PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 7265993

A simple method for measuring total calcium in small tissue samples.

A Johns
View original source ↗Export metadata ↓

Abstract

A simple and accurate method for determining total calcium in small tissue samples using aequorin is described. The method involves measuring the calcium-induced luminescence of aequorin with the tritium channel of a liquid scintillation counter. The sensitivity of the method and specificity of the method for calcium have been determined and, theoretically, this method could determine calcium in tissue samples weighing 3 mg or as little as 2 nmol of calcium could be measured.

Explore related subjects

AequorinAnimalsCalciumFemaleIn Vitro TechniquesMethodsRabbitsScintillation CountingSpectrophotometry, AtomicUterusJournal Article

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

A Johns. 1981. A simple method for measuring total calcium in small tissue samples.. https://doi.org/10.1016/0160-5402(81)90083-8

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed1997

Coelenterazine analogs as chemiluminescent probe for superoxide anion.

Eleven new coelenterazine analogs containing the 3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one structure were synthesized. The superoxide-triggered chemiluminescence of these compounds was investigated using the hypoxanthine-xanthine oxidase system in comparison with four known compounds. The results showed that an alkyl substitution at the position 5 of the imidazopyrazinone ring causes a drastic decrease in the superoxide-dependent chemiluminescence intensity, whereas a dimethylene bridge added between the position 5 and the phenyl group bound to the position 6 dramatically increases the luminescence intensity, indicating the potential usefulness of this type of compound as a probe for superoxide anion. The luminescence intensity of the bridged analog was 33 times greater than that of MCLA [2-methyl-6-(4-methoxyphenyl)-3, 7-dihydroimidazo[1,2-alpha]pyrazin-3-one], the most sensitive superoxide probe of Cypridina luciferin-type. Two of the analogs synthesized, each with a covalently bound cyclodextrin, had a good solubility in water, an advantage in actual use. Moreover, one of them having a beta-cyclodextrin group showed a unique property; its luminescence was little affected by various substances in the environment.

Aequorin↗
PubMed1997

The use of Renilla luciferase, Oplophorus luciferase, and apoaequorin as bioluminescent reporter protein in the presence of coelenterazine analogues as substrate.

To investigate the use of various luciferases as reporter protein, the substrate specificity of recombinant Renilla luciferase, Oplophorus luciferase and recombinant apoaequorin was examined using 23 kinds of coelenterazine analogues as the substrate. The intensity of luminescence was generally highest with Oplophorus luciferase and lowest with apoaequorin, but varied widely by the substrate used. A very high level of light intensity was obtained when the luminescence reactions of e-coelenterazine and v-coelenterazine were catalyzed by Renilla luciferase, strongly suggesting the usefulness of recombinant Renilla luciferase as a highly sensitive reporter protein. Oplophorus luciferase can be an equally sensitive reporter protein when its gene is obtained.

Aequorin↗
PubMed1997

Domains of high Ca2+ beneath the plasma membrane of living A7r5 cells.

Theoretical models and indirect experimental observations predict that Ca2+ concentrations at the inner surface of the plasma membrane may reach, upon stimulation, values much higher than those of the bulk cytosol. In the past few years, we have shown that the Ca2+-sensitive photoprotein aequorin can be intracellularly targeted and utilized for specifically monitoring the [Ca2+] of various organelles. In this work, we extend this approach to the study of the cytoplasmic rim beneath the plasma membrane. We have constructed a new aequorin chimera by fusing the photoprotein with SNAP-25, a neuronal protein which is recruited to the plasma membrane after the post-translational addition of a lipid anchor. The SNAP-25-aequorin chimera, expressed in the rat aortic smooth muscle cell line A7r5, appears correctly sorted as revealed by immunocytochemistry. Using this probe, we demonstrate that the mean [Ca2+] of this cytoplasmic region ([Ca2+]pm) can reach values >10-fold higher than those of the bulk cytosol ([Ca2+]c) upon activation of Ca2+ influx through plasma membrane channels. In unstimulated cells, the mean [Ca2+]pm appears also to be higher than the bulk cytosol, presumably reflecting the existence of microdomains of high [Ca2+].

Aequorin↗