Edward J. Herbst

Spermidine in Regenerating Liver: Relation to Rapid Synthesis of Ribonucleic Acid.

The spermidine in rat liver increases after partial hepatectomy, and the rate of polyamine accumulation closely approximates the increased rate of synthesis of RNA in regenerating liver. The uptake by the liver of intravenously injected putrescine and the biosynthesis of spermidine are accelerated within 2 hours after the operation. The uptake of spermidine also increases during early regeneration.

POLYAMINE CHANGES DURING DEVELOPMENT OF Drosophila Melanogaster

The significance of polyamines in biological processes has been recognized since the initial discovery of the requirement by Hemophilus parainfluenzae for either putrescine, spermidine or spermine as an essential growth factor.l3 Since this initial observation, the polyamines have been reported to be growth factors for various microorganisms,g plants,2 cultured mammalian cells,13 and insects.F+ In addition the polyamines have been implicated as prominent polycations, closely associated with nucleate biosynthesis in rapid growth systems including microorganisms,” regenerating rat The relatively rapid development and well-characterized biological parameters of Drosuphila melanogaster have facilitated this first report concerning polyamine levels throughout the life cycle of a multicellular organism. Larval, pupal, and adult stages of D . melanogaster have been previously demonstrated to contain spermidine and putrescine,x and the preferential association of spermidine with the cell nuclei of isolated larval salivary glands has been demonstrated by radioautographic procedures.* Also, the incorporation of labeled uridine into RNA of salivary gland nuclei is enhanced by exogenous spermidine, when added to organ-cultured salivary glands.8 The method of Seiler and Wiechmann,21 which relies on the conversion of spermidine and spermine to highly fluorescent I-dimethylaminonaphthalene-5sulfonamide (dansyl) derivatives, was employed for quantitative assays. However, the solvent system was modified to allow the simultaneous direct quantitation of dansyl putrescine, which could not be sufficiently resolved from dansyl ammonia by the original method. l.c and the chick

Analysis of polyamines by thin-layer chromatography☆

Abstract A sensitive method for the quantitative analysis of putrescine, spermidine, and spermine is described. Tissue extracts prepared with cold 5% trichloroacetic acid are freed of the acid by ether extraction and purified by either n -butanol extraction or a rapid cation-exchange column procedure. The n -butanol extracts or HCl eluates from the column containing the tissue putrescine, spermidine, and spermine are concentrated and chromatographed on thin-layer plates of Whatman cellulose CC41. The amines are reacted with ninhydrin and the colored thin-layer spots are scraped from the plate and extracted and the absorbance is measured at 575 mμ. The method is precise and is adapted to the analysis of polyamines in diverse biological materials.

The effect of polyamines on the enzymatic degradation of ribosomes

Abstract 1. Broken cell extracts of Escherichia coli and ribosomes isolated from such extracts were incubated in 0.1 M Tris buffer, pH 7.5–8.1, or in buffer containing 0.05 M EDTA. A rapid enzymatic degradation of rRNA occurred and the acid-soluble products of these reactions were identified as nucleoside 3′-phosphates. 2. Spermine and spermidine at low concentrations (0.1–1 mM polyamine) inhibited the autodegradation of the ribosomes in 0.1 M Tris but did not inhibit ribosome degradation when the polyamine concentration was increased. When EDTA was present, the degradation of the ribosomes was completely inhibited by 1–10 mM polyamine. 3. The inhibition by polyamines of the latent ribonuclease (ribonuclease I) released by EDTA could not be demonstrated with purified rRNA and ribonuclease I derived from E. coli ribosomes. 4. Low concentrations of the polyamines appear to stabilize the ribosomes and prevent the release of ribonuclease I while high concentrations of the polyamines destabilize the ribosomes and release ribonuclease I.

Stimulation of in vitro transcription of T4 DNA by the polyamine spermidine

Abstract The effect of the triamine spermidine [H 2 N-(CH 2 ) 4 NH(CH 2 ) 3 NH 2 ] on the in vitro transcription of T4 DNA by Escherichia coli DNA-dependent RNA polymerase has been studied. Under low ionic strength assay conditions, the incorporation of nucleoside triphosphates into RNA ceased by 15 min of incubation. The addition of spermidine to a final concentration of 2.5 m m delayed the appearance of this plateau by several hours. Analysis of transcription complexes by nitrocellulose filter binding and sedimentation through sucrose gradients revealed that nascent RNA is released in the presence or absence of added spermidine. Therefore, spermidine does not enhance in vitro transcription by facilitating the release of nascent transcripts from the DNA-enzyme-RNA complex. Also, sedimentation analysis of transcription products synthesized in the presence or absence of spermidine revealed no significant size differences. The presence of optimal concentrations of spermidine in the reaction mixture did enable the polymerase to dissociate from the DNA template and initiate new DNA chain synthesis. DNA-RNA hybridization studies revealed that the transcripts synthesized in the presence of spermidine throughout the reaction time course are faithful copies of the DNA template. Furthermore, spermidine prevented and reversed the inhibition of RNA synthesis by exogenous RNA.