Simone Beninati

Presence of di- and polyamines covalently bound to protein in rat liver.

Acid hydrolysis of trichloroacetic acid precipitate from rat tissue (liver, kidney and testis) homogenate released significant amounts of acid-insoluble putrescine, spermidine and spermine. Following incubation of liver homogenate with [1,4-14C]putrescine, 1.4% of total radioactivity and 1.0% of labelled diamine were recovered in the acid-insoluble fraction. Exhaustive digestion of acid-precipitable material with proteinases (Pronase, aminopeptidase M, carboxypeptidase A, B and Y) revealed the presence of di- and polyamines and of N1-(gamma-glutamyl)spermidine, N1-(gamma-glutamyl)spermine and N1,N12-bis(gamma-glutamyl)spermine. These derivatives were identified both by chromatographic analysis and by enzymatic digestion with purified gamma-glutamylamine cyclotransferase. The finding of di- and polyamine gamma-glutamyl derivatives in the proteinase-digested acid-insoluble fraction of homogenate may be considered as a proof of the in vivo transglutaminase-catalyzed binding of polyamines to proteins. This evidence suggests that di- and polyamines might have an important role in mammalian tissues through covalent binding to proteins by either one or both the primary amino groups.

Covalent incorporation of polyamines as γ-glutamyl derivatives into CHO cell protein

Abstract The possible role of polyamines in the covalent modification of proteins in CHO cells was investigated by metabolic labeling with [ 3 H]putrescine. A single radiolabeled protein band with an apparent relative molecular mass of 18 000 Da was observed by SDS-polyacrylamide gel electrophoresis. Almost all the radioactivity covalently linked to this protein was recovered as hypusine. The labeling of this protein was increased several-fold when cells were cultured with α-difluoromethylornithine (DFMO) or with this drug plus methylglyoxal bis(guanylhydrazone) (MGBG), as a result of increase in specific radioactivity of the hypusine immediate precursor, spermidine. Also labeled under the latter condition were other cellular proteins. These were aggregates on the top both of the stacking gel and of the running gel, and protein-like materials with relative molecular masses of 36 and 8 kDa. The radioactivity covalently associated with these proteins was recovered after acid hydrolofsis as polyamines. The identification of γ-glutamylputrescine and γ-glutamylspermidines in proteolytic digests of the acid-insoluble fraction of treated cells indicates that polyamines are covalently linked to these cellular protein. Several possible cellular functions of γ-glutamylpolyamine protein components are discussed.

High-performance liquid chromatographic method for the determination of ε-(γ-glutamyl)lysine and mono- and bis-γ-glutamyl derivatives of putrescine and spermidine

A sensitive, simple, and rapid high-performance liquid chromatographic (HPLC) method is reported for the determination of epsilon-(gamma-glutamyl)lysine and certain gamma-glutamylpolyamines in selected fractions from ion-exchange chromatograms of protein digests. The method involves pre-column derivatization of the gamma-glutamylamine conjugates with o-phthalaldehyde, linear-gradient reversed-phase HPLC separation, and fluorimetric detection. The gradient used was designed to provide a means of avoiding a desalting step, while maintaining proper chromatographic performance. gamma-Glutamylamines in amounts from 0.1 to 1 nmol display linear concentration-response relationships. The detection limits are approximately 10 and 200 pmol per mg of protein for the gamma-glutamylpolyamines and for epsilon-(gamma-glutamyl)lysine, respectively. The use of the method is exemplified by an analysis of the epidermal cell envelope from the skin of a newborn mouse.

Inhibition of deoxyhypusine hydroxylase by polyamines and by a deoxyhypusine peptide

The inhibition of deoxyhypusine hydroxylase was studied in vitro. Of the polyamines tested, spermine and its homologue thermine exhibited the strongest inhibition against the enzyme from rat testis. Kinetic analysis revealed that the inhibition by spermine was competitive (Ki, 0.25 +/- 0.02 mM) with respect to the deoxyhypusine protein substrate. Spermidine and its homologue caldine were also inhibitors, but less potent ones than spermine. The spermidine analogues with one or both primary amino groups replaced by the cyano group did not inhibit. A number of diamines, including putrescine, were found to display little or no inhibition. The observed effects of naturally occurring polyamines on deoxyhypusine hydroxylase activity is consistent with a suggestion of regulation of this enzymic activity by cellular levels of polyamines. A synthetic peptide Lys-Thr-Gly-deoxyhypusine-His-Gly-His-Ala-Lys, the amino acid sequence of which corresponds to that surrounding hypusine in eukaryotic initiation factor 4D, was found to display competitive-type inhibition (Ki, 0.44 +/- 0.02 mM) against deoxyhypusine hydroxylase from Chinese hamster ovary cells. Free hypusine and deoxyhypusine, on the other hand, possessed no inhibitory properties. A peptide analogous to the deoxyhypusine nonapeptide with lysine in place of deoxyhypusine had little effect on enzyme activity. The preparation of a derivative of deoxyhypusine, suitably protected for use in the solid-phase synthesis of deoxyhypusine peptides, is described.

Covalent Polyamine-Protein Conjugates: Analysis and Distribution

Most suggestions as to the biological functions of the polyamines, putrescine, spermidine, and spermine, are based on the observed noncovalent binding of these polycations to nucleic acids, proteins, and phospholipids.1–3 It was recently shown that polyamines are also present in mammalian tissues and body fluids in covalent association with proteins. Protein modifications in which structural elements of polyamines are involved occur by two pathways. In one of these the amino acid hypusine [Ne-(4-amino-2-hydroxybutyl)lysine] is formed through transfer of the butylamine moiety of spermidine to the e-amino group of a protein lysine residue and through subsequent hydroxylation.4 In the other polyamines are attached in covalent amide linkage to the γ-carboxyl groups of protein glutamic acid residues. Conjugation of the amines in this manner is catalyzed by transglutaminases, Ca2+ -dependent enzymes which promote exchange of primary amines for ammonia at the carboxamide groups of certain glutaminyl residues.5, 6 A number of transglutaminases have been identified and they are found widely distributed in mammalian cells and in biological fluids.7, 8 These enzymes are responsible for production of e-(γ-glutamyl)lysine crosslinks that connect protein chains and play a central role in such extracellular events as fibrin clot stabilization and seminal plug formation.7, 8

High-performance liquid chromatographic method for determination of hypusine and deoxyhypusine.

A selective and sensitive reversed-phase high-performance liquid chromatographic method is described for the determination of the amino acid hypusine which occurs ubiquitously in mammalian cells and for the simultaneous measurement of its immediate precursor deoxyhypusine. These amino acids, after their ion-exchange separation from the bulk of other amino acids in protein hydrolysates, are derivatized with o-phthalaldehyde and the fluorescent derivatives are separated by reverse-phase liquid chromatography. The sensitivity of this method allows detection of less than 5 pmol of each of these unusual amino acids. The method is applied to the determination of hypusine and deoxyhypusine in acid hydrolysates of cultured cells and tissues.

A novel transglutaminase-catalyzed posttranslational modification of HIV-1 aspartyl protease

We demonstrate that HIV-1 aspartyl protease (AP), the enzyme essential for the maturation of the AIDS virus, covalently incorporates spermidine catalyzed by guinea pig liver transglutaminase (TGase) and human coagulation factor XIIIa. Preliminary evidence indicates that there are at least three reactive glutamyl and lysyl residues in AP which act as acyl donor and acceptor respectively in a TGase reaction. SDS-PAGE and chromatographic analyses indicate that the two TGases tested catalyze the incorporation of radioactive spermidine into pure HIV-1 AP. The chemical identification and quantitation of (gamma-glutamyl) spermidine isopeptide provide conclusive evidence that the formation of this derivative is catalyzed by TGase. These results imply that TGase-catalyzed post-translational modification of HIV-1 AP may take place in a manner similar to the ones demonstrated in porcine pancreatic phospholipase A2.

AGE-RELATED CHANGES OF GLYCOSYLATION PATTERN IN ISOLATED RAT HEPATOCYTES

The glycosylation pattern in isolated rat hepatocytes during pre- and post-natal development and senescence has been studied by following: the [14C]glucosamine and [3H]galactose incorporation into cellular glycoproteins and glycolipids and the activity of two microsomal enzymes, N-acetyl-glucosaminyl-1-P transferase and galactosyl transferase. The data show a lowered precursor incorporation into lipids and proteins in the fetus, newborn and old rats versus the adult. Only the galactosyl transferase activity is enhanced on the 19th and 22nd day of fetal life. The glucosamine and N-acetyl-glucosamine content in both soluble and protein bound fractions was increased, while the galactose content in lipids and proteins decreased in the fetal stage. The different sugar composition of the proteins, and the decreased glucosamine and galactose incorporation into the proteins, observed in the fetus, newborn and old rat, suggest a post-translational modification which may cause alterations in functions usually mediated by glycoproteins.

Stimulation of phospholipases A2 by transglutaminases.

Among the enzymes catalyzing post-translational modifications of proteins, transglutaminases (TG; EC 2.3.2.13) have been extensively characterized from the enzymological point of view (1–4). Nevertheless, the physiological role(s) of these enzymes, particularly the intracellular TGs, are still poorly understood. TGs are a class of enzymes which catalyze a Ca++-dependent acyl-transfer reaction in which the γ-carboxamide group of a peptide-bound glutamine residue is the acyl-donor (1–4). Primary amino groups of many low-molecular weight amines may act as acyl-acceptors with the formation of mono-substituted γ-carboxamides of peptide-bound glutamic acid. In the absence of small molecular weight amines, TGs catalyze the formation of an e-(γ-glutamyl) - lysine isopeptide bond between endo-γ-glutaminyl and endo-e-lysyl residues in polypeptides (1–4). The latter reaction results in the formation of inter or intramolecular covalent crosslinks. These enzymes have been detected both intra and extracellularly in higher animals including man. The best characterized extracellular TG is variously known as fibrin-stabilizing factor, Laki-Lorand factor or coagulation Factor XIII.

Polyamine Metabolism in Human Epidermal Keratinocytes Transformed with AD12-SV40, HPV16-DNA and K- ras Oncogene

The intracellular concentration of the polyamines, spermidine and spermine, and their precursor, putrescine, vary with the growth rate of the cell. Although the specific role of these amines is still not well understood at the molecular level, recent studies have shown that their concentration is highly regulated and that polyamines are necessary for normal cell growth and differentiation (see reviews 1 – 3). The pathway of polyamine biosynthesis from ornithine and methionine in mammalian tissues is well char-acterized (4). Biosynthesis is modulated by rapid induction of both ornithine decarboxylase (ODC) and Sadenosylmethionine decarboxylase (AdoMetDC) both of which are present in very small amounts in quiescent cells and both of which have very short time turnovers (5,6). Exposure of resting cells to growth-promoting stimuli results in a rapid rise in ODC activity which thereafter parallels the proliferation response. In addition to the possibility of rapidly changing their rate of polyamine synthesis, cells are equipped with an effective pathway for degradation of spermidine and spermine. The first, and rate-limiting, step in this degradation is an acetylation of the polyamines, which is catalyzed by the inducible enzyme, spermidine/ spermine N 1-acetyltransferase (7).This enzyme also has an extremely short half-life (8), is rapidly induced by various polyamines (9) and appears to play a role in cellular protection against the deleterious effects of too high intracellular polyamine concentrations.