Yutaka Kurogochi

Occurrence of a new polyamine, canavalmine, in the sword bean Canavalia gladiata

Abstract A new tetraamine was detected in the seed of sword bean Canavalia gladiata and named canavalmine. The chemical structure was determined to be NH2 (CH2) 4NH(CH2) 3NH(CH2) 4NH2 (1,13-diamino-5,9-diazatridecane) based on gas chromatography-mass spectrometry after derivatization of polyamines with pentafluoropropionic anhydride. The proof of identity was established by comparison of infrared and 1H-NMR spectra of the tetraamine isolated from sword bean with those of a synthetic compound.

α4 is a major acetylcholine binding subunit of cholinergic ligand affinity-purified nicotinic acetylcholine receptor from rat brains

Nicotinic acetylcholine receptor (nAChR) purified from rat brains by cholinergic ligand affinity chromatography was characterized. Monoclonal antibody 299, which binds an acetylcholine (ACh) binding subunit termed alpha 4, depleted more than 85% of [3H]ACh binding activity of the purified preparation. A number of cholinergic agonists strongly inhibited [3H]ACh binding to the purified nAChR, whereas potential antagonists were less effective than the agonists. These results show that most of the purified nAChR contains alpha 4 subunit and the pharmacological properties are preserved upon purification.

Participation of cytochrome P-450 in nicotine oxidation

Abstract Addition of nicotine to phenobarbital-inducible cytochrome P-450 caused a shift of maximum of Soret peak toward the red approximately 3 nm. The difference spectrum produced by nicotine showed a type 2 spectral change with a peak at 427 nm and a trough at 393 nm. A spectral dissociation constant of phenobarbital-inducible cytochrome P-450 was found to be 0.16 mM for nicotine. Nicotine oxidation in the reconstituted system depended on cytochrome P-450, NADPH-cytochrome P-450 reductase and NADPH. These results indicate that phenobarbital-inducible cytochrome P-450 participates in nicotine oxidation.

Cytochrome P-450-dependent nicotine oxidation by liver microsomes of guinea pigs. Immunochemical evidence with antibody against phenobarbital-inducible cytochrome P-450.

When guinea pigs were treated with phenobarbital (PB), the specific activity of liver microsomal nicotine oxidase increased by 42%. PB-inducible cytochrome P-450 (PB-P-450) was purified to homogeneity from liver microsomes of PB-treated guinea pigs. Purified PB-P-450 catalyzed nicotine oxidation when reconstituted with NADPH-P-450 reductase and phospholipid system. Antibody prepared against the purified PB-P-450 formed single precipitation lines with both purified PB-P-450 and microsomal components in livers of PB-treated guinea pigs, and both precipitation lines fused. The antibody against PB-P-450 strongly inhibited nicotine oxidation in the reconstituted system. The antibody also inhibited liver microsomal nicotine oxidase activities in PB-treated and untreated guinea pigs by about 30% and less than 5% respectively. About 45% of total P-450 in liver microsomes of PB-treated guinea pigs was precipitated by the antibody. These results show that PB-P-450 participates in liver microsomal nicotine oxidation in PB-treated guinea pigs but not in untreated control animals.

Heterogeneity of hepatic nicotine oxidase.

When rats were pretreated with 3-methylcholanthrene of beta-naphthoflavone, hepatic nicotine oxidase activity per cytochrome P-448 molecule decreased, but the specific activity of the enzyme remained unchanged. After phenobarbital pretreatment, the specific activity of nicotine oxidase increased while the activity of the enzyme per cytochrome P-450 molecule decreased. alpha-Naphthoflavone selectively inhibited the activities of phenobarbital-induced nicotine oxidase and constitutive form(s) of the enzyme. These results show that phenobarbital-induced cytochrome P-450 and constitutive form(s) of the enzyme may be active in hepatic nicotine oxidation.

Determination of 15NH3 by gas chromatography-mass spectrometry : Application to the measurement of putrescine oxidation by human plasma

A convenient and sensitive method for the determination of 15NH3 has been developed. Ammonia was purified from sample solutions by a modified microdiffusion method, derivatized with pentafluorobenzoyl chloride to pentafluorobenzamide (PFBA) and determined by gas chromatography-mass spectrometry using a multiple ion detector. PFBA was eluted from the gas chromatographic column within 2 min and resulted in a simple mass fragmentation pattern. The 15N/14N ratio was accurately determined with picomole amounts of PFBA by measuring the molecular ions of PFBA and [15N]PFBA. The method was applied to the assay of putrescine oxidation by human plasma. 15NH3 was produced by incubating 15N-labelled putrescine with plasma. The 15NH3 production was time dependent and strongly inhibited by the addition of aminoguanidine, an inhibitor of diamine oxidase. Exceedingly high 15NH3 production from [15N]putrescine was observed in the plasma from pregnant women. In contrast, only trace amounts of 15NH3 were formed in the plasma from normal men and non-pregnant women. The method seems to be applicable to various biological systems that produce ammonia as a metabolic product.

Determination of polyamines in human blood by electron-capture gas-liquid chromatography

The present work was undertaken to develop a sensitive and selective method for the estimation of putrescine, spermidine and spermine in human blood employing electron-capture gas--liquid chromatography. Polyamines were derivatized with heptafluorobutyric anhydride. The heptafluorobutyric derivatives of polyamines could be well resolved within 15 min under a temperature programme. The detection limit was 0.1 pmol for putrescine and cadaverine, and 0.02 pmol for spermidine and spermine. The method was applied to polyamine determinations in erythrocytes from human blood. For pre-separation of the polyamines from other compounds, a simple clean-up method utilizing an activated Permutit has been devised. Major interfering substances could be removed by the batchwise Permutit treatment. The mean values of spermidine and spermine concentrations, and the spermidine/spermine ratio in erythrocytes obtained from normal subjects (n = 11) were similar to reported values. The analytical procedure is thought to be applicable to various biological materials.

Effects of the spermine analogue canavalmine on proliferation of murine erythroleukemia cells in culture

The effects of canavalmine, a structural analogue of spermine, were studied in cultured murine erythroleukemia cells 745A. Canavalmine exerted an inhibition on murine erythroleukemia cell growth at concentrations over 50 microM. The cell proliferation was, however, restored when canavalmine was removed from the culture medium after 24 h. Treatment of the cells with 500 microM canavalmine blocked the accumulation of intracellular polyamines. Especially, both spermine and spermidine levels were reduced below 50% of those in control cells after 48 h and below 30% after 96 h. The decreased contents of spermine and spermidine were compensated for by the increased content of canavalmine incorporated within the cells. In these cells, RNA and protein contents also decreased. The degree of growth inhibition by canavalmine during the cell cycle was examined using synchronized cells. Serum-induced growth stimulation was inhibited by canavalmine most effectively in the cells at G1 phase prior to DNA synthesis. The antiproliferative effect decreased when canavalmine was added to the cells after commencement of DNA synthesis. The results suggest that the growth-inhibitory action of canavalmine on murine erythroleukemia cells is most likely due to an inhibition of early events of the cell cycle, possibly due to the interference of a structure-specific function of spermidine and/or spermine on DNA replication.

Erythroid differentiation of cultured murine erythroleukemia cells by the spermine analogue canavalmine

Canavalmine, an analogue of spermine, induced erythroid differentiation of murine erythroleukemia cells 745A, as evidenced by benzidine staining and heme content of cultured cells. Benzidine-positive cells synthesizing hemoglobin appeared on day 4 after addition of 250 microM canavalmine. The canavalmine-induced cell differentiation was inhibited by the addition of agents which alter the structure of the cell membrane, such as local anesthetics (procainamide and lidocaine) or Ca2+ antagonists (nifedipine and verapamil) at dosages not toxic for the cell growth. Canavalmine did not significantly affect the levels of conjugated polyamines in the acid-insoluble fraction of the cells. In contrast, the level of free spermidine in the acid-soluble fraction greatly decreased during the 18 h after canavalmine treatment. Putrescine and spermidine, when added externally to the growth medium, showed dose-dependent inhibition of canavalmine-induced cell differentiation. Neither cadaverine nor spermine had any significant effect. These results suggest that not only structural change of cell membrane but alteration of the polyamine metabolism, especially a regulation of the cellular level of free spermidine, might have a key importance in erythroid differentiation of murine erythroleukemia cells induced by canavalmine.