Purification and characterization of rat T-kininogens isolated from plasma of adjuvant-treated rats. Identification of three kinds of T-kininogens

Two T-kininogens (TI- and TII-kininogens) found in plasma of Freund's adjuvant-treated rats were purified by several chromatographic procedures. The isolated TI- and TII-kininogens showed different mobilities on polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate, but were indistinguishable in the presence of sodium dodecyl sulfate. They were also indistinguishable in amino acid composition and antigenicity, but differed in sialic acid content. The NH2- and COOH-terminal sequences were determined. In the 30 NH2-terminal residues, 2 were different. The kinin regions in the COOH-terminal portions of the two kininogens have sequences that demonstrate TI-kininogen contains a mixture of two kinin-containing regions, with substitution of 4 amino acid residues, one of which is identical to the COOH-terminal portion of alpha 1-major acute phase protein (Cole, T., Inglis, A. S., Roxburgh, C. M., Howlett, G. J., and Schreiber, G. (1985) FEBS Lett. 182, 57-61) and the other to the COOH-terminal portion of TI-kininogen (Furuto-Kato, S., Matsumoto, A., Kitamura, N., and Nakanishi, S. (1985) J. Biol. Chem. 260, 12054-12059), both predicted from cDNA sequences. The amino acid sequence of the kinin-containing region from TII-kininogen is the same as the COOH-terminal portion of TII-kininogen predicted from the cDNA. These results indicate that T-kininogens from the plasma of adjuvant-treated rats consist of a family of kininogens, that is, TI- and TII-kininogens (separable on DEAE-Sephadex A-50), and that TI-kininogen consists of at least two variants (TI alpha and TI beta) which correspond to the alpha 1-major acute phase protein reported by Cole et al. and TI-kininogen reported by Furuto-Kato et al., respectively. Immunoblotting studies with plasmas from non-inflamed and adjuvant-treated rats also indicate that T-kininogen which was previously isolated from non-inflamed rat plasma corresponds to TI-kininogen and that TII-kininogen is newly generated after treatment of rats with adjuvants.     

Cysteine proteinase inhibitor in the ascitic fluid of sarcoma 180 tumor-bearing mice is a low molecular weight kininogen. Partial NH2- and COOH-terminal sequences and susceptibility to various glandular kallikreins

It has been proposed that a cysteine proteinase inhibitor (CPI) found in the ascitic fluid of Sarcoma 180 tumor-bearing mice is a kind of kininogen (Itoh, N., Yokota, S., Takagishi, U., Hatta, A., and Okamaoto, H. (1987) Cancer Res. 47, 5560-5565). The first 40 NH2-terminal residues and 54 residues of the COOH-terminal sequence, including the bradykinin moiety of highly purified ascites CPI, were determined and compared with those of mammalian low molecular weight kininogens (LMWK). The significant identity between these amino acid sequences with those of other mammalian LMWKs suggests that ascites CPI corresponds precisely to mouse LMWK. This kininogen has a light chain composed of 43 amino acid residues, which contains a unique Met-Ala-Arg-bradykinin sequence. Hydroxyproline, which was recently identified in the bradykinin sequence of kininogen from the ascitic fluid of a cancer patient, was not found in the kinin moiety of this mouse kininogen. Among purified glandular kallikreins from human, hog, rat, and mouse, only mouse submaxillary gland kallikrein was able to release bradykinin from this kininogen. Kinetic studies using a newly synthesized fluorogenic substrate, N-t-butoxycarbonyl-Met-Ala-Arg-MCA, revealed that mouse kallikrein hydrolyzes this substrate approximately 80-fold faster than does hog kallikrein, suggesting that the unique Met-Ala-Arg-bradykinin sequence is responsible for the varied susceptibility of mouse kininogen to different kallikreins.     

T-kininogen--the major plasma kininogen in rat adjuvant arthritis

Total kininogen in plasma of Freund's adjuvant treated rats increased 20-fold 7 days following the injection. Analysis of the kininogens demonstrated that increases in T-kininogen was the major reason for the rise in kininogen. High molecular weight and low molecular weight kininogens showed little or no change. The increase in T-kininogen paralleled the inflammatory condition. Anti-inflammatory agents which reduced paw swelling also reduced plasma T-kininogen levels. Unidentified peaks on HPLC of kinin following plasma treatment by trypsin were shown to be oligopeptides containing T-kinin (Ile-serbradykinin). The relationship of T-kininogen to the inflammatory response is discussed.     

Primary structures of the mRNAs encoding the rat precursors for bradykinin and T-kinin. Structural relationship of kininogens with major acute phase protein and alpha 1-cysteine proteinase inhibitor

Three types of cloned cDNA sequences for rat low molecular weight prekininogens were isolated and determined by molecular cloning and sequence analysis. The deduced amino acid sequences indicated that one, termed K-prekininogen, represents the counterpart of the known low molecular weight prekininogen present in other mammals, while the other two, called T-prekininogens, contain a novel T-kinin sequence which was recently identified from rat plasma. Although T- and K-prekininogens are highly homologous with each other, both of the T-prekininogens contain methionine, instead of arginine or lysine, as an amino acid preceding T-kinin and exhibit two consecutive amino acid deletions in the preceding region of T-kinin as compared with K-prekininogen. The former finding accounts for the previous observation of strong resistance of T-kininogens to cleavage with trypsin or kallikreins, while the latter finding has been explained by the structural analysis of genomic clones in which T-kinin-coding exon is contracted at its intron junction. A partial nucleotide sequence reported recently for the rat major acute phase protein (alpha 1-MAP) mRNA was found to be extremely related to the corresponding portion of the rat T-prekininogen mRNA. Furthermore, consistent with the previous report of the structural identity of major acute phase protein and alpha 1-cysteine proteinase inhibitor, kininogen closely resembles not only the former but also the latter in the amino acid compositions. The interrelationship among the triad of these proteins has been discussed.     

Cathepsin L, but not cathepsin B, is a potential kininogenase

Although papain-like enzymes are strongly inhibited by their natural tight-binding inhibitors of the cystatin superfamily, cathepsins B and L may still retain some residual proteolytic activity toward Z-Phe-Arg-AMC in the presence of an excess of kininogen. This activity is abolished by adding E-64 or chicken cystatin. Cathepsins B and L show a single band of gelatinolytic activity when subjected to gelatin-SDS-PAGE. Adding high Mr kininogen, low Mr kininogen, T-kininogen, or chicken cystatin to cathepsin L results in additional intense bands of enzyme activity corresponding to the protease-inhibitor complexes. Cathepsin B does not produce these additional bands. This gelatinolytic activity was inhibited by E-64, but not by EDTA, PMSF or Pefabloc. Cathepsin L also specifically generated kinins from high and low molecular weight kininogens in vitro, but cathepsin B did not. T-kininogen did not release any immunoreactive kinins when complexed with cathepsin L, as previously observed using tissue kallikreins. The ability of cathepsin L to generate vasoactive peptides raises the question of the physiological significance of this mechanism during inflammation.     

Purification and characterization of rat low molecular weight kininogen

Low molecular weight (LMW) kininogen was isolated from pooled rat plasma by chromatography on DEAE-Sephadex A-50, CM-Sephadex C-50, Blue-Sepharose CL-6B, and Sephadex G-100. It was shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoelectrophoresis. The molecular weight of rat LMW kininogen was determined to be 72,000 by SDS-PAGE. The LMW kininogen contained 83.5% protein, 4.0% hexose, 5.5% hexosamine, and 2.7% sialic acid. Kinin liberated from LMW kininogen by trypsin treatment was identified as an Ile-Ser-bradykinin(T-kinin) by analysis involving ion exchange column chromatography on CM-Sephadex C-25 and high performance liquid chromatography on a reverse-phase column (ODS-120T). LMW kininogen formed kinin with rat submaxillary gland kallikrein, but the kinin liberated was only 14% of the total kinin content, that is, that released by trypsin. In order to determine the immunochemical properties of LMW kininogen, specific antiserum was prepared in rabbits. The antiserum cross-reacted with high molecular weight (HMW) kininogen, but spur formation was observed between the LMW and HMW kininogens. The kininogen level in rat plasma was estimated to be 433 microgram/ml by a quantitative single radial immunodiffusion test.     

Properties of chemically oxidized kininogens

Kininogens are multifunctional proteins involved in a variety of regulatory processes including the kinin-formation cascade, blood coagulation, fibrynolysis, inhibition of cysteine proteinases etc. A working hypothesis of this work was that the properties of kininogens may be altered by oxidation of their methionine residues by reactive oxygen species that are released at the inflammatory foci during phagocytosis of pathogen particles by recruited neutrophil cells. Two methionine-specific oxidizing reagents, N-chlorosuccinimide (NCS) and chloramine-T (CT), were used to oxidize the high molecular mass (HK) and low molecular mass (LK) forms of human kininogen. A nearly complete conversion of methionine residues to methionine sulfoxide residues in the modified proteins was determined by amino acid analysis. Production of kinins from oxidized kininogens by plasma and tissue kallikreins was significantly lower (by at least 70%) than that from native kininogens. This quenching effect on kinin release could primarily be assigned to the modification of the critical Met-361 residue adjacent to the internal kinin sequence in kininogen. However, virtually no kinin could be formed by human plasma kallikrein from NCS-modified HK. This observation suggests involvement of other structural effects detrimental for kinin production. Indeed, NCS-oxidized HK was unable to bind (pre)kallikrein, probably due to the modification of methionine and/or tryptophan residues at the region on the kininogen molecule responsible for the (pro)enzyme binding. Tests on papain inhibition by native and oxidized kininogens indicated that the inhibitory activity of kininogens against cysteine proteinases is essentially insensitive to oxidation.     

The Occluding Loop of Cathepsin B Prevents Its Effective Inhibition by Human Kininogens

Kininogens, the major plasma cystatin-like inhibitors of cysteine cathepsins, are degraded at sites of inflammation, and cathepsin B has been identified as a prominent mediator of this process. Cathepsin B, in contrast to cathepsins L and S, is poorly inhibited by kininogens. This led us to delineate the molecular interactions between this protease and kininogens (high molecular weight kininogen and low molecular weight kininogen) and to elucidate the dual role of the occluding loop in this weak inhibition. Cathepsin B cleaves high molecular weight kininogen within the N-terminal region of the D2 and D3 cystatin-like domains and close to the consensus QVVAG inhibitory pentapeptide of the D3 domain. The His110Ala mutant, unlike His111Ala cathepsin B, fails to hydrolyze kininogens, but rather forms a tight-binding complex as observed by gel-filtration analysis. K_i values (picomolar range) as well as association rate constants for the His110Ala cathepsin B variant compare to those reported for cathepsin L for both kininogens. Homology modeling of isolated inhibitory (D2 and D3) domains and molecular dynamics simulations of the D2 domain complexed with wild-type cathepsin B and its mutants indicate that additional weak interactions, due to the lack of the salt bridge (Asp22-His110) and the subsequent open position of the occluding loop, increase the inhibitory potential of kininogens on His110Ala cathepsin B.     

Bradykinin and kininogens in bovine milk

Two peptides exhibiting kinin activity in an isolated rat uterus assay were purified from pasteurized skim bovine milk. The amino acid sequence of the more prominent peptide was found to be that of bradykinin. Partially purified kinin preparations were also obtained from N-tosyl-L-phenylalanyl chloromethyl ketone-treated trypsin digests of non-fat dry milk and insoluble lactalbumin. The application of fast atom bombardment/mass spectrometry permitted detection of the bradykinin protonated molecular ion in each of these samples. Collision-activated decomposition of the ion of m/z 1061 confirmed it to be the protonated molecular ion of bradykinin. Fast atom bombardment/mass spectrometry analysis further confirmed the occurrence of bradykinin in a pancreatic kallikrein digest of a partially purified bovine milk kininogen preparation. In apparent contrast with bovine plasma kininogens, the forms of kininogen which occur in milk include a high Mr kininogen (Mr greater than 68,000) and a low Mr kininogen (Mr 16,000-17,000). Kinin formation from the high Mr kininogen is catalyzed by porcine pancreatic kallikrein or trypsin.     

Single-step purification of "kallikrein-resistant" kininogen from rat plasma using monoclonal-antibody immunoaffinity chromatography

Monoclonal antibody to rat plasma kininogen, obtained after immunization of mice with the kininogen prepared by conventional methods, was purified from ascites fluid and coupled to CNBr-activated Sepharose-4B. Monoclonal-antibody affinity adsorbant thus prepared provided a rapid single-step method of purifying to homogeneity plasma kininogen. Purified rat plasma kininogen showed identical molecular weight and immunological cross-reactivity to rat plasma low molecular weight (LMW) kininogen purified by conventional procedures. Rat plasma kininogen differed from LMW kininogen from other species by virtue of its resistance to cleavage by either plasma or glandular kallikreins.     

Differing expression patterns and evolution of the rat kininogen gene family

The present investigation using molecular cloning and sequence analysis concerns the examination of the molecular basis for different expression patterns of two types of the rat kininogen genes. We show that the low molecular weight and high molecular weight forms of K kininogens are produced from a single gene through alternative usage of two 3'-coding regions, whereas only the low molecular weight forms of T kininogens are generated as a result of several mutational changes in the high molecular weight-specifying regions of both T-I and T-II kininogen genes. The mutational changes include a nucleotide substitution at the polyadenylation/processing signal site, nucleotide deletions resulting in the frame-shift mutation, and an insertion of the type 2 Alu-equivalent sequence. Because kininogens represent a multifunctional protein comprising the proteinase-inhibitory activity, the kinin moiety, and the clotting activity, these results present evidence indicating the molecular basis for the disappearance of a part of the gene functions. We also show that the K and T kininogen genes as well as the two T kininogen genes are extremely homologous, excluding and including the above mutational changes, respectively. These structural relationships allow us to envisage evolutionary processes for the generation of the rat kininogen gene family, particularly for the disappearance of a part of the gene functions.     

Characterization of serine proteinases isolated from rat submaxillary gland: with special reference to the degradation of rat kininogens by these enzymes

From the homogenate of rat submaxillary gland, two kinds of serine proteinases, named tentatively proteinases A and B, were isolated and their chemical properties and activities toward rat kininogens were examined, in comparison with those of submaxillary kallikrein. Proteinase A with Mr of 28,200 rapidly cleaved high-molecular-weight (HMW) kininogen into a protein of 67 kDa, which retained thiol-proteinase inhibitory activity, but had lost the correcting activity of HMW kininogen on the prolonged clotting time of Fitzgerald trait plasma. It liberated bradykinin from HMW kininogen but did not liberate kinin from T-kininogen and did not degrade T-kininogen. On the other hand, proteinase B with Mr of 30,400 showed a very weak activity for the liberation of kinin from T-kininogen and the cleavage of T-kininogen at pH 8.0. However, the enzyme extensively degraded T-kininogen at pH 4.5. Proteinase B also degraded HMW kininogen at pH 4.5 and pH 8.0, but liberated bradykinin only at pH 8.0. Thiol-proteinase inhibitory activities of HMW kininogen and T-kininogen were inactivated after the incubation with proteinase B at pH 4.5 but not at pH 8.0, while the correcting activity of HMW kininogen on the Fitzgerald trait plasma was inactivated at pH 4.5 and 8.0. The NH2-terminal amino acid sequences of proteinases A and B were different from each other, and distinguishable with those of serine proteinases in rat submaxillary gland so far reported. These results provide evidence that in addition to the known kallikrein, there exist at least two kinds of serine proteinases in rat submaxillary gland, both of which liberate bradykinin from rat HMW kininogen at pH 8.0 and modulate the functional activities of HMW kininogen and T-kininogen, degrading these proteins at pH 8.0 or 4.5.     

Mapping of functional domains of human high molecular weight and low molecular weight kininogens using murine monoclonal antibodies

Thirty-four monoclonal antibodies directed against human high molecular weight (HMW) and low molecular weight (LMW) kininogens and their derivatives were obtained, and the specificities of the antibodies were assayed by enzyme-linked immunosorbent assay (ELISA). By use of HMW kininogen, kinin-free HMW kininogen, kinin-free and fragment 1.2 (fr 1.2) free HMW kininogen, fr 1.2-light chain of HMW kininogen, LMW kininogen, kinin-free LMW kininogen, heavy chain of LMW kininogen, and light chain of LMW kininogen, the monoclonal antibodies were characterized and classified into four groups: (A) 20 monoclonal antibodies reacting with only the heavy chain, a common region of HMW and LMW kininogens; each of these monoclonal antibodies possessed the specificity to domain 1 (2 monoclonal antibodies), domain 2 (2 monoclonal antibodies), domain 3 (7 monoclonal antibodies), and both domains 2 and 3 (7 monoclonal antibodies) of the heavy chain; (B) 7 monoclonal antibodies reacting with fr 1.2, a unique histidine-rich region; (C) 5 monoclonal antibodies reacting with the light chain of HMW kininogen; (D) 2 monoclonal antibodies reacting with the light chain of LMW kininogen. Two monoclonal antibodies in the first group (group A), designated HKG H7 and H12, effectively suppressed the thiol proteinase inhibitor activity of HMW kininogen to papain and calpains and of LMW kininogen to papain, but the others did not affect it. Further, all the monoclonal antibodies which recognized the fr 1.2 or light chain of HMW kininogen (groups B and C) suppressed the clotting activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct radioimmunoassay for rat T-kininogen

Antibodies raised in rabbits against pure rat T-kininogen did not cross-react with Ile-Ser-Bradykinin, bradykinin, nor with kininogens from other mammalian species. They presented a 1 to 15% cross-reaction with pure rat HMW kininogen, depending on the quantity of HMW kininogen. A direct radioimmunoassay for rat T-kininogen in plasma was developed and it enabled 89 fmol of the protein to be detected. A good correspondence was obtained between the direct RIA and the T-kinin generating assay. By the direct assay, it was found that T-kininogen is increased about ten fold in rats subcutaneously injected with turpentine. These data were confirmed by HPLC analysis of the plasma kinins released by trypsin which demonstrated that only T-kinins are increased, bradykinin being unchanged. It was possible according to the results obtained by the direct RIA and HPLC analysis to estimate that in the normal rat, HMW and LMW kininogen represent about 35% and T-kininogen 65%. In the turpentine-treated rat, T-kininogen reached 95%. This RIA will allow the study of the regulation of T-kininogen in the rat and the synthesis of this protein in cells in culture.     

Human high molecular weight kininogen as a thiol proteinase inhibitor: presence of the entire inhibition capacity in the native form of heavy chain

High molecular weight (HMW) kininogen was purified from fresh human plasma by two successive column chromatographies on DEAE-Sephadex A-50 and Zn-chelate Sepharose 4B. The purified HMW kininogen appeared to be a single band on sodium dodecyl sulfate (SDS)-polyacrylamide disc gel electrophoresis in both the presence and absence of beta-mercaptoethanol. However, it gave two bands on nonreduced SDS-polyacrylamide slab gel electrophoresis, a major band of dimeric form (Mr 200 000, ca. 95%) and a minor band of monomeric form (Mr 105 000, ca. 5%). Under reduced conditions, the dimeric form was converted stoichiometrically to a monomeric form (Mr 110 000), and the monomeric form observed under nonreduced conditions (Mr 105 000) was converted to a heavy chain (Mr 60 000) and a light chain (Mr 50 000). The formation of a dimer of HMW kininogen was also confirmed by an immunoblotting experiment. This unique property of intact HMW kininogen to form a dimer was further utilized in studies on the kininogens and their derivatives as thiol proteinase inhibitors. The purified HMW kininogen strongly inhibited the caseinolytic activities of calpain I, calpain II, and papain but not those of trypsin, chymotrypsin, and thermolysin, indicating that it was a group-specific inhibitor for thiol proteinases. When HMW kininogen was reduced with 0.14 or 1.4 M beta-mercaptoethanol, its inhibitory activity was partially or mostly inactivated, but on subsequent air oxidation its activity was almost completely recovered. In addition, kinin-free and fragment 1,2 free HMW kininogen showed higher inhibitory activity than the intact HMW kininogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat plasma high-molecular-weight kininogen. A simple method for purification and its characterization

High-molecular-weight kininogen has been isolated from rat plasma in three steps in a relatively high yield. The purified preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of 2-mercaptoethanol, and the apparent Mr was estimated as 100,000. On incubation with rat plasma kallikrein, rat high Mr kininogen yielded a kinin-free protein consisting of a heavy chain (Mr = 64,000) and a light chain (Mr = 46,000), liberating bradykinin. The kinin-free protein was S-alkylated, and its heavy and light chains were separated by a zinc-chelating Sepharose 6B column. The amino acid compositions of rat high Mr kininogen and its heavy and light chains were very similar to those of bovine high Mr kininogen and its heavy and fragment 1.2-light chains, respectively. A high histidine content in the light chain of rat high Mr kininogen indicated the presence of a histidine-rich region in this protein as in bovine high Mr kininogen, although this region was not cleaved by rat plasma kallikrein. Rat high Mr kininogen corrected to normal values the prolonged activated partial thromboplastin time of Brown-Norway Katholiek rat plasma known to be deficient in high Mr kininogen and of Fitzgerald trait plasma. The kinin-free protein had the same correcting activity as intact high Mr kininogen. Rat high Mr kininogen also accelerated approximately 10-fold the surface-dependent activation of rat factor XII and prekallikrein, which was mediated with kaolin, amylose sulfate, and sulfatide. These results indicate that rat high Mr kininogen is quite similar to human and bovine high Mr kininogens in terms of biochemical and functional properties.     

Myeloperoxidase-catalyzed oxidative inactivation of human kininogens: the impairment of kinin-precursor and prekallikrein-binding functions

Bradykinin-related vasoactive peptides (kinins) are important mediators of local and systemic inflammatory reactions. However, at local inflammatory foci, the production of kinins from proteinaceous precursors (kininogens) can be affected by reactive oxygen species released by phagocyte cells. One of the predominant oxidants at these places is hypochlorous acid which is formed from hydrogen peroxide and chloride ions by neutrophil myeloperoxidase. In this study, inactivation of human kininogens after oxidation with the myeloperoxidase-H?O?-chloride system was observed and analyzed by protein chemistry methods. The kinin release from oxidized kininogens by major kinin-producing enzymes, plasma and tissue kallikreins, proceed with a very low rate. This effect was assigned to apparent inability of kallikreins to process the kinin N-terminus owing to the conversion of the adjacent Met-361 residue to methionine sulfoxide. Additionally, the oxidized high-molecular mass kininogen lost its natural ability to bind plasma prekallikrein. This effect was assigned to the oxidation of Trp-569 residue within the prekallikrein-binding region which is subsequently destructed owing to cleavage of the peptide bond after that residue. One possible pathophysiological consequence of the described effects on kininogens could be the impairment of the normal assembly and triggering of the kinin-forming system on defense cell surfaces.     

Interaction of human calpains I and II with high molecular weight and low molecular weight kininogens and their heavy chain: mechanism of interaction and the role of divalent cations

Calpain I prepared from human erythrocytes was half-maximally and maximally activated at 23 and 35 microM calcium ion, and two preparations of calpain II from human liver and kidney were half-maximally activated at 340 and 220 microM calcium ion and maximally activated at 900 microM calcium ion, respectively. High molecular weight (HMW) and low molecular weight (LMW) kininogens isolated from human plasma and the heavy chain prepared from these proteins inhibited calpain I as well as calpain II. The molar ratios of calpains to HMW kininogen to give complete inhibition of calpains were 1.4 for calpain I and 2.0 for calpain II, and those of calpains to heavy chain were 0.40-0.66 for calpain I and 0.85 for calpain II. LMW kininogen did not completely inhibit the calpains even with an excess amount of kininogen. The apparent binding ratio of calpain to HMW kininogen estimated from the disc gel electrophoretic analysis, however, was found to be 2:1, whereas those of calpain to LMW kininogen and of calpain to heavy chain were found to be 1:1. Calpains and kininogens failed to form complexes in the absence of calcium ion. In the presence of calcium ion, however, they formed the complexes, which were dissociable by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The minimum concentrations of calcium ion required to induce complex formation between calpain I and kininogens and calpain II and kininogens were 70 and 100 microM, respectively. Some other divalent cations such as Mn2+, Sr2+, and Ba2+ were also able to induce the complex formation between calpains and kininogens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Canine Plasma Kininogen: Evidence for the Presence of Two Kininogens and Purification of High Molecular Weight Kininogen and Characterization as Multi-Functional Molecule

The ratio of kininogen that is substrate of plasma kallikrein to kininogen, which is not substrate of plasma kallikrein in canine plasma, was about 1:3.6 by differential assay of kininogens. When the plasma was gel-filtered through a column of Sephacryl S-300 superfine, two fractions, which released kinin by trypsin, were obtained. These results indicate that two kininogens with different molecular weights are present in the plasma and they show different susceptibility to plasma kallikrein. One kininogen was purified by ion-exchange and zinc-chelating affinity chromatographies. Purified kininogen showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition and its molecular weight was 125 kDa. Released kinin from the kininogen by trypsin was bradykinin. The kininogen inhibited papain and ficin but did not inhibit bromelain at the concentration used. The kininogen bound to carboxymethylated-papain and this binding was dissociated by 3M NaSCN. Canine plasma shortened the abnormal clotting time of human high molecular weight kininogen-deficint plasma. The kininogen also shortened the abnormal clotting time of the plasma. From these results, the purified kininogen was high molecular weight kininogen and it was multi-functional protein.     

The relationship between rat major acute phase protein and the kininogens

The rat major acute phase protein (alpha 1-MAP) is a cysteine protease inhibitor. The stoichiometry of the interaction between the inhibitor and enzyme was shown to be 1:2. A cDNA clone specific for rat alpha 1-MAP was isolated from a cDNA library prepared from an inflamed rat liver RNA template. The 1458-base pair insert was sequenced and positively identified by alignment with a partial amino acid sequence obtained by radiosequence analysis of the primary translation product for alpha 1-MAP. Complete sequence analysis determined the alpha 1-MAP cDNA coded for the entire protein with the exception of the first four amino acids of the signal peptide, all of which were identified by radiosequencing. The coding sequence spans 1282 nucleotides, followed by 115 base pairs of a 3' untranslated region. Two putative active sites, suggested by the enzyme-inhibitor ratio, have been identified by analysis of internal duplications of the alpha 1-MAP sequence and the alignment of these regions with the sequences of several low molecular weight cysteine protease inhibitors. A computer homology analysis of the protein sequence revealed a 59.3% overall identity between rat alpha 1-MAP and bovine low molecular weight (LMW) kininogen. The homology included the signal peptide regions. LMW kininogen is a precursor of bradykinin. alpha 1-MAP does contain a bradykinin sequence; the flanking amino acids are different, however. Evidence for the expression of the LMW and a high molecular weight kininogen from the same gene, and the high degree of homology between these proteins and the rat acute phase protein suggest that all three proteins belong to a precisely regulated gene family.