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)     

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.     

Structural organization of the human kininogen gene and a model for its evolution

The entire human kininogen gene has been isolated as a set of overlapping genomic DNA fragments, and the 11 exons encompassing approximately 27 kilobase pairs have been mapped by restriction enzyme analysis and nucleotide sequence determination. The nine 5'-terminal exons encode the 5'-untranslated region and the protein-coding region for the signal peptide and the heavy chain, which are common for high molecular weight (HMW) and low molecular weight (LMW) prekininogen mRNAs. Exon 10 consists of the common sequence for bradykinin and the immediately following unique sequence for HMW prekininogen mRNA. Exon 11 is then located following a 90-nucleotide sequence downstream from exon 10 and precisely specifies the sequence unique to LMW prekininogen mRNA. This, together with the hybridization analysis of total human cellular DNA, leads us to conclude that human HMW and LMW prekininogen mRNAs are produced from a single gene as a consequence of alternative RNA processing events. The structural analysis of the kininogen gene also shows that each of the nine 5'-terminal exons discretely specifies the nine protein domains observed in the amino-terminal portion of the kininogens. Furthermore, these nine genetic domains can be characterized by a thrice repeated pattern of three genetic segments, and two sets of these three domains, encompassing exons 3-5 and exons 6-8, are most closely related to each other. Therefore, we have proposed two successive duplication mechanisms as a model for the generation of the structure of the kininogen gene.     

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)     

Kininogenase activity of alpha and beta/gamma forms of bovine thrombin

The kininogenase activity of alpha- and beta/gamma-forms of bovine thrombin with respect to the high molecular weight (HMW) and low molecular weight (LMW) human kininogens was studied. It was shown that both forms of the enzyme split of bradykinin from these kininogens. The kininogenase activity of alpha-thrombin is completely blocked by the highly specific thrombin inhibitor Nalpha-dansyl-L-arginine-p-ethylpiperidineamide, but not by the soya bean trypsin inhibitor. The alpha- and beta/gamma-forms of thrombin hydrolyze HMW (Km(app) = 4.5 and 3.3 microM, respectively) and LMW (Km(app) = 10.1 and 4.7 microM, respectively). The specific constants (kcat/Km(app) ) for thrombin with respect to the substrates differ about 7-fold, predominantly due to the high catalytic rates of HMW as compared to LMW; the kcat values are 0.18 and 0.06 min-1, respectively. alpha-Thrombin upon a long-term (over 1 hour) exposure to HMW, besides bradykinin, splits off the product inhibiting the kininogenase activity of thrombin. No differences in the specificity of the beta/gamma-form of thrombin with resect to HMW and LMW were detected.     

J chain in Rana catesbeiana high molecular weight Ig

A polypeptide homologous to human and mouse J chain has been identified in the high molecular weight (HMW) Ig of the bullfrog, Rana catesbeiana. In previous studies, we had detected a component that was similar in size to mammalian J chains and that, relative to L chains, migrated rapidly to the anode in alkaline-urea PAGE; however, its mobility was less than that of mammalian J chains. We now demonstrate that this component is covalently linked to the H chain of R. catesbeiana HMW Ig. All of the disulfide bridges of this polypeptide, like those of human and mouse J chain, can be cleaved by reducing agents even in the absence of denaturing solvents. The putative frog J chain was isolated by a procedure that did not require preliminary purification of the HMW Ig. The chain differed in amino acid composition from L chains but resembled J chains from several other species. Tryptic peptides were isolated and sequenced. Except for a single heptapeptide, the peptides could be aligned by virtue of their similarity to segments of human and mouse J chain. Of the 116 residues that were placed, 55 were identical with residues in human J chain and 60 with residues in mouse J chain. The six cysteine residues identified in the frog J chain are at the same positions as six of the eight cysteines in the human and mouse J chains. The results indicate significant conservation in structure between amphibian and mammalian Ig J chains.     

Studies on human kininogens. I. Isolation, characterization, and cleavage by plasma kallikrein of high molecular weight (HMW)-kininogen.

1. Human high molecular weight (HMW)-kininogen was highly purified from human plasma by chromatographies on QAE-Sephadex A-50 and CM-Sephadex C-50. Human HMW-kininogen thus purified was a mixture of a single chain and a disulfide-linked pair of chains. Human HMW-kininogen is an acidic glycoprotein having a molecular weight of 120,000. The amino acid composition of human HMW-kininogen is quite similar to that of bovine HMW-kininogen. 2. We investigated whether the liberation of kinin from human HMW-kininogen by human plasma kallikrein was accompanied by liberation of histidine-rich fragments, as observed with bovine HMW-kininogen (Han et al. (1975) J. Biochem. 77, 55--68). After prolonged incubation of human HMW-kininogen and human plasma kallikrein followed by gel-filtration on Sephadex G-50, a fragment of molecular weight 8,000 was isolated together with bradykinin. However, the histidine content of the fragment was not as high as that in the bovine fragments. Most of the histidine in human HMW-kininogen was recovered in the kinin-free protein, and the light chain of kinin-free protein was found to be rich in histidine compared with the heavy chain. These results suggest that the histidine-rich sequence in human HMW-kininogen is not released by the action of human plasma kallikrein, but remains bound to the light chain of kinin-free protein.     

Localization of DNA sequences governing alternative mRNA production of rat kininogen genes

The two types of the rat kininogen genes show different modes of mRNA production. The K gene encodes two distinct mRNAs for high molecular weight (HMW) and low molecular weight (LMW) kininogens. These two mRNAs are generated by differential usage of the 3'-terminal exon (LMW exon) and the one next to this exon (HMW exon) through alternative polyadenylation and splicing. In contrast, the two T genes selectively generate the LMW form of the mRNA, although the T genes are extremely homologous to the K gene, including the sequence (psi HMW region) corresponding to the HMW exon of the K gene. In this study, we constructed a series of chimeric kininogen genes by exchanging equivalent restriction fragments of the K and T genes and examined the sequences and the mechanisms governing the different expression patterns of the kininogen genes by introducing the chimeric genes into heterologous COS cells. The results indicate that the formation of the two forms of the mRNA is controlled by two separate 3' sequences of the kininogen genes. One is located within the internal sequence of the HMW/psi HMW region, whereas the other is within the LMW exon and its preceding region. Our data also suggest that the different expression patterns of the kininogen genes are primarily governed by differing splicing efficiency.     

gamma and gamma' chains of human fibrinogen are produced by alternative mRNA processing

cDNAs and the genomic DNA coding for the gamma and gamma' chains of human fibrinogen have been isolated and characterized by sequence analysis. The cDNAs coding for the gamma and gamma' chains share a common nucleotide sequence coding for the first 407 amino acid residues in each polypeptide chain. The predominant gamma chain contains an additional four amino acids on its carboxyl-terminal end (residues 408-411). These four amino acids, together with the 3' noncoding sequences, are encoded by the tenth exon. Removal of the ninth intervening sequence following the processing and polyadenylation reactions yields a mature mRNA coding for the predominant gamma chain. The less prevalent gamma' chain contains 20 amino acids at its carboxyl-terminal end (residues 408-417). These 20 amino acids are encoded by the immediate 5' end of the ninth intervening sequence. This results from an occasional processing and polyadenylation reaction that occurs within the region normally constituting the ninth intervening sequence. Accordingly, the gene for the gamma chain of human fibrinogen gives rise to two mRNAs that differ in sequence on their 3' ends. These mRNAs code for polypeptide chains with different carboxyl-terminal sequences. Both of these polypeptides are incorporated into the fibrinogen molecule present in plasma.     

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.     

Isolation of a human cDNA for alpha 2-thiol proteinase inhibitor and its identity with low molecular weight kininogen

A lambda gt11 cDNA library containing DNA inserts prepared from human liver mRNA has been screened with an antibody to human alpha 2-thiol proteinase inhibitor that was isolated from fresh plasma. Eighteen positive clones were isolated from one million phage, and each was plaque purified. The cDNA insert of one of these phage was sequenced and shown to code for alpha 2-thiol proteinase inhibitor as identified by a partial amino acid sequence of the light chain of alpha 2-thiol proteinase inhibitor. This cDNA insert contained 1529 base pairs coding for the complete alpha 2-thiol proteinase inhibitor. It included 45 base pairs of 5' noncoding sequence, 1281 base pairs that code for pre alpha 2-thiol proteinase inhibitor, a stop codon, 160 base pairs of 3' noncoding sequence, and 40 base pairs of poly(A) tail. The noncoding sequence on the 3' end contained a potential recognition site (AATAAA) for processing and polyadenylation of precursor messenger RNA. The amino acid sequence of alpha 2-thiol proteinase inhibitor deduced from the cDNA showed a striking similarity (overall homology at 74%) to that of bovine low molecular weight (LMW) kininogen, including two internally repeated sequences and a nonapeptide sequence of bradykinin. These data clearly indicated that alpha 2-thiol proteinase inhibitor and LMW kininogen are identical. This was further supported by immunological cross-reactivity between alpha 2-thiol proteinase inhibitor and LMW kininogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Various properties and kinetics of interaction of high and low molecular weight human kininogens with human plasma kallikrein

A relatively simple procedure for isolation and purification of human blood plasma kallikrein (HPK) by QAE-Sephadex A-50 SP-Sephadex C-50 and affinity chromatography on Sepharose 4B with immobilized soybean trypsin inhibitor with the activity yield of about 40% has been developed. The method allows for simultaneous isolation of low (LMW) and high molecular weight (HMW) kininogens from the same HPK sample. HPK preparations are homogeneous upon 7.5% polyacrylamide gel electrophoresis in the presence of 0.1% SDS; its Mr is 90,000. After treatment with beta-mercaptoethanol, HPK dissociates into two fragments with Mr of 43,000 and 37,000. HPK preparations have high specific activities of esterase (31 microM/min), amidase (78 microM/min), and kininogenase (420 micrograms equiv. bradikinin/min). The high degree of protein purification was demonstrated by titration of active centers with 4-methylumbelliferylguanidine benzoate. The values of equilibrium dissociation constants for the HPK complex with aprotinin (Ki) equal to 1 X 10(-8) M (ethyl ester of N-alpha-benzoyl-L-arginine) and 1,5 X 10(-9) M (HMW) were determined. The kinetics of HPK-induced liberation of bradikinin from purified preparations of HMW and LMW was studied. The kinetic parameters (Km, kcat and kcat/Km) of this reaction suggest a high affinity of HPK for HMW, but not for LMW. LMW does not compete with HMW for the enzyme active center. It is assumed that LMW is not a physiological substrate for HPK.     

A set of U1 snRNA-complementary sequences involved in governing alternative RNA splicing of the kininogen genes

The rat K and T kininogen genes show different modes of mRNA production. The K gene encodes two distinct mRNAs for high molecular weight (HMW) and low molecular weight (LMW) kininogens. These two mRNAs are generated by differential usage of the 3'-terminal exon (LMW exon) and the exon next to and upstream from the LMW exon (HMW exon) through alternative splicing and polyadenylation. In contrast, the T gene generates one mRNA by using selectively the LMW exon, although the T gene is extremely homologous to the K gene. In this study, we constructed a series of chimeric kininogen genes by not only exchanging equivalent restriction fragments of the two genes but also replacing nucleotides that differ between the two genes. We then examined the sequences and the mechanisms governing the different expression patterns of the two genes by transfecting the chimeric genes into heterologous COS cells. The results indicated that the different expression patterns of the K and T genes are governed by two separate internal sequences of the HMW and LMW exons. The internal HMW sequence contains a set of five repetitive sequences, and these repetitive sequences are highly complementary to the 5' portion of U1 snRNA. Furthermore, the nucleotide differences in the U1 snRNA-complementary sequences between the K and T genes have marked effects on the relative formation of the HMW and LMW mRNAs; this indicates that the repetitive sequences complementary to U1 snRNA play a crucial role in determining the relative expression of the two mRNAs. Based on these findings, we discuss a novel mechanism for alternative RNA processing, in which splicing efficiency is controlled by the interaction of U1 small nuclear ribonucleoproteins and the U1 snRNA-complementary repetitive sequences of the kininogen pre-mRNA.     

Expression of the high molecular weight fibroblast growth factor-2 isoform of 210 amino acids is associated with modulation of protein kinases C delta and epsilon and ERK activation

The high molecular weight (HMW) fibroblast growth factor (FGF)-2 isoform of 210 amino acids initiated at a CUG start codon possesses a nuclear localization sequence and is not secreted. In contrast, the low molecular weight (LMW) isoform of 155 amino acids initiated at the AUG start codon can be secreted and activates the cell surface FGF receptors. The two isoforms possess different biological properties; however, little is known about the intracrine regulatory mechanisms involved in the biological effects of the HMW FGF-2 isoform. Using pancreatic cells stably transfected with cDNAs leading to the expression of either the HMW FGF-2 (A3 cells) or the LMW form (A5 cells), we provide evidence that the two FGF-2 isoforms differentially modulate PKC levels. The LMW FGF-2 up-regulated the PKC epsilon levels by 1.6-fold; by contrast the HMW isoform down-regulated the level of this PKC isotype by about 3-fold and increased the amount of PKC delta by 1.7-fold. PKC mRNAs were also modified, suggesting that PKC expression was regulated at a pretranslational level. Additionally, expression of different levels of the HMW FGF-2 with an inducible expression system confirmed the role of this isoform on PKC delta and epsilon expressions. Increased activation of ERK-1 and -2 was also observed in cells expressing the HMW FGF-2. By using different PKC inhibitors and a dominant negative PKC delta, it was found that ERK activation was PKC delta-dependent. These data indicate that expression of HMW FGF-2 can modify PKC levels by acting at the intracellular level and that the overexpression of PKC delta induces ERK-1/2 activation. The expression of a dominant negative FGFR1 did not reduce ERK-1/2 activation by the HMW FGF-2, suggesting that ERK activation does not require FGFR activity. The signaling cascade downstream of ERK might be involved in the known mitogenic effect exerted by this FGF-2 isoform.     

Kinin release from kininogens by calpains

During the investigation of inhibitory activity of kininogens toward calpains [EC 3.4.22.17], we found that lysyl-bradykinin was liberated from both high molecular weight (HMW) and low molecular weight (LMW) kininogens by the action of the calpains. The kinin liberation occurred in a limited range of calpain to kininogen molar ratios of 0.5:1 to 8:1, and in that condition calpains were simultaneously inhibited 20 to 80% by kininogens. The maximum level of kinin release from HMW and LMW kininogens by calpain I was about 25% and that by calpain II was 20%. These results suggest that in case of inflammation the kininogens play two physiologically distinct roles by interaction with calpains: to release lysyl-bradykinin and to inhibit proteinase activity of calpains derived from the damaged tissues.     

High molecular weight renin identified in cytosol fractions of mouse renal cortices

In an attempt to confirm that high molecular weight renin was indeed true renin, we used a specific renin antibody and high performance liquid chromatography to determine characteristics of this protein. In mouse renin granules, renin was stored in a low molecular weight form of 38,000 daltons (LMW renin) and this molecular weight remained unchanged with application 20 mM of sodium tetrathionate. In the cytosol fraction of the renal cortex, LMW renin was partially converted to high molecular weight renin (HMW renin) of 65,000 daltons, as determined using tetrathionate. In both the LMW and HMW renin, enzymatic activity was completely neutralized by application of a specific antiserum of renin and an absolute amount of renin was identified by direct radioimmunoassay. The Km values of HMW and LMW renin were similar. Thus, LMW renin probably binds with renin binding substance and forms HMW renin.     

Role of calpains and kininogens in inflammation.

Neutrophil chemotactic activity was found in the autodigest of calcium dependent cysteine proteinase (calpain) I purified from human erythrocytes, an active peptide was isolated, and its structure was determined. It was an N-acetyl nonapeptide with the sequence: N-acetyl Ser-Glu-Glu-Ile-Ile-Thr-Pro-Val-Tyr. This peptide was identical with the N-terminal amino acid sequence of the large subunit of calpain I deduced from cDNA sequence, except that the peptide was lacking a methionine residue and was acetylated at the N-terminus. A number of N-acetyl peptides with N-terminal amino acid sequences of large and small subunits of calpains I and II were synthesized and their chemotactic activity was estimated. In addition to the N-acetyl nonapeptide from calpain I large subunit, several peptides of different lengths from the small subunit showed dose-dependent migrations of neutrophils. They include N-acetyl tetra, hepta, octa, nona and larger size peptides. Further, it was also revealed that when calpain was incubated with high molecular weight (HMW) or low molecular weight (LMW) kininogen, kinin liberation occurred with simultaneous inhibition of calpains by kininogens. These data suggest that chemical mediators generated from the calpain-kininogen system may participate in migration and accumulation of neutrophils to the inflammatory locus.     

Purification and immunochemical properties of human low molecular weight kininogen.

A low molecular weight (LMW) kininogen was isolated from pooled human serum by chromatography on DEAE-Sephadex A-50, CM-Sephadex C-50, Sephadex G-150, and Sephadex G-100. It was shown to be homogeneous by ultracentrifugation, polyacrylamide gel electrophoresis, and immunoelectrophoresis. The sedimentation coefficient, S020,W, of purified LMW kininogen was 3.85 s, and its molecular weight was determined to be 78,000 by Sephadex G-100 gel-filtration. The LMW kininogen contained 79.3% protein, 8.0% hexose, 3.9% hexosamine, and 4.9% sialic acid. In order to determine the immunochemical properties of LMW kininogen, specific antiserum was prepared in rabbits. The antigenic determinant of LMW kininogen was not related to the sialic acid and kinin moieties in the kininogen molecule, but could not be distinguished from that of high molecular weight (HMW) kininogen. In the quantitative single radial immunodiffusion test, a sialic acid-free LMW kininogen reacted to a greater extent with the antiserum than the native LMW kininogen. The kininogen level in human serum was estimated by single radial immunodiffusion. The antiserum cross-reacted with monkey serum, but not with sera from dogs, rats, and mice, horses, pigs, guinea pigs, oxen, and rabbits.     

Structural comparisons between chicken low molecular weight immunoglobulin heavy chains and human γ chains

Comparisons between chicken low molecular weight immunoglobulin (LMW Ig) and human heavy chains as to molecular weights, amino acid compositions, tryptic peptide maps, and CHO-peptide sequences revealed significant differences. The molecular weight of the chicken heavy chain, 60,000, is approximately 10,000 more than that for human , indicating up to 100 more amino acids, including three more cysteine residues. Tryptic peptide map comparisons revealed no common peptides as to mobilities and amino acid compositions. The sequence of the chicken heavy chain CHO-peptide, Gly-Trp-Val-Ser-Asx-Thr-Cys, exhibits little homology with the CHO-peptides or cysteine peptides of human or heavy chains. The implications of these structural differences between chicken and human heavy chains with regard to evolutionary relatedness and secondary biological functions are discussed.