Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.13.12.5 (aequorin)
 1,451 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have examined in unstimulated and thrombin-stimulated human and rabbit platelets the localization and behavior of aequorin loaded by a variety of published methods. When platelets were suspended at 37 degrees C in Tyrode-albumin medium containing 2 mM Ca2+ and apyrase, we found with all preparations that total aequorin revealed by addition of Triton X-100 decreased by more than 50% over one hour. Incubation in the presence of 5 mM EGTA followed by addition of Ca2+ to restore the concentration to 2 mM showed that some aequorin had entered the medium; subsequent addition of Triton X-100 showed that the increase in aequorin in the medium matched the decrease in aequorin in the platelets, such that total aequorin remained unchanged. However, comparison of aequorin in platelets incubated in media with and without Ca2+ showed a larger decrease in platelets incubated in the presence of Ca2+; this finding may indicate the presence of an intracellular pool of Ca2+ which is more dependent on external Ca2+. Stimulation of platelets with thrombin in the presence of EGTA resulted in a smaller luminescent signal than in the presence of Ca2+. Subsequent addition of Ca2+ to 2 mM in the platelet suspension that originally contained EGTA or to its supernate (after centrifugation of the platelet suspension), resulted in a larger luminescent signal compared with controls, indicating that stimulation of the platelets had increased loss of the aequorin into the medium.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A significant portion of the aequorin luminescent signal from stimulated human and rabbit platelets is due to exposure of the aequorin to calcium in the suspending medium. 812 38

Mannose-binding lectin (MBL) is a key component of the innate immune system, and its deficiency is associated with increased susceptibility to various infections and autoimmune disorders. Since several nucleotide variations in the mannose-binding lectin 2 gene (MBL2) have been associated with the functional deficiency of MBL, there is a growing need to screen its allelic variants and develop genotyping methods for MBL2. In this context we propose a rapid, robust, cost-efficient, and automatable method for detecting all known allelic variants of MBL2. This report introduces for the first time the photoprotein aequorin as a reporter in genotyping by primer extension (PEXT) reactions. The method involves a single PCR amplification of a genomic region that spans all six variant nucleotide sites, i.e., three structural mutations in exon 1 (c.154C>T, pArg52Cys; c.161A>G, p.Gly54Asp; and c.170A>G, p.Gly57Glu), two single nucleotide polymorphisms (SNPs) at positions c.-619G>C and c.-290G>C (promoter region), and one SNP at position c.-66C>T of the 5' untranslated region. PCR is followed by PEXT reactions for each site. Biotin-dUTP is incorporated in the extended primer. The genotyping primers contain a poly(dA) segment at their 5' end. The products are captured by hybridization on the surface of microtiter wells that are coated with a poly(dT)-albumin. The extended primers only are detected by reaction with a streptavidin-aequorin conjugate. The bound photoprotein aequorin is measured within 3 sec by simply adding Ca2+. We carried out extensive optimization studies of the PEXT reaction and genotyped the six nucleotide variant sites using blood specimens from 27 normal DNA samples. The results of the proposed method agreed entirely with the sequencing data.
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PMID:Photoprotein aequorin as a novel reporter for SNP genotyping by primer extension-application to the variants of mannose-binding lectin gene. 1641 84