tryptic peptides indicated that Arg-269 and Arg-462 were the sites of phenylglyoxal modification. Neither a spectrally discernible 495-nm intermediate (characteristic of the native enzyme when substrates are added) nor its enhancement by the addition of tetrahydrofolate, was observed with the phenylglyoxal-modified enzyme. There was no enhancement of the rate of the exchange of the alpha-proton of glycine upon addition of tetrahydrofolate to the modified enzyme as was observed with the native enzyme. These results demonstrate the requirement of specific arginine residues for the interaction of tetrahydrofolate with sheep liver serine hydroxymethyltransferase. 相似文献
6.
Stacie J. Wood Yeji A. Park Naga Pooja Kanneganti Hareesh Reddy Mukkisa Lauren L. Crisman Sarah E. Davis James L. Vandenbosch Jamie B. Scaglione Deborah L. Heyl 《International journal of peptide research and therapeutics》2014,20(4):519-530
Due to increasing resistance of bacteria to traditional antibiotics, antimicrobial peptides are being investigated as a promising alternative. Tachyplesin, an antimicrobial peptide isolated from horseshoe crab, inhibits the growth of many different types of bacteria with its ability to permeabilize the cell membrane. Starting with a previously reported linear tachyplesin analog lacking cysteine (cysteine-deleted tachyplesin, CDT, KWFRVYRGIYRRR-CONH2), this study examines the systematic deletion of the C-terminal arginines and the N-terminal lysine, addition of positively charged N-and C-terminal residues, replacement of arginine with similarly-charged lysine, and replacement of hydrophobic residues with aliphatic, aromatic, fluoro-substituted aromatic, and bicyclic amino acids to examine effects on activity. The 16 modified CDT analogs were tested for their ability to disrupt model liposomes, and minimum inhibitory concentrations were determined for gram-positive and gram-negative bacterial strains. Hemolytic activity also was assessed. Overall, results indicate that elimination of two C-terminal arginine residues results in a peptide ([des-Arg12,13]CDT) with preserved antimicrobial activity but a reduction in hemolysis, a selectivity desirable for a therapeutic agent. Additional deletion was not tolerated, nor was addition of residues at the termini. Analysis of the 16 analogs also reveals the importance of hydrophobicity, not necessarily aromaticity, as an analog with hydrophobic isoleucine residues placed throughout the sequence ([Ile2,3,6,10]CDT) displayed comparable antimicrobial activity to CDT with lower hemolysis, representing a promising antimicrobial peptide with lowered toxicity. 相似文献
7.
8.
Ouzzine M Gulberti S Levoin N Netter P Magdalou J Fournel-Gigleux S 《The Journal of biological chemistry》2002,277(28):25439-25445
The human beta1,3-glucuronosyltransferase I (GlcAT-I) plays a key role in proteoglycan biosynthesis by catalyzing the transfer of glucuronic acid onto the trisaccharide-protein linkage structure Galbeta1,3Galbeta1,4Xylbeta-O-Ser, a prerequisite step for polymerization of glycosaminoglycan chains. In this study, we identified His(308) and Arg(277) residues as essential determinants for the donor substrate (UDP-glucuronic acid) selectivity of the human GlcAT-I. Analysis of the UDP-glucuronic acid-binding site by computational modeling in conjunction with site-directed mutagenesis indicated that both residues interact with glucuronic acid. Substitution of His(308) by arginine induced major changes in the donor substrate specificity of GlcAT-I. Interestingly, the H308R mutant was able to efficiently utilize nucleotide sugars UDP-glucose, UDP-mannose, and UDP-N-acetylglucosamine, which are not naturally accepted by the wild-type enzyme, as co-substrate in the transfer reaction. To gain insight into the role of Arg(277), site-directed mutagenesis in combination with chemical modification was carried out. Substitution of Arg(277) with alanine abrogated the activity of GlcAT-I. Furthermore, the arginine-directed reagent 2,3-butanedione irreversibly inhibited GlcAT-I, which was effectively protected against inactivation by UDP-glucuronic acid but not by UDP-glucose. It is noteworthy that the activity of the H308R mutant toward UDP-glucose was unaffected by the arginine-directed reagent. Our results are consistent with crucial interactions between the His(308) and Arg(277) residues and the glucuronic acid moiety that governs the specificity of GlcAT-I toward the nucleotide sugar donor substrate. 相似文献
9.
10.
Raijmakers R Zendman AJ Egberts WV Vossenaar ER Raats J Soede-Huijbregts C Rutjes FP van Veelen PA Drijfhout JW Pruijn GJ 《Journal of molecular biology》2007,367(4):1118-1129
Peptidylarginine deiminase (PAD) enzymes catalyze the conversion of arginine residues in proteins to citrulline residues. Citrulline is a non-standard amino acid that is not incorporated in proteins during translation, but can be generated post-translationally by the PAD enzymes. Although the existence of citrulline residues in proteins has been known for a long time, only a few proteins have been reported to contain this amino acid under normal conditions. These include the nuclear histones, which also contain a wide variety of other post-translational modifications, as for instance methylation of arginine residues. It has been suggested that citrullination and methylation of arginine residues are competing processes and that PAD enzymes might "reverse" the methylation of arginine residues by converting monomethylated arginine into citrulline. However, conflicting data have been reported on the capacity of PADs to citrullinate monomethylated peptidylarginine. Using synthetic peptides that contain either arginine or methylated arginine residues, we show that the human PAD2, PAD3 and PAD4 enzymes and PAD enzyme present in several mouse tissues in vitro can only convert non-methylated peptidylarginine into peptidylcitrulline and that hPAD6 does not show any deiminating activity at all. A comparison of bovine histones either treated or untreated with PAD by amino acid analysis also supported the interference of deimination by arginine methylation. Taken together, these data indicate that it is unlikely that methyl groups at the guanidino position of peptidylarginine can be removed by peptidylarginine deiminases, which has important implications for the recently reported role of these enzymes in gene regulation. 相似文献
11.
The in vivo application of the protein streptavidin is limited by its propensity to localize to kidney, particularly when it is used as a carrier of radionuclides in Targeted Radionuclide Therapy. Our previous studies demonstrated that modification of recombinant "core" streptavidin (rSAv) by reaction of lysine residues with succinic anhydride and arginine residues with 1,2-cyclohexanedione dramatically decreases the kidney concentrations over that obtained with wild-type rSAv. In this investigation, we explored the role of lysine and arginine residues in kidney localization further by evaluating site-directed mutants of rSAv. In the five mutants studied, the four lysine residues found in each subunit of rSAv were replaced (independently) with an alanine (K80A, K121A, K132A, K134A), and a specific arginine was replaced with a histidine (R59H). The rSAv mutants were prepared from a "core" rSAv construct produced by expression in E. coli that had 124 amino acids (residues 13-136). Another rSAv construct that had 127 amino acids (residues 13-139), used in most of our previous studies, was also included for comparison. As an additional comparison, succinylated rSAv was prepared and evaluated. The rSAv proteins were radioiodinated and injected into athymic mice that were on a biotin-free diet for 5-7 days prior, and biodistribution data were obtained (for most proteins) at 1, 4, 24, and 48 h postinjection. The data obtained show large differences in kidney localizations of the wild-type rSAv and some rSAv mutants. The largest difference in the kidney concentration was noted for the rSAv-K134A mutant (1.90 +/- 0.22%ID/g; 24 h pi) as compared to the wild-type rSAv (31.83 +/-5.26%ID/g) at the same time point. The concentration of rSAv-K134A mutant in kidney was slightly lower than that obtained with succinylated rSAv. At the 24 h time point, the kidney concentrations of the rSAv-R59H mutant (8.95 +/- 2.94%ID/g) and the rSAv-K121A mutant (11.86 +/- 1.61%ID/g) were lower than wild-type rSAv, but the rSAv mutants rSAv-K80A (27.95 +/- 1.82%ID/g) and rSAv-K132A (32.50 +/- 10.09%ID/g) were essentially the same. The data suggests that specific lysine and arginine residues are involved in kidney localization. Possible mechanisms for the observed kidney localization are discussed. 相似文献
12.
Recent studies have shown that selection of proteins for degradation by the ubiquitin system occurs most probably by binding to specific sites of the ubiquitin-protein ligase, E3. A free alpha-NH2 residue of the substrate is one important determinant recognized by the ligase. Selective binding sites have been described for basic and bulky-hydrophobic NH2 termini (Reiss, Y., Kaim, D., and Hershko, A. (1988) J. Biol. Chem. 263, 2693-2698) and for alanine, serine, and threonine at the NH2-terminal position (Gonda, D. K., Bachmair, A., Wünning, I., Tobias, J. W., Lane, W. S., and Varshavsky, A. (1989) J. Biol. Chem. 264, 16700-16712). Proteins with acidic NH2-terminal residues are degraded by the ubiquitin system only following conversion of the acidic residue to a basic residue by the addition of an arginine moiety (Ferber, S., and Ciechanover, A. (1987) Nature 326, 808-811). Although the enzymes involved in this post-translational modification have been characterized, the underlying mechanism has been obscure. By using a chemical cross-linking technique, we demonstrate that proteins with acidic NH2 termini do not bind to E3 without prior modification of this residue by the addition of arginine. In contrast, proteins with a basic NH2-terminal residue bind to the ligase without any modification. The recognition of acidic NH2-terminal substrates by E3 is dependent upon the addition of all the components of the modifying machinery, arginyl-tRNA-protein transferase, arginyl-tRNA synthetase, tRNA, and arginine. The ligase-bound modified proteins are converted to ubiquitin conjugates in a "pulse-chase" experiment, indicating that the binding is functional and that the enzyme-substrate complex is an obligatory intermediate in the conjugation process. Chemical modification of the carboxyl groups, which results in their neutralization, generates substrates that bind to E3 without modification. This finding suggests that the amino-terminal binding site of E3 is negatively charged, and only positively charged amino-terminal residues may bind to it. Negatively charged (acidic) NH2-terminal residues will bind only following neutralization or reversal of the charge. 相似文献
13.
Protein arginine methyltransferase 1 (PRMT1) catalyzes the mono- and dimethylation of certain protein arginine residues. Although this posttranslational modification has been implicated in many physiological processes, the molecular basis for PRMT1 substrate recognition is poorly understood. Most modified arginine residues in known PRMT1 substrates reside in repeating "RGG" sequences. However, PRMT1 also specifically methylates Arg3 of histone H4 in a region that is not glycine-arginine rich, suggesting that PRMT1 substrates are not limited to proteins bearing "RGG" sequences. Because a systematic evaluation of PRMT1 substrate specificity has not been performed, it is unclear if the "RGG" sequence accurately represents the consensus target for PRMT1. Using a focused peptide library based on a sequence derived from the in vivo substrate fibrillarin we observed that PRMT1 methylated substrates that had amino acid residues other than glycine in the "RX (1)" and "RX (1)X (2)" positions. Importantly, eleven additional PRMT1 substrate sequences were identified. Our results also illustrate that the two residues on the N-terminal side of the modification site are important and need not both be glycine. PRMT1 methylated the eukaryotic initiation factor 4A1 (eIF4A1) protein, which has a single "RGG" sequence. Methylation of eIF4A1 and the similar eIF4A3 could be affected using single site mutations adjacent to the modification site, demonstrating the importance of amino acid sequence in PRMT1 protein substrates. Dimethylation of the parent library peptide was shown to occur through a dissociative mechanism. In summary, PRMT1 selectively recognizes a set of amino acid sequences in substrates that extend beyond the "RGG" paradigm. 相似文献
14.
Parker AR 《Journal of enzyme inhibition and medicinal chemistry》2003,18(5):453-462
The interaction between the "electron transferring flavoprotein" (ETF) and medium chain acyl-CoA dehydrogenase (MCAD) enables successful flavin to flavin electron transfer, crucial for the beta-oxidation of fatty acids. The exact biochemical determinants for ETF binding to MCAD are unknown. Here we show that binding of human ETF, to MCAD, was inhibited by 2,3-butanedione and diethylpyrocarbonate (DEPC) and reversed by incubation with free arginine and hydroxylamine respectively. Spectral analyses of native ETF vs modified ETF suggested that flavin binding was not affected and that the loss of ETF activity with MCAD involved modification of one ETF arginine residue and one ETF histidine residue respectively. MCAD and octanoyl-CoA protected ETF against inactivation by both 2,3-butanedione and DEPC indicating that the arginine and histidine residues are present in or around the MCAD binding site. Comparison of exposed arginine and histidine residues among different ETF species, however, indicates that arginine residues are highly conserved but that histidine residues are not. These results lead us to conclude that this single arginine residue is essential for the binding of ETF to MCAD, but that the single histidine residue, although involved, is not. 相似文献
15.
Brian D. Hudson Irina G. Tikhonova Sunil K. Pandey Trond Ulven Graeme Milligan 《The Journal of biological chemistry》2012,287(49):41195-41209
Free fatty acid receptors 2 and 3 (FFA2 and FFA3) are G protein-coupled receptors for short chain free fatty acids (SCFAs). They respond to the same set of endogenous ligands but with distinct rank-order of potency such that acetate (C2) has been described as FFA2-selective, whereas propionate (C3) is non-selective. Although C2 was confirmed to be selective for human FFA2 over FFA3, this ligand was not selective between the mouse orthologs. Moreover, although C3 was indeed not selective between the human orthologs, it displayed clear selectivity for mouse FFA3 over mouse FFA2. This altered selectivity to C2 and C3 resulted from broad differences in SCFAs potency at the mouse orthologs. In studies to define the molecular basis for these observations, marked variation in ligand-independent constitutive activity was identified using a [35S]GTPγS assay. The orthologs with higher potency for the SCFAs, human FFA2 and mouse FFA3, displayed high constitutive activity in this assay, whereas the orthologs with lower potency for the agonist ligands, mouse FFA2 and human FFA3, did not. Sequence alignments of the second extracellular loop identified single negatively charged residues in FFA2 and FFA3 not conserved between species and predicted to form ionic lock interactions with arginine residues within the FFA2 or FFA3 agonist binding pocket to regulate constitutive activity and SCFA potency. Reciprocal mutation of these residues between species orthologs resulted in the induction (or repression) of constitutive activity and in most cases also yielded corresponding changes in SCFA potency. 相似文献
16.
The N-terminal glycine of the A-chain in insulin is reported to be one of the residues that binds to the insulin receptor. Modifications near this region lead to variations in the biological activity of insulin. One such modification viz., an addition of an arginine at the N-terminal A-chain, was reported to possess two-thirds the activity of native insulin. The crystal structure of 2 zinc human arg (A0) insulin has been elucidated to 2A resolution to understand the mechanism of reduction in insulin activity. A conformational transition from T6 to T3R3(f) and a decrease in the surface accessibility of residues in the so called receptor binding region have been observed. The presence of arginine has also induced distortions in the A chain N-terminal helix. The subtle conformational alterations like decrease in surface accessibility, alterations in the charge surface and changes in the relative orientation of the two helices in the A chain may be responsible for the reduction in activity. 相似文献
17.
1. Reaction of 1,2-cyclohexanedione with arginine residues of egg white riboflavin-binding protein results in a loss of the binding activity. 2. In borate buffer pH 8.0, with 0.15 M cyclohexanedione, the inactivation proceeds with a pseudo-first-order rate constant 0.084 hr.-1. 3. At least 65% of lost riboflavin binding capacity can be recovered on 12 hr incubation in 0.5 M hydroxylamine pH 7.0. 4. All 5 arginine residues are modified, 2-3 of them seem to react much easier than others. 5. The correlation between modification of arginines and protein inactivation, as analyzed by kinetic and statistical methods, suggests that one of low-reactivity residues is "essential" for riboflavin binding. 6. In the holoprotein, one arginine residue is almost completely protected from 1,2-cyclohexanedione modification. 7. Riboflavin does not dissociate from holoprotein, even on prolongated incubation with the reagent. 8. The protected arginine residue seems to be located in the riboflavin binding pocket of protein macromolecule. 相似文献
18.
Pietzsch J 《Biochemical and biophysical research communications》2000,270(3):852-857
An alteration of apolipoprotein (apo) B-100 structure by direct oxidative modification is supposed to be an important mechanism involved in atherogenesis. There is difficulty in quantifying this type of modification owing to a lack of specific assays. We evaluated a methodology based on the oxidation of protein arginine and proline to gamma-glutamyl semialdehyde which by reduction forms 5-hydroxy-2-aminovaleric acid (HAVA). We determined HAVA by using derivatization to N(O)-ethoxycarbonyl ethyl esters and gas chromatography-mass spectrometry in different low-density lipoprotein preparations subjected to oxidative damage in the presence of iron. Results suggest that apoB-100 proline and arginine residues are highly reactive toward oxygen radicals ex vivo. Femtomole levels of HAVA can be reproducible measured. HAVA determination compares well with the measurement of carbonyl group formation used as a generally accepted but nonspecific index of protein oxidation. Thus, HAVA could prove to be a sensitive assay for studying specific modification of apoB-100. 相似文献
19.
Appropriately fixed preparations stained by a modification of the Guard (1959) reaction for "sex chromatin" display selective staining of interphase chromatin and mitotic or meiotic chromosomes. This is a regressive staining method which seems to depend on the selective displacement of an acidic dye from less basic structures, and retention of the dye at more basic sites. The results obtained with the reaction can be controlled by the length of time that the preparations are "differentiated" in solutions containing phosphomolybdic and phosphotungstic acids (polyacids). After three- or four-hour exposures to polyacid solutions, all chromatin is stained. However, with longer differentiation, "condensed" chromatin can be stained preferentially. Of a number of fixatives investigated, only 10% formalin, ethanol-acetic acid (3:1), and Bouin's solution proved useful. Others resulted in diminished specificity or a total loss of selectivity. The most intense staining was obtained after formalin fixation. Less intense dyebinding was observed after fixation in 3:1 - probably due to extraction of some histone fractions-and the least amount of dye was bound in Bouin's-fixed chromatin - probably due to blockage of arginine residues by picric acid. The reaction was not affected by enzymatic removal of nucleic acids or the extraction of lipids. It was diminished by treatment with trypsin or weak acetylation, and it was completely prevented by strong acetylation, deamination, or extraction of basic proteins with HCl. The results presented suggest that the modified Guard (1959) procedure selectively demonstrates basic nucleoproteins. Further, by the use of regressive differentiation in polyacid solutions, the retention of dye in more condensed chromatin can be favored. 相似文献
20.
Arginine residues are critical for the heparin-cofactor activity of antithrombin III. 总被引:1,自引:0,他引:1
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A dilution/quench technique was used to monitor the time course of chemical modification on the heparin-cofactor (a) and progressive thrombin-inhibitory (b) activities of human antithrombin III. Treatment of antithrombin III (AT III) with 2,4,6-trinitrobenzenesulphonate at pH 8.3 and 25 degrees C leads to the loss of (a) at 60-fold more rapid rate than the loss of (b). This is consistent with previous reports [Rosenberg & Damus (1973) J. Biol. Chem. 248, 6490-6505; Pecon & Blackburn (1984) J. Biol. Chem. 259, 935-938] that lysine residues are involved in the binding of heparin to AT III, but not in thrombin binding. Treatment of AT III with phenylglyoxal at pH 8.3 and 25 degrees C again leads to a more rapid loss of (a) than of (b), with the loss of the former proceeding at a 4-fold faster rate. The presence of heparin during modification with phenylglyoxal significantly decreases the rate of loss of (a). Full loss of (a) correlates with the modification of seven arginine residues per inhibitor molecule, whereas loss of (b) does not commence until approximately four arginine residues are modified and is complete upon the modification of approximately eleven arginine residues per inhibitor molecule. This suggests that (the) arginine residue(s) in AT III are involved in the binding of heparin in addition to the known role of Arg-393 at the thrombin-recognition site [Rosenberg & Damus (1973) J. Biol. Chem. 248, 6490-6505; Jörnvall, Fish & Björk (1979) FEBS Lett. 106, 358-362]. 相似文献