Search PubMed⌕ Search

Biomedical subjects

S Kojima

Publications and source records attributed to S Kojima.

At least 541 records · Page 30Linked to original sources
PubMed1993

Further study of effects of chelating agents on excretion of inorganic mercury in rats.

The effects of three chelating agents, N-benzyl-D-glucamine dithiocarbamate (BGD), 2,3-dimercaptopropanol (BAL) and D-penicillamine (D-PEN), on the excretion of mercury in rats exposed to mercuric chloride (HgCl2), the chemical forms of mercury compounds excreted in the bile and urine and the intestinal reabsorption of mercury compounds in the bile were studied. Rats were injected intraperitoneally with 203HgCl2 (300 micrograms Hg and 74 kBq of 203Hg/kg) and 24 h later, they were injected intraperitoneally with a chelating agent (a quarter of an LD50). The injection of the chelating agents significantly enhanced the biliary and urinary excretions of mercury. The enhancing effect of BGD on the excretions of mercury was almost the same as that of BAL and much larger than that of D-PEN. The major chemical form of mercury in the bile and urine of rats injected with BGD after HgCl2 treatment was Hg-BGD compounds. The chemical form of mercury in the bile and urine of rats injected with BAL after HgCl2 treatment was mainly Hg-GSH compound. The mercury after HgCl2 and D-PEN treatment was excreted mainly via the urine in the form of Hg-D-PEN compound. The intestinal reabsorption of mercury from the bile of rats injected with BGD or D-PEN was only 0.18% or 0.38% of the dose, respectively. The intestinal reabsorption of mercury from the bile of rats injected with BAL was 27.38% of the dose. It was suggested that the Hg-GSH compound excreted in the bile after HgCl2 and BAL treatment is partly degraded to Hg-cysteine (Cys) by the intestinal membranous enzymes and that the ligand of Hg-Cys is replaced by BAL in the bile, resulting in the effective reabsorption of Hg-BAL compound from the intestine.

Animals↗
PubMed1993

Phase studies of a urethane model compound and polyether macroglycols by infrared spectroscopy and the relationship between eutectic composition of soft segment and blood compatibility.

The binary mixtures of a urethane model compound, diethyl 4,4'-methylenebis(N-phenylcarbamate) (MDU), and various polyether macroglycols have been investigated mainly by infrared (IR) spectroscopy. The mixtures of macroglycol and MDU showed two C=O bands (free C=O at higher wave number, hydrogen bonded C=O at lower wave number), and the intensity of free C=O increased while that of hydrogen bonded C=O decreased linearly with increasing molar ratio of macroglycol/MDU. The slope of the increase or decrease suddenly changed at the specific molar ratio around the eutectic composition. The eutectic molar composition for PTMO1000/MDU was determined as 1.15 or 1.1 by differential scanning calorimetry (DSC) or infrared (IR) studies while that for PTMO3000/MDU as 0.345 or 0.55 by DSC or IR, respectively. The eutectic compositions for other sets were determined by IR studies as follows: PEO1000/MDU, 0.4; PEO3000/MDU, 0.16; PPO1000/MDU, 1.4; PPO3000/MDU, 0.45. The number of ethylene oxide, tetramethylene oxide, and propylene oxide units to form a eutectic with MDU calculated from these values were 15-20, 10-11, and 23-24, respectively. The similar DSC or IR changes were observed in the various kinds of polyurethanes such as MDI/BD, MDI/PTMO/BD, and MDI/PTMO polymers. The relationship between the eutectic compositions of soft segments (MDI/macroglycol) and the ideal Mn of the macroglycols in blood compatibility of segmented polyurethanes are discussed.

Benzyl Compounds↗
PubMed1993

Mechanism of retinoid-induced activation of latent transforming growth factor-beta in bovine endothelial cells.

Cell-associated plasmin is a putative physiological activator of latent transforming growth factor-beta (LTGF-beta). Since retinoids enhance the production of plasminogen activator (PA) and thereby increase cell-associated plasmin activity, we tested the possibility that retinoids might induce the activation of LTGF-beta using bovine endothelial cells (ECs) as a model system. ECs treated with physiological concentrations of retinol or retinoic acid formed active TGF-beta in the culture media in a dose- and time-dependent fashion. Cells were treated with 2 microM retinol for 24 h, and the amount of TGF-beta produced during a subsequent 12-h incubation period was measured. Out of a total of 14 pM LTGF-beta secreted, 0.7 pM was converted to active TGF-beta. Northern blot analyses showed that mRNA levels for TGF-beta 2 but not for TGF-beta 1 increased in cells treated with retinol. Inclusion of either inhibitors of PA or of plasmin or antibody against PA in the culture medium as well as depletion of plasminogen from the serum blocked the formation of TGF-beta, suggesting that PA, plasminogen, and the resulting plasmin are essential for activation of LTGF-beta in retinoid-stimulated cells. Antibody against the LTGF-beta binding protein blocked activation implying that localization of LTGF-beta through its binding protein may be important. However, inhibition of binding of LTGF-beta to the cell surface mannose 6-phosphate receptor did not prevent activation. These data indicate that retinoids up-regulate the production of LTGF-beta in ECs and induce activation of LTGF-beta, perhaps, by increasing PA and plasmin levels. Thus, TGF-beta might be a local mediator of some of the biological activities of retinoids both in vivo and in vitro.

Animals↗
PubMed1993

Anti-tumor activity of T-2 toxin-conjugated A7 monoclonal antibody (T-2-A7 MoAb) against human colon carcinoma.

Monoclonal antibody A7 (A7 MoAb), from splenocytes of a mouse immunized against human colorectal carcinoma, was used as a T-2 toxin (T-2) carrier targeting colon cancer. T-2 was converted to T-2 hemiglutarate by glutaric anhydride treatment, and T-2-A7 MoAb conjugates containing up to 20 T-2 per antibody molecule were obtained from the antibody and T-2 hemiglutarate activated with N-hydroxysuccinimide. The in vitro cytotoxicity against human colon cancer (LS174T) cells indicated that the conjugates were markedly less toxic than the toxin itself. The immunoreactivity was evaluated from the in vitro binding activity of A7 MoAb with LS174T cells, and from the in vivo localization in LS174T-bearing nude mice; it remained essentially intact after conjugation with T-2. The efficacy of the T-2-A7 MoAb conjugate was tested against LS174T-bearing nude mice. The conjugate significantly suppressed the growth of the tumor in comparison with both phosphate-buffered saline and free T-2. These results suggest that the conjugate of T-2 with A7 MoAb might be useful as a selective immunotoxin for cancer immunotherapy, with less serious side effects than T-2.

Animals↗
PubMed1993

Simple 24-hour preservation of rabbit hearts with hexanol and pyruvate cardioplegia.

The effectiveness of a newly developed crystalloid cardioplegic solution containing 4 mM 1-hexanol and 10 mM pyruvate was compared with St Thomas' Hospital solution. After control perfusion with a Langendorff method, rabbit hearts (n = 7 for each group) were arrested and stored in these solutions (4 degrees C) for 24 h and subsequently reperfused for 45 min. The hexanol solution preserved both left ventricular systolic and diastolic function significantly better than St Thomas' Hospital solution. Developed pressure was significantly higher after preservation with the hexanol solution than with the St Thomas' Hospital solution at left ventricular balloon volumes larger than the mid volume (P < 0.05). The maximum developed pressure values after storage with the hexanol and St Thomas' Hospital solution were 77% and 42% of the maximum control values, respectively. Values for end-diastolic pressure at the maximum ventricular volume were 26.1 +/- 9.8 and 66.7 +/- 24.6 mmHg for the hexanol and St Thomas' Hospital solution, respectively (P < 0.05). Creatine kinase release during the first 15 min of reperfusion was significantly lower with the hexanol solution as compared to St Thomas's Hospital solution (28.3 +/- 8.3 vs 92.9 +/- 52.6 IU/g wet weight, P < 0.05). In conclusion, a hexanol cardioplegic solution may be suitable for long-term cardiac preservation. Further evaluation of the effectiveness and safety of this solution is warranted.

Animals↗
PubMed1993

Close phylogenetic relationship between Vestimentifera (tube worms) and Annelida revealed by the amino acid sequence of elongation factor-1 alpha.

To clarify the phylogenetic position of Vestimentifera (tube worms), 346-bp fragments of the elongation factor-1 alpha (EF-1 alpha) gene (939-1286 according to the numbering of the human gene) of a vestimentiferan, Lamellibrachia sp., a sternaspid polychaete, Sternaspis scutata, an earthworm, Pheretima sp., and a gastropod, Alviniconcha hessleri, were sequenced. From the amino acid sequences of these EF-1 alpha, and those of two other vertebrates and two arthropods, phylogenetic relationships were deduced by the maximum likelihood (ML) method, by which the phylogenetic tree can be inferred without assuming constancy of the molecular evolutionary rate. For the ML tree and all of seven alternative trees, whose log-likelihoods could not be discriminated from that of the ML tree by the criterion of the standard error, the vestimentiferan, the polychaete, and the oligochaete formed a clade, excluding the arthropods and the gastropod as outgroups. This result is convincing evidence that Vestimentifera are protostomes that are closely related to Annelida. The ML tree suggests that Vestimentifera are more closely related to Polychaeta than to Oligochaeta, though the data were not sufficient to discriminate these three groups at a significant level. From recent evidence such as morphological characteristics and molecular information, it may safely be said that vestimentiferans should be included in the Annelida provided this phylum contains polychaetes and oligochaetes.

Amino Acid Sequence↗
PubMed1993

Severe pulmonary pneumocystosis in simian acquired immunodeficiency syndrome induced by simian immunodeficiency virus: its characterization by the polymerase-chain-reaction method and failure of experimental transmission to immunodeficient animals.

Pneumocystis carinii (Pc) infection was observed in three of five rhesus monkeys infected with simian immunodeficiency virus (SIVmac251). They showed severe symptoms similar to those associated with human acquired immunodeficiency syndrome (AIDS). Histopathology revealed severe pulmonary pneumocystosis in one of three Pc-positive monkeys, and anti-Pc antibodies were detected in sera from two of the three monkeys. Localization of Pc organisms in various organs of the monkeys was examined by the polymerase-chain-reaction (PCR) method, and Pc-specific bands of DNA amplification were detected in the liver, kidney, spleen, adrenal gland, testis, brain, and other organs examined, but no Pc organism was found in these organs by histopathologic examination. These results suggest that the activation of a latent infection of Pc occurs in SIV-infected rhesus monkeys as well as in human AIDS. Experimental transmission of Pc derived from a simian was attempted in severe combined immunodeficiency (SCID) mice and athymic nude (rnu/rnu, F344) rats. These animals were inoculated intranasally with 10(4) Pc cysts, but neither histopathologic changes nor Pc organisms were detected in SCID mice at 4 months after inoculation or in nude rats at 2 months postinoculation, suggesting that simian Pc is species-specific.

Animals↗
PubMed1993

Minimal residual disease status in pre-B acute lymphoblastic leukemia patients after chemotherapy and bone marrow transplantation: assessment of the anti-leukemic effects of chemotherapy and BMT.

We prospectively analyzed minimal residual disease (MRD) in four patients with B-cell precursor acute lymphoblastic leukemia who had been in complete remission for more than one year after chemotherapy and allogenic or autologous bone marrow transplantation (BMT). MRD was quantitatively estimated using polymerase chain reaction amplification to detect the complementarity-determining region III of the immunoglobulin heavy chain gene at limiting dilution DNA samples. Our study showed that remission induction chemotherapy reduced at most 2-logs of leukemia cells, and that subsequent consolidation chemotherapy induced further reduction of leukemia cells. In two cases, 10(-5) levels of MRD were detected two months after BMT. However, no MRD was detected four months after BMT. We also showed the effectiveness of ex vivo purging with anti-CALLA monoclonal antibodies which eliminated at least 2-logs of leukemia cells in autologous BMT. Our results suggest that this detection system is useful for assessing the reduction of the original leukemia clone, and that the presence of MRD within three months after BMT is not related to clinical outcome.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1993

Prevention of renal toxicity of cis-diamminedichloroplatinum by dithiocarbamates in rats.

The protective effects of various dithiocarbamates such as N-benzyl-D-glucamine dithiocarbamate (BGD), N-p-hydroxymethylbenzyl-D-glucamine dithiocarbamate (HBGD), N-p-carboxybenzyl-D-glucamine dithiocarbamate (CBGD), and N-methyl-D-glucamine dithiocarbamate (MGD) on DDP-induced renal toxicity in rats were studied. The rats received the simultaneous i.v. injection of DDP (20 mumol/kg) and a chelating agent (40 mumol/kg). Significant increase in blood urea nitrogen (BUN) level was observed 5 days after DDP injection. The increase in BUN level was completely prevented by only HBGD and CBGD among these chelating agents. Treatment with CBGD completely prevented against DDP-induced body weight loss. BGD and MGD treatment did not prevent the increase in BUN level or body weight loss. HBGD and CBGD were the most effective in decreasing the renal platinum content, resulting in maximum protection against the DDP-induced renal damage. The antitumor efficacy of DDP in the Walker 256 carcinoma-bearing rats was not affected by CBGD administration.

Animals↗
PubMed1993

Eighteen-hour preservation of rat hearts with hexanol and pyruvate cardioplegia.

OBJECTIVES: The aim of this study was to evaluate the effectiveness of 1-hexanol as an arresting agent and pyruvate as a substrate in a cardioplegic solution. BACKGROUND: Heart transplantation is limited in part by the short preservation time of donor hearts. Better preservation techniques would improve patient survival and the time and geographic area for using donor hearts. We previously showed that a cardioplegic solution containing ethanol and pyruvate was superior to a conventional high potassium cardioplegic solution in 24-h cold storage of hamster hearts. Hexanol, a more potent arresting agent than ethanol, might be a more suitable alcohol. METHODS: Rat hearts were arrested and stored for 18 h at 4 degrees C with an ethanol (3 vol% = 510 mmol/liter) or 1-hexanol (4 mmol/liter) and pyruvate (10 mmol/liter) cardioplegic solution, St. Thomas' Hospital solution and Stanford solution and subsequently reperfused for 1 h at 35 degrees C. In other groups of hearts, basal oxygen consumption and rest intracellular calcium (Indo 1 technique) were evaluated during ethanol-, hexanol- and potassium-induced cardiac arrest. RESULTS: The percent recovery of left ventricular developed pressure and rate-pressure product were significantly better with the hexanol cardioplegic solution (67 +/- 21% and 58 +/- 19%, respectively; p < 0.05 for all comparisons) compared with the ethanol (10 +/- 7% and 5 +/- 4%), St. Thomas' Hospital (14 +/- 6% and 10 +/- 5%) and Stanford solutions (2 +/- 2% and 2 +/- 1%, respectively). Exclusion of ethanol and hexanol from storage solutions did not influence functional recovery. Values for oxygen consumption after 15- and 30-min ethanol- and hexanol-induced arrest were significantly lower than those after potassium-induced cardiac arrest. There was no difference in the rest intracellular calcium during cardiac arrest induced by the three arresting agents. CONCLUSIONS: A hexanol and pyruvate cardioplegic solution was more favorable than ethanol or conventional solutions for long-term cold storage of rat hearts. The beneficial effects of hexanol may have been provided in part by lower energy consumption during hexanol-induced cardiac arrest. These results may have implications for preservation of hearts for heart transplantation.

Animals↗
PubMed1993

Comparative study of intact A7 MoAb and F(ab')2 fragments for radioimmunoimaging of human colon cancer in nude mice.

Differences of pharmacokinetics and tumor imaging ability between intact monoclonal antibody A7 (A7 MoAb) and F(ab')2 fragments were studied in human colon cancer (LS-174T)-bearing nude mouse. First of all, we examined the yield and the immunoreactivity of F(ab')2 fragments after treatment with ficin as a function of time. The yield of F(ab')2 fragments reached about 50% after ficin treatment for 8 h, and the F(ab')2 retained about 80% of the immunoreactivity of the corresponding MoAb. Longer digestion with ficin produced smaller fragments (less than 92 kDa) with a lower yield and most of the immunoreactivity was lost. In pharmacokinetics studies, the F(ab')2 was preferentially taken up by the tumor, was cleared more rapidly from the blood circulation and seemed to have less non-specific tissue binding than intact A7 MoAb. In addition, the tumor image obtained at an early time using 131I-F(ab')2 was much superior in quality to that with intact 131I-A7 MoAb. The use of F(ab')2 fragments may be effective for tumor diagnosis and therapy.

Animals↗
PubMed1993

Differences between A7 antibody and anti-CEA antibody.

The specificity of A7 monoclonal antibody (A7 MoAb), raised against a human colon cancer, was investigated in detail and compared with that of anti-CEA MoAb. Firstly, the biodistributions of radiolabeled A7 MoAb and anti-CEA MoAb were examined in human colon cancer (LS-174T)-, glioblastoma- and lung cancer (Lu-65)-bearing nude mice. 125I-A7 MoAb was highly concentrated into LS-174T and also, to a lesser extent, in glioblastoma, whereas 125I-anti-CEA MoAb was significantly taken up only by LS-174T. Both of these antibodies were taken up at only very low concentrations into Lu-65. Secondly, in vitro binding studies of 125I-A7 MoAb and 125I-anti-CEA MoAb with LS-174T cells and glioblastoma cells revealed high binding activity of these MoAbs to LS-174T cells, though A7 MoAb had a much higher affinity than that of anti-CEA MoAb. Neither of them showed high affinity for glioblastoma cells. Thirdly, competitive binding assay using A7 MoAb and anti-CEA MoAb to LS-174T cells showed that the binding of each 125I-MoAb was not inhibited by the other MoAb. Finally, in radioimmuno-imaging studies of LS-174T- and glioblastoma-bearing mice with 131I-A7 MoAb and -anti-CEA MoAb, both tumors were clearly visualized with the former, while the latter visualized only LS-174T. The A7 MoAb is clearly different from anti-CEA MoAb and may be useful for the in vivo radioimmunodetection and treatment of colon cancer.

Animals↗
PubMed1993

Requirement for transglutaminase in the activation of latent transforming growth factor-beta in bovine endothelial cells.

A hitherto unknown function for transglutaminase (TGase; R-glutaminyl-peptide: amine gamma-glutamyltransferase, EC 2.3.2.13) was found in the conversion of latent transforming growth factor-beta (LTGF-beta) to active TGF-beta by bovine aortic endothelial cells (BAECs). The cell-associated, plasmin-mediated activation of LTGF-beta to TGF-beta induced either by treatment of BAECs with retinoids or by cocultures of BAECs and bovine smooth muscle cells (BSMCs) was blocked by seven different inhibitors of TGase as well as a neutralizing antibody to bovine endothelial cell type II TGase. Control experiments indicated that TGase inhibitors and/or a neutralizing antibody to TGase did not interfere with the direct action of TGF-beta, the release of LTGF-beta from cells, or the activation of LTGF-beta by plasmin or by transient acidification. After treatment with retinoids, BAECs expressed increased levels of TGase coordinate with the generation of TGF-beta, whereas BSMCs and bovine embryonic skin fibroblasts, which did not activate LTGF-beta after treatment with retinoids, did not. Furthermore, both TGase inhibitors and a neutralizing antibody to TGase potentiated the effect of retinol in enhancing plasminogen activator (PA) levels in cultures of BAECs by suppressing the TGF-beta-mediated enhancement of PA inhibitor-1 (PAI-1) expression. These results indicate that type II TGase is a component required for cell surface, plasmin-mediated LTGF-beta activation process and that increased expression of TGase accompanies retinoid-induced activation of LTGF-beta.

Animals↗
PubMed1993

Increased responsiveness of left ventricular apical myocardium to adrenergic stimuli.

OBJECTIVE: The aim was to determine whether left ventricular apical myocardium has mechanisms to compensate for sparse sympathetic innervation. METHODS: Contractile and metabolic responses to various adrenergic stimuli and beta adrenergic receptor density were compared between left ventricular basal and apical regions in 26 anaesthetised mongrel dogs, weight 12-28 kg. RESULTS: Regional contractile changes in response to graded cardiac sympathetic nerve stimulation were compared among three basal (anterior, middle, and posterior) regions, and between basal middle and apical regions. There were significant differences in contractile changes among the three basal regions with distinct regions of innervation from right and/or left sided sympathetic ganglia, but not between apical and basal regions. Constant infusion of noradrenaline (0.2-0.4 microgram.kg-1.min-1) produced a greater response in normalised end systolic length in the apical myocardium than in the basal region, at 9.86(SEM 0.06) mm v 10.14(0.04) mm (n = 5, p < 0.025), and a greater increase in tissue cyclic AMP: 1.04(0.20) v 0.60(0.08) pmol.mg-1 (n = 5, p < 0.05). Giving a forskolin derivative (30 micrograms.kg-1, n = 5) produced a greater increase in cyclic AMP in the apical region than in the basal region: 1.26(0.18) v 0.88(0.19) pmol.mg-1 (p < 0.02). beta Adrenergic receptor density in the apical region was greater than in the basal region: 455(45) v 341(35) fmol.mg-1 protein (n = 5, p < 0.05). CONCLUSIONS: Greater beta adrenergic receptor density and/or increased myocardial responsiveness to adenylate stimulation in apical myocardium compensates, at least in part, for its sparse sympathetic innervation.

Animals↗
PubMed1993

Acute effects of ethanol on cardiac function and intracellular calcium in perfused rat heart.

OBJECTIVE: The aim was to evaluate effects of ethanol on cardiac function and intracellular Ca2+ ([Ca2+]i) in perfused rat hearts. METHODS: A Langendorff perfused rat heart preparation was used. Changes in [Ca2+]i were evaluated by surface fluorometry in hearts loaded with Indo 1-AM. RESULTS: Clinically relevant concentrations of ethanol (0.2 or 0.4% vol/vol) had no significant haemodynamic effects. High concentrations of ethanol (1, 2, 3, and 4% vol/vol) showed dose dependent decreases in developed pressure and the systolic peak and overall amplitude of the Indo 1 fluorescence transients (identical to [Ca2+]i), that were partially antagonised by high extracellular Ca2+ ([Ca2+]o = 4 mM). The ethanol concentrations that decreased developed pressure by 50% were 1.4 and 2.6% in the low (1.5 mM) and high [Ca2+]o, respectively. Four per cent ethanol decreased the amplitude of Indo 1 fluorescence transients to 54.5(SD 3.1) and 64.6(7.9)% of control values in the low and high [Ca2+]o, respectively. A relationship between the amplitude of Indo 1 fluorescence and developed pressure was fitted to a single sigmoid curve irrespective of [Ca2+]o. During ethanol washout, there was a dose dependent overshoot of the fluorescence ratio. CONCLUSIONS: Only high concentrations of ethanol depressed left ventricular function in a dose dependent manner by decreasing the amplitude of [Ca2+]i transients. High [Ca2+]o partially antagonised acute alcoholic cardiac depression by increasing the amplitude of [Ca2+]i transients. [Ca2+]i is a mediator of the acute cardiac effects of ethanol in perfused intact rat hearts.

Animals↗
PubMed1993

Interaction of subtilisin BPN' and recombinant Streptomyces subtilisin inhibitors with substituted P1 site residues.

Kinetic analysis was performed on the interaction between subtilisin BPN' and recombinant species of a proteinaceous proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), of which the P1 site amino acid residue, Met73, was replaced by site-directed mutagenesis. The inhibitor constant, Ki, was determined from the residual enzyme activity by using a peptide substrate. The rate constant of binding, kon, and the rate constant of dissociation, koff, were determined from a progress curve of the substrate hydrolysis in the presence of the inhibitor by using newly derived equations. A recombinant SSI in which Met73 was replaced by Ile showed an affinity (1/Ki) toward subtilisin BPN' of only about 7% of that of the wild-type SSI, and the kinetic analysis revealed that the increase of koff was responsible for this difference. The affinity of other SSI mutants in which Met73 was replaced by Glu or Asp decreased significantly as pH became increasingly alkaline. The decrease in the affinity of these recombinants was due to the decrease of kon rather than the increase of koff. Stopped-flow studies revealed that the binding reaction was reconcilable with a two-step mechanism, and the kinetic parameters for each step were obtained for the binding of the enzyme and recombinant SSIs.

Bacterial Proteins↗
PubMed1993

Interaction of Streptomyces subtilisin inhibitor (SSI) with Streptomyces griseus metallo-endopeptidase II (SGMP II).

We have unexpectedly found that Streptomyces subtilisin inhibitor (SSI) and some other similar serine protease inhibitors produced by Streptomycetes strongly inhibit Streptomyces griseus metallo-endopeptidase II (SGMP II) [Kajiwara, K. et al. (1991) J. Biochem. 110, 350-354]. In order to elucidate the mode of their unusual interaction with SGMP II in more detail, we prepared twelve kinds of SSI analogues, in which one or two amino acid residues in the peptide segment from Thr64 to Val74 of wild-type SSI had been replaced or deleted by site-directed mutagenesis, and determined the dissociation constants of their complexes with SGMP II. Six analogues among them showed dissociation constants one order of magnitude lower than that of the wild type. Three had higher values. The results suggest that at least some residues in this segment are interacting with SGMP II in the complex. We also prepared an SSI mutant in which the disulfide bridge between Cys71 and Cys101 had been eliminated by replacing the two Cys residues with Ser residues. This mutated SSI inhibited SGMP II as strongly as the wild-type SSI did. While peptide bonds in the wild-type molecule did not suffer from the hydrolytic action of SGMP II except those at the amino-terminal fragile portion, the Pro72-Met73 bond of the mutant was specifically cleaved by the enzyme. This peptide bond, therefore, seems to play the role of the reactive site in the interaction of SSI with SGMP II.

Amino Acid Sequence↗