Primary structure of human fibrinogen and fibrin. Structural studies on NH2-terminal part of B beta chain.

The cyanogen bromide fragment, N-DSK, containing the NH2-terminal portions of the three chains of fibrinogen, was found to exist in dimeric and polymeric forms. These different forms gave rise to identical chain fragments on reduction and alkylation. The B beta chain of N-DSK from fibrinogen and the beta chain of N-DSK from fibrin were isolated and characterized. The B beta chain fragment has a blocked NH2-terminal residue, and fibrinopeptide B is released on digestion with thrombin. The beta chain fragment has glycine as NH2-terminal residue. The molecular weight of the B beta chain fragment is 12200 as determined by ultracentrifugal analysis. Gel electrophoresis in sodium dodecyl sulphate gave the molecular weights of 14000 and 13000 for the B beta chain and beta chain fragments, respectively. The NH2-terminal B beta chain fragment consists of 118 amino acid residues and the beta chain fragment of 104 residues. The amino acid sequence of beta chain fragment is identical to B beta chain fragment except for the fibrinopeptide B portion. The isolation of a B beta-related fragment (B beta +), with a molecular weight of 30000, is also reported. The presence of B beta + was explained on the basis of incomplete cleavage at the Met-118 residue during treatment with cyanogen bromide. Some functional aspects of the B beta chain fragment are discussed.     

Iodination of Ricin D

Approximately five tyrosine residues of ricin D were iodinated preferentially under appropriate conditions probably forming diiodotyrosine. Iodination of this toxin carried out in 0.1 m phosphate buffer at pH 7.0 and 0°C for 60 min with a 20 fold molar excess of iodine per mole of protein, yielded a main component which appeared as a single band on polyacrylamide gel disc electrophoresis. Analysis of protein-bound radioactivity and the content of diiodotyrosine of ¹⁸¹I-labeled ricin D revealed that two tyrosine residues in the isoleucyl chain and three in the alanyl chain were substituted. The toxicity of iodinated ricin D decreased to one hundredth of that of native protein, However, the hemagglutinating activity of this protein was not affected by the iodination reaction.     

The effect of thrombomodulin on the cleavage of fibrinogen and fibrinogen fragments by thrombin

Thrombomodulin acts as a linear competitive inhibitor of thrombin with respect to the substrate fibrinogen. In the present study the effect of thrombomodulin on the activity of thrombin with fragments of the A alpha and B beta chain of fibrinogen has been examined. The cleavage of fibrinopeptide A from the N-terminal disulphide knot, fragment 1-44 and fragment 1-51 of the A alpha chain was inhibited by thrombomodulin. The average value for the inhibition constant obtained with these substrates was 0.83 +/- 0.09 nM, which was in good agreement with the values obtained previously for the inhibition of thrombin by thrombomodulin with native fibrinogen as the substrate [Hofsteenge, J., Taguchi, H. & Stone, S. R. (1986) Biochem. J. 237, 243-251]. In contrast, the cleavage of fibrinopeptide A from fragment 1-23 and fragment 1-29 of the A alpha chain was not affected by thrombomodulin. Although the cleavage of the B beta chain in the intact fibrinogen molecule was inhibited by thrombomodulin [Hofsteenge, J., Taguchi, H. & Stone, S. R. (1986) Biochem. J. 237, 243-251], the release of fibrinopeptide B from the N-terminal disulphide knot and the N-terminal 118-residue fragment of the B beta chain was not inhibited by thrombomodulin. In addition, we determined the second-order rate constants of cleavage of these substrates using human thrombin. Fragments of the A alpha chain whose cleavage was inhibited by thrombomodulin were found to have values for kcat/Km that were within one order of magnitude of that for the native fibrinogen, whereas those for A alpha chain fragments whose cleavage was not inhibited by thrombomodulin were found to be more than two orders of magnitudes lower. From these results we conclude that only a relatively small portion of the A alpha chain of the fibrinogen molecule is responsible for the specific binding to thrombin that is affected by thrombomodulin. Moreover, residues 30-44 of the A alpha chain play an important role in this thrombin-fibrinogen interaction.     

Determination of the sidedness of the C-terminal region of the gastric H,K-ATPase alpha subunit.

It cannot be predicted from hydropathy analysis whether the C-terminal end of the alpha subunit of the gastric H,K-ATPase is cytoplasmic or extracytoplasmic. The sideness of the C-terminal amino acids was determined by taking advantage of the two C-terminal tyrosines in the primary sequence of the enzyme. Intact, cytoplasmic side out vesicles derived from hog gastric mucosa or detergent solubilized vesicles were iodinated by the lactoperoxidase method and then the C-terminal amino acids hydrolyzed by carboxypeptidase Y. The alpha and beta subunits were separated by SDS gel electrophoresis. The level of iodination of the alpha subunit following solubilization was about three fold greater than when intact vesicles were iodinated, and the beta subunit was iodinated only when solubilized enzyme was used. Carboxypeptidase Y removed 28 +/- 4% of the radioactivity from the alpha subunit iodinated in intact vesicles. These data are consistent with a cytoplasmic location of the C-terminal amino acids of the alpha subunit and with a mostly extracytoplasmic location of the amino acids of the beta subunit.     

Complexes of the N-terminal disulfide branch point of fibrin with fibrinogen

Investigation of fibrin N-terminal disulphide knot (N-DSK) binding with fibrinogen (F) showed, that the F-N-DSK-complex represents growing polymer structure which is soluble at early polymerization stage and forms a solid phase during the further growth. This complex is characterized by constant stoichiometry expressed by formula F (N-DSK)2. A model of the complex structure as a regular copolymer of fibrinogen and N-DSK is proposed, in which neighbouring fibrinogen molecules are clamped with two N-DSK molecules. Such copolymer was never described. Since its formation is caused by specific D-E interdomain binding, it may be considered as a peculiar analogue of fibrin polymer.     

Role of histone tyrosines in nucleosome formation and histone-histone interaction

We have studied the functional properties of iodinated histones. Isolated, denatured histones were iodinated at trace levels and then renatured together with carrier histones and high molecular weight DNA to form nucleohistone. Nucleosomes were prepared from the reconstitute using micrococcal nuclease, and the relative representations of the individual iodinated tyrosines of the histones in the reconstituted nucleosomes were determined. Our principal findings are 1) that denatured histones can be iodinated at any tyrosine without interfering in subsequent nucleosome reconstitution and 2) that the resulting reconstituted nucleosomes nevertheless possess histone cores of altered stability, being either more or less stable depending on the particular tyrosine which is iodinated. We show that tyrosines 37, 40, and 42 of H2B are protected from iodination in intact core particles, as expected since these tyrosines lie within the H2B-H2A binding site. Yet iodination of these tyrosines in denatured H2B does not interfere with nucleosome assembly. However, the histone cores isolated from these reconstituted nucleosomes are of diminished stability as assayed by Sephadex column chromatography in 2 M salt. In contrast, iodination of tyrosines 83 and 121 of H2B, as well as iodination of the tyrosines of H2A, increases the stability of the histone octamer core. Iodination of H4 tyrosine 72 is without effect on histone octamer stability. Tyrosine iodination constitutes a profound amino acid alteration in the context of the absolute evolutionary conservation of most histone tyrosines. For example, all H2Bs sequenced to date, from fungi to mammals, possess tyrosines at positions 37, 40, and 42. Our results suggest that the immutability of these tyrosines reflects some sophisticated function of the nucleosome histone core beyond the assembly and mere maintenance of a compact structure.     

Iodination of αs1-casein and its effect on the calcium-induced aggregation reaction of the modified protein

The iodination of the tryosyl residues of α_s1-casein has been studied and a spectrophotometric procedure for differentiating monoiodotyrosine, diiodotyrosine and tyrosine is described. The calcium-induced aggregation behaviour of the iodinated caseins has been investigated. The initial stages of the reaction are shown to be dominated by electrostatic charge effects. The iodinated caseins behave as native α_s1-casein when the extent of conversion to diiodotyrosine is taken into account. The later stages of the reaction are found to be influenced by effects of the iodination not related to the change in charge.     

Effect of peptides--structural analogs of NH2-terminal sites of fibrin alpha- and beta-chains--on specific binding of the NH2-terminal disulfide bond of fibrin with fibrinogen

Peptides Gly-Pro-Arg-Pro and Gly-His-Arg-Pro (fibrin alpha- and beta-chain NH2-terminal analogs, respectively) are studied for their effect on fibrinogen (F) and fibrin NH2-terminal disulphide knot (N-DSK) specific binding. Both peptides are found to inhibit the formation of soluble and insoluble F-N-DSK-complexes through inhibition of the interdomain D-E-binding. Gly-Pro-Arg-Pro is much more potent inhibitor than Gly-His-Arg-Pro. Lowering the insoluble F-N-DSK-copolymer quantity by concentration-dependent way these peptides do not change its composition described by the formula [F(N-DSK)2]n. Invariability of fibrinogen and N-DSK copolymer structure is asserted. In this structure neighbouring fibrinogen molecules are bound by two N-DSK molecules via the D1-E1 and D2-E2 binding sites.     

Catalytic hydrogenolysis of poly-iodinated recombinant human insulin-like growth factor II (IGF-II): a potentially useful method for the tritiation of IGF-II.

A method has been developed to prepare, purify, and fully characterize poly-iodinated insulin-like growth factor II (IGF-II) which can then be catalytically deiodinated to produce IGF-II with its native disulfide bonded structure. This method can potentially be adapted to prepare tritiated IGF-II with the use of tritium gas in the hydrogenolysis step. IGF-II was iodinated at all three tyrosines using lactoperoxidase with a three-fold excess of sodium iodide. The iodinated products were purified using reversed-phase HPLC and characterized by peptide mapping. The tyrosine-containing peptides generated by pepsin digestion were characterized by amino acid sequence analysis. Mono- and di-iodinated phenylthiohydantoin tyrosine derivatives were synthesized and used to identify the iodination state of the modified tyrosine residues in the sequence analysis. Purified poly-iodinated IGF-II was deiodinated by hydrogenolysis, over a prereduced palladium (II) oxide catalyst to form IGF-II with its native disulfide bonds intact, as shown by peptide mapping.     

Relations of iodination, hormonosynthesis and proteolytic cleavage in human thyroglobulin

Normally iodinated thyroglobulin (Tg) contains low molecular weight hormone-rich peptides associated to the bulk of the molecule by disulfide bridges. It is shown, with the assistance of in vitro iodination experiments using different iodine concentrations and various incubation times, that the proteolytic cleavage giving rise to the 26 K hormonopeptide in human Tg is part of a coupling reaction rather than iodination. This cleavage may be a preliminary event related to a facilitation in the release of thyroid hormones.     

Conformation of the carboxy-terminal region of the A alpha chain of fibrinogen as elucidated by immunochemical analyses

The conformation of the carboxy-terminal aspects of the A alpha chain of human fibrinogen has been assessed by immunochemically characterizing the A alpha 239-476 and A alpha 518-584 regions of the molecule. Two peptides, corresponding to these regions, were isolated from cyanogen bromide digests of the A alpha chain by molecular exclusion and high-performance liquid chromatography. Each peptide reacted with antibodies elicited by immunization with the A alpha chain and intact fibrinogen. A alpha 239-476 appears to be a relatively immunodominant region of the molecule. Competitive inhibition analyses confirmed the accessibility of these regions to antibody in native fibrinogen. Each peptide, however, contained one or more epitopes, which was occult in the native molecule. These occult epitopes were expressed by the intact A alpha chain and became accessible when fibrinogen was cleaved with plasmin. With plasmic degradation the epitopes expressed by fibrinogen and contained within these two peptide regions became significantly more reactive with antibody. This change occurred in concert with release of the A alpha 518-584 region from the core of the molecule but did not require the generation of free A alpha 239-476. Ultimately the epitopes within both regions were shed from the plasmin-resistant core of fibrinogen. Peptide epitopes were expressed in a similar manner by prolonged plasmic degradation of fibrinogen and fibrin with alpha chain cross-linking. These results are generally consistent with models depicting the carboxy-terminal aspects of the A alpha chain as being surface-oriented but suggest a systematic ordering of structure when these regions are integrated into the native molecule. Plasmic cleavage significantly relaxes the conformational restraints on the organization within this region.     

Localization of a fibrin polymerization site

The formation of a fibrin clot is initiated after the proteolytic cleavage of fibrinogen by thrombin. The enzyme removes fibrinopeptides A and B and generates fibrin monomer which spontaneously polymerizes. Polymerization appears to occur though the interaction of complementary binding sites on the NH2-terminal and COOH-terminal (Fragment D) regions of the molecule. A peptide has been isolated from the gamma chain remnant of fibrinogen Fragment D1 which has the ability to bind to the NH2-terminal region of fibrinogen as well as to inhibit fibrin monomer polymerization. The peptide reduces the maximum rate and extent of the polymerization of thrombin or batroxobin fibrin monomer and increases the lag time. The D1 peptide does not interact with disulfide knot, fibrinogen, or Fragment D1, but it binds to thrombin-treated disulfide knot with a Kd of 1.45 X 10(-6) M at approximately two binding sites per molecule of disulfide knot. Fibrin monomer formed either by thrombin or batroxobin binds approximately two molecules of D1 peptide per molecule of fibrin monomer, indicating that the complementary site is revealed by the loss of fibrinopeptide A. The NH2-terminal sequence (Thr-Arg-Trp) and COOH-terminal sequence (Ala-Gly-Asp-Val) of the D1 peptide were determined. Therefore the gamma 373-410 region of fibrinogen contains a polymerization site which is complementary to the thrombin-activated site on the NH2-terminal region of fibrinogen.     

Mechanism of functioning of specific sites of interdomain D-E binding of fibrin molecules: interaction of fibrinogen with the N-terminal disulfide node of fibrin

The intermolecular noncovalent binding of complementary fibrin polymerization sites localized in fibrin domains D and E was investigated in the model system. In this system fibrinogen molecules represent the active D domains and the N-terminal disulphide knot of fibrin (N-DSK) represents the active E domain. Quantitative definition of insoluble fibrinogen and N-DSK copolymer and light scattering data of their mixtures before the appearance of visible precipitate show that complexing of these structures decreases with an increase of the temperature and ionic strength. The character of this dependence permits certain conclusions to be made on the functioning mechanism for two types of the D-E binding sites. These conclusions are based on an idea of their different affinity. The interdomain binding is primarily realized by D1-E1 sites which are characterized by a high affinity and work mainly on the basis of electrostatic forces. This binding directs the D2-E2 binding which is characterized by lower affinity and which determines the final degree of fibrinogen and N-DSK complexing. These sites function mainly by the H-binding.     

Plasminogen activation by tissue activator is accelerated in the presence of fibrin(ogen) cyanogen bromide fragment FCB-2

Fibrin, in contrast to fibrinogen, strongly accelerates the plasminogen activation by extrinsic activator (tissue-type plasminogen activator, t-PA). However, when fibrin and fibrinogen are digested with cyanogen bromide, both digests potentiate the t-PA-mediated plasminogen activation equally well. In this report, evidence is presented that this potentiating activity resides in CNBr fragment FCB-2 (= Ho1-DSK) and that a polymeric structure such as fibrin is not a prerequisite for the potentiation.     

Conservation of human fibrinogen conformation after cleavage of the B beta chain NH2 terminus

Human fibrinogen exposed to protease III from Crotalus atrox venom is cleaved near the NH2 terminus of the B beta chain yielding a species of Mr 325,000 (Fg325) with impaired thrombin clottability. The derivative was compared with intact fibrinogen in a number of ways to determine whether the functional defect resulted from a conformational change or from the loss of a polymerization site. NH2-terminal amino acid sequencing of isolated A alpha, B beta, and gamma chains showed that Fg325 contained intact A alpha and gamma chains, but differed from fibrinogen by the absence of the first 42 residues of the B beta chain. Fibrinopeptide A was present and was cleaved at the same rate in both fibrinogen and Fg325. The rate and extent of A alpha and gamma cross-linking by factor XIIIa was also indistinguishable. In contrast, the thrombin-catalyzed coagulation of Fg325 was 46% less in extent and 180-fold slower than observed for intact fibrinogen. A conformational comparison of Fg325 and fibrinogen was made using immunochemical and spectroscopic approaches. Antisera specific for different regions of the fibrinogen molecule were used to characterize the epitopes in Fg325. The only significant differences were found in the NH2-terminal region of the B beta chain, probed with antiserum to B beta 1-118. The conformational similarity of Fg325 and fibrinogen was confirmed by the identity of both near and far UV CD spectra of the two proteins. Structural, functional, and immunochemical results imply that cleavage of 42 NH2-terminal residues from the B beta chain is not accompanied by a measurable conformational change. The residues of this B beta chain segment, which are evidently located on the surface of the molecule, in conjunction with the NH2-terminal part of the A alpha chain appear to play an important role in the expression of a fibrin polymerization site.     

Free diiodotyrosine effects on protein iodination and thyroid hormone synthesis catalyzed by thyroid peroxidase.

Free diiosotyrosine exerts two opposite effects on the reactions catalyzed by thyroid peroxidase, thyroglobulin iodination and thyroid hormone formation. 1. Inhibition of thyroglobulin iodination catalyzed by thyroid peroxidase was observed when free diiodotyrosine concentration was higher than 5 muM. This inhibition was competitive, suggesting that free diiodotyrosine interacts with the substrate site(s) of thyroid peroxidase. Free diiodotyrosine also competively inhibited iodide peroxidation to I2. 2. Free diiodotyrosine, when incubated with thyroid peroxidase in the absence of iodide was recovered unmodified; in the presence of iodide an exchange reaction was observed between the iodine atoms present in the diiodotyrosine molecule and iodide present in the medium. Using 14C-labelled diiodotyrosine, 14C-labelled non-iodinated products were also observed, showing that deiodination occurred as a minor degradation pathway. However, no monoiodo[14C]tyrosine or E114C]tyrosine were observed. Exchange reaction between free diiototyrosine and iodide is therefore direct and does not imply deiodination-iodination intermediary steps. Thyroglobulin inhibits diiodotyrosine-iodide exchange and vice versa, again suggesting competition for both reactions. These results support, by a different experimental approach, the two-site model for peroxidase previously described by us in this journal. 3. Free diiodotyrosine when present at a very low concentration, 0.05 muM, exerts a stimulatory effect on throid hormones synthesis. The relationship between diiodotyrosine concentration and thyroid hormone synthesis give an S-shaped curve, suggesting that free diiodotyrosine acts as a regulatory ligand for thyroid peroxidase. Evidence is also presented that free diiodotyrosine is not incorporated into thyroid hormones. Therefore, thyroid peroxidase catalyzes only intra-molecular coupling between iodotyrosine hormonogenic residues. 4. Finally, although no direct proof exists that these free diiodotyrosine effects upon thyroglobulin iodination and thyroid hormone synthesis are physiologically significant, such a possibility deserves further investigation.     

Carboxyl-terminal residues of mammalian fibrinogen and fibrin

The carboxyl-terminal residues of mammalian fibrinogens of six different species and the chain peptides, alpha(A), beta(B) and gamma, isolated from these fibrinogens were determined by hydrazinolysis, digestion with carboxypeptidases and selective tritium labelling. The C-terminal ends of bovine fibrinogen and fibrin were identified as proline and valine, in the molar ratio of approximately 1:2. Proline was identified as the C-terminus of the alpha(A)-chain, and C-terminal valine was found on both the beta(B)- and gamma-chains. On hydrazinolysis after selective tritium labelling of fibrinogen, radioactive C-terminal valine was also identified. The same C-terminal ends as those of bovine fibrinogen were found on the corresponding chain peptides isolated from sheep fibrinogen. The C-terminal residues of all the chain peptides of human and horse fibrinogens, however, were valine. In hog and dog fibrinogens, proline was identified at the C-termini of the alpha(A)-chains, and C-terminal valine and isoleucine were found on the beta(B)- and gamma-chains, respectively. Thus, the C-terminal amino acid residues of the fibrinogens of all mammalian species tested were very similar. It should be noted that hydrophobic amino acids, like isoleucine, valine and proline, are mainly located in the C-terminal ends of all three chain peptides in the fibrinogen molecule.     

Identification of tyrosine residues that are susceptible to lactoperoxidase-catalyzed iodination on the surface of Escherichia coli 30S ribosomal subunit

The detailed surface topography of the Escherichia coli 30S ribosomal subunit has been investigated, with iodination catalyzed by immobilized lactoperoxidase as the surface probe. Under mild conditions, only proteins S3, S7, S9, S18, and S21 were iodinated to a significant and reproducible extent. These proteins were isolated from the iodinated subunits, and in each case, the individual tyrosine residues that had reacted were identified by standard protein sequencing techniques. The targets of iodination that could be positively established were as follows: in protein S3 (232 amino acids), the tyrosines at positions 167 and 192; in S7 (153 amino acids), tyrosines 84 and 152; in S9 (128 amino acids), tyrosine 89; in S18 (74 amino acids), tyrosine 3 (tentative); in S21 (70 amino acids), tyrosines 37 and 70. The results represent part of a broader program to investigate ribosomal topography at the amino acid-nucleotide level.     

The effect of adjacent residues on the reactivity of tyrosyl with iodine

The effect of the adjacent amino acid side chain groups on the iodination rate of the tyrosine was studied. The model peptides used were Gly-Tyr-Gly, Leu-Tyr-Leu, Glu-Tyr-Glu, and Lys-Tyr-Lys, in which the tyrosine is sandwiched between two hydrophobic, two negatively charged, or two positively charged residues. The results show only minor differences in the iodination rate of tyrosine in these four peptides. These differences are very small in comparison with those previously observed between the tyrosines of kappa Bence-Jones proteins.