The Pharmacokinetics of the Kininogens

Recent evidence has shown that plasma high molecular weight kininogen and both kininogens have the ability to modulate prekallikrein activation and thrombin-induced platelet activation, respectively. However, nothing is known about the plasma clearance and tissue distribution of these proteins. We examined the in vivo pharmacokinetics of high (HK) and low (LK) molecular weight kininogens in rats. ¹²⁵I-HK and -LK molecular weight kininogens’ clearance in rats best-fitted a biexponential model. For HK, the t_1/2 and t_1/2β were 0.6 and 9.5 h and for LK, 0.78 and 7.4 h, respectively. ¹²⁵I-kinin-free HK (cleaved HK) was cleared with a t_1/2 and t_1/2β of 0.45 and 9.9 h, respectively. ¹²⁵I-Domain 3 of kininogens was cleared with a t_1/2β and t_1/2c of 0.99 and 13.3 h, respectively. HK was mostly concentrated in lung; LK, domain 3, and cleaved HK were mostly concentrated in kidney. The kininogens were also concentrated in liver, spleen, and skin. These studies indicate that protein size rather than form is the major determinant of its clearance. Furthermore, the distribution of the kininogens is where bradykinin metabolism and activity are well described.     

Effect of bovine high molecular weight kininogen and its fragments on fitzgerald trait plasma

Fitzgerald trait, a deficiency of high molecular weight (HMW) kininogen, is characterized by a prolongation of both the activated partial thromboplastin time (aPTT) and of the kaolin-activated euglobulin lysis time. To study the role of HMW kininogen in coagulation and fibrinolysis, highly purified bovine HMW kininogen, kinin-free HMW kininogen, a histidine-rich fragment of HMW kininogen, and low molecular weight (LMW) kininogen were obtained. The effects of these proteins on the aPTT and kaolin-activated euglobulin lysis time of Fitzgerald trait plasma were studied. It was found that whole bovine plasma and HMW kininogen had identical capacities to partially correct the prolonged aPTT. Kinin-free HMW kininogen had only 30% of the correcting capacity of intact HMW kininogen. LMW kininogen had no effect on the aPTT of Fitzgerald plasma. The histidine-rich fragment prolonged the aPTT of Fitzgerald plasma and of normal human plasma without affecting the one-stage prothrombin time. HMW kininogen also corrected the kaolin-activated euglobulin lysis time of Fitzgerald plasma. Kinin-free HMW kininogen and LMW kininogen had weak capacities to correct the Fitzgerald kaolin-activated euglobulin lysis time. The histidine-rich fragment significantly prolonged the kaolin-activated euglobulin lysis time of Fitzgerald and normal plasmas. The histidine-rich fragment appears to inhibit the activation of Hageman factor (Factor XII) by kaolin or ellagic acid. Our data suggest that intact bovine HMW kininogen corrects the deficiency of Fitzgerald trait plasma; but the hydrolysis of HMW kininogen by plasma kallikrein yields peptides with decreased correcting activity, and at least one with inhibitory activity in contact-mediated coagulation and fibrinolysis.     

Characterization of kininogens in human malignant ascites

Ascites from seven patients with advanced cancer were studied to characterize the kininogens. Immunological quantification of low molecular weight kininogen (L-kininogen) and high molecular weight kininogen (H-kininogen) by rocket immunoelectrophoresis showed values of 42% and 39%, respectively, compared to control plasma. Release of kinin from the ascites samples was assayed on an isolated rat uterus. The total kinin released from the kininogens was 39% of the value in control plasma, while release selectively from H-kininogen amounted to 25% of plasma. This indicates about 30% of the bradykinin in H-kininogen to be released in vivo in ascites, and points to kinins as possible mediators of the increased vascular permeability causing accumulation of ascites. The function of kininogens as cysteine protease inhibitors (CPIs) was assayed as well, indicating that both L- and H-kininogen function as cysteine protease inhibitors in human ascitic fluid. The proteolytic cleavage of H-kininogen in ascites was studied by polyacrylamide gel electrophoresis and subsequent immunoblotting. H-kininogen was extensively cleaved in ascites compared to control plasma, with large amounts present of a degraded form with M_r of 99 kDa. The bands observed compared well with those described in plasma, and are consistent with contact activation taking place in ascites.     

Demonstration of the third kininogen in high and low molecular weight kininogens-deficient brown norway katholiek rat

A gel filtration profile of the plasma of Brown Norway Katholiek (B/N-Ka) rat was compared with those of B/N-Kitasato (B/N-Ki) and Sprague-Dawley (SD) rats. In the chromatograms of B/N-Ki and SD rat plasmas, high-molecular weight (HMW) kininogen was eluted together with pre-kallikrein. In lower molecular weight fractions, there were two kininogens, one of which released kinin by urinary kallikrein, snake venom kininogenase (SVK) and trypsin, and the other released kinin only by trypsin. In the chromatogram of B/N-Ka rat plasma, there was no fraction which released kinin by plasma kallikrein, urinary kallikrein or SVK. However, the kinin-release only by trypsin was found in the lower molecular weight fraction, which corresponds to the third peak of kininogen in the chromatograms of B/N-Ki and SD rat plasmas. These results indicate that B/N-Ka rat plasma is deficicent in HMW kininogen, and also deficient in the LMW kininogen susceptible to urinary kallikrein and SVK, but it contains the third kininogen responsive only to trypsin.     

Isolation and nucleotide sequence of a cDNA clone encoding bovine adrenal tyrosine hydroxylase: comparative analysis of tyrosine hydroxylase gene products

Investigations into the structure and mechanisms regulating the expression of the genes involved in catecholamine biosynthesis have led to the isolation of a cDNA coding for bovine adrenal tyrosine hydroxylase (TH). The 1,722 bp cDNA contains the complete coding sequence and 3' untranslated region of the TH mRNA. The nucleotide sequence of the cDNA and the deduced amino acid sequence were compared to those reported for rat and human TH. Bovine TH shares 85% and 84% amino acid sequence identity with that of rat and human TH, respectively. Alignment of the amino acid sequences of rat, bovine, and human TH reveals that 79% of the residues are identical in all three species, indicating a strong evolutionary conservation of enzyme structure. Moreover, three of the four putative phosphorylation sites located in the N-terminal region of TH are conserved in these animal species. There are, however, some interspecies differences in TH gene products. The 3' untranslated region of bovine TH mRNA is 56 and 97 nucleotides shorter than rat and human TH mRNA, respectively. Additionally, the bovine protein is 7 and 6 amino acids smaller than its rat and human homologues. All of the absent amino acid residues of bovine TH are missing from an alanine-rich region in the N-terminal portion of the rat and human proteins (amino acids 51-68). Comparison of the size of bovine and rat TH mRNA and protein by northern blot and immunoblot analyses yielded differences consistent with those predicted from the nucleotide sequence data.     

mRNA for kininogens and thiostatins (major acute phase alpha 1-proteins) in the liver of kininogen-deficient rats

mRNAs for low and high molecular weight kininogens (1.6 and 3.0 kb in size, respectively) and for two thiostatins (1.6 kb in size) were found in the liver of kininogen-deficient Brown-Norway (BN/Mai Pfd) rats. The levels of mRNAs for thiostatins, but not those for low and high molecular weight kininogens (arising from a single kininogen gene), increased strongly during acute inflammation. The pattern of DNA restriction sites for the kininogen gene and the thiostatin genes in the mutant rat strain was identical to that in at least four other rat strains.     

Kininogen in factor VIII-deficient plasma (haemophilia A)

The HM_r and LM_r molecular forms of kininogen were found to occur in normal distribution in plasma from a patient with severe haemophilia A. Present findings indicate that lack of Factor VIII does not influence, the normal content and distribution of immunoreactive kininogens determined by single radial immunodiffusion and radioimmunoassay. The partially purified HM_r and LM_r kininogens were antigenically identical with the kininogens in normal human plasma determined using the monospecific antisera raised in rabbits against purified kininogen antigens. Low kallikrein activity on H-D-Pro-Phe-Arg-pNA in the haemophilic plasma was apparently due to preparative procedures. This concept is supported by the normal binding ratio of trypsin to pure α₂-macroglobulin isolated from the haemophilic plasma.     

Modulated expression of a nuclear-associated glycoprotein during normal rat liver development and in various hepatoma cells

Liver plays a major role in systemic detoxification and drug metabolism. NF-164, a protein of 164 kDa predominantly localized in hepatocyte nuclei, was found to be present in increasing amounts during liver maturation. In addition, fetal rat hepatocytes had ten times, and neonatal five times less of this protein than adult hepatocytes. It was also detected in an albumin producing hepatoma cell line, but not in three other lines that have lost several differentiated functions. These data suggest that NF-164 expression is development-dependent and that it may be a marker for both normal and malignant hepatocyte differentiation. NF-164 seems to be liver-specific, since it was not detected in rat brain, spleen, kidney, lung and bovine thymus. It was purified from adult rat hepatocyte nuclei. Its estimated pI is 6.8. Its total amino acid composition and partial amino acid sequence is also being reported. Despite major differences between their respective contents in amino acids, partial sequences showed homologies with carbamyl phosphate synthetase I (CPSI). These observations may suggest that NF-164 also shares some functional features with this enzyme.     

Isolation and sequence of cDNA clones coding for the precursor to the gamma subunit of mouse muscle nicotinic acetylcholine receptor

cDNA libraries have been constructed in plasmid (pBR322) and bacteriophage lambda gammagt10) vectors with poly (A+) RNA isolated from the nonfusing mouse muscle cell line BC3H-1. The libraries were screened with a restriction fragment derived from a genomic clone coding for a human acetylcholine receptor gamma subunit. Several clones were obtained whose cDNA inserts possessed nucleotide and deduced amino acid sequence homology with acetylcholine receptor gamma subunits from Torpedo californica, chick, calf, and human. One isolate, lambda BMG419, has 88 nucleotides of 5'-untranslated sequence, an open reading frame of 1,557 nucleotides coding for the precursor to the mouse acetylcholine receptor gamma subunit, and 144 nucleotides of 3'-untranslated sequence. Alignment of the lambda BMG419-deduced amino acid sequence with homologs from other species predicts a precursor peptide of 519 amino acids and a mature protein of 497 amino acids, with nonglycosylated molecular weights of 58,744 and 56,424 daltons, respectively. Comparison of the deduced amino acid sequence of the mouse gamma subunit with Torpedo, chick, calf, and human sequences showed overall homologies of 54%, 67%, 90%, and 90%, respectively; however, significantly higher homologies were found in several putative functional domains. Radiolabeled lambda BMG419 has been used to identify homologous RNA species, one of approximately 2 kb and one of about 3.5 kb, in poly (A+) RNA prepared from BC3H-1 cells and denervated mouse limb muscle. gamma Subunit-coding RNA species are considerably more abundant in denervated than in innervated muscle, suggesting that neural regulation of the abundance of the gamma subunit is exerted through regulation of the amount of its mRNA.     

The immune response to myelin proteolipid protein in the Lewis rat: identification of the immunodominant B cell epitope.

The polyclonal antibody response of the Lewis rat to bovine myelin proteolipid protein (PLP) has been investigated following immunisation with either the purified protein or bovine central nervous system myelin. In both situations, the carboxyl-terminal sequence of PLP was identified as the immunodominant domain of this protein and epitope mapping demonstrated that the carboxyl-terminal amino acid, phenylalanine276, was a critical requirement for antibody recognition of this epitope. This single epitope accounted for approximately 78% and 56% of the antibody response to PLP in rats immunised with PLP or bovine myelin, respectively. Polyclonal rat antibodies specific for this carboxyl terminal epitope of PLP were also obtained following immunisation with a synthetic 20 amino acid oligopeptide analogue of the carboxyl-terminal sequence of PLP. Western blotting demonstrated this antibody response was specific for the PLP and DM-20 components of mammalian central nervous system myelin. In contrast, no major PLP epitopes were detected within the PLP amino acid sequences 35-58 and 91-150, the other major hydrophilic domains of this protein.     

Activation of factor XII and prekallikrein with cholesterol sulfate

Cholesterol sulfate was found to display a strong ability to trigger the activation of Factor XII and prekallikrein in the presence of HMW kininogen. Other sulfate ester derivatives of testosterone, estrone, pregnenolone and dehydroepiandrosterone and cholesterol tested did not show any effect on the activation of Factor XII and prekallikrein. The activity of cholesterol acetate and sulfodeoxycholic acid was very weak. Cholesterol sulfate markedly shortened the partial thromboplastin time of normal human plasma, but not plasmas deficient in Factor XII, Factor XI and HMW kininogen. Upon prolonged incubation, the partial thromboplastin time of prekallikrein-deficient plasma was also shortened. Moreover, as well as kaolin and sulfatide, cholesterol sulfate shortened the partial thromboplastin time of plasmas from monkey, dog, rat, guinea pig, sheep, cow, hog and horse, but not from duck and chicken. Since cholesterol sulfate is distributed in erythrocytes, various organs and body fluids, it may play an important role in the activation of the intrinsic blood coagulation system.     

Prolonged activated partial thromboplastin time and deficiency of high molecular weight kininogen in brown Norway rat mutant (Katholiek strain)

Activated partial thromboplastin time (APTT) was examined in Brown Norway (B/N) Katholiek rat, which was previously reported as high molecular weight kininogen deficient. APTT of B/N Katholiek was prolonged to 35 sec in comparison with B/N Kitasato and SD rat, showing APTT of 22-24 sec. The mixture of B/N Katholiek plasma and B/N Kitasato plasma (1:1) showed normal APTT value. B/N Katholiek plasma corrected the abnormally prolonged human coagulation factor deficient plasmas, such as XI, XII and prekallikrein deficient plasmas, while it did not correct the APTT of HMW kininogen deficient, Fitzgerald plasma. Intravenous injection of bromelain, which was previously reported to produce prolonged hypotension through the activation of factor XII to release bradykinin, induced slight effect in Katholiek rat, while in Kitasato rat it showed prolonged hypotension in similar degree as SD rat. Contents of coagulation factors in B/N Katholiek thus measured as well as the values of prekallikrein and HMW kininogen previously reported were summarized and suggested that B/N Katholiek rat could be similar deficiency as Fitzgerald trait.     

Recent evolutionary divergence of plasma prekallikrein and factor XI

The evolution in mammals of the zymogens of the contact activation system of coagulation (factor XII, prekallikrein and factor XI) has been investigated. The NH2-terminal sequences of human plasma prekallikrein and the heavy and light chains of kallikrein have been determined and compared with those of bovine prekallikrein and of human and bovine factors XII and XI. The human and bovine NH2-terminal sequences of the light chains (catalytic polypeptide) show striking similarities both among themselves and with those of the catalytic polypeptide chains of other coagulation and digestive proteases, indicating a common origin. Comparison of the NH2-terminal sequences of human prekallikrein with those of the bovine prekallikrein and human bovine factors XIa and XIIa indicates a common origin of the heavy chain of kallikrein and factor XIa, different from that of either factor XIIa or other known amino acid sequences. Ancestral sequences for human and bovine prekallikrein and factor XI, deduced by genetic analysis of the minimum number of base changes indicate that the NH2-terminus of prekallikrein and factor XI have evolved at about the same rate. The estimated time for the gene duplication was about 124 million years ago, a value consistent with the age of the mammals.     

Giant neural systems in the inner retina and optic nerve of small whales

Giant neural cell systems (dendrites, cell bodies, and axons) are present among more usual structures in the retina and optic nerve of the small whale (dolphin) Tursiops truncatus retina. Giant cell body dimensions range up to 75 μm in diameter. Nuclei of the cells are frequently larger (>20 μm) than nearby ganglion, bipolar, and receptor cell bodies. The presence of the giant cell system and giant elements in the nerve fiber layer agree with the unusually broad fiber spectrum of the dolphin optic nerve where more than 6% of the axons are >15 μm in diameter. Smaller axons in the size distribution are typical of dimensions found in terrestrial mammals. The axon estimate totaled 157,000 per optic nerve. The giant cell-axon systems of the whale retina may be a unique expression of the large ganglion cell-axon (transient or “Y” functional unit) systems recently identified in terrestrial mammals.     

Bovine arylalkylamine N-acetyltransferase activity correlated with mRNA expression in pineal and retina

Arylalkylamine N-acetyltransferase (AA-NAT, E. C. 2.3.1.87) is the enzyme that catalyzes the transfer of an acetyl group from acetyl-CoA to serotonin to form N-acetylserotonin (NAS) in the indoleamine biosynthetic pathway. Bovine pineal AA-NAT, partially purified on an anion exchange column, displayed an 8-fold higher enzymatic activity in pineals from animals killed in early morning (0800) compared to an afternoon group (1430). Poly A(+) mRNA was isolated from early morning bovine pineals, used to construct a mammalian expression cDNA library (lambdaZAP Express), and then screened with a rat AA-NAT cDNA to isolate a 924 basepair cDNA that encodes the bovine pineal AA-NAT. The amino acid sequence alignment reveals that bovine AA-NAT shares 94.20%, 78.54%, 76.33% and 56.3% identity to ovine, rat, human and chicken sequences, respectively. Northern blot analysis demonstrates a 0.7-fold higher mRNA level in pineal glands taken from animals from the 0800 time-point compared with mRNA from the 1430 time-point. AA-NAT mRNA was expressed at high levels in pineal and retina, but the message was undetectable in adrenal, cerebellum, cortex, small intestine, testis and thyroid. Based on the significant identity of amino acid sequence and the similar mRNA expression pattern, these data suggest that the bovine AA-NAT is more analogous to the ovine rather than either the rat, human or chicken AA-NAT.     

Functionally active high molecular weight-kininogen was found in the liver, but not in the plasma of brown Norway Katholiek rat

Plasma and liver levels of high molecular weight (HMW-) kininogen were assessed by a newly developed radioimmunoassay in B/N-Katholiek strain rats, which is congenitally deficient in plasma HMW- and low molecular weight (LMW-) kininogens. The plasma level of immuno-reactive HMW-kiniogen in this strain was about 4% of that of the normal strain, B/N-Kitasato, whereas its level in B/N-Katholiek liver was approximately 60% of that in normal strain liver. There was no significant difference of half-life of HMW-kininogen in the circulating blood between the deficient and normal rats. When secretion of HMW-kiniogen from the liver cells of the two strains was examined by primary culture of their hepatocytes, the hepatocytes from the deficient strain did not secrete HMW-kininogen in the medium. HMW-kininogens were isolated from the liver microsomal fractions of both strains by use of an immuno-affinity column. The isolated protein from B/N-Katholiek liver showed similar mobility on SDS-PAGE to that from normal rat liver, and had biological activities of HMW-kininogen purified from normal rat plasma, such as intrinsic blood clotting cofactor, thiol-proteinase inhibitor, and kinin precursor. These results indicate that the plasma deficiency in the B/N-Katholiek strain is due to a defect of HMW-kininogen secretion from the liver.     

A radioimmunoassay for bradykinin based on monoclonal antibodies

We describe two monoclonal antibodies with recognise the peptides bradykinin and kallidin. These antibodies were used in a study to assess the feasibility of using monoclonal antibodies in place of conventional sera for radioimmunoassay. Antibody SBK1 recognises bradykinin with a Kd of 0.67 +/- 0.17 nM. It fails to recognise bradykinin from which any carboxyl terminal amino acid has been removed. Antibody SBK2 recognises bradykinin with a Kd of 58.11 +/- 7.55 nM; it recognises 1-8 and 1-7 bradykinin about 30% as effectively as the full-length sequence, and shorter carboxyl terminal truncated sequences with a progressively declining efficiency. In this model system, bradykinin concentrations of 0.2 nM could be reliably measured, and carboxyl truncated versions of the peptide could be distinguished from the parent molecule. The feasibility of using monoclonal antibodies to assay bradykinin and other small peptides is discussed in the light of these results.     

Purification and partial amino acid sequence of bovine adrenal phenylethanolamine N-methyltransferase: a comparison of nucleic acid and protein sequence data

Recently, we have reported the isolation and characterization of a putative genomic DNA clone encoding bovine adrenal phenylethanolamine N-methyltransferase (PNMT) (Batter et al., 1988). However, the lack of primary amino-acid sequence data for this enzyme precluded a definitive proof of the authenticity of this clone. To establish identity, the amino acid sequence of several peptides generated by chemical and enzymatic hydrolysis of purified PNMT was compared to that predicted from the nucleotide sequence of the exons of the putative PNMT gene. Bovine adrenomedullary PNMT was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Treatment with 70% formic acid cleaved the protein at a single Asp-Pro bond near the N-terminus. The purified protein was also cleaved at a single methionine residue near the C-terminus by treatment with cyanogen bromide. N-terminal amino acid sequence analysis identified 8 and 10 amino acid residues, respectively, following each of the scissile peptide bonds. Four tryptic peptides, generated by complete enzymatic digestion, were isolated by reverse-phase HPLC and subjected to sequence analysis. Combined, the amino acid sequences of these six peptides represent 20% of the PNMT protein. These amino acid sequences matched exactly the sequences predicted from the exons of the putative PNMT genomic clone.     

Human plasma prekallikrein and high molecular weight kininogen decrease during parturition

Prekallikrein and high molecular weight kininogen were measured in plasma taken from nine women during parturition. Prekallikrein decreased significantly (p<0.01) from 1.49 ± 0.15 S-2302 U/ml (mean ± SEM) in early labor to 1.26 ± 0.13 S-2302 U/ml in the immediate postpartum period. Immunoreactive high molecular weight kininogen also significantly decreased from 76 ± 5 μg/ml to 68 ± 5 μg/ml one day postpartum (p<0.OL). Both proteins rose to normal levels within two days. The data suggest that the kallikrein-kinin system is utilized during parturition.