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S Beninati

Publications and source records attributed to S Beninati.

52 records · Page 3Linked to original sources
PubMed1988

High-performance liquid chromatographic method for the determination of epsilon-(gamma-glutamyl)lysine and mono- and bis-gamma-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.

Animals↗
PubMed1988

Free and protein-conjugated polyamines in mouse epidermal cells. Effect of high calcium and retinoic acid.

We have investigated polyamine metabolism in primary cultures of mouse epidermal cells. These cells, which grow at low Ca2+ levels as a monolayer with characteristics of basal cells, terminally differentiate when the extracellular Ca2+ level is raised above 1 mM. The cellular levels of free polyamines were measured, and, after incubation of cell cultures with [3H]putrescine, the distribution of label in both acid-soluble and acid-insoluble cellular components was examined. Free polyamine levels were reduced in cells induced to differentiate. Treatment with retinoic acid, which prevents differentiation and causes increased proliferation, resulted in an increase in free putrescine. Upon adjustment of the calcium concentration to a level that induces differentiation, the enzyme transglutaminase was activated, and a concomitant increase in the level of both protein-bound mono- and bis-gamma-glutamyl derivatives of putrescine and spermidine was observed. Isolation of a material of apparent molecular weight about 6000 which contains only mono-gamma-glutamylpolyamines and the finding of both mono- and bis-gamma-glutamylpolyamines in the protein fraction containing cornified cell envelopes provided the basis for speculation on polyamines in envelope formation. Our data suggest that polyamines play a role during epidermal cell differentiation through transglutaminase-mediated post-translational modification.

Animals↗
PubMed1988

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

The possible role of polyamines in the covalent modification of proteins in CHO cells was investigated by metabolic labeling with [3H]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 alpha-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 hydrolysis as polyamines. The identification of gamma-glutamylputrescine and gamma-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 gamma-glutamylpolyamine protein components are discussed.

Animals↗
PubMed1988

gamma-Glutamylamine derivatives in isolated rat hepatocyte proteins.

Freshly isolated rat hepatocytes were found to contain a 3-fold higher level of putrescine than perfused liver. The bulk of this diamine was recovered in the acid-insoluble fraction of the cell. In order to determine the nature of the amine binding, the levels of gamma-glutamylamine derivatives were measured. The method used involves exhaustive proteolytic digestion of the acid-insoluble fraction of hepatocytes, followed by ion-exchange chromatography. For N1-(gamma-glutamyl)putrescine, a combined ion-exchange chromatographic and reverse-phase h.p.l.c. procedure was adopted. This allowed for the direct detection of less than 50 pmol of this derivative in enzymic hydrolysates. Several of the gamma-glutamylamines reported previously [Beninati, Piacentini, Argento-Ceru', Russo-Caia & Autuori (1985) Biochim. Biophys. Acta 841, 120-126] in the whole organ were found in the isolated liver cells. The elevated level of N1-(gamma-glutamyl)putrescine and the absence of bis-(gamma-glutamyl)spermine was noteworthy. The results suggest that, in rat hepatocytes, both polyamine-dependent post-translational modification of some proteins and cross-linking between proteins involving the glutamine and lysine residues occurs.

Animals↗
PubMed1986

Ornithine decarboxylase, transglutaminase, diamine oxidase and total diamines and polyamines in maternal liver and kidney throughout rat pregnancy.

Ornithine decarboxylase (ODC; EC 4.1.1.17), transglutaminase (EC 2.3.2.13), diamine oxidase (DAO; EC 1.4.3.6) and total di- and poly-amines were studied in rat liver and kidney cortex throughout pregnancy. In liver, ODC activity exhibited two major peaks (4.5-5 times the control activities) on days 15 and 17. Also putrescine and spermidine increased biphasically (3-4-fold), but no variation in spermine content was observed. Transglutaminase activity showed slight variations only near the end of gestation. In kidney, ODC activity did not fluctuate significantly during pregnancy, whereas both transglutaminase activity and putrescine content showed three major increases, in very early, middle and late pregnancy. No significant variations in spermidine and spermine were observed. In both organs, DAO activity, very low or undetectable until day 10, dramatically increased (10- and 20-fold in kidney and liver respectively) in the second half of pregnancy, reaching maxima on days 16-17 and 19. The results obtained for transglutaminase, ODC and total di- and poly-amines are interpreted on the basis of hyperplastic and hypertrophic events in the liver and kidney respectively. The behaviour of DAO suggests that the enzyme plays an important role in the control of intracellular diamine concentration.

Amine Oxidase (Copper-Containing)↗
PubMed1985

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.

Animals↗
PubMed1983

Mechanism of release of integral proteins from rat liver microsomal membranes.

The release of three integral enzymatic activities (NADH- and NADPH-cytochrome c reductase and 5'-nucleotidase) and total protein from washed rat liver microsomal membranes, upon simple incubation at 37 degrees C in aqueous media, was investigated. Release does not depend on contaminating proteases and is enhanced by alkaline pH. Total protein and enzyme release is consistent with a loss of phospholipids which are not recovered in the soluble phase. Following incubation at pH 9.0 large amounts of free fatty acids were recovered in the soluble phase, accounting for a ratio of 1/1 (w/w) with released protein. This evidence, together with the data available about densities (1.07-1.08 g/ml) and molecular weights (1 700 000-700 000) of the released enzymes, suggests that they are solubilized from microsomal membranes in the form of mixed micelles mostly formed by free fatty acids and integral proteins, probably owing to the activity of endogenous phospholipases on membrane lipids. Release of total protein and enzymatic activities is decreased by Ca2+, whose possible role in the phenomenon is discussed.

5'-Nucleotidase↗
PubMed1980

Urinary polyamine excretion in man. II. Influence of menstrual cycle.

24 hours urinary excretion of putrescine, spermidine and spermine was analysed at various phases of the menstrual cycle in 10 regularly menstruating women aged 20-40 years. After determining the ovulation time, by means of the basal temperature, the menstrual cycle was divided into five stages. The urinary excretion of putrescine and spermidine appears more marked during the follicular phase. During the luteal phase spermine excretion levels may be as much as 50 times higher than those found during the other stages of the cycle. Mean values show a wide fluctuation, which depends upon the individual. Moreover, the mean values of urinary excretion of di- and polyamines of 3 menstrual cycles in a 37 year old subject, showed little variation in this person. These results suggest that a relationship exists between the phases of the menstrual cycle and the variations in urinary levels of di- and polyamines.

Adult↗
PubMedDate not supplied

A gas chromatographic method for the determination of di- and polyamines in human urine.

A gas chromatographic method for the determination of di- and polyamines in human urine has been developed. The di- and polyamines have been extracted from standard solutions or from urine by a butanolic method modified to improve its sensitivity in relation to the low concentration of the amines in human normal urine. The quantitation is controlled by using two internal standards: 1,6-diaminohexane for Putrescine and Cadaverine, l-ephedrine for Spermidine and Spermine. Distilled water was the final solvent injected into the GLC column and the detector response was linear between 5 and 100 ng.

Cadaverine↗
PubMedDate not supplied

Urinary polyamine excretion in man: I. Influence of sex and age.

Urinary di- and polyamines were analysed by gas-liquid chromatography in 63 normal volunteers. Subjects were divided into two age groups for each sex (males: from 18 to 40 years, and 40 to 70; females from 15 to 35 and 35 to 50), in order to establish age and sex dependent variations in the normal excretion of polyamines. Age appears to have some influence only in the excretion of spermine in male subjects. Sex on the contrary, has a considerable influence on urinary polyamines. Mean values of excretion for putrescine and spermine in female subjects are higher than those in males and show a wide variation in standard deviations.

Adolescent↗