Agmatine uptake by cultured hamster kidney cells.

Agmatine, the product of arginine decarboxylation, has been recently found in a wide variety of animal tissues. In spite of the emergent interest on agmatine in animals, the mechanism of agmatine uptake in mammalian cells has been scarcely studied. An analysis of radiolabeled agmatine uptake was carried out by using a classical, kinetic approach with BHK-21 hamster kidney cells in culture. A high affinity, temperature- and energy-dependent agmatine transport system in BHK-21 kidney cells is here kinetically characterized which seems to be a "general" transporter shared by di- and triamines and different to a highly specific carrier for the tetraamine spermine.

Structure/function relationship studies on the T/S residues 173-177 of rat ODC.

A well-conserved T/S cluster was detected among vertebrate ornithine decarboxylase by computer analysis (E. Viguera, O. Trelles, J.L. Urdiales, J.M. Matés, F. Sánchez-Jiménez, Trends Biochem. Sci. 19 (1994) 318-319). In the present report we studied the role of these residues (173, 176 and 177 in rat ornithine decarboxylase (ODC)) in enzymic activity and stability by in vitro expression, kinetic characterization and in vitro degradation of site-directed mutants. These T/S residues are substituted by a D/E-enriched fragment in other lower eukaryotic ODCs. The substitution of the T/S-enriched fragment (TLKTS) of rat ODC by the negative charged fragment of T. brucei ODC (KVEDC) did not affect protein stability, but increased Km values of the mutant enzyme. The substitution of the T/S residues by alanine also has a similar effect on rat ODC kinetic values. However, results indicate that polarity of the fragment must be an important factor for protein conformation, since the latter mutant, having no T/S or D/E residue in the fragment (ALKAA), showed reduced stability in vitro.

Involvement of essential cysteine and histidine residues in the activity of isolated glutaminase from tumour cells.

The pH dependence of the phosphate-activated glutaminase isolated from Ehrlich tumour cells suggests a functional role for two prototropic groups with apparent pKa of 9.3 and 7.7 at the active site of the protein; these pKa values are compatible with cysteine and histidine residues, respectively. This possibility was investigated by chemical modification studies of the purified enzyme. N-Ethylmaleimide fully inactivated the purified glutaminase; the reaction order was very close to 1.0, suggesting that N-ethylmaleimide modifies glutaminase at a single essential site. Spectrophotometric studies of the isolated protein treated with diethyl pyrocarbonate indicate that two histidine residues are modified. Since glutaminase is loosely associated to the inner mitochondrial membrane, modification experiments were also carried out using mitochondrial membrane fractions. N-Ethylmaleimide and diethyl pyrocarbonate gave similar results in mitochondria membrane-bound enzyme to those obtained with purified enzyme. Glutamate, which behaves as a competitive inhibitor of the enzyme, partially protected the inactivation caused by N-ethylmaleimide in membrane-bound experiments. The results suggest the existence of a critical histidine residue(s) in the tumour glutaminase, and strongly support the notion that a cysteine residue, which is located at (or near) the active site, is involved in the catalytic mechanism as well.