First evidence for polyamine conjugation mediated by an enzymic activity in plants.

An enzyme activity, found for the first time in plants, mainly located in the 22,000g supernatant of the crude extract of sprout apices of Helianthus tuberosus L. cv OB1 tubers, is able in vitro to covalently bind polyamines to endogenous substrates of different molecular weights. The major assay parameters, such as pH, dithiothreitol, and extract concentrations were optimized. The time course of the reaction, the dependence on putrescine concentration, its competition with histamine, the capacity to bind spermidine and spermine better than putrescine, the stability of the reaction product and analysis of the latter by sodium dodecyl sulfate polyacrylamide gel electrophoresis and thin-layer chromatography suggest that putrescine is linked to endogenous substrates by means of an enzyme reaction that shows some similarities with transglutaminase activities detected in animals. However, the activities of the crude extract and of a fraction eluted from a Sephadex G25 column were not affected by CaCl(2) concentrations lower or equal to 5 millimolar; 4 or 10 millimolar EGTA caused a very small reduction; higher concentrations of CaCl(2) and 15 millimolar or more of EDTA were inhibitory. N,N'-dimethylcasein was not recognized as a substrate. These data indicate that this activity also displays some characteristics which are different from those of animal transglutaminases.

In vitro interactions between polyamines and pectic substances.

Putrescine, spermidine and spermine induce a decrease in the pH value of 1 mM polygalacturonic acid or pectin solutions; spermidine and spermine also cause the precipitation of the polymers. The association constants between polyamines and polygalacturonic acid were in the order of 10(5) for putrescine and spermidine, and 10(6) for spermine. The number of galacturonic units per binding sites are proportional to the number of positive charges on the polyamine molecule. Low affinity binding sites appear at high polyamine concentrations. Calcium ions seem to compete weakly with spermine by lowering the association constant 4- to 6-fold. Two natural pectins tested, showed that methylation of the carboxylic groups influences only the number of galacturonic units per site but not the association constant.

Ornithine decarboxylase activity and polyamines level from red cells lysate of uraemic patients and normal healthy subjects.

We studied the ornithine decarboxylase (ODC) activity and polyamines (PAs) content in the red cells lysate before and after haemodialysis of 15 uremic patients and 15 healthy subjects. ODC activity is significantly increased after haemodialysis but the PAs concentrations (particularly spermine and spermidine) do not increase. This could be because of a minimal loss of PAs surplus production. On the contrary the increase of ODC activity could be explained in several ways, e.g. by stimuli during haemodialysis (hypoxia of the first hour of treatment, decrease of plasmatic osmolarity and biological stimulation of the blood during its passage through the filter).