RESUMO
Recombinant bovine cardiac sodium-calcium exchange (NCX1) in a baculovirus construct was used to infect cabbage looper larvae (Trichoplusia ni). Infected larvae were homogenized and larvae membrane vesicles were purified. Western blot analysis indicated the presence of recombinant NCX1 protein in vesicles from infected larvae but not in controls. Vesicles from infected larvae expressed high levels of NCX1 activity (1.7 nmol Ca2+/mg protein/s) while vesicles from control larvae had no activity. NCX1 in larvae vesicles was bidirectional. Kinetic analysis yielded a Vmax of 3.6 nmol Ca2+/mg protein/s and a Km for Ca of 4.2 microM. NCX1 activity was inhibited by the exchange inhibitory peptide with an IC50 of 4 microM. These data demonstrate a novel and efficient method for the expression of large amounts of active recombinant NCX1 protein that has general application for expression and analysis of recombinant membrane proteins.
Assuntos
Mariposas/genética , Miocárdio/metabolismo , Sarcolema/metabolismo , Trocador de Sódio e Cálcio/genética , Sequência de Aminoácidos , Animais , Baculoviridae , Cálcio/metabolismo , Bovinos , Cinética , Larva , Dados de Sequência Molecular , Peptídeos/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Trocador de Sódio e Cálcio/biossíntese , Trocador de Sódio e Cálcio/metabolismo , Transfecção/métodosRESUMO
The exchange inhibitory peptide (XIP; RRLLFYKYVYKRYRAGKQRG) is a potent inhibitor of cardiac Na-Ca exchange activity. This study attempted to identify the XIP binding site on the Na-Ca exchange protein. Bovine cardiac sarcolemmal vesicles were proteolyzed and fractionated by XIP-affinity column chromatography. A 24 kDa fragment was purified and subjected to amino acid sequence analysis. A negatively charged region of intracellular loop f of the Na-Ca exchange protein (IDDDIFEEDEN; aa 445-455) was identified. The affinity and specificity of XIP interaction with peptides IDDDIFEEDEN and GEDDDDDECGEE (another negatively charged region of the Na-Ca exchange protein) were examined. XIP cross-linked to peptide IDDDIFEEDEN but not GEDDDDDECGEE in a pH-dependent manner. Fluorescence titration binding studies indicated that binding of IDDDIFEEDEN with XIP was saturable (Kd=5 microM) while binding with GEDDDDDECGEE was not specific. These data suggest that amino acids 445-455 on Na-Ca exchange loop f are involved in XIP binding.
