Different molecular forms of acid-stable trypsin inhibitor in normal and uremic plasma.
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Adult↗
Biomedical subjects
H Mihara
Publications and source records attributed to H Mihara.
Studies on blood coagulation and fibrinolysis in patients bitten by Bothrops jararaca (jararaca).
The blood coagulation and the fibrinolytic systems of nine patients envenomed by Bothrops jararaca in São Paulo (Brazil) were studied. Five of the accidents were caused by young snakes (less than 50 cm). On admission, four patients had non-clotting and three partially-clotting blood. Fibrinogen levels were decreased due to the thrombin-like activity of the venom as expected. Consequent secondary activation of the fibrinolytic system was evident from the low levels of alpha-2-antiplasmin and the high titres of fibrin(ogen) degradation products. High titres of cross-linked fibrin fragment D (D-dimer) in seven patients together with decreased platelet counts and/or factor V, and/or factor VIII in some, suggests intrinsic thrombin formation as these factors are not consumed in the defibrinogenation induced by venom thrombin-like fractions such as Ancrod and Batroxobin. However, normal or increased levels of antithrombin III in all and normal levels of factor II in eight patients do not support this interpretation. The existence of variable concentrations of other proteins in the venom of B. jararaca such as botrocetin and thrombocytin isolated from B. jararaca and B. atrox or crotalocytin from Crotalus horridus venom should be considered. Such proteins are known to activate factors V, VIII, XIII, and platelets without affecting prothrombin (factor II) and antithrombin III. Slower recovery of the haemostatic disturbances after antivenom administration to patients bitten by young snakes suggests a more severe coagulopathy in such accidents. This is supported by clinical observations.
Adolescent↗
Design and synthesis of basic peptides having amphipathic beta-structure and their interaction with phospholipid membranes.
Basic amphipathic beta-structural peptides, Ac-(Ser-Val-Lys-Val)n-NHCH3 (1n, n = 1-3) and Ac-(Lys-Val)n-NHCH3 (2n, n = 2-4), were synthesized and their interaction with DPPC and DPPC-DPPG (3:1) bilayers was studied by CD, dye-leakage and fluorescence experiments. The CD data indicated that oligopeptides consisting of more than eight residues with alternating hydrophobic (Val) and hydrophilic amino acids (Ser and Lys) were able to form an amphipathic beta-structure in acidic phospholipid bilayers, but not or weakly in aqueous solution and in neutral phospholipid bilayers. The dye-leakage experiment showed that the basic amphipathic beta-structural peptides interact with acidic phospholipid bilayers to perturb them, but less effectively compared with basic amphipathic alpha-helical peptides. Fluorescent spectroscopic data suggest that hydrophobic side of the amphipathic peptides may immerse into membrane without deep penetration. Based on these results, we postulate that the formation of the basic amphipathic beta-structure on acidic lipid bilayers may be due to the combined effect of electrostatic and hydrophobic interactions between basic peptides and acidic lipid bilayers.
1,2-Dipalmitoylphosphatidylcholine↗
Carboxypeptidase activity in human urine from healthy subjects and renal disease patients.
We have attempted to estimate the carboxypeptidase activity in unconcentrated human urine by means of a colorimetric method in which Bz-Gly-Lys is employed as a substrate. Upon addition of both 1.6 mmol/l CoCl2 and 0.18 g/l bovine serum albumin to the assay solution at pH 5.6, the carboxypeptidase activity in human urine can be evaluated. Using our method, the daily excretion of carboxypeptidase in both healthy subjects and patients with renal diseases has been examined. The level is increased in patients with nephrotic glomerulonephritis (24.9 +/- 14.9 (SD) U/day; n = 8) and patients with nephritic glomerulonephritis (11.4 +/- 7.55 (SD) U/day; n = 14) as compared to that in healthy subjects (5.10 +/- 1.88 (SD) U/day; n = 18). However, it is decreased in patients with chronic renal failure (2.70 +/- 2.13 (SD) U/day; n = 12).
Cadmium↗
Low molecular weight alanine aminopeptidase of human serum: separation and some characteristics.
An alanine aminopeptidase, which has characteristics different from those of known alanine cleaving aminopeptidases, was partially purified from human serum by Sephadex G-200 gel chromatography and DEAE Sepharose column chromatography. The enzyme exhibited a molecular weight of 58,000 by gel chromatography. The pI of the enzyme was 5.0, and it was inactivated at 60 degrees C in 20 min. The enzyme readily hydrolyzed L-alanine beta-naphthylamide, but hardly hydrolyzed the other tested beta-naphthylamides. The Km value for L-alanine beta-naphthylamide was 0.29 mM, the pH optimum 7.5. The activity of the enzyme was enhanced by chloride ions and by sulfhydryl ethylenediaminetetraacetic compounds, and was inhibited by sulfhydryl blocking ethylenediaminetetraacetic agents, and bestatin. Furthermore, 1.10 phenanthroline and ethylenediaminetetraacetic acid were inhibitory, and the activity was restored by CoCl2 and ZnCl2. The enzyme is a chloride-enhanced thiol dependent metalloaminopeptidase.
Aminopeptidases↗
The spectroscopic analysis for binding of amphipathic and antimicrobial model peptides containing pyrenylalanine and tryptophan to lipid bilayer.
The binding of basic amphipathic fluorescent peptides to lipid bilayers was studied in relation to their antimicrobial activity. Four fluorescent peptides containing pyrenylalanine or tryptophan in an amphipathic basic peptide (4(4] consisting of four repeated units of tetrapeptide, -L-Leu-L-Ala-L-Arg-L-Leu-, were found to have antimicrobial activities against Gram-positive bacteria and to take conformations with fairly high alpha-helical content both in aqueous solutions and liposomes. The fluorescence spectroscopic data suggested that the pyrenylalanine-peptide existed as a monomer in methanol or liposomes but as an oligomer in aqueous solutions to form an excimer between pyrenylalanyl residues. Upon binding with liposomes, the fluorescence spectra of the tryptophan-containing peptide shifted to a shorter wavelength, indicating the change in the state of tryptophan from hydrophilic environment to hydrophobic one. The analytical data for the quenching of tryptophan fluorescence by I- anion suggest that the tryptophan residue in the peptide is not deeply buried in the hydrophobic core of the bilayers. Based on these findings, it is suggested that the peptides may interact with liposomes in such a manner that they lie parallel to the surface of the lipid bilayers with their hydrophobic regions shallowly in the amphipathic moiety of the bilayers.
Alanine↗
Distribution of acid stable trypsin inhibitor immunoreactivity in normal and malignant human tissues.
The distribution and localization of acid stable trypsin inhibitor (ASTI) in normal and malignant human tissues from various organs were examined using immunohistochemical techniques that used goat antibody raised against highly purified ASTI from human urine. Tissues were assessed as positive only when they were stained by both the biotin-avidin-peroxidase complex system and biotin-streptavidin-beta-galactosidase complex system, and the staining was abolished by absorption with purified ASTI. Under normal conditions, ASTI immunoreactivity was observed in only a few organs. Positive tissues for ASTI immunoreactivity included the kidney proximal tubules, glial cells of the cerebrum, fibrillar structures of the lamina propria of the stomach and colon, and bronchial epithelial cells. No ASTI immunoreactivity was observed in the cardiovascular system, reproductive system, or other tissues examined. As is not the case for normal tissues, ASTI immunoreactivity was found to be widely distributed in malignant tumors. Staining was observed in the extracellular space, i.e., in the stroma of the tumor and in connective tissues around the tumor invasion, whereas no ASTI immunoreactivity was detected in the malignant cells. Considering the identity of the first 36 NH2-terminal residues of ASTI purified from plasma or urine with a recently reported endothelial cell growth factor, the present findings suggest that ASTI could play an important role, not limited to its function as a protease inhibitor, in the invasive growth of malignant neoplasms.
Digestive System↗
Peptide and protein synthesis by segment synthesis-condensation.
The chemical synthesis of biologically active peptides and polypeptides can be achieved by using a convergent strategy of condensing protected peptide segments to form the desired molecule. An oxime support increases the ease with which intermediate protected peptides can be synthesized and makes this approach useful for the synthesis of peptides in which secondary structural elements have been redesigned. The extension of these methods to large peptides and proteins, for which folding of secondary structures into functional tertiary structures is critical, is discussed. Models of apolipoproteins, the homeo domain from the developmental protein encoded by the Antennapedia gene of Drosophila, a part of the Cro repressor, and the enzyme ribonuclease T1 and a structural analog have been synthesized with this method.
Amino Acid Sequence↗
Clearance and distribution of acid-stable trypsin inhibitor (ASTI).
The clearance, organ distribution and metabolic pathway of the acid-stable trypsin inhibitor (ASTI) were studied in mice using 125I-labeled urinary trypsin inhibitor (UTI), the most typical ASTI in the urine. Following intravenous injection of 125I-UTI, the radioactivity disappeared rapidly from the circulation with a half-life of 4 min for the initial part of the curve. Gel filtration of plasma samples revealed that the rapid disappearance of the radioactivity was due to elimination of free inhibitor from the plasma. 125I-UTI was cleared primarily in the kidney. Gel filtration of urine samples showed that part of the radioactivity in the urine appeared at the same elution volume as 125I-UTI in the plasma, indicating that the origin of UTI was ASTI in the plasma.
Animals↗
A chemically synthesized Antennapedia homeo domain binds to a specific DNA sequence.
A peptide 60 residues in length that corresponds to the homeo domain of Antennapedia (Antp), a protein governing development in Drosophila, was synthesized by segment condensation with protected peptide segments prepared on an oxime resin. A footprinting assay showed that the homeo domain binds specifically to a TAA repeat DNA sequence in the Antp gene. Thus the Antp homeo domain has a sequence-specific DNA binding property. The circular dichroism spectra of the homeo domain peptide showed the presence of a significant amount of alpha-helical structure in aqueous solution and in 50 percent trifluoroethanol. The alpha helicity measured in water appears to depend on the peptide concentration, which suggests that the peptide aggregates. These results support the hypothesis that the homeo domain binds to DNA through a helix-turn-helix motif.
Amino Acid Sequence↗
Strong activity of acid-stable trypsin inhibitor in bovine thrombin for clinical use.
Strong activity of acid-stable trypsin inhibitor (ASTI) was confirmed in some clinical thrombin preparations. Thrombin preparations of human plasma origin had no detectable ASTI activity, whereas some preparations of bovine plasma origin revealed more than 5,000 U/vial (5,000 thrombin units), indicating a higher content of ASTI than of thrombin in terms of protein concentration. Contamination by other biologically active substances was also suggested by variations in amidolytic activity with several synthetic substrates (S-2238, S-2251, S-2444, S-2266 and Bz-L-Arg-pNA). On isoelectric focussing, the ASTI activities migrated in acidic positions with pI values of 3.9, 4.5, 5.0, 5.9 and 6.5, respectively. They were almost parallel to the thrombin Bz-L-Arg-pNA hydrolytic activity, and differed from that of the purified thrombin preparation (pI = 7.0). By gel filtration on Sephadex G-100, the molecular weights of the inhibitors as calculated using standard proteins were 140,000 (main), 70,000 and less than 10,000 (minor), respectively. An immunological difference between the main inhibitor (pI = 3.9, mol wt 140,000) and previously reported plasma ASTI was also confirmed with goat anti-UTI serum by the double immunodiffusion and ELISA methods. The inhibitor exerted a strong inhibitory effect not only on trypsin and chymotrypsin, but also on non-plasmic fibrinolysis with human leukocyte elastase, and to a lesser extent on the blood coagulation system (lengthening of APTT and PT). Clearly, when using thrombin preparations and analyzing the data obtained after their administration, the effects of this and other contaminant biologically active substances must be taken into account.
Animals↗
Acid-stable trypsin-plasmin inhibitors formed enzymatically from plasma precursor protein.
Enzymatic formation of acid-stable trypsin-plasmin inhibitors (ASTPIs) in human plasma with several proteinases, particularly SH-proteinases, was demonstrated. The maximal activity obtained with bromelain was 40 U/ml plasma, which corresponded to about a 10-fold increase as compared to the untreated control plasma (4.2 U/ml). Gel filtration revealed at least two ASTPI activity peaks of molecular weight 16,000 (main peak) and 8000 (minor peak). The main ASTPI was further purified by trypsin-Sepharose affinity chromatography, isoelectric focusing and gel filtration on Sephadex G-75 superfine. The purified inhibitor was found to be identical to the active fragment of plasma ASTPI or urinary trypsin inhibitor (UTI) formed by bromelain treatment. It had an isoelectric point (pI) of 3.7, a molecular weight of 16,000 by SDS-polyacrylamide gel electrophoresis and was a glycine- and glutamic acid-rich protein lacking histidine. The NH2-terminal amino acid sequence was H2N-(Lys)-Glu-Asp-Ser-X-Gln-Leu-Gly-Tyr-Ser-Ala-Gly-Pro-X-Met-Gly-Met-Th r-X-Arg - Tyr-Phe-Tyr-... COOH, which was homologous to the Lys22-Met36 part (or Glu23-Met36 part; 30% of the total) of the plasma ASTPI or UTI molecule (molecular weight 70,000-80,000 by gel filtration). The purified ASTPI displayed the same antigenicity as UTI and exerted strong inhibitory effects on trypsin, chymotrypsin and plasmin amidolysis, but had a much lesser effect on plasmin fibrinolysis. It also strongly inhibited non-plasmic fibrinolysis with human leukocyte proteinase and earthworm proteinase.
Amino Acid Sequence↗
Urokinase-like plasminogen activator increased in plasma after alcohol drinking.
After taking the local Japanese spirit 'Shochu', 'Sake' and beer, increased plasma fibrinolysis was confirmed in normal persons at about 1 hr after for both the pyro-Glu-Gly-Arg-pNA amidolytic and Glu-plasminogen activating activities in eluates from [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester;)]-Sepharose affinity column. The activity was highest in the Shochu group, followed by the Sake and beer groups with an approximately 2.1-, 1.7- and 1.5-fold increase in fibrinolytic activity, respectively, compared to the control. The enzyme could be further purified using urokinase-IgG-Sepharose immunoadsorbent column from the Shochu group and it reacted with and was inhibited by urokinase specific antibody. The main molecular weight of the active enzyme was about 30,000, and those of minor components were about 50,000 and 100,000, as determined by zymography. These findings suggest that some of the enzymes appearing in the plasma after drinking are related to 'urokinase-like plasminogen activator', being endogenously produced.
Adult↗
Cysteine proteinase inhibitors in human melanoma transplanted into nude mice.
Cysteine proteinase inhibitors in the human melanoma tissue transplanted into nude mice were found to increase in concentration during tumor growth. The activity (unit/g) at 8 weeks was about 3 times higher than the activity at 4 weeks after transplantation. The inhibitors were separated into two main forms (Mr about 76,000 and 10,000) with Sephacryl S-200 and/or Sephadex G-75 gel chromatography. The activities of the inhibitors of both molecular masses increased parallely during tumor growth. The high molecular mass inhibitor fractions reacted with antisera made against alpha-cysteine proteinase inhibitor (alpha-CPI, human kininogen) and against neutral low-molecular mass proteinase inhibitor (cystatin B). Free cystatin B appeared to be liberated in SDS-polyacrylamide gel electrophoresis following electroimmunoblotting with an antiserum to cystatin B. Similarly, free cystatin B was detected in gel chromatography on Sephadex G-75 after alkali treatment at pH 11.5. It may thus represent a cystatin B--cysteine proteinase complex mixed with alpha-CPI. The low molecular mass inhibitor fractions reacted with antisera made against cystatin A and cystatin B. When the low-molecular mass inhibitor fraction was subjected to isoelectric focusing, it was separated into three peaks with pIs 8.0, 7.4, and 6.0. The inhibitors with pI 8.0 and 7.4 reacted with antisera made against cystatin B, while the inhibitor with pI 6.0 reacted with antisera made against cystatin B and cystatin A.
Animals↗
A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet.
A strong fibrinolytic activity was demonstrated in the vegetable cheese Natto, which is a typical soybean food eaten in Japan. The average activity was calculated at about 40 CU (plasmin units)/g wet weight. This novel fibrinolytic enzyme, named nattokinase, was easily extracted with saline. The mol. wt and pI were about 20,000 and 8.6, respectively. Nattokinase not only digested fibrin but also the plasmin substrate H-D-Val-Leu-Lys-pNA (S-2251), which was more sensitive to the enzyme than other substrates tried. Diisopropyl fluorophosphate and 2,2,2-trichloro-1-hydroxyethyl-o,o-dimethylphosphate strongly inhibited this fibrinolytic enzyme.
Chromatography, Gel↗
Development and application of an enzyme-linked immunosorbent assay for acid stable trypsin inhibitor (ASTI) in human plasma.
An enzyme-linked immunosorbent assay (ELISA) for acid-stable trypsin inhibitor was developed using goat antibodies raised against urinary trypsin inhibitor purified from normal human urine. The assay was highly sensitive and precise, and gave reproducible results in the range of 1-1,000 ng/ml. The specificity and sensitivity of this assay permitted measurement of a low content of acid stable trypsin inhibitor in different tissue extracts and body fluids. Using the new method, the mean level in plasma samples taken from healthy individuals was found to be 54.9 +/- 17.4 (mean +/- SD) micrograms/ml and that of acid-treated plasma was 11.5 +/- 3.0 micrograms/ml (n = 27). The calculated specific activity of acid stable trypsin inhibitor in acid treated plasma was 300 U/mg, while that of highly purified inhibitor is 2,100 U/mg. These results suggest the presence of both acid unstable protein having the same antigenicity as urinary trypsin inhibitor and the inactive form of acid stable trypsin inhibitor.
Enzyme-Linked Immunosorbent Assay↗
Interaction of synthetic fragments of the extension peptide of cytochrome P-450(SCC) precursor with phospholipid bilayer.
Six peptide fragments, SEP6-11, SEP6-15, SEP1-11, SEP1-15, SEP1-20, and SEP25-39, corresponding to the residues 6-11, 6-15, 1-11, 1-15, 1-20, and 25-39, respectively, of the extension peptide of cytochrome P-450(SCC) precursor were chemically synthesized by the solution method. CD spectra of the peptides indicated that SEP1-15 and SEP1-20, which inhibit the import of the precursor, held a random conformation in a Tris-HCl buffer and a partially ordered conformation (like alpha-helix or type II' beta-turn) in a buffer containing acidic liposomes. Slightly changed spectra were observed for SEP1-15 and SEP1-20 in the buffer containing neutral liposomes and in MeOH, respectively. Other peptides which show weak or no inhibitory effect had almost random conformations in these solvents. The hydrophobic moments of SEP1-15 and SEP1-20 when they take alpha-helical conformation are very small, suggesting that amphiphilic helical property is not always necessary for the import of the precursor, although partially ordered conformation seems to be required. The ability of SEP1-15 and SEP1-20 to induce leakage of carboxyfluorescein from the inside of dipalmitoyl-DL-alpha-phosphatidylcholine (DPPC) or DPPC-dipalmitoyl-DL-alpha-phosphatidylglycerol (3:1) vesicles was much greater than that of other peptides. The leakage induction ability of the peptides qualitatively parallels the degree of their inhibition of the import of the precursor. Perturbation of the membranes caused by the action of the peptides in their partially ordered form may be important for the import of the precursor into mitochondria.
Amino Acids↗
Synthesis, receptor binding activity and fluorescence property of fluorescent enkephalin analogs containing L-1-pyrenylalanine.
The novel fluorescent amino acid, L-1-pyrenylalanine (L-Pya), was prepared by the asymmetric hydrogenation of cyclic dehydrodipeptide. Fluorescent enkephalins containing one or two Pya residues at position 1,4 or 5 of [D-Ala2, Leu5]enkephalin were synthesized by the solution method. Mono-Pya-enkephalins showed strong fluorescence intensities and potent binding affinities with specificity and selectivity for opiate receptors. However, di-Pya-enkephalins showed markedly decreased receptor binding affinities. These results indicate that the incorporation of two Pya residues into enkephalin makes the peptide unable to interact with the opiate receptors, although introduction of one Pya residue is effective to elicit a specific receptor interaction. Di-Pya-enkephalins showed intramolecular excimer spectra, indicating that the peptides are able to take possible folded conformations.
Animals↗