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Biomedical subjects

H Sumi

Publications and source records attributed to H Sumi.

At least 73 records · Page 4Linked to original sources
PubMed1988

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↗
PubMed1988

Dynamics of protein conformational fluctuation in enzyme catalysis with special attention to proton transfers in serine proteinases.

We have provided a quantum mechanical model for proteinase-catalyzed peptide, amide and ester hydrolysis. The model rests on electron and atom transfer theory, but incorporates the dynamics of conformational nuclear modes as a new element. The model is applied to acylation, but can straightaway be extended to deacylation, and is substantiated by recent structural and kinetic data for proteinase enzyme catalysis. The role of the conformational modes is found to be two-fold. First, the crystallographic distances for the proton transfers involved are far too large for direct transfer. His-57 mobility, handled stochastically, to bring the donor and acceptor groups within suitable reach, is therefore a crucial element of the theory. Secondly, the charge alignment in the Asp-102/His-57/tetrahedral intermediate system implies that the curvature of the potential surface along the conformational coordinates in this state is much lower than in the initial enzyme-substrate and final acyl states. A consequence of this is that the activation energy liberated after the first proton transfer is not dissipated, but stored in the conformational system and used in the second proton transfer step.

Binding Sites↗
PubMed1988

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↗
PubMed1988

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↗
PubMed1987

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↗
PubMed1987

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↗
PubMed1987

Trinitrophenyl-derivative of urinary trypsin inhibitor: a strong antifibrinolytic agent.

A trinitrophenyl derivative of urinary trypsin inhibitor (TNP-UTI) was found to demonstrate approximately 50 times stronger antiplasmin fibrinolysis than the intact UTI. Almost no change in antitrypsin or antichymotrypsin activity occurred as a result of the UTI modification. TNP-UTI was also shown to be a strong inhibitor of nonplasmin mediated fibrinolysis.

Antifibrinolytic Agents↗
PubMed1987

[Pharmacokinetic, bacteriological and clinical studies of cefuzonam in the field of obstetrics and gynecology].

To study the transfer of cefuzonam (CZON, L-105) into female genital organs, concentrations of the compound in pelvic dead space exudate were measured in cases of radical hysterectomy due to cervical cancer and analyzed by the two-compartment model. When CZON 1 g was drip-infused intravenously, the concentration in the cubital vein blood was 46.95 micrograms/ml at 1 hour after the start of infusion. Concentrations in the pelvic dead space exudate reached the peak of 11.29 micrograms/ml at 2.44 hours after the start, were higher than 4 micrograms/ml after 8 hours and were higher than 1.7 micrograms/ml after 12 hours. The area under the concentration-time curve in the pelvic dead space exudate was 77.85 micrograms X hr/ml. From these results CZON was considered to be effective when administered at 1 g against infections of Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, and haemophilus influenzae, but increased dose levels seemed necessary against infections of Staphylococcus epidermidis and Bacteroides fragilis. In 3 cases of obstetric and gynecological infections the efficacy of CZON was good in 2 cases and unknown in the other case.

Adult↗
PubMed1986

Acid stable trypsin inhibitor in bile.

Acid stable trypsin inhibitor having the same antigenicity as urinary trypsin inhibitor was first identified in the bile of patients with malignant tumors (biliary tract carcinoma or pancreas head carcinoma) and gallstones. Bile trypsin inhibitor from malignant tumor patients was partially purified by DEAE cellulose ion exchange column chromatography. Two molecular forms of the inhibitor were identified. The main form had a molecular weight of about 86,000 and the minor one a molecular weight of 31,000 as determined by gel filtration. Using isoelectric focussing, the larger molecular form gave a pI value of 2.0 and the smaller form, a pI value of 5.1. The isolated larger form migrated on the slightly cationic side of human serum albumin by analytical polyacrylamide gel electrophoresis. The larger form reacted and fused with anti-urinary trypsin inhibitor serum and strongly inhibited trypsin, partially inhibited chymotrypsin and plasmin, but did not inhibit urokinase. The clinical significance of acid stable trypsin inhibitor is discussed.

Bile↗
PubMed1986

Purification and amino acid analysis of plasma acid stable trypsin inhibitor.

Acid stable trypsin inhibitor (ASTI), with a molecular weight of about 85,000 by gel filtration, specific activity of 1,498 U/mg protein and pI of 1.6, from renal failure patient plasma was first purified. The amino acid composition of the purified ASTI was found to be that of a Gly- and Glu-rich protein which lacked His, closely resembling that of urinary trypsin inhibitor. The NH2-terminal amino acid sequence was Ala-Val-Leu-Pro-Gln-Glu- Glu-Glu-Gly-X-Gly-Gly-Gly-Gln-Leu-Val-Thr-Glu-Val-Thr-Lys-Lys-Glu- Asp-Ser-Ser-Gln-Leu-Gly-Tyr-Ser-Ala-Gly-Pro.

Amino Acid Sequence↗
PubMed1985

Elastase digested urokinase (ED-UK).

Elastase digested urokinase (ED-UK) was prepared from human high mol. wt urokinase (HMW-UK). It resembled low mol. wt urokinase (LMW-UK) in its mol. wt, specific activity, and active sites. The steady-state kinetic parameters of each enzyme for the activation of human Glu-plasminogen also resembled each other, as did their amidase parameters (with pyro-Glu-Gly-Arg-pNA).

Chromatography, Affinity↗
PubMed1985

Immunochemical determination of the serum protein reacting with antibody against human urinary trypsin inhibitor by single radial immunodiffusion: use of polyethylene glycol.

A single radial immunodiffusion method is described for determining the serum protein which reacts with antibody against the urinary trypsin inhibitor, but does not react with anti-inter-alpha-trypsin inhibitor antibody. Since the amount of this protein could not be determined with an agar plate containing antibody alone, we first prepared an agar plate containing 4% polyethylene glycol 6 000 and 1 mg of anti-urinary trypsin inhibitor gamma-globulin and confirmed that the amount of this protein could be measured accurately from the sizes of precipitin rings after 72 h incubation at room temperature. Levels of this serum protein, alpha 1-antitrypsin, alpha 2-macroglobulin and inter-alpha-trypsin inhibitor were measured in normal human serum by the single radial immunodiffusion method, and it was confirmed that the level of this serum protein did not correlate with the levels of these 3 other proteins.

Antibodies↗
PubMed1985

Fibrinolytic activity of middle ear fluid in otitis media with effusion.

In order to investigate the prolonged factors of otitis media with effusion, hydrolytic activities for specific substrates of plasmin and plasminogen activator (P.G.-activator) and the activity for fibrin degradation (fibrinolysis) were measured. Materials were middle ear fluids of 36 cases diagnosed as unilateral otitis media with effusion. Age of the patients were from 28 to 88 years old (mean: 61.3). Using specific substrates, determinations of P.G.-activator activity and plasmin activity were made by the method of Claeson et al. and fibrinolysis was determined by the standard fibrin plate. The results are shown below: 1) Both activities for decomposition of amide against the specific substrates of plasmin and of P.G.-activator were revealed. 2) Concerning fibrinolysis in all cases, we divided them into 3 groups; fibrinolysis (-) group of 8 cases (22.2%) which could not dissolve fibrin at all, fibrinolysis (+) group of 17 cases (47.2%) which could dissolve fibrin. Fibrinolysis (+/-) group of 11 cases (30.6%) of which the presence of fibrin dissolution was unknown. 3) The progress of fibrinolysis was observed by serial collection of middle ear fluid in the same side ear of 5 cases out of 17 cases having much activity for fibrin degradation, and lowering of fibrinolytic activity was revealed in 4 out of these 5 cases as the result. On the basis of these results, we discussed on the process of prolongation in this disease from the standpoint of the presence of fibrinolytic activity in the middle ear fluid.

Adult↗
PubMed1985

Fibrin-binding urokinase (or precursor form of urokinase) with a molecular weight of about 100,000 in fresh human urine.

Fresh human urine was found to contain at least three different molecular forms of fibrin-binding urokinase (UK) or its precursor, all of which were absorbed on a fibrin/Celite column at neutral pH, and could be eluted with 0.3-1.0 mol/l NaCl in phosphate buffer, followed by 0.2 mol/l, Arg, 2 mol/l KSCN, and 2 mol/l urea, respectively. The main molecular form isolated revealed a molecular weight (MW) of approximately 100,000 (UK-100), and the minor ones were estimated to have MW of 150,000-200,000 and 45,000. In contrast, commercially obtained UK preparations contained mostly active enzymes with MW of 53,000 and 32,000, respectively, and the remaining high molecular forms represented less than 2.0% of the total amount. Rabbit monospecific antibody (IgG) against UK subcomponent (active heavy chain; H-chain UK) reacted and inhibited the fibrinolytic activity of all the active UK molecules. The UK-100 isolated was relatively stable in solution at neutral pH and resistant to mild reduction, without molecular change. Although the preparation had a very low specific activity (ca. 300 IU/mg protein), both the pyro-Glu-Gly-Arg-pNA amidolytic and plasminogen activating activities could be partially enhanced by the addition of trace amounts of plasmin. In this process, the appearance of two additional active enzymes of MW 53,000 and 32,000 was also confirmed by zymography.

Adolescent↗
PubMed1985

Transport of urokinase across the intestinal tract of normal human subjects with stimulation of synthesis and/or release of urokinase-type proteins.

Oral administration of clinical-type high molecular weight urokinase (HMW-UK) in a single dose of 30,000-60,000 International Units (IU) in enteric-coated capsules, in a group of normal human subjects, induced a plasma fibrinolytic state suggesting transport of HMW-UK across the intestinal tract. Other groups of human subjects were given a single dose of 120,000 IU daily of pure HMW-UK for 1 d and 7 d together with a placebo dose, all of the ingredients except urokinase (UK), daily for 7 d. UK-type proteins were isolated from the plasma of blood samples drawn 6 h after administration of the final dose, by a sequential two-step affinity chromatography method first with [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester]-Sepharose and second with a specific rabbit anti-HMW-UK-IgG-Sepharose. The yield of UK-type proteins from the 7-d group, 0.79 mg/dl, was approximately twofold greater than that obtained from either the placebo or 1-d groups. The specific plasminogen activator activity of the 1-d and 7-d groups were similar, 508 and 537 IU/mg protein, respectively; negligible plasminogen activator activity could be detected in the placebo group. The kinetics of activation parameters of human Glu-plasminogen of the UK-type protein, isolated from the 1-d group, were similar to those obtained with urinary HMW-UK. The UK-type proteins isolated only from the 7-d group showed, in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a major protein band of molecular weight 53,000 in the same position as HMW-UK. In addition, two other major protein bands of approximately 140,000 and approximately 120,000 mol wt were found in the 7-d placebo-, and 1-d groups, and also in the 7-d group. The 53,000 mol wt protein, about 50% of the total protein in the 7-d group, was further purified by preparative SDS-polyacrylamide gel electrophoresis, and found to be a two-chain protein with a specific activity of 1,241 IU/mg protein. The protein showed common antigenic determinants with urinary HMW-UK. The oral administration of 120,000 IU HMW-UK to human subjects for 7 d stimulated the synthesis of a UK-type protein of 53,000 mol wt, probably the zymogen, from either the liver or vascular endothelium, which was released into the circulation, and converted into active UK by circulating plasmin. The induction of the fibrinolytic state produced the conversion of native circulating Glu-plasminogen into the degraded Lys-plasminogen form, which was found in large amounts in the plasma of the 7-d group. The new plasma components, e.g., the 53,000-mol wt UK-zymogen and Lys-plasminogen, could play an important role in clot resolution of the fibrin-thrombus in thromboembolic diseases. Oral administration of HMW-UK has been shown to be clinically effective in patients with cerebral thrombosis in a multicenter double blind study.

Adolescent↗
PubMed1985

Cysteine protease inhibitors isolated from human malignant melanoma tissue.

Cysteine protease inhibitors that specifically reacted with several cysteine proteases were found in KSCN extract of human melanoma tissue. From 30 gm of the tissue, approximately 593.5 U inhibitor was obtained. The inhibitors were adsorbed on a papain-Sepharose column and could be eluted with 10 mmol/L phosphate buffer, pH 6.0, containing NaCl or KCl, or with 20 mmol/L acetate buffer, pH 4.0, containing KSCN. They revealed a strong inhibitory activity for cysteine proteases such as ficin, papain, and cathepsin B, but did not react with cysteine protease bromelain or serine protease trypsin. No immunologic relationship was confirmed between the inhibitor and other well-known plasma inhibitors such as alpha 1-antitrypsin, alpha 2-macroglobulin, alpha 1-antichymotrypsin, antithrombin III, C1-in-activator, and alpha 2-plasmin inhibitor. With Sephadex G-100, two main peaks of molecular weight 40,000 and 10,000 were detected in the KSCN extract of the human melanoma tissue. However, the inhibitors revealed three molecular weights of 10,000, 25,000, and 80,000 when estimated by Sephadex G-100 gel filtration after papain-Sepharose affinity chromatography. On the other hand, the molecular weights of the inhibitors changed to two peaks of 25,000 and 10,000 on rechromatography with a papain-Sepharose column.

Adsorption↗
PubMed1985

The transport of 125I-labeled human high molecular weight urokinase across the intestinal tract in a dog model with stimulation of synthesis and/or release of plasminogen activators.

In an attempt to elucidate the mechanism of fibrinolytic enhancement by orally administered urokinase, studies on the intestinal transport of urokinase were carried out, using 125I-labeled human high mol wt urokinase, administered intraduodenally in the experimental dog model with a saphenous vein thrombus. Using the plasma sample obtained from blood 45 minutes after intraduodenal administration of the urokinase, protein fractions were isolated by a sequential two-step affinity chromatography method, first with [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester]-Sepharose followed by a specific anti-human low mol wt urokinase rabbit IgG-Sepharose (adsorbed-eluted and unadsorbed). Each of the isolated protein fractions was further purified by gel filtration on Sephacryl S-300. The proteins isolated by the two-step affinity chromatography method were transported human urokinase with radioactivity in the adsorbed-eluted fraction, and newly synthesized and/or released dog plasminogen activators, probably urokinase-type and tissue-activator type, without radioactivity. In an antibody quenching assay, dog urokinase and the immuno-affinity unadsorbed fraction were not neutralized, but the immuno-affinity adsorbed-eluted fraction was completely neutralized by the specific anti-urokinase IgG antibody. Proteins isolated from control plasma (after administration of saline) by the two-step affinity chromatography method in the unadsorbed fraction had negligible amounts of activator activity. In these studies, we were able to show that synthesis of plasminogen activators was stimulated, with the activators being released, from either the liver or the vascular endothelium. Also we showed that urokinase is transported across the intestinal tract in the dog model.

Animals↗