Limited proteolysis of T-kininogen (thiostatin). Release of comparable fragments by different endopeptidases

Limited proteolysis of T-kininogen by heterologous and homologous endopeptidases (bovine trypsin, human leukocyte elastase, rat submaxillary gland endopeptidase k, and rat mast cell chymase) produced similar fragmentation. Amino-terminal sequence analysis of whole T-kininogen lysates and purified proteolytic fragments identified four susceptible regions which contained all the preferential cleavage sites for these proteinases. Two of these susceptible regions were close to the junction between heavy chain cystatin-like domains, the third was in the kinin-containing region, and the fourth was close to the carboxyl terminus of the T-kininogen light chain. There was only one primary site for each proteinase in the kinin-containing region, which explains why catalytic amounts of these proteinases did not release immunoreactive kinin from this kininogen. However, preferential cleavage of T-kininogen close to the junction between cystatin-like domains released fragments which, provided they included cystatin-like domains 2 and/or 3, strongly inhibited papain and cathepsin L. The fragments were inhibitory even when parts of the amino-terminal ends of the domains were lacking. The highly conserved glycyl residue, thought to be involved in the inhibitory reactive site of cystatin-like inhibitors, was not required in purified domain 3 for inhibition of cathepsin L.     

Studies on human high molecular weight (HMW) kininogen. II. Structural change of HMW kininogen by the action of human plasma kallikrein

We have investigated in detail the cleavage of human high molecular weight (HMW) kininogen by human plasma kallikrein and revealed the formation of a nicked kininogen and a novel kinin-free protein (KFP) as intermediate cleavage products. The cleavage of a single chain HMW kininogen (Mr=120,000) by plasma kallikrein was a three-step reaction. The first cleavage yielded a nicked kininogen composed of two disulfide-linked 62,000 and 56,000 daltons chains. The second cleavage yielded kinin and an intermediate kinin-free protein, KFP-I, which was apparently of equal size to the nicked kininogen. The third cleavage yielded a stable kinin-free protein, KFP-II, composed of two disulfide-linked 62,000 and 45,000 daltons chains. The liberation of an 8,000 daltons fragment was identified when the 56,000 daltons chain isolated by SP-Sephadex C-50 chromatography of reduced and alkylated KFP-I was cleaved by plasma kallikrein into the 45,000 daltons chain. Although the antiserum against HMW kininogen cross-reacted with low molecular weight (LMW) kininogen, the antiserum against the 45,000 daltons chain was specific for HMW kininogen. These results suggest that the antigenic determinant groups common to HMW and LMW kininogens are located in the 62,000 daltons heavy chain, while those specific for HMW kininogen are located in the 45,000 daltons light chain, which is known to retain blood coagulation activity.     

The hyaluronan-binding serine protease from human plasma cleaves HMW and LMW kininogen and releases bradykinin

The influence of the hyaluronan-binding protease (PHBSP), a plasma enzyme with FVII- and pro-urokinase-activating potency, on components of the contact phase (kallikrein/kinin) system was investigated. No activation or cleavage of the proenzymes involved in the contact phase system was observed. The pro-cofactor high molecular weight kininogen (HK), however, was cleaved in vitro by PHBSP in the absence of any charged surface, releasing the activated cofactor and the vasoactive nonapeptide bradykinin. Glycosoaminoglycans strongly enhanced the reaction. The cleavage was comparable to that of plasma kallikrein, but clearly different from that of coagulation factor FXIa. Upon extended incubation with PHBSP, the light chain was further processed, partially removing about 60 amino acid residues from the N-terminus of domain D5 of the light chain. These cleavage site(s) were distinct from plasma kallikrein or FXIa cleavage sites. PHBSP and, more interestingly, also plasma kallikrein could cleave low molecular weight kininogen in vitro, indicating that domains D5H and D6H are no prerequisite for kininogen cleavage. PHBSP was also able to release bradykinin from HK in plasma where the pro-cofactor circulates predominantly in complex with plasma kallikrein or FXI. In conclusion, PHBSP represents a novel kininogen-cleaving and bradykinin-releasing enzyme in plasma that shares significant catalytic similarities with plasma kallikrein. Since they are structurally unrelated in their heavy chains (propeptide), their similar in vivo catalytic activities might be directed at distinct sites where PHBSP could induce processes that are related to the kallikrein/kinin system.     

High-molecular-weight kininogen from horse plasma. Isolation, characterization and comparison with bovine high-Mr kininogen

High-molecular-weight (high-Mr) kininogen was purified from horse plasma by chromatography on columns of DEAE-Sephadex A-50, CM-Sephadex C-50, p-chlorobenzylamine-Sepharose and Sephadex G-150. The yield was about 150 mg from 81 of fresh plasma. The purified material gave a single band on sodium dodecylsulfate/polyacrylamide gel electrophoresis and a single precipitin line on immunodiffusion and immunoelectrophoresis. The molecular weight of horse high-Mr kininogen was estimated to be 78000 by dodecylsulfate gel electrophoresis using the Ferguson plot. Its polypeptide content was determined to be 86% by amino acid analysis and there was a total of 581 amino acid residues/molecule of protein. The kininogen contained a total of 13.9% carbohydrates, consisting of hexoses (7.8%), glucosamine (1.9%), galactosamine (0.6%) and sialic acid (3.6%). On incubation of horse high-Mr kininogen with bovine and horse plasma kallikreins, several fragments which contained extremely high levels of histidine, were liberated, in addition to kinin. After the liberation of kinin and histidine-rich fragments, a protein free of kinin and its fragments was isolated. This protein consisted of two polypeptide chains, heavy chain and light chain, which are bridged by disulfide bonds. The molecular weight and amino acid composition of the heavy chain and the light chain from horse high-Mr kininogen were very similar to those of the heavy and light chains from bovine high-Mr kininogen, respectively. From these results, it was revealed that horse high-Mr kininogen is quite similar to bovine high-Mr kininogen in terms of their physicochemical and chemical properties, although they are immunologically distinguishable.     

Human high molecular weight kininogen. Studies of structure-function relationships and of proteolysis of the molecule occurring during contact activation of plasma.

Human high Mr kininogen was purified from normal plasma in 35% yield. The purified high Mr kininogen appeared homogeneous on polyacrylamide gels in the presence of sodium dodecyl sulfate and mercaptoethanol and gave a single protein band with an apparent Mr = 110,000. Using sedimentation equilibrium techniques, the observed Mr was 108,000 +/- 2,000. Human plasma kallikrein cleaves high Mr kininogen to liberate kinin and give a kinin-free, two-chain, disulfide-linked molecule containing a heavy chain of apparent Mr = 65,000 and a light chain of apparent Mr = 44,000. The light chain is histidine-rich and exhibits a high affinity for negatively charged materials. The isolated alkylated light chain quantitatively retains the procoagulant activity of the single-chain parent molecule. 125I-Human high Mr kininogen undergoes cleavage in plasma during contact activation initiated by addition of kaolin. This cleavage, which liberates kinin and gives a two-chain, disulfide-linked molecule, is dependent upon the presence of prekallikrein and Factor XII (Hageman factor) in plasma. Addition of purified plasma kallikrein to normal plasma or to plasmas deficient in prekallikrein or Factor XII in the presence or absence of kaolin results in cleavage of high Mr kininogen and kinin formation.     

Selective removal of the carboxyl-terminal tail end of the Dictyostelium myosin II heavy chain by chymotrypsin

Dictyostelium myosin II is a conventional myosin consisting of two heavy chains of 243,000 Da and two pairs of light chains of 16,000 and 18,000 Da. In this paper, we show that the heavy chain of myosin II can be rapidly and selectively cleaved by chymotrypsin to yield two fragments with molecular weights of 195,000 and 38,000 Da as estimated from sodium dodecyl sulfate-polyacrylamide gels. Chymotryptic cleavage at this site occurs most readily in the absence of salt and is greatly inhibited as the salt concentration is increased from 0 to 60 mM. Amino acid sequence analysis of the small fragment demonstrates that its amino terminus corresponds to lysine 1826 of the myosin II heavy chain. If the fragment extends to the carboxyl terminus of the myosin II heavy chain, it would have a molecular weight of 33,700. Upon digestion of myosin II which has been phosphorylated with a high molecular weight Dictyostelium myosin heavy chain kinase (C?té, G.P., and Bukiejko, U. (1987) J. Biol. Chem. 262, 1065-1072), all of the phosphate is recovered on the 33,700-Da tail-end fragment. Chymotrypsin-cleaved myosin II is shown to be capable of forming filaments at salt concentrations between 20 and 100 mM as judged by its ability to be sedimented by centrifugation. Only the large fragment of myosin II is found in the pellet; the 33,700-dalton fragment remains soluble. Chymotrypsin-cleaved myosin II can bind to actin and displays a high Ca2+-activated ATPase activity but has very low actin-activated ATPase activity.     

Proteinase-sensitive sites on isolated rabbit dystrophin.

Dystrophin was isolated from the purified large oligomeric dystrophin complex with its associated proteins (DC) of rabbit skeletal muscle by alkaline dissociation followed by gel filtration to remove the associated proteins. Isolated dystrophin and DC were subjected to digestion with calpain or alpha-chymotrypsin, and the generated polypeptide fragments were studied by immunoblot analysis using seven kinds of antibodies raised against antigens corresponding to various regions from the N- to the C-terminal of human dystrophin. For some fragments, the amino acid sequences at the N-termini were determined. Two proteinases, which bear distinct specificities, generated very similar fragments from purified dystrophin with or without the associated proteins. The cleavage sites found by mapping the fragments onto the dystrophin molecule were similar to those found in a previous study using crude mouse muscle cell membrane fraction [Koenig, M. & Kunkel, L.M. (1990) J. Biol. Chem. 265, 4560-4566]. On the basis of these results, we concluded that dystrophin has several unique proteinase-sensitive sites.     

Mapping of the discontinuous H-kininogen binding site of plasma prekallikrein. Evidence for a critical role of apple domain-2.

Plasma prekallikrein, a zymogen of the contact phase system, circulates in plasma as heterodimeric complex with H-kininogen. The binding is mediated by the prekallikrein heavy chain consisting of four apple domains, A1 to A4, to which H-kininogen binds with high specificity and affinity (K(D) = 1.2 x 10(-8) M). Previous work had demonstrated that a discontinuous kininogen-binding site is formed by a proximal part located in A1, a distal part exposed by A4, and other yet unidentified portion(s) of the kallikrein heavy chain. To detect relevant binding segment(s) we recombinantly expressed single apple domains and found a rank order of binding affinity for kininogen of A2 > A4 approximately A1 > A3. Removal of single apple domains in prekallikrein deletion mutants reduced kininogen binding by 21 (A1), 64 (A2), and 24% (A4), respectively, whereas deletion of A3 was without effect. Transposition of homologous A2 domain from prekallikrein to factor XI conferred high-affinity kininogen binding from the former to the latter. The principal role of A2 for H-kininogen docking to the prekallikrein heavy chain was further substantiated by the finding that cleavage of a single peptide bond in A2 drastically diminished the H-kininogen binding affinity. Furthermore, the epitope of monoclonal antibody PKH6 which blocks kallikrein-kininogen complex formation with an IC(50) of 8 nM mapped to the center portion of domain A2. Our data indicate that domain A2 and two flanking sequence segments of A1 and A4 form a discontinuous binding platform for H-kininogen on the prekallikrein heavy chain. Domain-specific antibodies directed to these critical sites efficiently interfered with contact phase-induced bradykinin release from H-kininogen.     

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)     

Cloning and sequence analysis of human genomic DNA encoding gamma subunit precursor of muscle acetylcholine receptor

The limited proteolysis of human low-molecular-mass kininogen by kallikrein from tissue sources has been studied. Porcine pancreatic kallikrein applied in catalytic amounts split the kininogen molecule (apparent mass 68 kDa) with the release of lysyl-bradykinin (1 kDa). This generated a nicked kininogen molecule with a heavy chain and light chain interconnected via disulfide bridging. Following reductive cleavage of the disulfide bonds, the heavy chain of apparent mass 62 kDa was isolated by preparative sodium dodecyl sulfate electrophoresis, and the light chain of 5 kDa by reversed-phase high-performance liquid chromatography. The light chain was found to be composed of 38 amino acids with a single half-cystine residue. Amino-terminal sequence analysis revealed that the light chain is derived from the carboxy terminus of the kininogen molecule [Lottspeich et al. (1984) Eur. J. Biochem. 142, 227-232]. Immunological characterization of the isolated L chain indicated that it harbours antigenic site(s) unique for low-Mr kininogen as well as sites common to high-Mr and low-Mr kininogen.     

Fragmentation of the human transplantation antigen heavy chain by limited proteolysis, acid cleavage, and cyanogen bromide treatment.

Highly purified, papain-solubilized HLA-A, -B, and -C antigens comprising a mixture of a great number of allelic forms from at least three loci have been fragmented by limited proteolysis, acid cleavage, and cyanogen bromide treatment. Limited proteolysis of 125I-labeled HLA-A, -B, and -C antigens with trypsin, chymotrypsin, thermolysin, and pepsin resulted in the production of two large fragments. One fragment was associated with beta 2-microglobulin and contained all of the carbohydrate. The other fragment, which had a molecular weight of about 13,000, is most probably derived from the COOH-terminal part of the heavy chain. Acid cleavage of the HLA antigen heavy chain gave rise to two main fragments with molecular weights of 22,000 and 11,000. Both fragments contained disulfide bonds. Two minor components, representing further cleavage products of the 22,000-dalton fragment, were also observed. Cleavage of the HLA antigen heavy chain at methionyl residues gave rise to one carbohydrate-containing, cysteine-free 14,000-dalton fragment and one 20,000-dalton fragment that contained all cysteines but no carbohydrate. NH2-terminal amino acid sequence analyses demonstrated that the 22,000-dalton acid cleavage fragment and the 14,000-dalton cyanogen bromide fragment were derived from the NH2-terminal part of the HLA antigen heavy chain.     

Fine mapping of the high molecular weight kininogen binding site on blood coagulation factor XI through the use of rationally designed synthetic analogs.

Using immunological and chemical cleavage techniques, we have previously identified a domain contained within residues Phe56-Ser86 in the first tandem repeat (A1) of the heavy chain of factor XI which binds high Mr kininogen (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1990) J. Biol. Chem. 265, 4149-4154). We have now chemically synthesized peptides from corresponding homologous regions in the second (A2), third (A3), and fourth (A4) tandem repeats of the heavy chain (A2: Asn145-Ala176; A3: Asn235-Arg266; and A4: Gly326-Lys357). These peptides had no effect on the binding of factor XI to high Mr kininogen. Because of a lack of detailed structural information for the A1 domain, a molecular model of this region was constructed. This hypothetical model made distinct and testable predictions regarding potential surfaces and concomitant secondary structure. Specifically, the resulting structure depicted two juxtaposed beta-stranded stem-loops that, in conjunction with biological information, constitute a candidate surface for contact with high Mr kininogen. The hypothetical A1 model was, consequently, used as a predictive template in the rational design of two synthetic peptides (Val59-Arg70 and Asn72-Lys83). When both these peptides were added together and the binding of factor XI to high Mr kininogen was examined, a synergistic inhibitory effect was observed compared with each peptide added individually. Our data are consistent with the notion that the sequence of amino acids from Val59-Lys83 of the heavy chain of factor XI contains two antiparallel beta-strands connected by beta-turns that together comprise a continuous surface utilized for the binding of high Mr kininogen.     

Dynein isoforms in sea urchin eggs

Biochemical and immunological analysis of unfertilized sea urchin eggs has revealed the presence of at least two distinct isoforms of cytoplasmic dyneins, one soluble and the other microtubule-associated. The soluble enzyme is a 20 S particle with a MgATPase activity that can be activated 5-fold by nonionic detergents. It contains heavy chain polypeptides that 1) comigrate with the dynein heavy chains of embryonic cilia; 2) cross-react with antibodies against flagellar dynein; and 3) are cleaved by UV irradiation in the presence of MgATP and sodium vanadate into specific peptide fragments. The soluble egg dynein is, therefore, closely related to axonemal dynein and may be a ciliary precursor. Egg microtubule preparations contain a distinct dynein-like polypeptide, previously designated HMr-3 (Scholey, J.M., Neighbors, B., McIntosh, J.R., and Salmon, E.D. (1984) J. Biol Chem. 259, 6516-6525). HMr-3 binds microtubules in an ATP-sensitive fashion; it sediments at 20 S on sucrose density gradients, and it is susceptible to vanadate-sensitized UV cleavage. However, HMr-3 can be distinguished from the soluble cytoplasmic dynein on the basis of its weak cross-reactivity with antiflagellar dynein antibodies, its heavy chain composition on high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis, its low specific ATPase activity, and the molecular weight of its vanadate-induced UV cleavage fragments. HMr-3 may represent a dynein-like polypeptide that is distinct from the pool of ciliary dynein precursors.     

The fragments of bovine high molecular weight kininogen promote osteoblast proliferation in vitro

High molecular weight (HMW) kininogen is known to be a large plasma protein and cleaved by plasma proteinase kallikrein, then it generates four fragments in the blood coagulation cascade: heavy chain, bradykinin, fragment 1.2, and light chain. The fragment 1.2 has also been found in the basic protein fraction of bovine milk as a bioactive protein which promotes osteoblast proliferation. The milk basic protein has been shown to be a multi functional edible protein which promotes bone formation and inhibits bone resorption. In the present study, we purified the fragment 1.2 from bovine plasma and assessed it could promote osteoblast proliferation and posses the activity after pepsin digestion. Purified plasma HMW kininogen did not promote the proliferation, however, the kallikrein-cleaved HMW kininogen promoted the proliferation. The fragment 1.2, purified from the proteolysate, also promoted the proliferation. The pepsin digestion was performed according to the method of the assessment of allergenesity of genetically modified crops. After pepsin digestion, the fragment 1.2 generated resistant fragments and showed the promoting activity of osteoblast proliferation. These results suggest that the enzymatically-digested fragments of bovine HMW kininogen are able to be a naturally occurred active protein that promotes the bone formation by oral administration.     

Immunochemical analysis of the domain structure of CAD, the multifunctional protein that initiates pyrimidine biosynthesis in mammalian cells

CAD, is a multidomain polypeptide, with a molecular weight of over 200,000, that has glutamine-dependent carbamyl-phosphate synthetase, aspartate transcarbamylase, and dihydroorotase activity as well as regulatory sites that bind UTP and 5-phosphoribosyl 1-pyrophosphate. The protein thus catalyzes the first three steps of de novo pyrimidine biosynthesis and controls the activity of the pathway in higher eukaryotes. Controlled proteolysis of CAD isolated from Syrian hamster cells, cleaves the molecule into seven major proteolytic fragments that contain one or more of the functional domains. The two smallest fragments, which had molecular weights of 44,000 and 40,000, corresponded to the fully active dihydroorotase (DHO) and aspartate transcarbamylase (ATC) domains, respectively, but the larger fragments have not been previously characterized. In this study, enzymatic assays of partially fractionated digests and immunoblotting with antibodies specifically directed against the purified ATC domain, the purified dihydroorotase domain and an 80-kDa fragment of the putative carbamyl-phosphate synthetase domain established the precursor-product relationships among all of the major proteolytic fragments of CAD. These results indicate that 1) only the intact molecule had all of the functional domains, 2) a species with a molecular weight of 200,000 was produced in the first step of proteolysis which had glutamine-dependent carbamyl-phosphate synthetase and dihydroorotase activity, but neither aspartate transcarbamylase activity nor the antigenic determinants present on the isolated ATC domain, and 3) cleavage of the 200-kDa species produced a species, with a molecular mass of 150,000 which lacked both aspartate transcarbamylase and dihydroorotase domains. This 150-kDa species, containing the postulated carbamyl-phosphate synthetase, glutamine, and regulatory (UTP, 5-phosphoribosyl 1-pyrophosphate) domains, had two elastase-sensitive sites that divided this region of the polypeptide chain into 10-, 65-, and 80-kDa segments. The location of the functional sites on these segments has not yet been established. The immunochemical analysis also revealed the existence of possible precursors of the stable aspartate transcarbamylase and dihydroorotase domains, suggesting that the chain segments connecting the functional domains of CAD are extensive and that the overall size of the intact polypeptide chain has been underestimated. On the basis of these studies we have proposed a model of the domain structure of CAD.     

Characterization of fragments of human albumin purified by Cibacron blue F3GA affinity chromatography.

Controlled limited proteolysis of human plasma albumin (0.3 mM; 37 degrees C; 15 min; pH 3.7) with pepsin [pepsin/albumin, 1:1000 (w/w)] in the presence of octanoic acid (4.2 mM) yields at least 14 fragments in the range of 5000--56000 Da. By utilizing a combination of conventional and affinity-chromatographic procedures, two fragments with mol. wts. 25000 and 27000 were purified to more than 99% homogeneity. The larger fragment consists of a continuous polypeptide chain and has been shown to contain the primary bilirubin-binding site. The small fragment contains an internal cleavage site. On the basis of amino acid compositions, N-terminal sequences, C-terminal sequences, molecular weights and other internal markers the locations of these fragments within the known sequence of human albumin were determined to be residues 49--308 for the 27000 Da peptide and 309--585 for the 25000 Da peptide. Peptide 309--585 contains an internal cleavage site and appears to be missing residues 408--423. These non-overlapping fragments should be useful for investigations of individual ligand-binding sites and for the determination of antigenic sites.     

Characterization of a cDNA clone coding for human testis membrane cofactor protein (MCP,CD46)

Membrane cofactor protein (MCP) is a complement regulatory protein that acts as a cofactor for the cleavage of C3b and C4b by the serine protease factor I. We have previously reported the characterization of a functional MCP molecule on the acrosomal membrane. This protein migrated as a single band with a molecular weight of 40,000 Da, which is 10,000–20,000 Da smaller than the known MCP molecules, and is devoid of N-and O-linked sugars. We have proposed that the difference in molecular weight resulted from the lack of sugars. To investigate if this is due to the absence of glycosylation sites, we have characterized a cDNA clone from a human testis cDNA library. This cDNA corresponds to a peculiar MCP form previously described, which is characterized by the presence of the serine/threonine/proline-rich exon C (STP^C) and the cytoplasmic tail known as CYT², and we conclude that the absence of mature oligosaccharide of the sperm MCP cannot be totally attributed to a defect of N- and O-glycosylation sequences but rather reflects an alteration of the mechanisms of glycosylation in spermatozoa. The presence of functional MCP on the acrosomal membrane, as well as the other complement regulatory protein, decay-accelerating factor, strongly suggests that these proteins may act concomitantly to protect the acrosome-reacted spermatozoa from the attack of the complement present in the female genital tract. © 1993 Wiley-Liss, Inc.     

Qualitative analysis of skeletal myosin as substrate of Ca2+-activated neutral protease: comparison of filamentous and soluble, native, and L2- deficient myosin

Ca2+ -activated neutral protease (CAF) was capable of degrading myosin over a 200-fold range of protease concentrations. CAF selected the heavy chain of myosin, although either prolonged exposure to or high concentrations of the protease degraded the L1, but not the L2 or L3, light chains of myosin. The following results indicated that during the first hour of digestion, under conditions where native myosin was the substrate, CAF selected for the "head" region of the myosin heavy chain: (a) large heavy chain fragments of identical molecular weight were produced from filamentous and from soluble myosin; (b) light meromyosin was not a substrate; (c) agents known to bind to the head of myosin (actin, MgATP, and L2) had both a qualitative and quantitative effect on degradation; and (d) similar cleavage sites could be demonstrated for myosin and for heavy meromyosin (HMM) despite the fact that HMM was a much poorer substrate than myosin. This observation is interpreted as an indication that the conformation of myosin heavy chain is altered in the preparation of HMM. The principal cleavage sites on the heavy chain of myosin were 20,000, 35,000 and 50,000 D from the N-terminus, producing large fragments with molecular weights of 180,000, 165,000, and 150,000 which comprised a "nicked" species of myosin. This nicked species retained both normal solubility properties and normal hydrolytic activities. For this reason, it is concluded that "nicked myosin" is an important pathophysiological species.     

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)     

Determination of the bifunctional properties of high molecular weight kininogen by studies with monoclonal antibodies directed to each of its chains

In normal human plasma two forms of kininogen exist, low molecular weight kininogen (LMWK) and high molecular weight kininogen (HMWK). When these proteins are cleaved they are found to have a common heavy chain and bradykinin, but each has a unique light chain. Monoclonal antibodies to the heavy and light chains of HMWK have been developed, and the effects of each on the function of this protein are defined. Initial studies showed that an antibody, C11C1, completely neutralized the coagulant activity of plasma HMWK whereas another antibody, 2B5, did not. On a competitive enzyme-linked immunosorbent assay (CELISA) the C11C1 antibody was consumed by kininogen antigen in normal plasma but not by kininogen antigen in HMWK-deficient plasma. On immunoblot, the C11C1 antibody recognized one kininogen protein in normal plasma and did not recognize any kininogen antigen in HMWK-deficient plasma. These combined studies indicated that the C11C1 antibody was directed to an epitope on the unique 46-kDa light chain of HMWK. In contrast, the 2B5 antibody on a CELISA was consumed by kininogen antigen in both normal plasma and HMWK-deficient plasma but not by total kininogen-deficient plasma. On immunoblot, the 2B5 antibody recognized both kininogens in normal plasma but only LMWK in HMWK-deficient plasma. These combined studies indicated that the 2B5 antibody was directed to the common 64-kDa heavy chain of the plasma kininogens. Utilizing direct binding studies or competition kinetic experiments, the 2B5 and C11C1 antibodies bound with high affinity (1.71 and 0.77 nM, respectively) to their antigenic determinants on the HMWK molecule. The 2B5 antibody did neutralize the ability of HMWK to inhibit platelet calpain. These studies with monoclonal antibodies directed to each of the HMWK chains indicate that HMWK is a bifunctional molecule that can serve as a cofactor for serine zymogen activation and an inhibitor of cysteine proteases.