Biomedical subjects

S R Stone

Publications and source records attributed to S R Stone.

At least 73 records · Page 4Linked to original sources

PubMed1992

Interaction of the N-terminal region of hirudin with the active-site cleft of thrombin.

Site-specific substitutions of the first five amino acids of the thrombin inhibitor hirudin have been made and the effects of these substitutions on the kinetics of formation of the thrombin-hirudin complex evaluated. The effects of different substitutions of Val1 indicate that nonpolar interactions play a major role in the binding of this residue. In the second position (Val2), polar amino acids were better accommodated than in the first. The mutant with arginine in the second position bound particularly well to thrombin; its dissociation constant was 9-fold lower than that of wild-type recombinant hirudin. Comparison of the effects of single and double mutations involving Val1 and Val2 indicates that there was no cooperativity in the binding of these two residues. Elimination of the hydrophobic interactions made by the aromatic ring of Tyr3 of hirudin resulted in a large loss of binding energy (12.7 kJ mol-1). Replacement of Thr4 of hirudin by serine and alanine suggested that both the gamma-methyl and the hydroxyl group of the threonine were important in the stabilization of the thrombin-hirudin complex. Replacement of Asp5 of hirudin by alanine and glutamate caused about the same loss in binding energy (5 kJ mol-1). The effects of site-specific substitutions are discussed in terms of the crystal structure of the thrombin-hirudin complex. Molecular modeling provided plausible explanations for many of the observed effects. For instance, such studies suggested that the improved binding of the mutant with arginine in the second position could be due to an interaction of the arginine with the primary specificity pocket.

Amino Acid Sequence↗

PubMed1992

The interaction of thrombin with fibrinogen. A structural basis for its specificity.

The structure of the ternary complex of human alpha-thrombin with a covalently bound analogue of fibrinopeptide A and a C-terminal hirudin peptide has been determined by X-ray diffraction methods at 0.25 nm resolution. Fibrinopeptide A folds in a compact manner, bringing together hydrophobic residues that slot into the apolar binding site of human alpha-thrombin. Fibrinogen residue Phe8 occupies the aryl-binding site of thrombin, adjacent to fibrinogen residues Leu9 and Val15 in the S2 subsite. The species diversity of fibrinopeptide A is analysed with respect to its conformation and its interaction with thrombin. The non-covalently attached peptide fragment hirudin(54-65) exhibits an identical conformation to that observed in the hirudin-thrombin complex. The occupancy of the secondary fibrinogen-recognition exosite by this peptide imposes restrictions on the manner of fibrinogen binding. The surface topology of the thrombin molecule indicates positions P1'-P3', differ from those of the canonical serine-proteinase inhibitors, suggesting a mechanical model for the switching of thrombin activity from fibrinogen cleavage to protein-C activation on thrombomodulin complex formation. The multiple interactions between thrombin and fibrinogen provide an explanation for the narrow specificity of thrombin. Structural grounds can be put forward for certain congenital clotting disorders.

Amino Acid Sequence↗

PubMed1992

Peptidyldiazomethanes. A novel mechanism of interaction with prolyl endopeptidase.

Peptidyldiazomethanes with proline in the P1 position were found to be competitive slow-binding inhibitors of prolyl endopeptidase. Progress-curve experiments monitoring the increase in the degree of inhibition with time indicated that the kinetic mechanism involved an initial complex that isomerized to form a tighter complex. Reversibility of the inhibited complex was demonstrated by monitoring the regain of enzyme activity after removal of free inhibitor and dilution into an assay containing competing substrate. The kinetics of the reversal of inhibition indicated a more complicated inhibitory mechanism involving more than one pathway for reversal of the tight complex. A slow-binding mechanism of inhibition has not been previously observed with peptidyldiazomethanes. Incorporation of [3H]Ac-Ala-Ala-Pro-diazomethane into prolyl endopeptidase was observed after denaturation of the inhibited complex. The peptide labelled with [3H]Ac-Ala-Ala-Pro-diazomethane was isolated and found to contain the active-site serine residue.

Amino Acid Sequence↗

PubMed1992

Characterization of the heparin-binding site of glia-derived nexin/protease nexin-1.

The interaction of heparin with glia-derived nexin (GDN) has been characterized and compared to that observed between heparin and antithrombin III (ATIII). Heparin was fractionated according to its affinity for immobilized GDN, and the ability of various fractions to accelerate the inhibition rate of thrombin by either GDN or ATIII was examined. Fractions with different affinities for GDN accelerated the thrombin-GDN reaction to a similar extent; heparin with a high affinity for immobilized GDN stimulated the reaction only about 30% more than the fraction that did not bind to immobilized GDN. Slightly greater differences were observed for the effect of these fractions on the thrombin-ATIII reaction; heparin that did not bind to the GDN affinity column was about 60% more effective than heparin with a high affinity for GDN in accelerating the inhibition of thrombin by ATIII. The CNBr fragment of GDN between residues 63 and 144 was able to reduce the heparin-accelerated rate of inhibition of thrombin by GDN indicating that this region of GDN was able to bind the heparin molecules responsible for the acceleration. Shorter synthetic peptides within this sequence did not significantly reduce the rate, suggesting that the heparin-binding activity of fragment 63-144 depends on a specific conformation of the polypeptide chain. Fragment 63-144 was less effective in decreasing the heparin-accelerated rate of inhibition of thrombin by ATIII. The results are discussed in terms of the heparin species that are responsible for the acceleration of the GDN- and ATIII-thrombin reactions and the heparin-binding sites of GDN and ATIII.

Amino Acid Sequence↗

PubMed1992

Primary structure and function of novel O-glycosylated hirudins from the leech Hirudinaria manillensis.

Hirudin from the leech Hirudo medicinalis is a most powerful anticoagulant, and many isoforms have been described. In the present work, the primary structure of two hirudins from the leech Hirudinaria manillensis has been elucidated. The antithrombotic activity is similar to that of H. medicinalis hirudins although the sequence identity is below 60%. Surprisingly, the hirudins were found to be glycosylated at one site. Sugar analysis after methanolysis yielded fucose, galactose, and N-acetylgalactosamine. These results combined with data from matrix-assisted laser desorption ionization mass spectrometry, plasma desorption mass spectrometry, capillary zone electrophoresis, and lectin-binding tests indicate that the sequence is Fuc-Gal beta 1-3GalNAc-(O-threonine). This structure shows an interesting similarity to human blood group H determinants.

Amino Acid Sequence↗

PubMed1992

pH dependence of the interaction of hirudin with thrombin.

The kinetics of the inhibition of human alpha-thrombin by recombinant hirudin have been studied over the pH range from 6 to 10. The association rate constant for hirudin did not vary significantly over this pH range. The dissociation constant of hirudin depended on the ionization state of groups with pKa values of about 7.1, 8.4, and 9.2. Optimal binding of hirudin to thrombin occurred when the groups with pKa values of 8.4 and 9.0 were protonated and the other group with a pKa of 7.1 was deprotonated. The pH kinetics of genetically engineered forms of hirudin were examined in an attempt to assign these pKa values to particular groups. By using this approach, it was possible to show that protonation His51 and ionization of acidic residues in the C-terminal region of hirudin were not responsible for the observed pKa values. In contrast, the pKa value of 8.4 was not observed when a form of hirudin with an acetylated alpha-amino group was examined, and, thus, this pKa value was assigned to the alpha-amino group of hirudin. The requirement for this group to be protonated for optimal binding to thrombin is discussed in terms of the crystal structure of the thrombin-hirudin complex. Examination of this structure allowed the other pKa values of 7.1 and 9.2 to be tentatively attributed to His57 and the alpha-amino group of Ile16 of thrombin.

Hirudins↗

PubMed1992

Electrostatic interactions in the association of proteins: an analysis of the thrombin-hirudin complex.

The role of electrostatic interactions in stabilization of the thrombin-hirudin complex has been investigated by means of two macroscopic approaches: the modified Tanford-Kirkwood model and the finite-difference method for numerical solution of the Poisson-Boltzmann equations. The electrostatic potentials around the thrombin and hirudin molecules were asymmetric and complementary, and it is suggested that these fields influence the initial orientation in the process of the complex formation. The change of the electrostatic binding energy due to mutation of acidic residues in hirudin has been calculated and compared with experimentally determined changes in binding energy. In general, the change in electrostatic binding energy for a particular mutation calculated by the modified Tanford-Kirkwood approach agreed well with the experimentally observed change. The finite-difference approach tended to overestimate changes in binding energy when the mutated residues were involved in short-range electrostatic interactions. Decreases in binding energy caused by mutations of amino acids that do not make any direct ionic interactions (e.g., Glu 61 and Glu 62 of hirudin) can be explained in terms of the interaction of these charges with the positive electrostatic potential of thrombin. Differences between the calculated and observed changes in binding energy are discussed in terms of the crystal structure of the thrombin-hirudin complex.

Amino Acid Sequence↗

PubMed1992

Thrombin causes neurite retraction in neuronal cells through activation of cell surface receptors.

The mechanism by which thrombin induces neurite retraction was studied in NB2a mouse neuroblastoma cells. The rapid effect of thrombin (completed within minutes) appears to involve an interaction between its anion-binding exosite and the thrombin receptor. Structural alterations of this site increase the EC50 for thrombin-mediated retraction, and a hirudin C-terminal peptide that blocks this site inhibits the response. The thrombin effect was mimicked by a 14 amino acid peptide starting with Ser-42, at the proposed cleavage site of the human thrombin receptor. The protein kinase inhibitors staurosporine and H-7 blocked thrombin-induced retraction. It is therefore proposed that thrombin-mediated neurite retraction is caused by cleavage-induced activation of the thrombin receptor and involves stimulation of a protein kinase(s).

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1991

Studies on the interaction of ribonuclease inhibitor with pancreatic ribonuclease involving differential labeling of cysteinyl residues.

Ribonuclease inhibitor (RI) is a protein that forms a very tight complex with ribonucleases (RNases) of the pancreatic type. RI contains 30 thiol groups, some of which are important for the enzyme-inhibitor interaction. To examine which thiols are affected by the binding of RNase, differential labeling experiments were performed. Reaction of porcine RI with the cysteine-specific labeling reagent 4-N,N-dimethylaminoazobenzene-4'-iodoacetamido-2'-sulfonic acid resulted in labeling of an average of 7.4 of the 30 cysteinyl residues. Binding of bovine pancreatic RNase A caused a 3.2-fold reduction in the extent of modification. Peptide mapping showed that in free RI, Cys-57, -371, and -404 were labeled to the greatest extent (yield, 0.4-0.6 mol/mol). RNase A did not protect Cys-57 against modification, whereas the labeling of Cys-371 and -404 was reduced by more than 90%. A second group of residues was labeled to a lesser extent in free RI (yield, 0.04-0.2 mol/mol). Within this group 11 residues were protected by RNase A by more than 90%, 2 were not affected at all, and 7 were protected between 10 and 90%. Seven cysteinyl residues in RI that were protected in the RI.RNase A complex were no longer protected in the RI.S-protein complex. These residues were mainly present in the N-terminal region of RI. However, when the S-peptide was included to yield the RI.RNase S complex, the same pattern of labeling was obtained as with the RI.RNase A complex. Addition of the S-peptide alone had no effect on the labeling. The implications of these observations with respect to RNase binding areas of RI are discussed in relation to the results obtained from the analysis of active RI molecules that contain deletions.

Amino Acid Sequence↗

PubMed1991

Mechanistic studies on thrombin catalysis.

The kinetic mechanism of the cleavage of four p-nitroanilide (pNA) substrates by human alpha-thrombin has been investigated by using a number of steady-state kinetic techniques. Solvent isotope and viscosity effects were used to determine the stickiness of the substrates at the pH optimum of the reaction; a sticky substrate is defined as one that undergoes catalysis faster than it dissociates from the Michaelis complex. Whereas benzoyl-Arg-pNA could be classified as a nonsticky substrate, D-Phe-pipecolyl-Arg-pNA was very sticky. The other two substrates (tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA) were slightly sticky. The pH profiles of kcat/Km were bell-shaped for all substrates. The pKa values determined from the pH dependence of kcat/Km for benzoyl-Arg-pNA were about 7.5 and 9.1. Similar pKa values were determined from the pH profiles of kcat/Km for tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA and for the binding of the competitive inhibitor N alpha-dansyl-L-arginine-4-methylpiperidine amide. The groups responsible for the observed pKa values were proposed to be His57 and the alpha-amino group of Ile16. The temperature dependence of the pKa values was consistent with this assignment. The pKa values of 6.7 and 8.6 observed in the pH profile of kcat/Km for D-Phe-pipecolyl-Arg-pNA were displaced to lower values than those observed for the other substrates. The displacement of the acidic pKa value could be attributed to the stickiness of this substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

Role of interactions involving C-terminal nonpolar residues of hirudin in the formation of the thrombin-hirudin complex.

The role of interactions involving C-terminal nonpolar residues of hirudin in the formation of the thrombin-hirudin complex has been investigated by site-directed mutagenesis. The residues Phe56, Pro60, and Tyr63 of hirudin were replaced by a number of different amino acids, and the kinetics of the inhibition of thrombin by the mutant proteins were determined. Phe56 could be replaced by aromatic amino acids without significant loss in binding energy. While substitution of Phe56 by alanine decreased the binding energy (delta G degrees b by only 1.9 kJ mol-1, replacement of this residue by amino acids with branched side chains caused larger decreases in delta G degrees b. For example, the mutant Phe56----Val displayed a decrease in delta G degrees b of 10.5 kJ mol-1. Substitution of Pro60 by alanine or glycine resulted in a decrease in delta G degrees b of about 6 kJ mol-1. Tyr63 could be replaced by phenylalanine without any loss in binding energy, and replacement of this residue by alanine caused a decrease of 2.2 kJ mol-1 in delta G degrees b. Substitution of Tyr63 by residues with branched side chains resulted in smaller decreases in delta G degrees b than those seen with the corresponding substitutions of Phe56; for example, the mutant Tyr63----Val showed a decrease in binding energy of 5.1 kJ mol-1. The effects of the mutations are discussed in terms of the crystal structure of the thrombin-hirudin complex.

Amino Acid Sequence↗

PubMed1991

Specificity determinants for tyrosine protein kinase. A study with recombinant hirudin mutants.

Hirudin, the powerful anticoagulant agent of leech (Hirudo medicinalis) saliva, was readily phosphorylated by two spleen tyrosine protein kinases (TPK-IIB and TPK-III) at Tyr63 with Km values of 238 microM and 74 microM, respectively. The synthetic tridecapeptide DGDFEEIPEEYLQ, corresponding to the hirudin 53-65 C-terminal fragment, was phosphorylated even more efficiently than hirudin itself. Four hirudin mutants, in which one or more of the glutamic acids at positions 57, 58, 61 and 62 have been replaced by glutamines, were poorer substrates than hirudin. The mutant in which all four glutamates were substituted, [Gln57,58,61,62]hirudin, was virtually not phosphorylated by either TPK-IIB and TPK-III. Substitution of Glu57 and Glu58 was less deleterious than substitution of the two glutamic acids adjacent to Tyr63: [Gln61,62]hirudin exhibited a 20-fold lower phosphorylation efficiency with TPK-IIB. With TPK-III, however, the Km value of [Gln61,62]hirudin was slightly lower, while the Vmax decreased sixfold. The substitution of Glu62 alone was also more detrimental with TPK-IIB than with TPK-III. The behaviour of a third spleen TPK, named lyn TPK-I and belonging to the src family, was markedly different in that it did not phosphorylate hirudin but exhibited significant activity towards [Gln57,58,61,62]hirudin. Taken together, these data confirm and extend with a protein substrate the results obtained with short model peptides which indicated the stringent substrate requirements of TPK-IIB (and of TPK-III to a lesser extent) for N-terminal acidic residues. In contrast, such residues are deleterious with lyn TPK-I. These observations also support the concept that tyrosine protein kinases recognize specificity determinants situated in the vicinity of the target residue rather than requiring higher-order structural features.

Amino Acid Sequence↗

PubMed1991

Interaction of hirudin with the dysthrombins Quick I and II.

The interaction of hirudin with the dysfunctional enzymes thrombin Quick I and II has been investigated. Natural and recombinant hirudin caused nonlinear competitive inhibition of thrombin Quick I. The results were consistent with thrombin Quick I existing in two forms that have different affinities for hirudin. The affinities of these forms for natural hirudin were respectively 10(4)- and 10(6)-fold lower than that of alpha-thrombin. In contrast, truncated hirudin molecules lacking the C-terminal tail of the molecule caused linear inhibition of thrombin Quick I. These results indicate that different modes of interaction of the two forms of thrombin Quick I with the C-terminal tail of hirudin were the cause of the nonlinear inhibition. Comparison of the dissociation constants of thrombin Quick I with the truncated and full-length forms of hirudin suggested that the interactions that normally occur between the C-terminal tail of hirudin and thrombin were completely disrupted with the low-affinity form of thrombin Quick I. Thrombin Quick II displayed an affinity for natural hirudin that was 10(3)-fold lower than that observed with alpha-thrombin. In contrast, it bound a mutant hirudin with altered N-terminal amino acids only 16-fold less tightly. These results are discussed in terms of structural alterations in the active-site cleft in thrombin Quick II.

Amino Acid Sequence↗

PubMed1991

Inactivation of prolyl endopeptidase by a peptidylchloromethane. Kinetics of inactivation and identification of sites of modification.

The kinetics of inactivation of prolyl endopeptidase by acetyl-Ala-Ala-Pro-CH2Cl were studied by progress-curve methods in the presence of substrate. The kinetic mechanism was found to involve the formation of an initial complex between the enzyme and the chloromethane followed by an inactivation step. The substrate was shown to compete for the formation of the initial complex, indicating that binding at the active site was a prerequisite for inactivation. After reaction of the enzyme with [3H]acetyl-Ala-Ala-Pro-CH2Cl, it was possible to isolate five labelled peptides. Four of these peptides contained a cysteine residue as the site of modification, whereas the fifth peptide contained no cysteine and a histidine residue was identified as the site of modification. This residue (His-680) probably represents the active-site histidine of prolyl endopeptidase.

Alkylation↗

PubMed1991

Ionic interactions in the formation of the thrombin-hirudin complex.

Site-directed mutagenesis has been used to examine the importance of each of the acidic C-terminal residues of hirudin in the formation of its complex with alpha-thrombin. The contribution to binding energy of acidic residues in the 11 C-terminal amino acids varied from 2.3 to 5.9 kJ.mol-1. The differences between the contributions of individual residues were smaller than would be expected from the crystal structures of the thrombin-hirudin complex. In particular, the small effect (2.4 kJ.mol-1) for the replacement of Asp-55 was surprising in view of the two salt bridges made by this residue. The results of studies involving multiple mutations indicated that the additivity of the effects varied with the position of the mutation. Whereas the effect of mutations involving the glutamic acid residues at positions 61 and 62 were additive, non-additivity was observed with the glutamic acid residues at positions 57 and 58.

Amino Acid Sequence↗

PubMed1991

Basis for the reduced affinity of beta T- and gamma T-thrombin for hirudin.

Partial proteolysis of human alpha-thrombin by trypsin results in the formation of beta T-thrombin and gamma T-thrombin which have a reduced affinity for the inhibitor hirudin and the cell-surface cofactor thrombomodulin as well as reduced activity with fibrinogen. The basis of the reduction in affinity of these thrombin derivatives for hirudin has been investigated by examining their kinetics of interaction with a number of hirudin mutants differing in their C-terminal charge properties as well as with a truncated form of hirudin. The results indicate that the reduced affinity of beta T-thrombin for hirudin is most likely due to a decrease in the strength of nonionic interactions between thrombin and the C-terminal region of hirudin. No decrease in the strength of ionic interactions was observed with beta T-thrombin. In contrast, the reduced affinity of gamma T-thrombin was due to a decrease in the strength of both ionic and nonionic interactions. The N-terminal core region of hirudin, which interacts predominantly with the active-site cleft of thrombin, exhibited similar affinities for alpha-, beta T-, and gamma T-thrombin, indicating that thrombin-hirudin interactions within the active site are largely preserved in beta T- and gamma T-thrombin.

Amino Acid Sequence↗

PubMed1991

Structure of the 55-kDa regulatory subunit of protein phosphatase 2A: evidence for a neuronal-specific isoform.

The trimeric form of protein phosphatase 2A (PP2A1 or polycation-stimulated protein phosphatase H1) was purified to homogeneity from rabbit skeletal muscle. Preparative SDS-polyacrylamide gel electrophoresis was used to purify the individual subunits with relative molecular masses of 36, 55, and 65 kDa. Sequence analysis of five peptides from the 65-kDa regulatory subunit (PR65) suggested that it was identical with the PR65 subunit derived from the dimeric protein phosphatase 2A2. Amino acid sequences derived from the 55-kDa regulatory subunit (PR55) were used to clone human and rabbit cDNAs encoding this protein. The PR55 subunit was found to be encoded by two genes, termed alpha and beta. The open reading frames of the PR55 alpha and beta cDNAs spanned 1341 and 1329 nucleotides, respectively, and predicted proteins with a molecular mass of about 52 kDa that are 86% identical. Comparison of the human PR55 amino acid sequences with the data obtained from the rabbit skeletal muscle protein and a partial rabbit PR55 beta cDNA clone indicated a high degree of conservation. Analysis of the mRNA expression in human cell lines revealed that the PR55 alpha isoform was encoded by two transcripts of about 2.3 and 2.5 kb and a less abundant 4.4-kb mRNA. Whereas a PR55 beta transcript of about 2.3 kb was detected at high levels in the neuroblastoma derived cell line LA-N-1, the level of the mRNA was very low in the other human cell lines analyzed. Interestingly, the PR55 sequence showed limited homology to the catalytic domain (domains VI-IX) of the c-abl protein tyrosine kinase.

Amino Acid Sequence↗

PubMed1991

Purification and characterization of truncated ribonuclease inhibitor.

A recombinant pig ribonuclease inhibitor (delta r-RI) lacking 90 or 93 N-terminal amino acid residues was isolated from a preparation of recombinant inhibitor. The kinetic parameters for the inhibition of ribonuclease A by delta r-RI were determined and found to be only slightly altered in comparison with the full-length inhibitor. The deletion did, however, affect the surface properties of RI. The results are discussed in relation to those obtained by Lee & Vallee [(1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1879-1883].

Amino Acids↗