Polymerization of Hemoglobin S

Abstract

Hemoglobin S (HbS) Hoshida and three substituted forms of HbS Hoshida (the substituents being on the amide nitrogen of Gln-43(beta) have been prepared by the amidation of Glu-43(beta) of HbS with ammonia, methylamine, glycine ethyl ester, and galactosamine. The O2 affinity of HbS is increased slightly on amidation of Glu-43(beta). All the four amidated derivatives exhibited nearly the same oxygen affinity. On the other hand, the influence of amidation on the solubility exhibits some sensitivity to the chemical nature of the substituent on the Gln-43(beta). The solubility of HbS Hoshida (a case with no substitution on Gln-43(beta), and the methyl-substituted derivatives are about 33 and 36% higher than that of HbS. The solubility of the HbS modified with the glycine ethyl ester or galactosamine is increased to 41 and 47%, respectively. The first derivative UV spectra of HbS Hoshida and its methyl derivative reflect very little perturbations in their alpha 1 beta 2 interface as compared with that of HbS, whereas the amidated derivatives with larger substituents on Gln-43(beta) reflected noticeable difference. Thus, the increase in the solubility and the oxygen affinity of HbS on the amidation of Glu-43(beta) is primarily a consequence of the loss of the negative charge at 43(beta), a residue proximal to the alpha 1 beta 2 interface. The copolymerization studies of amidated HbS with HbA, and HbS with amidated HbA demonstrate that cis Glu-43(beta) is the "active" residue. This assignment is discrepant with the earlier implication of a trans configuration for this residue in the polymer (Edelstein, S. J. (1981) J. Mol. Biol. 150, 557-575). However, it is consistent with the solution studies of Nagel et al. (Nagel, R. L., Bookchin, R. M., Johnson, J., Labie, D., Wajcman, H.., Isaac-Sodeye, W. A., Honig, G. R., Schiliro, G., Crookstan, J. H., and Matsutomo, K. (1979) Proc. Nat. Acad. Sci. U.S.A. 76, 670-672) and McCurdy et al. (McCurdy, P. R., Lorkin, P. A., Casey, R., Lehmann, H., Uddin, D. E., and Dickson, L. G. (1974) Am. J. Med. 57, 665-760).

Proteolytic Cleavage of the Linker Region of the Human P-glycoprotein Modulates Its ATPase Function

“…Although Pgp contains two ABDs, no significant basal ATPase activity was noticed in Pgp preparations made from Sf9 insect cells (25)(26)(27)(28). However, basal ATPase activity was detected in Pgp preparations from mammalian cells (29 -32).…”

“…The Sf9 insect cells were cotransfected with these cDNAs and BaculoGold DNA (BD PharMingen) to prepare the corresponding recombinant baculoviruses according to the instructions provided by the manufacturer. Preparation of a baculovirus carrying the wild-type human Pgp cDNA was reported previously (25).…”

“…Fraction-The monolayers of Sf9 insect cells growing in tissue culture flasks were infected with the above recombinant baculoviruses, and the total membrane fractions were prepared as described previously (25). The membrane fractions were resuspended in Tris/ glycerol buffer (50 mM Tris, 50 mM mannitol, 2 mM EGTA, 2 mM 2-mercaptoethanol, and 30% (w/v) glycerol (pH adjusted to 7.0 with HCl) and stored at Ϫ20°C for later use.…”

“…In the presence of transport substrates, Pgp exhibits high ATPase activity, which is known as drug (transport substrate)-stimulated ATPase activity (20, 25-27, 30, 31, 33). Because of the ease with which the ATPase measurements can be carried out, drug-stimulated ATPase activity has been employed by several laboratories to monitor drug transport by Pgp (19,22,23,25,26,30,(33)(34)(35)(36)(37)(38).…”

“…Other Methods-SDS-PAGE, Western blotting, and protein estimations were carried out as described (25,26). Characterization of the NH 2 11 antibody was described previously (28).…”