Primary structure of guinea pig plasma prekallikrein

A full length guinea pig plasma prekallikrein (PK) cDNA was cloned from a liver cDNA library. The nucleotide sequence with 2242 bp was analyzed and the amino acid sequence with 618 residues was deduced. Kallikrein was purified from guinea pig plasma and cleavage site in the activation was determined. The amino acid sequence around the cleavage site -368Ile-Asp-Ala-Arg-Ile-Val-Gly-375Gly- differed from that of the human PK -368Thr-Ser-Thr-Arg-Ile-Val-Gly-375Gly-. Protease substrates containing penta-peptides which mimicked the sequence of the cleavage sites from P3 to P2' of guinea pig Hageman factor (HF) and PK were synthesized, and kinetic analyses of the hydrolysis by guinea pig activated HF (HFa) and kallikrein were carried out. The combination between HFa and the PK mimicking peptide provided the best kinetics. These results in part explain why the cascade activation of PK by HFa is predominant in the guinea pig system.     

Binding and dissociation of Hageman factor, prekallikrein and high molecular weight kininogen in human plasma during contact activation

A kaolin pellet was incubated with human plasma at room temperature and immediately washed to remove all unbound proteins. Whereas in normal plasma, 154 mU(PPAN) of kallikrein was bound to the kaolin surface, in Hageman factor (HF)-deficient plasma or normal plasma preincubated with polybrene the surface-bound kallikrein was undetected, while a trace of prekallikrein was bound to the kaolin surface. Dissociation of kinin and kallikrein from the kaolin surface occurs during varying periods of incubation of the kaolin suspension alone. The dissociation of the surface-bound kallikrein revealed two phases in a 15 min incubation: the first phase of dissociation which rapidly progressed until the kinin liberation reached a plateau was followed by the second phase where the kallikrein was slowly dissociated and kinin was no longer liberated. Treatment of the kaolin suspension with trypsin, plasmin or plasma kallikrein enhanced the kinin liberation and dissociation of kallikrein from the kaolin surface. Kallikrein-kinin-free high molecular weight (HMW) kininogen-activated HF complex, kalli-krein-kinin-free HMW kininogen complex, kallikrein, kinin-free HMW kininogen and two activated HFs were found on Sephacryl S-300 and Sephadex G-100 gel filtrations of the supernatant obtained after a 60 min incubation of the kaolin suspension alone. The ternary complex of kallikrein, kinin-free HMW kininogen and activated HF suggests the presence of prekallikrein-HMW kininogen-HF complex on the kaolin surface.     

Guinea pig or rabbit lung flavin-containing monooxygenases with distinct mobilities in SDS-PAGE are allelic variants that differ at only two positions.

Both guinea pig and rabbit express two variants of the 'lung' flavin-containing monooxygenase (FMO), observed as three distinct phenotypes based on mobility differences in SDS-PAGE. Samples of messenger RNA prepared from lungs of the two homozygous phenotypes of the guinea pig were used for the construction of two cDNA libraries. The libraries were screened with a cDNA encoding the rabbit lung FMO, and positive clones for each guinea pig lung FMO variant were isolated and sequenced. A full length clone from each library was found to encode a protein of 535 amino acids containing two pyrophosphate binding sites. Comparison of the sequences of the guinea pig and rabbit lung FMOs shows that their primary structures are 86% identical. The coding region sequences of the guinea pig variants differ at only two positions, and both differences result in amino acid substitutions. Sequence analysis has also been completed on a partially characterized variant of the rabbit lung FMO. As with the guinea pig, the nucleotide and amino acid sequences of the rabbit variants differ at only two positions. The cDNAs encoding the guinea pig variants were expressed in yeast. The activities of the enzymes are characteristic of the lung FMO, and the mobilities of the expressed enzymes are the same as those observed for the variants present in guinea pig pulmonary microsomal preparations. Similar to findings for the rabbit, analysis of genomic DNA indicates that the guinea pig lung FMO is associated with a single gene. The results of cDNA sequence analysis, expression in yeast, and analysis of genomic DNA indicate that the multiple lung FMOs in guinea pig and rabbit are allelic variants whose mobilities in SDS-PAGE are markedly altered by minimal changes in primary structure.     

Studies on kininogen bioassay in human and rat plasma by means of trypsin and guinea pig ileum

We studied further plasma kininogen bioassay, which is based on the cleavage of bradykinin from kininogen by trypsin and on bradykinin assay by guinea pig ileum. Bradykinin-like activities released from human and rat plasma in incubation did not reach constant levels at any trypsin concentrations from 200 to 2.500 micrograms/ml of plasma. With high trypsin concentrations bradykinin activity released, like the spontaneous formation of bioactivity when incubation is performed without trypsin, may be partly due to other bioactive materials than bradykinin. On the other hand, with trypsin concentration less than 500 micrograms/ml of plasma the formation of bradykinin from human plasma may be incomplete if the substrate concentration is increased. The sensitivity of guinea pig ileum to bradykinin in Tyrode medium is highest near the pH 7, while the response to unspecific bioactive material produced by trypsin rises when pH increases from 7 to 8. The temperature of Tyrode medium bath does not seem to be very critical. Though the method described here gives no absolute activities, in stable conditions we can obtain well comparable results on different kininogen concentrations in human and rat plasma.     

A new direct radioimmunoassay of rat urinary kininogen

A protein-binding radioimmunoassay (RIA) of rat low molecular weight (LMW) kininogen with the following characteristics has been developed: sensitivity, 2.5 ng/tube; inter-assay coefficient of variation, 12.4% (N = 28); and intra-assay coefficient of variation, 9.4% (N = 11). The new assay correlated (r = 1) with the determination of kinin equivalence of kininogen after trypsinization. The cross-reactivity of rabbit anti-rat LMW kininogen antibody was 2.5% with bovine LMW kininogen, 5.8% with rat plasma high molecular weight (HMW) kininogen, and none with kinin. Although the antibody appears to partially recognize des-kinin-kininogen, the low degree of cross-reactivity and the extremely low levels of kinin-free-kininogen allow accurate determination of total LMW kininogen in rat urines. The LMW kininogen formed 20% kinins with salivary kallikrein when compared with trypsin, suggesting that the preparation consists of both K- and T-kininogens (K = kallikrein susceptible; T = trypsin susceptible). The newly developed protein-binding RIA recognizes LMW kininogen of rat urine which consists of both K- and T-kininogens.     

Rodent kinin-forming enzyme systems—I: Purification and characterization of plasma kininogen

Low molecular weight (LMW) kininogen was purified 70-fold with a 16% yield from fresh rat plasma by DEAE-Sephadex chromatography, ammonium sulfate precipitation, Sephadex G-200 gel filtration, SP-Sephadex chromatography, CM-cellulose chromatography, and Sephadex G-200 gel filtration. Ferguson plots of polyacrylamide gel electrophoretic patterns revealed four bands with relative molecular weights of 64,000, 123,500, 252,436 and 357,900 (ratio of 1:2:4:6). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis provided a single protein band with a molecular weight of 72,000, suggesting that the four kininogen bands had been caused by the aggregation of a single oligomeric protein. The purified LMW rat kininogen Fraction B (3.9 μg bradykinin/mg) was used to elicit an antiserum in the rabbit. Monospecificity of the antiserum was demonstrated by immunoelec-trophoresis (Laurell rocket and Grabar methods) and, thus, the homogeneity of the kininogen was also. The purified kininogen (both Fractions A and B) formed kinin with human urinary kallikrein, rat urinary kallikrein and hog pancreatic kallikrein. Murphy-Sturm lymphosarcoma acid protease also formed kinin when incubated with the kininogen at pH 3.0. The isoelectric point for both fractions was at pH 4.3. Amino acid analyses showed the two kininogen fractions to be rich in acidic amino acids and to have a total carbohydrate content of 8.5% consisting of galactose (1.2 to 1.5%), mannose (1.9 to 2.1%), N-acetylglucosamine (4.3 to 5.1%), N-acetylgalactosamine (0.3%), and sialic acid (0.68%).     

Isolation of two functionally different kininogens from human plasma--separation from proteinase inhibitors and interaction with plasma kallikrein

A high (HMW) and a low (LMW) molecular weight kininogen were isolated in highly purified form from human plasma, using QAE-Sephadex chromatography, followed by ammonium sulfate precipitation, gel filtration through Sephadex G-200, re-precipitation with ammonium sulfate, CM-Sephadex and SP-Sephadex chromatography. The initial preparative step was done at room temperature and the remaining procedures at 4°. In aqueous media, the apparent molecular size of the HMW-kininogen was about four times the size of the LMW-kininogen (200,000 vs 50,000). During the process of purification, proteinase inhibitors were separated from the two kininogens: α₁-antitrypsin and α₂-macroglobulin from the LMW-kininogen preparations: Cl-inactivator and inter-α-trypsin inhibitor from the HMW-kininogen preparations. There was a well defined functional difference between the two kininogens with respect to kinin generation by plasma kallikrein. This enzyme released kinin at a much faster rate from the HMW-kininogen than from the LMW-kininogen. When equipotent preparations of kininogens were incubated for 10 min with kallikrein, 60 times more enzyme was required to release the same amount of kinin from the LMW-kininogen as from the HMW-kininogen.     

Characterization and cDNA cloning of halyxin, a heterogeneous three-chain anticoagulant protein from the venom of Agkistrodon halys brevicaudus.

We report upon the isolation, characterization, and cDNA cloning of an anticoagulant protein, halyxin from Agkistrodon halys brevicaudus venom. The protein exists as a 29kDa protein, and is separated into three chains on SDS-PAGE under reducing conditions. However, we cloned only two cDNAs encoding halyxin from the cDNA library of the snake venom gland, on the basis of the determined amino acid sequences. The complete amino acid sequences were deduced from their nucleotide sequences and named halyxin A (129 amino acid residues) and B chain (123 amino acid residues). The deduced amino acid sequence of halyxin A chain corresponds to the two smaller chains. Thus, it is considered that halyxin A chain could be synthesized as a single-chain protein that is subsequently cleaved to yield the mature two-chain protein. The amino acid sequence of halyxin is similar to that of other snake venom proteins of the C-type lectin superfamily, and prolongs plasma-clotting time. In the presence of Ca(2+) ions, halyxin binds to coagulation factors IX, X, IXa, and Xa, but not to other vitamin K-dependent coagulation factors. It also inhibits factor Xa in a non-competitive manner but does not affect other activated coagulation factors.     

Nucleotide sequences of putative cDNAs for guinea-pig monoamine oxidase

To study the molecular structure of guinea pig monoamine oxidase (MAO) and its phylogenetic relationship with other mammalian MAOs, we determined nucleotide sequences of putative MAO cDNAs isolated from guinea pig tissues. Both the 5- and 3-ends of the cDNAs were amplified using the RACE (rapid amplification of cDNA ends) method. The sequence (1924 bp) of a putative guinea-pig MAO-B cDNA covers a complete coding region that corresponds to 521 amino acids. We also analyzed a partial sequence of a putative guinea-pig MAO-A cDNA, which corresponds to 506 amino acids, but have left the region of 66 bp at the 3-end undetermined. The nucleotide and deduced amino-acid sequences of the putative guinea-pig MAO cDNAs showed the highest homology with that of human MAO cDNAs among the known mammalian MAO sequences. These results suggest that guinea-pig MAOs are structurally similar to human MAOs. Our molecular phylogenetic data support the idea that guinea pigs and rodents diverged before the separation between rodents and other lineage leading to Primates and Artiodactyla.     

我国近期分离的6株狂犬病病毒的糖蛋白基因序列分析

目的  分析我国近期分离的6株代表性狂犬病病毒（rabies virus,RV）的糖（G）蛋白基因序列,了解RV街毒株与疫苗株在核苷酸和氨基酸序列水平的差异。方法  对6个RV街毒株G基因进行逆转录和PCR扩增,经纯化、克隆、测序后获得6条G基因全长序列,采用生物信息学软件对G基因序列进行分析。结果  6个街毒株的G蛋白在核苷酸水平的同源性为97.7％～100％,在氨基酸水平的同源性为97.9%~100%;与CTN-181疫苗株的核苷酸序列同源性最高,为86.7%~87.2%,氨基酸序列同源性为92.7%~93.1%。结论  6个RV街毒株的G蛋白在核苷酸及氨基酸水平上均有变异,但与CTN-181疫苗株关系最近,表明CTN-181疫苗株可能为我国境内流行的狂犬病提供良好的预防作用。     

Characterization of coagulase from Staphylococcus intermedius

A protein coagulase was isolated from Staphylococcus intermedius 6131 using bovine prothrombin-Sepharose 4B and Bio-gel P-4 column chromatographies. Homogeneity was demonstrated by the formation of a single band in polyacrylamide gel electrophoresis and isoelectric focusing. The purified preparation possesses a molecular weight of 64,500, an isoelectric point of 4.1, consists of 615 total amino acid residues and demonstrates coagulase activity for human and rabbit fibrinogen, but does not show the activity for rat or guinea pig fibrinogens. This purified protein contains galactose and fucose, and the amino-terminal amino acid sequence was determined. The coagulase activity is inhibited by N-bromosuccinimide (NBS), suggesting that tryptophan is involved in this activity. The coagulase was heat stable to 80 degrees C and stable to pH over the range of 7-9. This is the first report of coagulase from Staphylococcus intermedius.     

Amino acid analysis of human von Willebrand factor fragments cleaved by porcine calpain II

Porcine calpain II (Ca2+-dependent cysteine proteinase) was found to cleave human von Willebrand Factor (HvWF) into two major fragments (approximately 165 kDa and 145 kDa) in sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). But, the cleavage was not complete even with a high enzyme/substrate ratio (w/w) of 1:10 for 20 hours. The two main fragments were isolated by electroelution, and the amino-terminal sequences as well as amino acid compositions were determined. Amino acid compositions of these two fragments were similar to those of equivalent parts of whole HvWF. The amino-terminal sequence of the 165 kDa fragment was SLSCRPPMVK corresponding to the amino-terminal residues of whole HvWF amino acid sequence which was recently determined by Titani et al. (Biochemistry 25:3171-84, 1986). The amino-terminal sequence of the 145 kDa fragment was SHRVNCDRGL beginning from 1151th amino acid of whole HvWF.     

Cleavage of porcine high molecular weight kininogen by the action of porcine pancreatic kallikrein

Pancreatic kallikrein (KK), converts the single chain high molecular weight kininogen (HK) into a two-chain molecule accompanied by kinin release. Further degradation was not observed. Molecular weights of the heavy (H)-chain and light (L)-chain were estimated to be 61 kDa and 56 kDa, respectively, by SDS-PAGE. The amino (N)-terminal sequences of intact HK, reduced and carboxymethylated (RCM)-H-chain, and RCM-L-chain were determined. These results indicate that porcine pancreatic KK initially cleaved the Arg-Ser bond of HK, to form a two chain molecule. Then pancreatic KK cleaved the Met-Lys bond yielding kallidin and a kinin-free protein, which was apparently equal in size to the nicked kininogen.     

A genomic region encoding stonefish (Synanceja horrida) stonustoxin beta-subunit contains an intron

The polymerase chain reaction (PCR) has been used to amplify a 1899 base pair fragment from stonefish genomic DNA. A comparison of the translated nucleotide sequence of this product with the separately determined N-terminal amino acid sequence of the β-subunit reveals the presence of a 416 bp intron at Gly 18. The nucleotide sequence following this intron encodes 476 amino acids whose sequence showed no homology to other known toxins. This region, however, contained amino acid sequences identical to internal peptide sequences determined separately from the toxin's β-subunit.     

Structure and other chemical characterizations of gila toxin,a lethal toxin from lizard venom

The complete primary structure of a lethal toxin, horridum toxin, from the venom of the lizard, Heloderma horridum horridum, was determined by Edman degradation. The amino acid sequence was deduced by overlapping peptide fragments generated by chemical and enzymatic digestions. Horridum toxin causes hemorrhage in internal organs and particularly in the eye, leading to exophthalmia, an effect that has not been observed for other toxins. It is a glycoprotein with a total of 210 residues. Examination of the primary sequence revealed that horridum toxin has considerable homology to tissue-type kallikrein and trypsin. Furthermore, synthetic substrates for trypsin, such as tosyl-l -arginine methyl ester, benzoyl-l -arginine ethyl ester and other p-nitroanilide substrates, were hydrolyzed. The toxin released bradykinin upon hydrolysis of kininogen. This enzymatic behavior is similar to that of plasma kallikrein; however, the presence of a characteristic “kallikrein-like” loop at 91-100 (GTIYNCNYVN) in the primary structure and other features similar to tissue kallikrein suggest that horridum toxin is more like tissue kallikrein. This toxin degraded all three chains of fibrinogen but did not form a clot, which suggests that it is different from thrombin. Moreover, it differs from another lethal factor from H. horridum horridum, gila toxin, which has 245 amino acid residues and does not cause exophthalmia.     

Purification and characterization of antithrombotics from Syzygium aromaticum (L.) MErr. & PERRY

Two antithrombotic polysaccharides with relatively high molecular weight (HMW) and low molecular weight (LMW) were isolated from the flower buds of Syzygium aromaticum (L.) MERR. & PERRY (clove) by anion-exchange chromatography, hydrophobic interaction column chromatography and size exclusion chromatography (LMW: EC-2B-IIIa-2, M.W. ca. 34000; HMW: EC-2C-Ia-2, M.W. ca. 103000). The LMW polysaccharide was mainly composed of Rha, Gal, GalA and Ara (molar %: 24.1, 18.9, 18.0 and 17.9, respectively) with 10.8% of sulfate and 18.2% of protein. The HMW fraction consisted of Ara, Gal, Glc and Rha (molar %: 26.0, 23.7, 17.5 and 12.4, respectively) with 15.4% of sulfate and 8.0% of protein. Both polysaccharides had the backbone of type I rhamnogalacturonan and the side chain of arabinan. Also, most of the sulfates were attached at the position 6 of 3-linked galactosyl residues. Compared to the antithrombotic activity of the HMW fraction (plasma clotting time of 145 s in APTT assay), the LMW fraction displayed a slightly low activity (90 s). However, animal studies indicated that crude LMW polysaccharide did not show acute toxicity, while the acute LD50 of the HMW fraction was approximately 2-fold lower than that of heparin.     

Purification and characterisation of proteins with cardiac stimulatory and haemolytic activity from the anemone Actinia tenebrosa

Three new proteins with cardiac stimulatory and haemolytic activity, designated tenebrosins-A, -B and -C, have been purified from the Australian sea anemone Actinia tenebrosa. These proteins are basic (pI greater than or equal to 9.4), have mol. wt of about 20,000, and have very similar amino acid compositions and N-terminal amino acid sequences. None of the proteins contains cysteine or cystine residues. On isolated, spontaneously beating guinea pig atria they exhibit at 1-2 nM strong positive inotropic and slight to moderate chronotropic effects. In some cases a transient negative inotropic effect occurs prior to onset of the positive inotropic response. The proteins are also haemolytic, producing 50% haemolysis of guinea pig erythrocytes at concentrations similar to those showing positive inotropic effects.     

Cloning and sequence of guinea pig interleukin 2 (IL-2)

Interleukin 2 (IL-2) is a T-cell proliferation factor released from TH0- and TH1-type helper T-cells and is an essential cytokine for certain immune responses. We reported here cloning and sequence of IL-2 cDNA in guinea pigs, which have been used for a long time in various immunological experiments and in vivo screening tests for skin sensitization potential of chemicals. Consequently, a cDNA clone was obtained encoding guinea pig IL-2 of 520 bp in length, which contained a complete open reading frame. Alignment of the amino acid sequence with human IL-2 indicates that guinea pig IL-2 is composed of 20 amino acids (aa) of a signal peptide and 132 aa of a mature peptide with a predicted molecular weight of 15 133. Guinea pig IL-2 has an amino acid homology of 62% with human IL-2, 52% with murine IL-2, and 55% with rat IL-2. In addition, guinea pig IL-2 has a possible N-linked glycosylation site as seen in bovine and porcine IL-2. Received: 8 April 1998 / Accepted: 1 July 1998