RESUMO
The Trypanosoma brucei mitochondrial F(1)-ATPase has been previously isolated and characterized. It is composed of five subunits of molecular weights 55000, 42000, 32000, 22000, and 17000 [1]. We have identified the alpha and beta subunits of the T. brucei F(1)-ATPase by N-terminal sequence determination together with analysis of cDNA and genomic clones. The genes for both subunits are homologous to the same subunits from other organisms. They contain the Walker A and B boxes of homology and a putative mitochondrial import sequence. The isolated T. brucei alpha subunit is unusually small at 42 kDa. The alpha cDNA clone encodes a protein of predicted size 59 kDa with a mitochondrial import presequence at the N-terminus. The predicted size was confirmed by expression of a 59 kDa protein from the cDNA clone in vitro. These results suggest that the alpha subunit may have an unusually large mitochondrial presequence of 159 amino acids. In contrast, the estimated size of the native beta subunit (55 kDa) correlates well with the size predicted from the cDNA clone, 57 kDa, from which a 21 amino acid presequence has been removed in vivo. The size of the beta subunit was confirmed by expression in an in vitro and an Escherichia coli expression system. The purified recombinant beta subunit, like the native F(1)-ATPase, can be labeled by the photoaffinity nucleotide analogue 8-azido ATP. Binding of the 8-azido ATP probe is best competed by the natural substrate ATP, and is significantly reduced by pretreatment with the inhibitor 7-chloro-4-nitrobenzo-2-oxa-1,3-diazide as has been shown with beta subunits of other organisms. The differential binding of this photoaffinity analogue was used to resolve the identities of the alpha and beta subunits of the ATP synthase from T. brucei. These results are in contrast to results previously obtained for a related trypanosomatid Crithidia fasciculata.
Assuntos
Domínio Catalítico/genética , Clonagem Molecular , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Domínio Catalítico/fisiologia , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Marcadores de Fotoafinidade , ATPases Translocadoras de Prótons/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Trypanosoma brucei brucei/genéticaRESUMO
This research examines 23 different laws passed by state governments in an effort to restrict the number of abortions. It assesses both laws passed and laws actually enforced after the Supreme Court permitted states to restrict access to abortion in 1989. None of the policy actions by state governments has had a significant impact on the incidence of abortion from 1982 to 1992. Abortion rates continue to reflect past abortion rates, the number of abortion providers, whether the state funds abortions for Medicaid-eligible women, urbanism, and racial composition of the population. Recent restrictive policies have not affected these trends.
