RESUMO
In previous work (Sankaran, B., Osterhout, J., Wu, D., and Smrcka, A. V. (1998) J. Biol. Chem. 273, 7148-7154), we showed that overlapping peptides, N20K (Asn(564)-Lys(583)) and E20K (Glu(574)-Lys(593)), from the catalytic domain of phospholipase C (PLC) beta2 block Gbetagamma-dependent activation of PLC beta2. The peptides could also be directly cross-linked to betagamma subunits with a heterobifunctional cross-linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. Cross-linking of peptides to Gbeta(1) was inhibited by PLC beta2 but not by alpha(i1)(GDP), indicating that the peptide-binding site on beta(1) represents a binding site for PLC beta2 that does not overlap with the alpha(i1)-binding site. Here we identify the site of peptide cross-linking and thereby define a site for PLC beta2 interaction with beta subunits. Each of the 14 cysteine residues in beta(1) were altered to alanine. The ability of the PLC beta2-derived peptide to cross-link to each betagamma mutant was then analyzed to identify the reactive sulfhydryl moiety on the beta subunit required for the cross-linking reaction. We find that C25A was the only mutation that significantly affected peptide cross-linking. This indicates that the peptide is specifically binding to a region near cysteine 25 of beta(1) which is located in the amino-terminal coiled-coil region of beta(1) and identifies a PLC-binding site distinct from the alpha subunit interaction site.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Isoenzimas/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Sítios de Ligação , Reagentes de Ligações Cruzadas , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/genética , Isoenzimas/química , Isoenzimas/genética , Mutação , Fosfolipase C beta , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fosfolipases Tipo C/química , Fosfolipases Tipo C/genéticaRESUMO
When the beta(5) (short form) and gamma(2) subunits of heterotrimeric G proteins were expressed with hexahistidine-tagged alpha(i) in insect cells, a heterotrimeric complex was formed that bound to a Ni-NTA-agarose affinity matrix. Binding to the Ni-NTA-agarose column was dependent on expression of hexahistidine-tagged alpha(i) and resulted in purification of beta(5)gamma(2) to near homogeneity. Subsequent anion-exchange chromatography of beta(5)gamma(2) resulted in resolution of beta(5) from gamma(2) and further purification of beta(5). The purified beta(5) eluted as a monomer from a size-exclusion column and was resistant to trypsin digestion suggesting that it was stably folded in the absence of gamma. beta(5) monomer could be assembled with partially purified hexahistidine-tagged gamma(2) in vitro to form a functional dimer that could selectively activate PLC beta2 but not PLC beta3. alpha(o)-GDP inhibited activation of PLC beta2 by beta(5)gamma(2) supporting the idea that beta(5)gamma(2) can bind to alpha(o). beta(5) monomer and beta(5)gamma(2) only supported a small degree of ADP ribosylation of alpha(i) by pertussis toxin (PTX), but beta(5) monomer was able to compete for beta(1)gamma(2) binding to alpha(i) and alpha(o) to inhibit PTX-catalyzed ADP ribosylation. These data indicate that beta(5) functionally interacts with PTX-sensitive GDP alpha subunits and that beta(5) subunits can be assembled with gamma subunits in vitro to reconstitute activity and also support the idea that there are determinants on beta subunits that are selective for even very closely related effectors.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Proteínas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Adenosina Difosfato Ribose/antagonistas & inibidores , Adenosina Difosfato Ribose/metabolismo , Animais , Baculoviridae/genética , Cromatografia em Gel , Cromatografia por Troca Iônica , Detergentes , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP/biossíntese , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/isolamento & purificação , Histidina/genética , Hidrólise , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Fosfolipase C beta , Fosfoproteínas/metabolismo , Ligação Proteica/genética , Proteínas RGS , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Spodoptera/genética , Tripsina/metabolismo , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
A 5-mg dose of cocaine free base applied to the volar forearm skin surface of a volunteer resulted in a maximal urinary benzoylecgonine concentration of 55 ng/mL at 48 h, using discrete urine specimens. A total of 58 micrograms of benzoylecgonine, representing 1.2% of the dose, was excreted in the 96-h urine. An identical trial using 5 mg of cocaine hydrochloride resulted in a maximal urinary benzoylecgonine concentration of 15 ng/mL at 24 h. We conclude that dermal absorption of cocaine represents a minor, but significant route of exposure to this drug that needs to be considered when interpreting low-level urine drug testing results.
