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Biomedical subjects

M Nakane

Publications and source records attributed to M Nakane.

At least 127 records · Page 7Linked to original sources
PubMed1980

Production and properties of antibody to soluble guanylate cyclase purified from bovine brain.

Guanylate cyclase was purified 12,700-fold from bovine brain supernatant, and the purified enzyme exhibited essentially a single protein band on polyacrylamide gel electrophoresis. Repeated injection of the purified enzyme into rabbits produced an antibody to guanylate cyclase. The immunoglobulin G fraction from the immunized rabbit gave only one precipitin line against the purified guanylate cyclase and the crude supernatant of bovine brain on double immunodiffusion and immunoelectrophoreis. The antibody completely inhibited the soluble guanylate cyclase activity from bovine brain, various tissues of rat and mouse and neuroblastoma N1E 115 cells, whereas the Triton-dispersed particulate guanylate cyclase from these tissues was not inhibited by the antibody.

Animals↗
PubMed1979

Guanylate cyclase in neuroblastoma N1E 115 cells: presence of endogenous activator.

Guanylate cyclase in cultured neuroblastoma N1E 115 cells was readily solubilized. MgCl2 as well as MnCl2 served as a metal cofactor of the guanylate cyclase. The maximal guanylate cyclase activity obtained with MgC12 was 80% of that with MnCl2. When the supernatant of cell homogenate was adjusted to pH 5.2, all of enzyme activity was precipitated. The guanylate cyclase activity recovered in the pH 5.2 precipitate was reduced to about 10% of the original supernatant. Combination of the pH 5.2 supernatant and precipitate fractions, however, restored guanylate cyclase activity, indicating that the pH 5.2 supernatant contains an endogenous activator for guanylate cyclase. The activating factor in the pH 5.2 supernatant remained in the aqueous phase after proteins were removed by perchloric acid. The factor was filterable through Diaflo ultrafilter membranes UM 2 and UM 10 indicating that the factor is a small molecule. The activation by the endogenous activator was prevented by N-methylhydroxylamine and lysolecithin.

Cell Fractionation↗
PubMed1978

Purification and properties of guanylate cyclase from the synaptosomal soluble fraction of rat brain.

Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) was purified 2250-fold from the synaptosomal soluble fraction of rat brain. The specific activity of the purified enzyme reached 41 nmol cyclic GMP formed per min per mg protein at 37 degrees C. In the purified preparation, GTPase activity was not detected and cyclic GMP phosphodiesterase activity was less than 4% of guanylate cyclase activity. The molecular weight was approx. 480 000. Lubrol PX, hydroxylamine, or NaN3 activated the guanylate cyclase in crude preparations, but had no effect on the purified enzyme. In contrast, NaN3 plus catalase, N-methyl-N'-nitro-N-nitrosoguanidine or sodium nitroprusside activated the purified enzyme. The purified enzyme required Mn2+ for its activity; the maximum activity was observed at 3-5 mM. Cyclic GMP activated guanylate cyclase activity 1.4-fold at 2 mM, whereas inorganic pyrophosphate inhibited it by about 50% at 0.2 mM. Guanylyl-(beta,gamma-methylene)-diphosphonate and guanylyl-imidodiphosphate, analogues of GTP, served as substrates of guanylate cyclase in the purified enzyme preparation. NaN3 plus catalase or N-methyl-N'-nitro-N-nitrosoguanidine also remarkably activated guanylate cyclase activity when the analogues of GTP were used as substrates.

Animals↗
PubMed1978

Supercoiled DNA folded by nonhistone proteins in cultured mouse carcinoma cells.

Upon gentle lysis of exponentially growing mouse carcinoma cells FM3A by sodium dodecyl sulfate, DNA was released as a "DNA-protein complex" in a folded conformation. No histones could be detected in the DNA-protein complex. The proteins bound to DNA were found to be composed of several kinds of nonhistone proteins with a molecular weight range of 50,000 to 60,000; they appear to play a key role in stabilizing and maintaining the compact and folded structure of the complex. Removal of the proteins by Pronase or 2-mercaptoethanol produced a more relaxed structure sedimenting about half as fast as the original complex in a neutral sucrose gradient. DNA in the folded complex is supercoiled, as indicated by the characteristic biphasic response of its sedimentation rate to increasing concentration of various intercalating agents, actinomycin D, ethidium bromide and acriflavine, with which the cells were treated before lysis. Pronase- or 2-mercaptoethanol-treated relaxed DNA still possessed the characteristic of closed-circular structure as judged from its response to intercalating agents. Nicking with gamma-ray or 4NQO broke these superhelical turns and relaxed the folded complex to slower sedimenting forms equivalent to the relaxed DNA obtained on treatment with Pronase or 2-mercaptoethanol. Viscometric observations of DNA-protein complex were consistent with the above results. A tentative model for the structure of this DNA-protein complex is proposed in which supercoiled DNA is folded into loops by several kinds of nonhistone proteins. Autoradiographic examination of the complex appeared to support this model.

4-Nitroquinoline-1-oxide↗
PubMed1976

Subcellular distribution and activation by non-ionic detergents of guanylate cyclase in cerebral cortex of rat.

Non-ionic detergents stimulated particulate guanylate cyclase activity in cerebral cortex of rat 8- to 12-fold while stimulation of soluble enzyme was 1.3- to 2.5-fold. Among various detergents, Lubrol PX was the most effective one. The subcellular distribution of guanylate cyclase activity was examined with or without 0.5% Lubrol PX. Without Lubrol PX two-thirds of the enzyme activity was detected in the soluble fraction. In the presence of Lubrol PX, however, two-thirds of guanylate cyclase activity was recovered in the crude mitochondrial fraction. Further fractionation revealed that most of the particulate guanylate cyclase activity was associated with synaptosomes. The sedimentation characteristic of the particulate guanylate cyclase activity was very close to those of choline acetyltransferase and acetylcholine esterase activities, two synaptosomal enzymes. When the crude mitochondrial fraction was subfractionated after osmotic shock, most of guanylate cyclase activity as assayed in the absence of Lubrol PX was released into the soluble fraction while the rest of the enzyme activity was tightly bound to synaptic membrane fractions. The total guanylate cyclase activity recovered in the synaptosomal soluble fraction was 6 to 7 times higher than that of the starting material. The specific enzyme activity reached more than 1000 pmol per min per mg protein, which was 35-fold higher than that of the starting material. The membrane bound guanylate cyclase activity was markedly stimulated by Lubrol PX. Guanylate cyclase activity in the synaptosomal soluble fraction, in contrast, was suppressed by the addition of Lubrol PX. The observation that most of guanylate cyclase activity was detected in synaptosomes, some of which was tightly bound to the synaptic membrane fraction upon hypoosmotic treatment, is consistent with the concept that cyclic GMP is involved in neural transmission.

Animals↗
PubMed1975

[Acute epidural spinal abscess--A case of complete recovery by surgical treatment (author's transl)].

Acute spinal epidural abscess is found to be relatively rare and may be getting rarer since antibiotics have been widely used. An early diagnosis and prompt surgical treatment are still important to prevent severe neurological deficit such as paraplegia. A characteristic case is presented. Eighteen years old boy showed acute progression of paraplegia and rectourinary difficulty with complete spinal block at T 6-7 intervertebral space on myelography, who had suffered from multiple infectious lacerations on his face and hands two months previously. There was complete recovery of neurological signs following removal of epidural abscess with granulation tissue and spinal decompression by lamineotomy through T 5-7, and supplimentary antibiotic therapy for the organism (Staphylococcus aureus). To our knowlodge, this will be the first case report in this country, which has been obtained successful recovery after surgical treatment in such an acute case.

Abscess↗
PubMed1975

Recovery of a DNA-protein complex in cultured mammalian cells from damage caused by 4-nitroquinoline 1-oxide.

Treatment of FM3A cells with 4-nitroquinoline 1-oxide caused a decrease in the sedimentation velocity of a DNA-protein complex, but did not cause a dissociation of the complex, as revealed by neutral sucrose gradient centrifugation. Microscopic autoradiography of the complex spread on a Millipore filter, demonstrated that treatment of the cells with 4-nitroquinoline 1-oxide, or of the complex with Pronase E, gave rise to a relaxed mass of DNA fibers, in contrast to a compact mass of DNA from control cells. The damage to the DNA-protein compelx was repaired completely by incubation of the cells in a medium without 4-nitroquinoline 1-oxide. The following metabolic inhibitors had no effect on the repair of the complex: inhibitors of nucleic acid synthesis, alpha-amanitine, cordycepin, 2-mercapto-1-(beta-4-pyridethyl)benzimidazol, 1-beta-D-arabinofuranosylcytosine, 5-fluorodeoxyuridine, and hydroxyurea; inhibitors of protein synthesis, cycloheximide and puromycin; an inhibitor of the dark repair process in a variety of biological systems, caffeine; inhibitors of the microtubular and microfilament system, Colcemid and cytochalasin B, respectively; and inhibitors of energy metabolism, 2,4-dinitrophenol, KCN, iodoacetic acid, ouabain, and an atmosphere of nitrogen. Acriflavine and actinomycin D, which are known to intercalate into DNA, caused a decrease in the sedimentation velocity of the DNA-protein complex; therefore, the effects of these agents on the recovery process remained unsolved. The repair process of the complex was, however, demonstrated to be temperature dependent. The process was inhibited at 10 degrees, retarded at 28 degrees, but accelerated at 40 degrees as compared with the rate at 37 degrees.

4-Nitroquinoline-1-oxide↗