Activation of human plasminogen by equimolar levels of streptokinase.

Native Glu-human plasminogen (Mr approximately 92,000 with NH2-terminal glutamic acid) is able to combine directly with streptokinase in an equivalent molar ratio, to yield a stoichiometric complex. The plasminogen moiety in the complex then undergoes streptokinase-induced conformational changes. As a result of such, an active center develops in the plasminogen moiety of the complex. This proteolytically active complex then activates plasminogen in the complex to plasmin and at least two peptide bonds are cleaved in the process. The data presented in this paper reveal that initially an internal peptide bond of plasminogen (in the complex) is cleaved to yield a two-chain, disulfide-linked plasmin molecule. The heavy chain (Mr approximately 67,000 with NH2-terminal glutamic acid) of this plasmin molecule has an identical NH2-terminal amico acid as the native plasminogen. The light chain (Mr approximately 25,000 with NH2-terminal valine) of plasmin is known to be derived from the COOH-terminal portion of the parent plasminogen molecule. A second peptide is then cleaved from the NH2-terminal end of the heavy chain of plasmin producing a proteolytically modified heavy chain (Mr =60.000 with NH2-terminal lysine). This cleavage of the NH2-terminal peptide from the heavy chain of plasmin is shown to be mediated by the dissociated free plasmin present in the activation mixture. Plasmin in the streptokinase-plasmin complex is unable to cleave this NH2-terminal peptide. This same NH2-terminal peptide can also be cleaved from native Glu-plasminogen or from the Glu-plasminogen-streptokinase complex by free plasmin and not by a complex of streptokinase-plasmin. From these studies we conclude (a) in the streptokinase-plasminogen complex, the NH2-terminal peptide need not be released prior to the cleavage of the essential Arg-Val peptide bond which leads to the formation of a two chain plasmin molecule and (b) that this peptide is cleaved from the native plasminogen or from the heavy chain of the initially formed plasmin in the streptokinase complex by free plasmin and not by the plasmin associated with streptokinase. In agreement with this, plasmin associated with streptokinase was unable to cleave the NH2-terminal peptide from the isolated native heavy chain possessing glutamic acid as the NH2-terminal amino acid; whereas free plasmin readily cleaved this peptide from the same isolated Glu-heavy chain.     

Chemical structure of two fragments of human serum albumin and their location in the albumin molecule.

1. 'Inhibitor fragment' isolated from human serum albumin degraded by rabbit cathepsin D is composed of one peptide chain with two intrachain disulphide bonds. There are two kinds of inhibitor molecules having different N-terminal amino acids: one is threonine and the other glutamine. 2. Fragment F1, isolated from inhibitor degraded by trypsin, is composed of two chains linked by a disulphide bond. There are three kinds of fragment F1. All have one alpha chain in common, which has an intrachain disulphide bond. They differ by the nature of the chain, which is linked to the alpha chain by a disulphide bond. The epsilon chain is present in trace amounts. The two other chains, beta and gamma, differ by their C-terminal amino acid, which is respectively arginine and lysine. 3. Inhibitor is composed of the last 92 or 89 residues of the human albumin molecule and fragment F1 is composed of two parts of this C-terminal portion of the albumin molecule.     

Modification of specific lysine residues in E. coli methionyl-tRNA synthetase by crosslinking to E. coli formylmethionine tRNA

A protein affinity labeling derivative of E. coli tRNAfMet has been prepared which carries an average of one reactive side chain per molecule, distributed over four structural regions. Each side chain contains a disulfide bond capable of reaction with cysteine residues and an N-hydroxysuccinimide ester group capable of coupling to lysine epsilon-amino groups in proteins. Reaction of the modified tRNA with E. coli methionyl-tRNA synthetase leads to crosslinking only by reaction with lysine residues in the protein. Examination of the tRNA present in the crosslinked complex reveals that the enzyme is coupled to side chains attached to the 5' terminal nucleotide, the dihydrouridine loop, the anticodon and the CCA sequence. Digestion of the crosslinked enzyme with trypsin followed by peptide mapping reveals that the major crosslinking reactions occur at four specific lysine residues, with minor reaction at two additional sites. Native methionyl-tRNA synthetase contains 90 lysine residues, 45 in unique sequences of the dimeric alpha 2 enzyme. Crosslinking of the protein to different regions in tRNAfMet thus occurs with the high degree of selectivity necessary for use in determining the peptide sequences which are near specific nucleotide sequences of tRNA bound to the protein.     

Thrombin specificity. Selective cleavage of antibody light chains at the joints of variable with joining regions and joining with constant regions

Selective cleavage of polypeptides by alpha-thrombin can be reasonably predicted [Chang, J.Y. (1985) Eur. J. Biochem. 151,217-224]. This knowledge was applied to the selective cleavage of antibody light chains with the aim of producing intact fragments of both variable region and constant region. (a) Mouse kappa light chains 10K26 and 10K44 from anti-(azobenzene arsonate) antibodies contain 20 Arg/Lys-Xaa bonds. Only two of them, one ProArg-Thr bond located at the joint of the variable region with the joining peptide and one ValLys-Ser bond located near the carboxyl-terminal end of the constant region, were selectively cleaved by alpha-thrombin. The ProArg-Thr bond has a 50% cleavage time of about 10 min under the designated conditions, whereas the ValLys-Ser has a 50% cleavage time approx. 9-10 h. A single selective cleavage at the joining position of the variable region and joining peptide can be achieved by short-time thrombin digestion. Fragments containing intact variable region (1-96) and intact joining peptide-constant region (97-214) obtained from both denatured and native light chains of 10K26 can be separated by gel filtration. (b) lambda light chains from both human and mouse all begin with the GlnProLys-(Ala/Ser) structure (positions 108-111) at their constant regions. This ProLys-Ala/Ser bond is also susceptible to specific thrombin cleavage. Four human lambda chain (KERN, NEI, NEW, VOR) and one mouse lambda chain (RPC20) were shown to be selectively cleaved by thrombin at these ProLys-Ala/Ser bonds. For human lambda chains, the 50% cleavage time at this ProLys-Ala bond was approx. 3-4 h under the designated conditions. Six additional thrombin specific cleavages were also detected within the variable regions of NEI, VOR and RPC-20. (c) Heparin inhibits thrombin cleavage of Arg/Lys-Xaa bonds located near the center of the antibody light chain, but slightly activates thrombin cleavage of those located near the amino or carboxyl-terminal ends of the protein. The significance of these findings is threefold. (a) It demonstrates that selective cleavage of large polypeptides by alpha-thrombin can also be reasonably predicted. (b) It provides a useful method for light chain fragmentation which can greatly facilitate amino acid sequencing of antibodies. (c) It serves to generate fragments containing intact variable regions and constant regions from antibody light chains of human and mouse. Such fragments may be useful for chemical semisynthesis of a human-mouse light chain chimeras.     

Staphylocoagulase-binding region in human prothrombin

A staphylocoagulase-binding region in human prothrombin was studied by utilizing several fragments prepared from prothrombin by limited proteolysis. Bovine prothrombin, prethrombin 1, prethrombin 2, and human diisopropylphosphorylated alpha-thrombin strongly inhibited formation of the complex ("staphylothrombin") between human prothrombin and staphylocoagulase, but bovine prothrombin fragment 1 and fragment 2 had no effect on the complex formation, indicating that the binding region of human prothrombin for staphylocoagulase is located in the prethrombin 2 molecule. To identify further the staphylocoagulase-binding region, human alpha-thrombin was cleaved into the NH2-terminal large fragment (Mr = 26,000) and the COOH-terminal fragment (Mr = 16,000) by porcine pancreatic elastase. Of these fragments, the COOH-terminal fragment, which includes Asn-200 to the COOH-terminal end of the alpha-thrombin molecule, partially inhibited the complex formation between staphylocoagulase and human prothrombin. In contrast, the NH2-terminal large fragment did not show any inhibitory effect on the staphylothrombin formation. These results suggest that the staphylocoagulase interacts with human prothrombin through the COOH-terminal region of alpha-thrombin B chain. Other plasma proteins, factor X, factor IX, protein C, protein S, protein Z, all of which are structurally similar to prothrombin, did not inhibit the staphylothrombin formation at all, indicating that a specific interaction site with staphylocoagulase is contained only in the prothrombin molecule.     

Human prothrombin activation.

Human prothrombin has been purified from American Red Cross Factor IX concentrates. Studies of the activation of the human prothrombin with the use of sodium dodecyl sulfate electrophoretic analysis of activation products indicated that human prothrombin activation is similar to bovine prothrombin activation. Molecular weight analysis of human prothrombin and intermediated by sodium dodecyl sulfate co-electrophoresis with bovine prothrombin and its intermediates resulted in molecular weights of 70,000 for prothrombin, 51,000 for intermediate 1, 41,000 for intermediate 2, 23,000 for intermediate 3, and 13,000 for intermediate 4. Amino acid compositions of human prothrombin and intermediates are similar to those for bovine prothrombin and intermediates. NH2-terminal sequence studies of human prothrombin, intermediates, and alpha-thrombin A and B chains placed the intermediates in the parent human prothrombin molecule as described for the bovine system. Intermediate 3 is the NH2-terminal of prothrombin, and intermediate 1 is the COOH-terminal segment of the zymogen. Intermediate 4 is the NH2-terminal of intermediate 1. Intermediate 2', the immediate precursor of alpha-thrombin, is the COOH-terminal segment of intermediate 1. In general, a high degree of homology in the primary structure of prothrombin and intermediates was observed between the human and bovine system. The NH2-terminal sequences of human intermediate 2' and alpha-thrombin A chain are identical. However, human intermediate 2' isolated in a manner identical with that used for the isolation of bovine intermediate 2 is homologous with bovine intermediate 2, beginning with residue 14.     

Enzymatic properties of proteolytic derivatives of human alpha-thrombin

The use of derivatives of alpha-thrombin obtained by limited proteolysis, that have only a single peptide bond cleaved, allowed the unequivocal correlation between the change in covalent structure and alteration of the enzymatic properties. beta T-Thrombin contains a single cleavage in the surface loop corresponding to residues 65-83 of alpha-chymotrypsin [Birktoft, J. J., & Blow, D. M. (1972) J. Mol. Biol. 68, 187-240]. Compared with alpha-thrombin, this modification had a minor effect on the following: (1) The Michaelis constant (Km) for two tripeptidyl p-nitroanilide substrates increased 2-3 fold, whereas the catalytic constant (k cat) remained unaltered. (2) A 2-3 fold increase in the binding constant (KI) of a tripeptidyl chloromethane inhibitor was observed, but the inactivation rate constant (k i) was the same, which indicated that the nucleophilicity of the active-site histidyl residue had not changed. (3) The second-order rate constant for the inhibition by antithrombin III decreased 2-fold. Heparin accelerated the inactivation, and the degree of acceleration was similar to that obtained with alpha-thrombin. Pronounced effects of the cleavage of this loop were found. (1) The cleavage of fibrinogen was approximately 80-fold slower than that with alpha-thrombin. This was mainly due to a 40-fold decrease in k cat. In contrast, only a 1.9-fold increase in the Michaelis constant was observed. (2) The affinity for thrombomodulin had decreased 39-fold compared to alpha-thrombin. epsilon-Thrombin contains a single cleaved peptide bond in the loop corresponding to residues 146-150 in alpha-chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Croonian Lecture, 1980. The complex proteases of the complement system

The assembly and activation of the early components of complement, after their interaction with antibody-antigen complexes, are described in terms of the structures of the different proteins taking part. C1q, a molecule of unique half collagen--half globular structure, binds to the second constant domain of the antibody molecules through its six globular heads. A tetrameric complex of C1r2-C1s2 binds to the collagenous tails and leads to formation of the serine-type proteases C1r and C1s. C1s activates C4, which forms a covalent bond between its alpha' chain and the Fab section of the antibody. C2 is also activated by C1s and associates with the bound C4 molecule to form C42, a labile protease that activates C3, but which loses activity as the C2 peptide chains dissociate from C4. C2, by analogy with factor B, the equivalent component of the alternative pathway of activation, appears to be a novel type of serine protease with a similar catalytic site but different activation mechanism to the serine proteases that have been described previously.     

Covalent structure of protein C. A second major low molecular weight protein degraded during germination of Bacillus megaterium spores

The complete covalent structure of protein C, a protein degraded during germination of Bacillus megaterium spores, has been determined. The intact protein was cleaved with a highly specific spore protease into two peptides, residues 1 to 30 and 31 to 71. The intact protein was also cleaved by cyanogen bromide into two peptides, residues 1 to 27 and 28 to 71. Cleavage of the larger cyanogen bromide peptide with trypsin allowed isolation of the COOH-terminal peptide, residues 59 to 71. Automated sequenator analysis of the intact protein and peptide fragments, together with previously published partial sequence data on this protein and carboxypeptidase A digestion of the intact protein provided data from which the following unique sequence was deduced: (formula: see text). The primary sequence of the C protein shows an extremely high degree of homology with that of the A protein--another protein degraded during germination of B. megaterium spores.     

Interaction between the heavy and the regulatory light chains in smooth muscle myosin subfragment 1.

The interaction between the heavy and the regulatory light chains within chicken gizzard myosin heads was investigated by using a zero-length chemical cross-linker, 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide (EDC). The chicken gizzard subfragment 1 (S-1) used was treated with papain so that the heavy chain was partly cleaved into the NH2-terminal 72K and the COOH-terminal 24K fragments and the regulatory light chain into the 16K fragment. S-1 was reacted with EDC either alone or in the presence of ATP or F-actin. In all cases, the 16K fragment of the regulatory light chain formed a covalent cross-link with the 24K heavy chain fragment but not with the 72K fragment. The 38K cross-linked peptide, which was the product of cross-linking between the 16K light chain and the 24K heavy chain fragments, was isolated and further cleaved with cyanogen bromide and arginylendopeptidase. Smaller cross-linked peptides were purified by reverse-phase HPLC and then characterized by amino acid analysis and sequencing. The results indicated that cross-linking occurred between Lys-845 in the heavy chain and Asp-168, Asp-170, or Asp-171 in the regulatory light chain. The position of the cross-linked lysine was only three amino acid residues away from the invariant proline residue mapped as the S-1-rod hinge by McLachlan and Karn [McLachlan, A. D., & Karn, J. (1982) Nature (London) 299, 226-231]. We propose that the COOH-terminal region of the regulatory light chain is located in the neck region of myosin and that this region and the phosphorylation site of the regulatory light chain together may play a role in the phosphorylation-induced conformational change of gizzard myosin.     

Structural relationship between "glutamic acid" and "lysine" forms of human plasminogen and their interaction with the NH2-terminal activation peptide as studied by affinity chromatography.

Urokinase digestion of maleinated plasminogen results in cleavage of the single peptide bond Arg-68-Met-69, which is one of the bonds normally cleaved during the first step of the activation procedure. The inactive intermediate compound formed in this way was subjected to NH2-terminal amino acid sequence analysis, which clearly demonstrates the structural relationship between the forms of plasminogen with different NH2-terminal amino acids. It is thus shown that lysine-78 and valine-79 in the "glutamic acid" plasminogen actually are the NH2-terminal amino acids in "lysine" and "valine" plasminogen respectively. The forms with glutamic acid in NH2-terminal position are called plasminogen A, while all other forms lacking the NH2-terminal part of the molecule and which can be activated in a single step are called plasminogen B. By affinity chromatographic studies of the NH2-terminal activation peptide on insolubilized plasminogen B, it was demonstrated that this peptide has specific affinity for plasminogen B. It was also shown that this noncovalent interaction is broken by 6-aminohexanoic acid in two concentration. The tryptic heptapeptide (Ala-Phe-Gln-Tyr-His-Ser-Lys) which occupies the positions number 45 to 51 in the NH2-terminal activation peptide (as well as in the intact plasminogen molecule) is importance for the conformational state of the plasminogen molecule.     

Specificity of alpha 2-macroglobulin covalent cross-linking for the active domain of proteinases

The reaction of alpha 2-macroglobulin (alpha 2M) with the two-chain enzyme plasma kallikrein results in covalent bond formation between the catalytic subunit and the inhibitor. We have recently published a model of alpha 2M which suggests that this phenomenon may be a general mechanism when multisubunit proteinases are inactivated by alpha 2M. In order to test this hypothesis, we studied the reactions of factor Xa, plasmin, streptokinase-plasmin and alpha-thrombin with alpha 2M. In the case of factor Xa the catalytic heavy chain demonstrated greater than 99% covalent incorporation while over 97% of the light chain failed to crosslink to the inhibitor. Preferential binding of the catalytic light chains of plasmin (70% covalent incorporation) and plasmin in complex with streptokinase (79% covalent incorporation) was also observed. Finally, 82% covalent incorporation of the catalytic heavy chain of alpha-thrombin was found. These studies demonstrate that in the case of multisubunit proteinases, the chain containing the active site demonstrates preferential binding as predicted by the model supporting placement of the site of covalent binding close to the "bait region" of alpha 2M.     

Proteolytic processing sites producing the mature form of human cathepsin D.

1. The proteolytic processing sites of human lysosomal aspartic protease cathepsin D at which the intermediate single-chain form was converted into the mature two-chain form were determined. 2. The two chains were isolated by reversed-phase HPLC in order to investigate the cleavage sites of the enzyme. 3. Protein sequencing of the heavy chain, which was presumed to be derived from the C-terminal side in the single-chain enzyme, gave an N-terminal Leu 105. In addition, it revealed that there were also minor sequences, which commenced with Gly 106 and Gly 107. 4. A small C-terminal peptide was isolated from the light chain, which had been digested with two kinds of exogenous proteases. Sequence determination of this peptide, which was characterized as a nonapeptide by mass spectrometry, suggested that the C-terminus of the light chain was Ser 98. 5. These results indicate that a Ser 98-Ala 99 bond and an Ala 104-Leu 105 bond are cleaved to release 6 amino acid residues between the two chains.     

Molecular structure of L-lysyl-L-alanyl-L-alanine: a tripeptide found in histone H1

The crystal structure of L -lysyl-L -alanyl-L -alanine hydrochloride has been determined by x-ray diffraction. The peptide is in zwitterionic form with the carboxylic group deprotonated, and with positive charges both in the amino terminal and ?-amino groups of lysine. Crystals are monoclinic, space group P2₁ and Z = 4, with two peptide molecules in the asymmetric unit, which show different conformations. While one molecule has torsional angles for the Lys-Ala peptide bond (φ₂, φ₂) in the β-pleated sheet region, the values for the other molecule are close to those for the α-helix. This molecular flexibility is of interest for the study of H1 histone, which contains this sequence repeated several times. The two lysine residues show fully extended side chains. Two methanol molecules and two acetonitrile molecules are also present in the unit cell. An extensive network of hydrogen bonds and ionic interactions stabilize the crystal structure.     

The structural integrity of anion binding exosite I of thrombin is required and sufficient for timely cleavage and activation of factor V and factor VIII

Alpha-thrombin has two separate electropositive binding exosites (anion binding exosite I, ABE-I and anion binding exosite II, ABE-II) that are involved in substrate tethering necessary for efficient catalysis. Alpha-thrombin catalyzes the activation of factor V and factor VIII following discrete proteolytic cleavages. Requirement for both anion binding exosites of the enzyme has been suggested for the activation of both procofactors by alpha-thrombin. We have used plasma-derived alpha-thrombin, beta-thrombin (a thrombin molecule that has only ABE-II available), and a recombinant prothrombin molecule rMZ-II (R155A/R284A/R271A) that can only be cleaved at Arg(320) (resulting in an enzymatically active molecule that has only ABE-I exposed, rMZ-IIa) to ascertain the role of each exosite for procofactor activation. We have also employed a synthetic sulfated pentapeptide (DY(SO(3)(-))DY(SO(3)(-))Q, designated D5Q1,2) as an exosite-directed inhibitor of thrombin. The clotting time obtained with beta-thrombin was increased by approximately 8-fold, whereas rMZ-IIa was 4-fold less efficient in promoting clotting than alpha-thrombin under similar experimental conditions. Alpha-thrombin readily activated factor V following cleavages at Arg(709), Arg(1018), and Arg(1545) and factor VIII following proteolysis at Arg(372), Arg(740), and Arg(1689). Cleavage of both procofactors by alpha-thrombin was significantly inhibited by D5Q1,2. In contrast, beta-thrombin was unable to cleave factor V at Arg(1545) and factor VIII at both Arg(372) and Arg(1689). The former is required for light chain formation and expression of optimum factor Va cofactor activity, whereas the latter two cleavages are a prerequisite for expression of factor VIIIa cofactor activity. Beta-thrombin was found to cleave factor V at Arg(709) and factor VIII at Arg(740), albeit less efficiently than alpha-thrombin. The sulfated pentapeptide inhibited moderately both cleavages by beta-thrombin. Under similar experimental conditions, membrane-bound rMZ-IIa cleaved and activated both procofactor molecules. Activation of the two procofactors by membrane-bound rMZ-IIa was severely impaired by D5Q1,2. Overall the data demonstrate that ABE-I alone of alpha-thrombin can account for the interaction of both procofactors with alpha-thrombin resulting in their timely and efficient activation. Because formation of meizothrombin precedes that of alpha-thrombin, our findings also imply that meizothrombin may be the physiological activator of both procofactors in vivo in the presence of a procoagulant membrane surface during the early stages of coagulation.     

Covalent structures of beta and gamma autolytic derivatives of human alpha-thrombin

Nonclotting beta- and gamma-thrombins have been prepared by autolysis of human alpha-thrombin at pH 8.6 in the presence of 0.4 M NaCl and purified on BioRex 70. Reduced and carbamidomethylated A and B chains fragments were separated by gel filtration and reverse phase high performance liquid chromatography. Structural characterization of these fragments demonstrated that alpha to beta conversion results from two cleavages at Arg 62 and Arg 73 in the B chain, releasing an intact 11-residue peptide. beta to gamma conversion corresponds to the additional loss of a fragment of the B chain stretching from Ile 124 to Lys 154. Autolysis is not accompanied by cleavages in the A chain. Loss of clotting activity is therefore related solely to the excision of residues 63 to 73 in the B chain. With the exception of cleavage at Arg 73, these results differ from a proposed model for alpha to gamma conversion of bovine thrombin.     

Covalent structure of protein A. A low molecular weight protein degraded during germination of Bacillus megaterium spores.

The complete covalent structure of Protein A, a protein degraded during bacterial spore germination, has been determined. The intact protein was cleaved with a highly specific spore protease into two peptides, residues 1 to 21 and 22 to 61. The larger peptide was further cleaved into two fragments with either cyanogen bromide or by trypsin cleavage following arginine modification with cyclohexanedione. The peptides derived from cyanogen bromide fragmentation encompassed residues 22 to 53 and 54 to 61 while trypsin hydrolysis yielded overlapping fragments comprising residues 22 to 48 and 49 to 61. Automated sequenator analysis together with carboxypeptidase Y digestion of the intact protein and the peptide fragments provided data from which the following unique amino acid sequence was deduced. NH2-Ala-Asn-Thr-Asn-Lys-Leu-Val-Ala-Pro-Gly10-Ser-Ala-Ala-Ala-Ile-Asp-Gln-Met-Lys-Tyr20-Glu-Ile-Ala-Ser-Glu-Phe-Gly-Val-Asn-Leu30-Gly-Pro-Glu-Ala-Thr-Ala-Arg-Ala-Asn-Gly40-Ser-Val-Gly-Gly-Glu-Ile-Thr-Lys-Arg-Leu50-Val-Gln-Met-Ala-Glu-Gln-Gln-Leu-Gly-Gly60-Lys-COOH.     

Activation of human prothrombin by a procoagulant fraction from the venom of Echis carinatus. Identification of a high molecular weight intermediate with thrombin activity.

In the presence of a procoagulant fraction (Echis carinatus procoagulant) isolated from the venom of the saw-scaled viper Echis carinatus sochureki, purified human prothrombin (P1) is completely converted to thrombin. The first step is the removal of an NH2-terminal peptide (F1) representing approximately one-third of the prothrombin molecule. The remaining peptide (P2) is then cleaved by the action of E.c. procoagulant to yield a two-chain, disulfide-bridged protein (P'2) which has the same molecular weight as P2. P'2 has enzymic (thrombin) activity, as evidence by incorporation of radiolabeled diisopropylphosphate into its heavy chain (TB), hydrolysis of p-toluenesulfonylarginine methyl ester, and clotting of fibrinogen. Relative to thrombin, its esterolytic activity greatly exceeds its clot-promoting activity. Examination of the polypeptide chains obtained by reducing P'2 has shown that its larger chain (TB) is indistinguishable from the heavy chain of thrombin. Its other chain (F2TA) consists of the light chain (TA) of thrombin bound by peptide linkage to the protion of the prothrombin molecule which had been adjacent to F1. Removal of this portion (F2) is catalyzed by thrombin (and, evidently, by P'2), but not by the E.c. procoagulant. When F2 is removed from P'2, the remaining two-chian protein is indistinguishable from thrombin by any of the criteria applied--molecular weight, subunit chain composition, or enzymic activity. Polyacrylamide gel electrophoresis was carried out in sodium dodecyl sulfate before and after disulfide reduction of samples generated in the presence and in the absence of diisopropylphosphorofluoridate, which inhibits thrombin but not the E.c. procoagulant. Such experiments showed that thrombin (and probably P'2), as well as E.c. procoagulant, catalyzes the release of F1. Furthermore, thrombin brings about the cleavage of F1 to yield a two-chain, disulfidebridged protein (F'1). These observations, particularly those made in the course of characterizine P'2, have led to the conclusion that cleavage of the peptide bond connecting the TA and TB portions of the prothrombin molecule (or its derivatives) produces a serine active center and, hence, a molecule possessing thrombin activity. This cleavage is catalyzed by the E.c. procoagulant but not by thrombon itself.     

The binding of human complement component C4 to antibody-antigen aggregates.

The binding of human complement component C4 to antibody-antigen aggregates and the nature of the interaction have been investigated. When antibody-antigen aggregates with optimal C1 bound are incubated with C4, the C4 is rapidly cleaved to C4b, but only a small fraction (1-2%) is bound to the aggregates, the rest remaining in the fluid phase as inactive C4b. It has been found that C4b and th antibody form a very stable complex, due probably to the formation of a covalent bond. On reduction of the C4b-immunoglobulin G (IgG) complex, the beta and gamma chains, but not the alpha' chain, of C4b are released together with all the light chain, but only about half of the heavy chain of IgG. The reduced aggregates contain two main higher-molecular-weight complexes, one shown by the use of radioactive components to contain both IgG and C4b and probably therefore the alpha' chain of C4b and the heavy chain of IgG, and the other only C4b and probably an alpha' chain dimer. The aggregates with bound C1 and C4b show maximal C3 convertase activity, in the presence of excess C2, when the alpha'-H chain component is in relatively highest amounts. When C4 is incubated with C1s in the absence of aggregates, up to 15% of a C4b dimer is formed, which on reduction gives an alpha' chain complex, probably a dimer. The apparent covalent interaction between C4b and IgG and between C4b and other C4b molecules cannot be inhibited by iodoacetamide and hence cannot be catalysed by transglutaminase (factor XIII). The reaction is, however, inhibited by cadaverine and putrescine and 14C-labelled putrescine is incorporated into C4, again by a strong, probably covalent, bond. It is suggested that a reactive group, possibly an acyl group, is generated when C4 is activated by C1 and that this reactive group can react with IgG, with another C4 molecule, or with water.     

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.