The specificity of the IgA receptor purified from human neutrophils.
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ABSTRACT:
SUBMITTER: Mazengera RL
PROVIDER: S-EPMC1149671 | biostudies-other | 1990 Nov
REPOSITORIES: biostudies-other
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Project description:Humans cannot synthesize vitamin A and thus must obtain it from their diet. ?-Carotene 15,15'-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15-15' double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (kcat/Km). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of ?-carotene with a Vmax = 197.2 nmol retinal/mg BCO1 × h, Km = 17.2 ?M and catalytic efficiency kcat/Km = 6098 M(-1) min(-1). The enzyme also catalyzed the oxidative cleavage of ?-carotene, ?-cryptoxanthin, and ?-apo-8'-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of ?-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of ?-carotene. The shorter ?-apocarotenals (?-apo-10'-carotenal, ?-apo-12'-carotenal, ?-apo-14'-carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-?-carotene. Our results show that BCO1 favors full-length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.
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Project description:The guanine nucleotide binding properties of rap1 protein purified from human neutrophils were examined using both the protein kinase A-phosphorylated and the non-phosphorylated forms of the protein. Binding of GTP[S] (guanosine 5'-[gamma-thio]triphosphate) or GDP was found to be slow in the presence of free Mg2+, but very rapid in the absence of Mg2+. The binding of guanine nucleotides was found to correlate with the loss of endogenous nucleotide from the rap1 protein, which was rapid in the absence of Mg2+. The relative affinities of GTP and GDP for the binding site on rap1 were modulated by the presence of Mg2+, with a preferential affinity (approx. 15-fold) for GTP observed only in the absence of this bivalent cation. The dissociation of GDP from rap1 was not affected by the G-protein beta/gamma-subunit complex. Phosphorylation of rap1 in vitro by protein kinase A did not modify any of the observed nucleotide-binding parameters. Furthermore, the ability of a cytosolic rap1 GTPase-activating protein to stimulate neutrophil rap1 GTP hydrolysis was not modified by phosphorylation. These data suggest that the activation of rap in vivo may be regulated by the release of endogenous GDP, but that phosphorylation by protein kinase A does not affect guanine nucleotide binding or hydrolysis.
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