Satoru Maekawa

Purification by affinity chromatography and characterization of porcine liver cytoplasmic polyamine oxidase

1. Polyamine oxidase was purified from the soluble fraction of porcine liver by more than 70,000-fold to electrophoretic homogeneity using N8-acetylspermidine-Sepharose 4B affinity chromatography. 2. The molecular weight and isoelectric point of this enzyme were 62,000 and pH 4.5, respectively. 3. Optimal pH for the catalytic activity was close to 10.0. 4. The enzyme activity was enhanced by 5 mM dithiothreitol or 5 mM benzaldehyde. 5. Preferential substrates for this cytoplasmic PAO were N1-acetylspermine, N1-acetylspermidine and spermine. 6. Spermidine was not virtually the substrate for this enzyme. 7. The present results suggested the physiological roles of cytoplasmic PAO, being coupled with the reaction of spermidine/spermine N1-acetyltransferase, in recycling the cellular polyamines to putrescine.

Induction of spermidine/spermine N1-acetyltransferase in needle-punctured rat lens as a model of traumatic cataract

Isolated rat lens was punctured with a needle at a single point in the equatorial region and was incubated at 37 degrees C. Spermidine/spermine N1-acetyltransferase activity was increased about 5-fold at 8 h after the puncture. Concomitantly, putrescine content in the lens increased markedly at 8-16 h after the puncture, while spermidine levels were slightly depressed. Pretreatment of the lens with actinomycin D or cycloheximide blocked the increases of spermidine/spermine N1-acetyltransferase activity and putrescine content. Ornithine decarboxylase, on the other hand, was not induced to a detectable degree by this stimulus and 5 mM difluoromethylornithine could not block the increase of putrescine content. Polyamine oxidase showed a relatively constant activity that was sufficient for the metabolism of newly formed N1-acetylspermidine. These results suggested that, in the punctured lens, the polyamine levels were regulated predominantly by the activity of spermidine/spermine N1-acetyltransferase, but not by the induction of ornithine decarboxylase.

Antitumor effect of methylglyoxal bis(butylamidinohydrazone), a new inhibitor of S-adenosylmethionine decarboxylase, against human erythroid leukemia K 562 cells

Methylglyoxal bis(butylamidinohydrazone) (MGBB) inhibited S-adenosylmethionine decarboxylase (SAMDC) activity competitively with S-adenosylmethionine (SAM) showing the Ki value of 1.8 X 10(-5) M. MGBB showed less SAMDC-stabilizing effect in rat liver in vivo than did methylglyoxal bis-(guanylhydrazone) (MGBG). MGBB inhibited the growth of human erythroid leukemia K 562 cells. Putrescine, spermidine and spermine concentrations in MGBB-treated cells were depressed to 56%, 58% and 88% of the values of control cells, respectively. [35S]Methionine incorporation into trichloroacetic acid-insoluble fraction was decreased in the inhibitor-treated cells.

Methylglyoxal bis(guanylhydrazone) analogs: multifunctional inhibitors for polyamine enzymes

Abstract Methylglyoxal bis(3-aminopropyl-amidinohydrazone) (MGBA), methylglyoxal bis(4-aminobutyl-amidinohydrazone) (MGBT), methylglyoxal bis(cyclohexyl-amidinohydrazone) (MGBC) and methylglyoxal bis(butyl-amidinohydrazone) (MGBB) were synthesized as potent competitive inhibitors for polyamine biosynthetic enzymes. Four compounds competitively inhibited S-adenosylmethionine decarboxylase. Ornithine decarboxylase was also inhibited by MGBA, MGBT and BGBB competitively with ornithine. Furthermore, MGBC and MGBB inhibited spermidine synthase competitively with the substrate putrescine. All the compounds showed anti-proliferative effects on human leukemia Molt4B cells. The intracellular contents of putrescine, spermidine and spermine analyzed in MGBB-treated Molt4B cells, were shown to be depressed simultaneously. These results suggest that the combination of effective moieties in a single inhibitor molecule affecting different enzymic steps of the pathway would provide an efficient way to block the metabolic pathways.

Ornithine decarboxylase and spermidine/spermine N1-acetyltransferase are induced in K562 cells by S-adenosylmethionine decarboxylase inhibitor methylglyoxal bis(guanylhydrazone) but not by analogous methylglyoxal bis(butylamidinohydrazone)

The activities of ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SAT) were increased by the addition of S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor methylglyoxal bis(guanylhydrazone) (MGBG) in cultured human erythroid leukemia K 562 cells. ODC activity began to increase 4 hr after the addition of the drug and attained a maximum at 12 hr. The increase of SAT activity lagged behind that of ODC activity. The increases of both ODC and SAT activities produced by MGBG were blocked by treatment with cycloheximide, suggesting that the increase of enzyme activity resulted from the synthesis of new enzyme proteins. The putrescine content in cells treated with MGBG increased markedly, whereas the levels of spermidine and spermine were depressed lower. On the other hand, methylglyoxal bis(butylamidinohydrazone) (MGBB), a derivative of MGBG inhibiting AdoMetDC effectively, did not induce ODC or SAT activities.

Active transport and metabolic characteristics of polyamines in the rat lens

Putrescine, spermidine and spermine were transported into the rat lens against a concentration gradient. This process appeared to be energy-dependent and involved a carrier system different from those for amino acids. Competition experiments suggested that the three polyamines were transported by the same system or very similar systems. Incorporated spermine was converted to spermidine and putrescine, and spermidine was converted to putrescine. In contrast, the conversion of putrescine to spermidine and spermine, or the conversion of spermidine to spermine was not observed. Furthermore, ornithine was not utilized for the synthesis of putrescine. These metabolic characteristics of the polyamines in the rat lens were correlated with the extremely low activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase. Other enzymes of polyamine metabolisms, however, were relatively active. In conclusion, the lens has a very low ability for the de novo synthesis of polyamines. The polyamines in the lens are considered to be supplied form the surrounding intraocular fluid by an active transport system specific for polyamines.

Synthesis of N-chlorosulfonyl dicyclohexylamine as a potent inhibitor for spermidine synthase and its effects on human leukemia molt4B cells

N-Chlorosulfonyl dicyclohexylamine (CSD) was synthesized as a potent inhibitor of spermidine synthase and analyzed for antiproliferative effects on leukemic cells. The compound specifically inhibited spermidine synthase in a competitive mode with the substrate putrescine (Ki, 1.8 X 10(-7) M). When human leukemia Molt4B cells were cultured in the presence of the inhibitor, the intracellular level of spermidine and the rate of cell proliferation were markedly depressed. In these polyamine depleted and growth retarded cells the synthesis of protein, but not of DNA or RNA, was found to be significantly diminished.

2-Mercaptoethylamine, a competitive inhibitor of spermidine synthase in mammalian cells

Spermidine synthase from rat ventral prostate was inhibited by 2‐mercaptoethylamine (MEA). Inhibition of spermidine synthase by MEA was competitive with respect to one of the substrates putrescine, but not competitive with respect to the other substrate decarboxylated S‐adenosylmethionine. MEA markedly depressed spermidine and spermine contents in human erythroid leukemia K562 cells, suggesting that these changes resulted from the inhibitory effect of MEA on spermidine synthase in situ.

Antitumor effect of methylglyoxal bis(3-aminopropyl-amidinohydrazone), a new inhibitor of S-adenosylmethionine and ornithine decarboxylases, on human erythroid leukemia K562 cells

SummaryMethylglyoxal bis(3-aminopropylamidinohydrazone (MGBA) inhibited S-adenosylmethionine decarboxylase (AdoMetDC) activity competitively with S-adenosylmethionine (AdoMet), showing a Ki value of 2.60x10-5M. It also inhibited ornithine decarboxylase competitively with ornithine, showing a Ki value of 3.80x10-5M. MGBA inhibited the growth of human erythroid leukemia K562 cells. Putrescine, spermidine, and spermine concentrations in MGBA-treated cells were depressed to 19%, 36%, and 66% of the values of control cells, respectively.

Inhibition by polyamines of methylthiopropylamine-induced ornithine decarboxylase in human lymphoid leukemia Molt 4B cells

Methylthiopropylamine (MTPA), an inhibitor of spermidine synthase, markedly induced ornithine decarboxylase (ODC) activity (about 30-fold of the basal level) in human lymphoid leukemia Molt 4B cells. This induction was blocked by the addition of spermidine, spermine or putrescine simultaneously with MTPA. Inhibition by spermidine or spermine of the MTPA-induced ODC activity was larger than that by putrescine. The increase of ODC activity by MTPA led to the large increase of cellular putrescine content. This increase of putrescine content was abolished drastically by the simultaneous addition of spermidine or spermine. The increase of ODC activity was almost completely blocked by the addition of cycloheximide or actinomycin D. This finding suggested that the increase of ODC activity was not due to activation of ODC preformed in Molt 4B cells. The ODC induction by MTPA was dose-dependently blocked by adding the calcium channel blockers (verapamil and nifedipine) or protein kinase C inhibitors (1-(5-isoquinolinesulfonyl)-2-methylpiperazine and palmitoyl carnithine). These results suggested that calcium and protein kinase C (PKC) were involved in MTPA-associated induction of ODC.