Differential effects of polyamines on the phosphorylation of chromatin-associated proteins

Studies are presented on the nature of chromatin-associated phosphoproteins whose phosphorylation is influenced by polyamines. After labelling with 32P, chromatin-associated proteins were separated into four fractions. Fraction I comprised neutral and basic non-histone phosphoproteins, including high-mobility-group non-histones; fraction II consisted mostly of histones; fraction III consisted of a class of (salt-soluble) acidic non-histone phosphoproteins; and fraction IV consisted of residual (salt-insoluble) acidic non-histone phosphoproteins. The average relative distribution of protein in the four fractions (I-IV) was about 1:4:2:1 for both liver and prostate. However, tissue-dependent differences were observed in the incorporation of 32P in various protein fractions. In the presence of polyamines (e.g. 1 mM-spermine or 2 mM-spermidine) maximal stimulation of phosphorylation was observed in non-histone proteins of fraction I (160-180%), followed by that in non-histone proteins of fraction III (80-110%). The phosphorylation of residual non-histone proteins in fraction IV, and the small extent of phosphorylation of histones in fraction II, remained unaltered in the presence of polyamines. Thus polyamines do not stimulate the phosphorylation of all non-histone proteins; their stimulative effect is most prominent in the phosphorylation of neutral and basic non-histone proteins and a class of salt-soluble acidic non-histone proteins. In accord with our hypothesis, these differential effects of polyamines on phosphorylation of endogenous non-histone proteins may relate to the conformation of these substrates rather than to endogenous kinases.     

Differential conjugation of polyamines to calf nuclear and nucleolar proteins

Nuclei and nucleoli isolated from calf liver contain acid-precipitable putrescine, spermidine and spermine conjugates. The polyamines are released upon peptide bond hydrolysis. All of the nuclear putrescine conjugate and a major portion of the polyamine conjugates are localized within the nucleolus. Nuclei and nucleoli also contain, in proportions consistent with the nucleolar/nuclear protein ratio, the putative conjugating enzyme, transglutaminase, as well as amine acceptor substrates to which radiolabeled putrescine can be conjugated by endogenous enzyme. Extraction of the isolated organelles with saline solutions of increasing ionic strength showed a differential distribution of the polyamine derivatives: all the covalently linked putrescine was associated with the less soluble components of the chromatin residue, while the spermidine and spermine conjugates were associated with several salt-extractable protein fractions as well as tightly bound to the chromatin pellet. Mono-gamma-glutamyl putrescine was detected after proteolytic digestion of the 600 mM NaCl fraction, further suggesting the enzymatic action of transglutaminase(s) in the conjugation process.     

Template-specific stimulation of RNA synthesis by phosphorylated non-histone chromatin proteins

Phosphorylated non-histone chromatin proteins are shown to stimulate the synthesis of RNA in a cell-free system using rat liver RNA polymerase and rat DNA as template. This stimulation is not observed when DNA's of other species are used as template, or when the phosphorylated proteins have been treated with alkaline phosphatase.     

Enhanced differential synthesis of proteins in a mammalian cell-free system by addition of polyamines.

Addition of the polyamines spermidine, spermine, or putrescine to a fractionated mammalian cell-free protein-synthesis system programmed by a variety of mRNAs results in a 3- to 5-fold stimulation of amino acid incorporation over that found in the absence of added polyamine. The mRNAs used as template were adenovirus mRNA, globin 9s mRNA, and RNA from the bacteriophages R17, Qbeta, and MS2. The relative amounts of 10 adenovirus polypeptides synthesized in vitro are altered by the addition of polyamines to the translation system to reflect more closely the relative amounts of these polypeptides synthesized in vivo. This qualititive improvement in translation products on addition of polyamines allow the analysis of a number of products which are at best only marginally synthesized in the absence of added polyamines. The low level of synthesis due to endogenous mRNA is stimulated by spermidine and spermine but a lesser extent by putrescine.     

Differential incorporation of biotinylated polyamines by transglutaminase 2

Polyamine incorporation or cross-linking of proteins, post-translational modifications mediated by transglutaminase 2 (TGase 2), have been implicated in a variety of physiological functions including cell adhesion, extracellular matrix formation and apoptosis. To better understand the intracellular regulation mechanism of TGase 2, the properties of biotinylated polyamines as substrates for determining in situ TGase activity were analyzed. We synthesized biotinylated spermine (BS), and compared the in vitro and in situ incorporation of BS with that of biotinylated pentylamine (BP), which is an artificial polyamine derivative. When measured in vitro, BP showed a significantly higher incorporation rate than BS. In contrast, in situ incorporation of both BS and BP was not detected even in TGase 2-overexpressed 293 cells. Cells exposed to high calcium showed a marked increase of BP incorporation but not of BS. These data indicate that the in situ activity of TGase 2 gives different results with different substrates, and suggest the possibility of overrepresentation of in situ TGase 2 activity when assayed with BP. Therefore, careful interpretation or evaluation of in situ TGase 2 activity may be required.     

Enhanced solubilization of membrane proteins by alkylamines and polyamines

Around 25% of proteins in living organisms are membrane proteins that perform many critical functions such as synthesis of biomolecules and signal transduction. Membrane proteins are extracted from the lipid bilayer and solubilized with a detergent for biochemical characterization; however, their solubilization is an empirical technique and sometimes insufficient quantities of proteins are solubilized in aqueous buffer to allow characterization. We found that addition of alkylamines and polyamines to solubilization buffer containing a detergent enhanced solubilization of membrane proteins from microsomes. The solubilization of polygalacturonic acid synthase localized at the plant Golgi membrane was enhanced by up to 9.9‐fold upon addition of spermidine to the solubilization buffer. These additives also enhanced the solubilization of other plant membrane proteins localized in other organelles such as the endoplasmic reticulum and plasma membrane as well as that of an animal Golgi‐localized membrane protein. Thus, addition of alkylamines and polyamines to solubilization buffer is a generally applicable method for effective solubilization of membrane proteins. The mechanism of the enhancement of solubilization is discussed.     

Identification of proteins whose synthesis is preferentially enhanced by polyamines at the level of translation in mammalian cells

In Escherichia coli, several proteins whose synthesis is enhanced by polyamines at the level of translation have been identified. We looked for proteins that are similarly regulated in eukaryotes using a mouse mammary carcinoma FM3A cell culture system. Polyamine deficiency was induced by adding an inhibitor of ornithine decarboxylase, α-difluoromethylornithine, to the medium. Proteins enhanced by polyamines were determined by comparison of protein levels in control and polyamine-deficient cells using two-dimensional gel electrophoresis, and were identified by Edman degradation and/or LC/MALDI-TOF/TOF tandem mass spectrometry. Polyamine stimulation of the synthesis of these proteins at the level of translation was confirmed by measuring levels of the corresponding mRNAs and proteins, and levels of the [³⁵S]methionine pulse-labeled proteins. The proteins identified in this way were T-complex protein 1, β subunit (Cct2); heterogenous nuclear ribonucleoprotein L (Hnrpl); and phosphoglycerate mutase 1 (Pgam1). Since Cct2 was most strongly enhanced by polyamines among three proteins, the mechanism of polyamine stimulation of Cct2 synthesis was studied using NIH3T3 cells transiently transfected with genes encoding Cct2-EGFP fusion mRNA with normal or mutated 5′-untranslated region (5′-UTR) of Cct2 mRNA. Polyamines most likely enhanced ribosome shunting on the 5′-UTR of Cct2 mRNA.     

Selecting high-affinity binding proteins by monovalent phage display

Variants of human growth hormone (hGH) with increased affinity and specificity for the hGH receptor were isolated using an improved phage display system. Nearly one million random mutants of hGH were generated at 12 sites previously shown to modulate binding to the hGH receptor or human prolactin (hPRL) receptor. The mutant hormones were displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. After three to six cycles of enrichment for hGH-phage particles that bound to hGH receptor beads, we isolated hGH mutants that exhibited consensus binding sequences for the hGH receptor. Residues previously identified as important for hGH receptor binding by alanine-scanning mutagenesis were more highly conserved by this selection method. However, other residues nearby were not optimal, and by mutating them, hormone variants having greater affinity and selectivity for the hGH receptor were isolated. This approach should be useful for those who wish to modify and understand the energetics of protein-ligand interfaces.     

RNA-directed cell-free synthesis of the galactose enzymes of Escherichia coli

Summary Conditions are described for the RNA-directed cell-free synthesis of the three galactose enzymes of Escherichia coli. Together with the DNA-directed synthesis described previously, this system permits the measurement of the three gene-products encoded by the gal operon as active enzymes synthesized in vitro in response to either gal-DNA or gal-RNA. The yield of enzyme is proportional to the amount of RNA added. Thus, the RNA-directed enzyme synthesis can serve as an assay system for functional mRNA. This test has been employed to determine the kinetics of synthesis and degradation of functional gal mRNA under the conditions of cell-free enzyme synthesis. The functional half-life of gal mRNA in this system is 6–7 minutes and is higher than expected from in vivo measurements.In contrast to the DNA-directed cell-free synthesis, the RNA-directed synthesis of the galactose enzymes is neither stimulated by cyclic adenosine-3:5-monophosphate nor by inducer.     

Polyamines stimulate natural RNA-directed DNA synthesis by Rauscher murine leukemia virus DNA polymerase

In the presence of optimal concentrations of Mg²⁺, spermine and spermidine were found to stimulate rabbit globin mRNA-directed cDNA synthesis by Rauscher murine leukemia virus (R-MuLV) DNA polymerase. Stimulation of DNA synthesis did not occur with the polyamines putrescine or cadaverine, nor could exogenously provided salt or ammonium ions duplicate the stimulation. Analysis of the mechanism of stimulation showed that inclusion of spermine in reaction mixtures a) increased Vmax and decreased apparent Km with respect to the globin mRNA-oligo(dT) tem?late-primer complex, and b) decreased the quantity of oligo (dT) required for optimal rates of cDNA synthesis on a fixed quantity of mRNA template. Genomic 70S RNA-directed cDNA synthesis was also stimulated by spermine addition to reaction mixtures, but only at supra-optimal RNA concentrations. Our results suggest that stimulation of R-MuLV DNA polymerase activity by polyamines is primarily due to stabilization of the enzyme-templateprimer initiation complex resulting in increased efficiency of initiation of cDNA synthesis.