ATP-Dependent human erythrocyte glutathione-conjugate transporter. I. Purification, photoaffinity labeling, and kinetic characteristics of ATPase activity

Dinitrophenyl S-glutathione (DNP-SG) ATPase is a 38 kDa membrane protein expressed in erythrocytes and other tissues. Although stimulation of ATP hydrolysis catalyzed by DNP-SG ATPase has been demonstrated in the presence of several structurally unrelated amphiphilic ions, structural and functional properties of this protein have not been well-defined. In the present study, we have developed an improved protocol for the purification of DNP-SG ATPase and investigated its kinetic and substrate-binding properties. The purification procedure was based on highly specific elution of the 38 kDa protein from DNP-SG affinity resin in the presence of ATP. The protein could not be eluted using either ADP or adenosine-5'-[beta,gamma-methylene]triphosphate (methylene-ATP), a nonhydrolyzable analogue of ATP. Doxorubicin (DOX), a weakly basic anthracycline chemotherapy agent, was found to be the preferred activator for stimulation of ATP hydrolysis by the enzyme. ATP binding to the enzyme was demonstrated using 8-azido-ATP photoaffinity labeling and binding of trinitrophenyl (TNP)-ATP, a fluorescent analogue of ATP. The photoaffinity labeling of DNP-SG ATPase (38 kDa) was saturable with respect to 8-azido ATP (Kd = 2 microM), indicating that the enzyme was capable of specific and saturable binding to ATP. DNP-SG binding was evident from the purification procedure itself and was also demonstrable by quenching of tryptophan fluorescence. Results of quenching of tryptophan fluorescence as well as radioactive isotope-binding studies indicated that DOX was bound to the purified protein as well.     

A computerized obstetric medical record

Duke University has utilized computerized obstetric medical records since 1971. System evolution is described. Deficiencies in the current system appear to evolve from the computer/human interface rather than from basic system design. Critical elements in system success are physician acceptance of the appearance of data collection sheets and printed notes and continual rapid response in programing modification to allow for physician individuality and changes in medical practice. The limiting factor in the potential usefulness of such a system is the rate of incomplete data collection. It is suggested that if the physician were to enter data directly into the computer through a terminal, data collection would be more accurate and complete.     

Dapsone poisoning

Two cases of suicidal dapsone ingestion are reported. These patients presented with marked cyanosis and had significant methaemoglobinaemia on admission. One patient also had acute psychosis and haemolytic anaemia. Both patients responded to intravenous methylene blue therapy and nasal oxygen inhalation and recovered without any sequelae.