n(1)-(gamma-glutamyl)spermidine and epsilon-(gamma-glutamyl)-lysine

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.

Topics: Animals; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Dipeptides; Humans; Mice; Putrescine; Spermidine

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.

Topics: Animals; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Dipeptides; Hydrolysis; Liver; Polyamines; Proteins; Putrescine; Rats; Rats, Inbred Strains; Spermidine; Spermine