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 developed a sensitive and simple method for assaying glycosyltransferase activities. This method makes use of solution-phase transferase reactions followed by capture to a microplate well coated with a substrate-specific monoclonal antibody. Sugar incorporation is quantitated by binding a saccharide-specific lectin and using bioluminescent aequorin for a reporter molecule. We demonstrate this method using the glycoprotein hormone-specific GalNAc-transferase and its acceptor substrate, agalacto-hCG. As little as 20 ng of agalacto-hCG with 32 nU of GalNAc-transferase gives a detectable signal with less than 10% of the acceptor sites substituted. In addition to this high sensitivity, by doing the transferase reactions in solution, we can assay up to 10 micrograms of agalacto-hCG. We show that this allows the determination of Km and Vmax kinetic constants that compare well to those obtained with radiolabeled nucleotide sugars.
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PMID:A microplate assay for analysis of solution-phase glycosyltransferase reactions: determination of kinetic constants. 181 92

1. Sugar transport in the giant muscle cells of Balanus nubilus is accelerated during contractile activity and exposure to porcine insulin. The characteristics of hexose-transfer regulation in the giant muscle cells have been examined by studying the transport of 3-O-methylglucose (a non-metabolized sugar) in both intact giant fibres and fibres subjected to internal solute control by internal dialysis.2. Sugar transport in barnacle muscle is mediated by a saturable process which is inhibited by both phloretin and cytochalasin B. Insulin increases the capacity of the transport system with little effect on its apparent affinity for sugar. Under the same conditions insulin increases 3-O-methylglucose-displaceable cytochalasin B binding. The effects of insulin on transport are half-maximal at 5 muM-insulin and are abolished by both insulin antibody and phloretin. The intact barnacle releases an insulin-like material in response to a rise in blood glucose levels.3. Insulin increases the cyclic GMP (cGMP) content and reduces the cyclic AMP (cAMP) content of barnacle muscle. Experiments with fibres injected with aequorin show that insulin also lowers cytosolic ionized Ca levels. The changes in cyclic nucleotide levels induced by insulin precede the effects on sugar transport and cytosolic ionized Ca. During repetitive contractile activity, cAMP, cGMP and ionized Ca levels are raised.4. Agents which raise the cAMP content of barnacle muscle normally inhibit sugar transport. Dibutyryl cAMP also inhibits transport. Alterations in cytosolic ionized Ca levels in intact fibres are without effect on sugar transport. Nevertheless, stimulation of transport by insulin is blunted when cytosolic ionized Ca is lowered by intracellular injection of the Ca-chelating agent, EGTA.5. Sugar uptake in the internally dialysed fibre is inhibited by intracellular application of cAMP. Internal application of Ca and cGMP stimulate sugar uptake in the dialysed fibre. Cyclic AMP reduces the capacity of the transport system whereas Ca and cGMP increase the capacity of the saturable transfer system. Cyclic AMP and cGMP act at kinetically independent sites. Internal ATP (2 mM) inhibits sugar uptake in the dialysed fibre by some 40%, possibly through the production of cAMP.6. External insulin stimulates sugar uptake in the dialysed fibre even when ionized Ca levels are buffered using EGTA. Stimulation by insulin requires the presence of cytosolic ATP and is potentiated by internal application of 1 mM-GTP. In the dialysed fibre stimulation of transport by insulin is greater than that brought about by Ca and cGMP.7. The stimulation of transport by insulin in the intact fibre and its inhibition by dibutyryl cAMP are abolished by intracellular injection of Gpp(NH)p. Injection of intact fibres with GTPgammaS potentiates the stimulation of transport by insulin and renders insulin-activation of transport irreversible. Injection of intact fibres with ATPgammaS leads to the irreversible inhibition of transport.8. Injection of intact fibres with cAMP phosphodiesterase lowers cAMP levels close to zero and stimulates sugar transport. Application of insulin to diesterase-injected fibres still stimulates transport in the absence of altered cytosolic cAMP.
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PMID:Insulin regulation of sugar transport in giant muscle fibres of the barnacle. 630 27

Extracellular ATP activates two distinct types of P2 purinoreceptor-mediated signaling pathways in macrophages, 1) the rapid formation of nonselective pores/channels in the plasma membrane and 2) a guanine nucleotide-binding protein-dependent stimulation of phosphotidylinositol-specific phospholipase C, with subsequent mobilization of intracellular Ca2+. Several studies have suggested that the pore-forming, or P2z, purinoreceptor may be involved in the cytolytic effects of ATP on macrophages and other cell types. We have identified 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) and UTP as selective agonists for the P2z purinoreceptor and Ca(2+)-mobilizing nucleotide receptor, respectively, in BAC1.2F5 macrophages. In this paper we demonstrate that BzATP and ATP (but not UTP) activate membrane depolarization in BAC1.2F5 cells and also stimulate appropriate electrophysiological responses, consistent with the expression of the P2z purinoreceptor, in Xenopus oocytes injected with poly(A)+ RNA derived from BAC1.2F5 cells. Micromolar concentrations of BzATP or millimolar concentrations of ATP induced a sustained increase in the membrane holding current in these voltage-clamped oocytes. This response was significantly potentiated in the absence of extracellular divalent cations, consistent with the specificity of the P2z purinoreceptor for tetrabasic nucleotides. The sustained currents induced by BzATP or ATP were distinct from the transient and/or oscillating increases in Ca(2+)-dependent Cl- currents that were stimulated by UTP but not BzATP. UTP-stimulated transient currents and nucleotide-dependent increases in aequorin luminescence in poly(A)+ RNA-injected oocytes were independent of extracellular Ca2+ and were correlated with the mobilization of intracellular Ca2+ stores. Sucrose fractionation of the poly(A)+ RNA from BAC1.2F5 cells resulted in the enrichment of mRNA species encoding the components of the P2z purinoreceptor, as well as the Ca(2+)-mobilizing nucleotide receptor, in fractions containing 2.5-4.0-kilobase species.
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PMID:Expression of the pore-forming P2Z purinoreceptor in Xenopus oocytes injected with poly(A)+ RNA from murine macrophages. 768 70

The gene encoding voltage-gated channel with high affinity for Ca(2+) permeation has not been cloned from plants. In the present study, we isolated a full-length cDNA encoding a putative Ca(2+ )channel (AtTPC1) from Arabidopsis. AtTPC1 has two conserved homologous domains, both of which contain six transmembrane segments (S1-S6) and a pore loop (P) between S5 and S6 in each domain, and has the highest homology with the two pore channel TPC1 recently cloned from rat. The overall structure is similar to the half of the general structure of alpha-subunits of voltage-activated Ca(2+) channels from animals. AtTPC1 rescued the Ca(2+) uptake activity of a yeast mutant cch1. Sucrose-induced luminescence, which reflects a cytosolic free Ca(2+) increase in aequorin-expressing Arabidopsis leaves, was enhanced by overexpression of AtTPC1 and suppressed by antisense expression of it. Sucrose-H(+) symporters AtSUC1 and 2, which depolarize membrane potential of cells receiving sucrose, also depressed a Ca(2+) increase by their antisense expression. These results suggest that AtTPC1 mediates a voltage-activated Ca(2+ )influx in Arabidopsis leaf cells.
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PMID:A putative two pore channel AtTPC1 mediates Ca(2+) flux in Arabidopsis leaf cells. 1157 83