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N Sugimoto

Publications and source records attributed to N Sugimoto.

At least 235 records · Page 13Linked to original sources
PubMed1985

Rapid, simplified method for production and purification of tetanus toxin.

A rapid, simplified method for production and purification of tetanus toxin from bacterial extracts was described. The extracts were prepared by stirring young cells (ca. 45-h culture) of Clostridium tetani in 1 M NaCl-0.1 M sodium citrate, pH 7.5, overnight at 0 to 4 degrees C. The toxin was purified by a combination of (i) ammonium sulfate fractionation (0 to 40% saturation), (ii) ultracentrifugation for removal of particulate materials, and (iii) gel filtration by high-pressure liquid chromatography on a TSK G3000 SW-type column. This method required 6 days as follows: (i) overnight incubation of the seed culture, (ii) 2 days for growing the bacteria for toxin production, (iii) overnight extraction of the toxin from the bacteria, (iv) overnight precipitation of the toxin with ammonium sulfate, (v) 2 h for ultracentrifugation of the ammonium sulfate concentrate of the bacterial extract, and (vi) 1 h for high-pressure liquid chromatography. The minimum lethal dose of the purified toxin preparations for mice was 1.4 X 10(7) to 1.5 X 10(7) per mg of protein and they showed 360 to 390 Lf (flocculating activity) per mg protein and a 280/260 nm absorbance ratio of 2.0 to 2.1. The final recovery of the toxin from bacterial extracts was 90 to 93%. The purified preparations gave a single band of toxin protein with a molecular weight of 150,000 +/- 5,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On crossed immunoelectrophoresis, the purified toxin preparations gave a single precipitation arc against anti-crude toxin serum.

Ammonium Sulfate↗
PubMed1984

Artificial electron transport mediated by trinitrophenyl groups introduced into protein molecules by means of chemical modification.

Spinach ferredoxin was found to lose its iron-sulfur center during trinitrophenylation. The loss of the iron-sulfur center resulted in disappearance of its activities as electron mediators in the NADP+ photoreduction and NADPH-cytochrome c reduction systems, whereas it did not affect the activity in the cytochrome c photoreduction system. Based on the experimental evidence that ovalbumin, originally inactive in electron transport, became active in the cytochrome c photoreduction system after trinitrophenylation, the retained activity of the modified ferredoxin was concluded to be due not to the original function of the iron-sulfur center, but to artificial electron transport mediated by TNP groups introduced into the protein molecule. Trinitrophenylated cytochrome c was also active as a mediator, but lost its ability as a terminal acceptor in the system.

Animals↗
PubMed1983

Tetanus toxin: a rapid and selective blockade of the calcium, but not sodium, component of action potentials in cultured neuroblastoma N1E-115 cells.

The slow action potential of mouse neuroblastoma N1E-115 cells, which was elicited in Na-free medium and whose amplitude was directly dependent on extracellular Ca concentrations, was blocked by tetanus toxin (TNTX) without change in resting membrane potential and resistance. The average value of action potential overshoot (peak potential level of spikes) in the absence and presence of TNTX was -1 and -11 mV, respectively. The concentration of TNTX required for 50% inhibition of Ca-dependent spikes (IC50) was about 65 ng/ml. The amplitude of spikes was decreased within 10-20 min after adding TNTX. The inhibition of Ca-dependent spikes was mimicked by 10 mM CoCl2 and MnCl2, but not by the boiled or neutralized toxin. The results indicate that TNTX inhibited electrogenesis of Ca, but not Na, components of action potentials of the neuroblastoma cells.

Animals↗
PubMed1983

Tetanus toxin blocks Ca spikes in neuroblastoma clone N1E-115 cells.

The effect of tetanus toxin on the Ca spike of the action potential evoked in neuroblastoma clone N1E-115 cells by constant current stimulation in Na+-free medium containing various concentrations of Ca2+(1.8-80 mM) was examined by intracellular recordings. Tetanus toxin (more than 140 ng/ml) blocked the Ca spikes. The resting membrane potentials and membrane input resistances of tetanus toxin-treated cells were similar to those of untreated cells. The blocking effect of tetanus toxin was evident in medium containing Ca2+ at concentrations of more than 30 mM. The difference between the peak levels of the action potentials of toxin-treated and untreated cells was statistically significant at Ca2+ concentrations of 7.2 mM and more.

Action Potentials↗
PubMed1983

Toyopearl HW-65C: ammonium sulfate as a new column chromatographic adsorbent for enzyme purification.

We found that Toyopearl HW-65C gel matrix adsorbed ferredoxin and ferredoxin-NADP+ reductase in the presence of concentrated ammonium sulfate. Ferredoxin was strongly adsorbed on the gel in 80% saturated ammonium sulfate, and ferredoxin-NADP+ reductase was adsorbed in 40% saturated ammonium sulfate. The phenomenon was utilized for purification of ferredoxin and the reductase on a Toyopearl HW-65C: ammonium sulfate column. The technique greatly simplified the early stage of purification of ferredoxin and the reductase. The improved purification methods further involved column treatments with DEAE-Toyopearl 650M and Matrex Red A. The effectiveness of the columns is reported. Since a number of other proteins such as cytochrome c, myoglobin, chymotrypsinogen A, ovalbumin, and glucose oxidase were also adsorbed well in an appropriately concentrated ammonium sulfate solution, the method may be of general use in enzyme purification.

Adsorption↗
PubMed1983

Action of tetanus toxin on cholinergic neuroblastoma X glioma hybrid cells: selective blockade of Ca spikes.

We examined the effect of tetanus toxin on clonal neuroblastoma X glioma hybrid cells, NG108-15, by intracellular microelectrode studies of passive membrane electrical properties and action potentials generated under various conditions. Binding of tetanus toxin to the surface of the cells was demonstrated by indirect immunofluorescent staining but no morphological alteration was observed in tetanus toxin-treated cells under a phase contrast microscope. These is no significant difference between the tetanus toxin-treated and untreated cells in their passive electrical membrane properties, i.e. resting membrane potentials, input resistances, time constants and input capacities. Cells in 120 mM Na+, 2 mM Ca2+ salt solution showed Na spikes, and cells in high Ca2+ (30 mM), Na+-free salt solution showed Ca spikes in response to depolarizing current pulses. While the Na spike was not affected by tetanus toxin, the Ca spike was blocked by the toxin. The minimum dose of tetanus toxin for maximum suppression of the peak potential level of the Ca spike was 250 ng/ml. Addition of tetraethyl ammonium (TEA) to extracellular fluid enhanced the Ca spike in untreated cells. In toxin-treated cells, TEA did not alter the effect of tetanus toxin on the Ca spike. Blockade of the Ca spike by tetanus toxin could be detected even at low extracellular Ca2+ concentration (10 mM) by adding TEA to the extracellular fluid and adjusting the membrane potential to a steady hyperpolarized level (-80 mV) to ensure optimal and uniform electrical responses. The usefulness of NG108-15 hybrid cells for in vitro investigations on the mechanism of action of tetanus toxin was discussed.

Action Potentials↗
PubMed1982

Ornithine oxoacid aminotransferase found in AH 130 ascites hepatoma cells.

We found heterogenous ornithine oxoacid aminotransferase (L-ornithine: 2-oxo-acid aminotransferase, EC 2.6.1.1.3) in rat ascites hepatoma AH 130 cells. Compared with enzymes from normal rat tissues, this heterogenous enzyme showed larger Km values for 2-oxoglutarate, a different elution-profile upon affinity chromatography with 2-oxoglutarate, more anionic mobility upon polyacrylamide gell electrophoresis, and a clearly different salting-out property upon ammonium sulfate fractionation. Similar heterogeneity of this aminotransferase was found in human cancer cells.

Animals↗
PubMed1982

Immobilized ferredoxin-NADP+ reductase: preparation and properties.

Immobilized ferredoxin-NADP+ reductase (FNR) was prepared by coupling reaction of CNBr-Sepharose 4B with the spinach enzyme. The immobilized FNR was found to retain the activity of complex formation with ferredoxin as well as the enzymatic activities such as NADPH-diaphorase and NADPH-cytochrome c reductase activities. The complex formation of immobilized FNR with ferredoxin was investigated by measuring reflex spectra of the immobilized FNR with or without ferredoxin and by titration with ferredoxin. The experimental results obtained for the dissociation constant, pH profile and effect of salts were coincidental with those reported for the free enzyme system.

Enzymes, Immobilized↗
PubMed1982

Neuromuscular blocking in acutely tetanus intoxicated mice.

The effects of tetanus toxin on neuromuscular transmission of mice in acute intoxication produced by intravenous injection of a large amount of the toxin were examined by (1) recording the phrenic nerve impulses, the electromyograms (EMGs) of the diaphragm and the electrocardiograms, and (2) the evoked EMGs of the gastrocnemius muscle in response to electrical stimulation of the sciatic nerve. The evoked EMGs of the gastrocnemius muscle were analyzed in terms of kinetic and tonic components by their different latencies. Just before death of animals, the EMGs of the diaphragm appeared with some delay relative to the corresponding phrenic discharges. Finally, the EMG of the diaphragm disappeared even in the presence of phrenic discharge, but cardiac electrical activities continued. The amplitudes of the evoked EMGs of the gastrocnemius muscle invariably became low before death, but the muscle action potential could be recorded by direct muscle stimulation for several minutes after death. The latencies of the evoked EMGs were constant until about the middle of the survival time when the latencies suddenly became prolonged. The longer latency was the same as that of the tonic action potentials. Thus, in acutely tetanus-intoxicated mice, neuromuscular transmission was blocked rapidly and the kinetic component of the muscle was blocked earlier than the tonic component.

Action Potentials↗