Role of Intramembrane Polar Residues in the YgfO Xanthine Permease: HIS-31 AND ASN-93 ARE CRUCIAL FOR AFFINITY AND SPECIFICITY, AND ASP-304 AND GLU-272 ARE IRREPLACEABLE

Using the YgfO xanthine permease of Escherichia coli as a bacterial model for the study of the evolutionarily ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family, we performed a systematic Cys-scanning and site-directed mutagenesis of 14 putatively charged (Asp, Glu, His, Lys, or Arg) and 7 highly polar (Gln or Asn) residues that are predicted to lie in transmembrane helices (TMs). Of 21 single-Cys mutants engineered in the background of a functional YgfO devoid of Cys residues (C-less), only four are inactive or have marginal activity (H31C, N93C, E272C, D304C). The 4 residues are conserved throughout the family in TM1 (His-31), TM3 (Asn-93/Ser/Thr), TM8 (Glu-272), and putative TM9a (Asp-304/Asn/Glu). Extensive site-directed mutagenesis in wild-type background showed that H31N and H31Q have high activity and affinity for xanthine but H31Q recognizes novel purine bases and analogues, whereas H31C and H31L have impaired affinity for xanthine and analogues, and H31K or H31R impairs expression in the membrane. N93S and N93A are highly active but more promiscuous for recognition of analogues at the imidazole moiety of substrate, N93D has low activity, N93T has low affinity for xanthine or analogues, and N93Q or N93C is inactive. All mutants replacing Glu-272 or Asp-304, including E272D, E272Q, D304E, and D304N, are inactive, although expressed to high levels in the membrane. Finally, one of the 17 assayable single-Cys mutants, Q258C, was sensitive to inactivation by N-ethylmaleimide. The findings suggest that polar residues important for the function of YgfO cluster in TMs 1, 3, 8 and 9a

Isolation and structure of goat prothymosin a and contribution to the investigation of the biological activity of prothymocin a

The primary sequence of goat prothymosin a exhibits a high degree of homology to the sequences of prothymosin a from the other mammalian species. The highest levels of prothymosin a are found in thymus and spleen, with significantly lower levels in non-lymphoid tissues. During development, the lymphoid tissue levels of rat prothymosin a are significantly altered with highest levels observed in newborn and prepubertal rats. Autoantibodies against prothymosin a were detected in 18% of the sera of patients with SLE. These antibodies recognize only the C-terminal portion of the molecule. Human monocytes incubated in complete culture medium with prothymosin a produce increased quantities of thymosin a, (the N-terminal peptide fragment of prothymosin a) in the culture supernatants.Η προθυμοσίνη α αιγός παρουσιάζει πολύ μεγάλη ομολογία με τις προθυμοσίνες άλλων θηλαστικών, ως προς την πρωτοταγή δομή. Μεγαλύτερες συγκεντρώσεις προθυμοσίνης α περιέχουν οι λεμφικοί ιστοί (θύμος, σπλήνα) και μικρότερες οι μη λεμφικοί (πνεύμονες, νεφροί, ήπαρ). Οι συγκεντρώσεις προθυμοσίνης α των λεμφικών ιστών μεταβάλλονται κατά την ανάπτυξη, με μέγιστες τιμές σε προεφηβικές ηλικίες (τα πυρώματα αυτά έγιναν σε επίμυς). Αυτοαντισώματα έναντι προθυμοσίνης α ανιχνεύθηκαν σε ποσοστό 18% των ορών ασθενών με συστηματικό ερυθηματώδη λύκο, με εξειδίκευση για την c-πλευρά του μορίου. Επίδραση προθυμοσίνης α in vitro σε μονοκύτταρα οδηγεί σε παραγωγή αυξημένων ποσοτήτων θυμοσίνης α, (Ν-πλευράς του μορίου) στο υπερκείμενο της καλλιέργειας