Glutathione S‐transferase P1‐1 isoforms, differing in a single amino acid residue (Ile104 or Val104), have been previously identified in human placenta [Ahmad, H., Wilson, D. E., Fritz, R. R., Singh, S. V., Medh, R. D., Nagle, G. T., Awasthi, Y. C. & Kurosky, A. (1990) Arch. Biochem. Biophys. 278, 398–408]. In the present report, the enzymic properties of these two proteins are compared. [I104]glutathione S‐transferase P1‐1 has been expressed from its cDNA in Escherichia coli and purified to homogeneity by affinity chromatography; the cDNA has been mutated to replace Ile104 by Val104, and [V104]glutathione S‐transferase P1‐1 was expressed and isolated as described for [I104]glutathione S‐transferase P1‐1. The two enzymes differed in their specific activity and affinity for electrophilic substrates (K_M values for 1‐chloro‐2,4‐dinitrobenzene were 0.8 mM and 3.0 mM for [I‐104]glutathione S‐transferase P1‐1 and [V‐104]glutathione S‐transferase P1‐1, respectively), but were identical in their affinity for glutathione. In addition, the two enzymes were distinguishable by their heat stability, with half‐lives at 45°C of 19 min and 51 min, respectively. The resistance to heat denaturation was differentially modulated by the presence of substrates. These data, in conjunction with molecular modeling, indicate that the residue in position 104 helps to define the geometry of the hydrophobic substrate‐binding site, and may also influence activity by interacting with residues directly involved in substrate binding.

Senescence of vascular smooth muscle cells (VSMCs) contributes to aging as well as age-related diseases of the cardiovascular system. Senescent VSMCs have been shown to be present in atherosclerotic plaques. Both replicative (RS) and stress-induced premature senescence (SIPS) accompany cardiovascular diseases. We aimed to establish the signature of RS and SIPS of VSMCs, induced by a common anticancer drug, doxorubicin, and to discover the so far undisclosed features of senescent cells that are potentially harmful to the organism. Most of the senescence hallmarks were common for both RS and SIPS; however, some differences were observed. 32 % of doxorubicin-treated cells were arrested in the G2/M phase of the cell cycle, while 73 % of replicatively senescing cells were arrested in the G1 phase. Moreover, on the basis of alkaline phosphatase activity measurements, we show that a 7-day treatment with doxorubicin (dox), does not cause precocious cell calcification, which is a characteristic feature of RS. We did not observe calcification even though after 7 days of dox-treatment many other markers characteristic for senescent cells were present. It can suggest that dox-induced SIPS does not accelerate the mineralization of vessels. We consider that detailed characterization of the two types of cellular senescence can be useful in in vitro studies of potential anti-aging factors.