Seiyu Hirose

Inhibition by polyamines of lipid peroxide formation in rat liver microsomes.

Abstract Both NADPH- and ascorbic acid-dependent lipid peroxidations were inhibited by spermine, the degree of inhibition being greater with the former peroxidation. The effective concentration of spermine required for inhibition was higher when larger amounts of microsomes were used. However, the activities of NADPH-cytochrome c reductase and NADPH-peroxidase were not influenced by spermine. These results suggest that spermine inhibits lipid peroxidation by binding to phospholipids in the microsomes.

Interaction between polyamines and nucleic acids or phospholipids

Abstract The binding of polyamines to DNA, RNA, and phospholipids has been studied by gel filtration and sucrose density gradient centrifugation. Spermine was found to bind more to a GC-rich DNA. Among RNAs containing double-stranded region [poly(AU), poly(GC), and ribosomal RNA], the binding of spermine was nearly equal. Among the single-stranded RNAs, the binding of spermine was in the order poly(U) > poly(C) > poly(A). An increase in K+ or Mg2+ concentration resulted in a great decrease in spermine binding to DNA and in a slight decrease in spermine binding to RNA. Therefore, in the presence of more than 2 m m Mg2+ and 100 m m K+, the binding of spermine to RNA was greater than that to DNA. No significant difference in spermine binding was observed between 16 S ribosomal RNA and 30 S ribosomal subunits, suggesting that ribosomal proteins did not affect significantly the binding of spermine to ribosomal RNA. The binding of spermine to microsomes was dependent on phospholipids. The binding strength was in the order phosphatidylinositol > phosphatidylethanolamine > phosphatidylcholine.

Beta-VLDL-induced cholesterol ester deposition in macrophages may be regulated by neutral cholesterol esterase activity.

We examined the role of enzyme activities involved in cholesterol ester metabolism in the accumulation of cholesterol ester in macrophages. When [3H]cholesterol linoleate-beta-very low density lipoproteins (beta-VLDLs) were incubated with rat resident peritoneal macrophages (RPMs), thioglycolate-elicited macrophages (TEMs), or alveolar macrophages (AMs), cholesterol ester accumulation was the greatest in TEMs and the least in AMs. The uptake of [3H]cholesterol linoleate-beta-VLDL into AMs was four times that into RPMs, and the uptake into TEMs was twice that into RPMs. The [3H]cholesterol released from [3H]cholesterol linoleate-beta-VLDL-loaded AMs was five times higher than from TEMs and RPMs, whereas those from RPMs and TEMs were almost the same. In studies using cell homogenates, the acid cholesterol esterase activity in AMs was 1.7 times that in TEMs and six times that in RPMs. The acyl-coenzyme A:cholesterol acyltransferase (ACAT) activities in AMs and TEMs were similar but were higher than that in RPMs. The neutral cholesterol esterase activity was seven times higher in AMs than in RPMs and TEMs. In the study using intact cells, the hydrolysis of loaded [3H]cholesterol linoleate-beta-VLDL in the presence of the ACAT inhibitor CL277,082 and the cholesterol [14C]oleate synthesis from [14C]oleate were enhanced in AMs about twofold compared with RPMs and TEMs. The reduction of de novo synthesized cholesterol [14C]oleate in the presence of CL277,082 was enhanced in AMs four times compared with TEMs or RPMs.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of polypeptide synthesis by spermidine at the level of initiation in rabbit reticulocyte and wheat germ cell-free systems

Abstract It is shown that the stimulation of eukaryotic polypeptide synthesis by spermidine is due to the stimulation at the level of initiation by following reasons. The incorporation of formylmethionine into polypeptides was stimulated by spermidine at the same degree to the incorporation of leucine into polypeptides. Fluorography of the polypeptides formed showed that the number of chains of individual protein synthesized was larger when spermidine was added. The formation of the complex of Met-tRNA f , globin mRNA and 40-S ribosomal subunits was stimulated by spermidine.

Comparative studies on the increase by polyamines of fidelity of protein synthesis in Escherichia coli and wheat germ cell-free systems

Abstract In the presence of polyamines, the fidelity of protein synthesis in a wheat germ cell-free system was increased significantly, while it was increased slightly in an E. coli cell-free system. The effective concentration of polyamines for the increase in fidelity of protein synthesis was nearly equal to that for the stimulation of protein synthesis in a wheat germ cell-free system.

Mechanism of stimulation of polyphenylalanine synthesis by spermidine

Abstract It is shown that the stimulation of polyphenylalanine synthesis by spermidine is due mainly to the stimulation of initiation of polypeptide synthesis by following reasons: 1) the binding of poly(U) to ribosomes was stimulated more by spermidine than the binding of Phe-tRNA to ribosomes, and 2) the number of polyphenylalanine chains was increased more by spermidine than the extension of the chain length. In addition, it is shown that 30S ribosomal subunits are responsible for the stimulation of polyphenylalanine synthesis by spermidine.

Polyamine and magnesium contents and polypeptide synthesis as a function of cell growth

Abstract The polyamine and ribosome contents during the logarithmic phase of growth were much higher than those in the late logarithmic or stationary phase of growth. On the other hand, the Mg 2+ content did not vary markedly throughout the different growth phases. These data, together with the results of a previous communication (1), suggest that increased polyamines during the logarithmic phase of growth may play significant roles not only in the neutralization of the negative charges of increased ribosomes but also in the increase of the velocity of polypeptide chain elongation by binding to the ribosomes. The polyphenylalanine synthetic activity of ribosomes from the logarithmic phase of growth was higher than that of ribosomes obtained from the stationary phase of growth in the presence of optimal concentrations of spermidine.

Differential stimulation by polyamines of phage RNA-directed synthesis of proteins

The effect of polyamines on Q beta and MS2 phage RNA-directed synthesis of three kinds of protein in an Escherichia coli cell-free system has been studied. With both phage RNAs, the degree of stimulation of protein synthesis by spermidine was in the order RNA replicase greater than A protein, while the synthesis of coat protein was not stimulated significantly by spermidine. The synthesis of RNA replicase was stimulated by 1 mM spermidine approx. 8-fold. From the results of Q beta RNA direct alanyl-tRNA and seryl-tRNA binding to ribosomes and initiation dipeptide synthesis, it is suggested that the preferential stimulation of the synthesis of RNA replicase by spermidine is due at least partially to the stimulation of the initiation of RNA replicase synthesis.

Effect of polyamines on polypeptide synthesis in rat liver cell-free system.

1. The effect of polyamines (spermine, spermidine and putrescine) on rat liver cell-free polypeptide synthesis was studied in the following three systems: (1) microsome-S100 fraction system, (2) polysome-S100 fraction system and (3) ribosome-poly(U)-S100 fraction system. n n2. In the presence of Mg2+ of sub-optimal concentration, the polypeptide synthesis was stimulated by polyamines in all three systems. The maximum incorporation of radioactivity into the polypeptide fraction under these conditions was almost equal to that obtained at optimal Mg2+ concentration without polyamines. n n3. In the presence of very low Mg2+, no significant level of polypeptide synthesis was observed regardless of the amount of polyamines added, thus showing that the sparing effect of polyamines on Mg2+ in rat liver polypeptide synthesis is only partial. n n4. Putrescine (at all concentrations tested) and spermidine (less than 1.0 mM) stimulated polypeptide synthesis even at optimal Mg2+ concentration when polysomes were attached to the endoplasmic reticulum.

Polyamine binding sites on Escherichia coli ribosomes

To determine the binding sites of polyamines on Escherichia coli ribosomes, ribosomal proteins crosslinked with polyamines through bifunctional reagents were analyzed. When 1,5-difluoro-2,4-dinitrobenzene was used as a crosslinking reagent, spermine was bound to S3, S8, S9, L1, L2, L3, L5, L6, L13, L18, L24, and L27 proteins. When dimethyl suberimidate was used, spermine was bound to S1, S3, S4, S5, S7, S8, S9, S15, L1, L2, L3, L6, L18, and L24 proteins. In addition to crosslinking with the above proteins, spermidine, when crosslinked with dimethyl suberimidate, bound to S2, S14, S20, L4, L5, L9, L13, and L16 proteins. The relationship between the binding site(s) of polyamines on ribosomes and the function of polyamines is discussed.