Abstract
The Mitochondrial Ascorbic Acid Transporter (MAT) from both rat liver and potato mitochondria has been reconstituted in proteoliposomes. The protein has a molecular mass in the range of 28–35 kDa and catalyzes saturable, temperature and pH dependent, unidirectional ascorbic acid transport. The transport activity is sodium independent and it is optimal at acidic pH values. It is stimulated by proton gradient, thus supporting that ascorbate is symported with H+. It is efficiently inhibited by the lysine reagent pyridoxal phosphate and it is not affected by inhibitors of other recognized plasma and mitochondrial membranes ascorbate transporters GLUT1(glucose transporter-1) or SVCT2 (sodium-dependent vitamin C transporter-2). Rat protein catalyzes a cooperative ascorbate transport, being involved two binding sites; the measured K0.5 is 1.5 mM. Taking into account the experimental results we propose that the reconstituted ascorbate transporter is not a GLUT or SVCT, since it shows different biochemical features. Data of potato transporter overlap the mammalian ones, except for the kinetic parameters non-experimentally measurable, thus supporting the MAT in plants fulfills the same transport role.
Highlights
Ascorbate is a multifaceted ubiquitous, small molecular weight, carbohydrate derivative that plays important roles in several cellular processes in plant and animal cells, being cofactor of many enzymes and involved in antioxidant cellular protection (Englard and Seifter, 1986; Patak et al, 2004; Mandl et al, 2009; Padayatty and Levine, 2016)
Mammals cells take up ascorbic acid by two high affinity/low capacity Na+-dependent transporters: SVCT1 (SLC23A1) and SVCT2 (SLC23A2), both constituted of about 600 aminoacids and with similar hydropathic profiles predicting 12 putative membrane-spanning domains, with different tissue distribution (Tsukaguchi et al, 1999)
It has been demonstrated that the oxidized form of ascorbate, dehydroascorbic acid (DHA), is taken up into mammalian cells by members of hexose transporters family glucose transporter (GLUT), and in the cytosol it is enzymatically reduced to ascorbic acid (Rumsey et al, 1997; Corti et al, 2010; Bánhegyi et al, 2014)
Summary
Ascorbate is a multifaceted ubiquitous, small molecular weight, carbohydrate derivative that plays important roles in several cellular processes in plant and animal cells, being cofactor of many enzymes and involved in antioxidant cellular protection (Englard and Seifter, 1986; Patak et al, 2004; Mandl et al, 2009; Padayatty and Levine, 2016). It has been demonstrated that the oxidized form of ascorbate, dehydroascorbic acid (DHA), is taken up into mammalian cells by members of hexose transporters family GLUTs, and in the cytosol it is enzymatically reduced to ascorbic acid (Rumsey et al, 1997; Corti et al, 2010; Bánhegyi et al, 2014). It has been demonstrated that human HEK-293 cells express a mitochondrial ascorbic acid transporter (MAT) that kinetically corresponds to a low-affinity form of the sodium-coupled ascorbic acid transporter-2 (SVCT2) (Muñoz-Montesino et al, 2014), responsible of ascorbate transport in U937 cell mitochondria and whose activity is proposed to be regulated by DHA (Fiorani et al, 2014, 2015; Cantoni et al, 2017).
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