Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1992 Jul;63(1):89–97. doi: 10.1016/S0006-3495(92)81597-7

Indo-1 binding to protein in permeabilized ventricular myocytes alters its spectral and Ca binding properties.

L Hove-Madsen 1, D M Bers 1
PMCID: PMC1262127  PMID: 1420876

Abstract

We have examined the binding of the fluorescent Ca indicator indo-1 to cellular protein in permeabilized ventricular myocytes and also to soluble and particulate myocyte protein. Using either a filtration technique or equilibrium dialysis, and conditions similar to those in a cardiac myocyte patch clamped with 100 microM indo-1 in the patch pipette, we found that 72% of the total indo-1 was bound to myocyte protein at a protein concentration of 100 mg/ml. This corresponds to a binding of 3.8 +/- 0.5 nmol indo-1/mg protein. Separation of the myocyte protein into a soluble and a particulate fraction showed that 63% of the bound indo-1 was bound to soluble protein, corresponding to a binding of 3.22 +/- 0.99 nmol/mg, whereas 37% of the bound indo-1 was bound to particulate protein (0.85 +/- 0.14 nmol/mg) at a low [Ca] (pCa approximately 9). Binding of indo-1 in permeabilized myocytes was approximately 60% higher at a saturating Ca concentration (pCa = 3), than under Ca free conditions (1 mM EGTA). Simultaneous measurements of free [Ca] with a Ca selective electrode and indo-1 fluorescence showed that, the dissociation constant (Kd) for Ca was increased 4-5 fold in the presence of permeabilized myocytes as compared to the value obtained in vitro. In agreement with the binding experiments we estimate that the true Kd and the apparent Kd (using ratiometric measurements) for Ca binding to indo-1 are increased approximately four fold, at a myocyte protein concentration of 100 mg/ml.

Full text

PDF

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baylor S. M., Chandler W. K., Marshall M. W. Dichroic components of Arsenazo III and dichlorophosphonazo III signals in skeletal muscle fibres. J Physiol. 1982 Oct;331:179–210. doi: 10.1113/jphysiol.1982.sp014369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baylor S. M., Hollingworth S. Fura-2 calcium transients in frog skeletal muscle fibres. J Physiol. 1988 Sep;403:151–192. doi: 10.1113/jphysiol.1988.sp017244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baylor S. M., Hollingworth S., Hui C. S., Quinta-Ferreira M. E. Properties of the metallochromic dyes Arsenazo III, Antipyrylazo III and Azo1 in frog skeletal muscle fibres at rest. J Physiol. 1986 Aug;377:89–141. doi: 10.1113/jphysiol.1986.sp016178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beeler T. J., Schibeci A., Martonosi A. The binding of arsenazo III to cell components. Biochim Biophys Acta. 1980 May 7;629(2):317–327. doi: 10.1016/0304-4165(80)90104-x. [DOI] [PubMed] [Google Scholar]
  5. Bers D. M. A simple method for the accurate determination of free [Ca] in Ca-EGTA solutions. Am J Physiol. 1982 May;242(5):C404–C408. doi: 10.1152/ajpcell.1982.242.5.C404. [DOI] [PubMed] [Google Scholar]
  6. Blatter L. A., Wier W. G. Intracellular diffusion, binding, and compartmentalization of the fluorescent calcium indicators indo-1 and fura-2. Biophys J. 1990 Dec;58(6):1491–1499. doi: 10.1016/S0006-3495(90)82494-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Borzak S., Kelly R. A., Krämer B. K., Matoba Y., Marsh J. D., Reers M. In situ calibration of fura-2 and BCECF fluorescence in adult rat ventricular myocytes. Am J Physiol. 1990 Sep;259(3 Pt 2):H973–H981. doi: 10.1152/ajpheart.1990.259.3.H973. [DOI] [PubMed] [Google Scholar]
  8. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  9. Hirota A., Chandler W. K., Southwick P. L., Waggoner A. S. Calcium signals recorded from two new purpurate indicators inside frog cut twitch fibers. J Gen Physiol. 1989 Oct;94(4):597–631. doi: 10.1085/jgp.94.4.597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ikenouchi H., Peeters G. A., Barry W. H. Evidence that binding of Indo-1 to cardiac myocyte protein does not markedly change Kd for Ca2+. Cell Calcium. 1991 Jun;12(6):415–422. doi: 10.1016/0143-4160(91)90067-o. [DOI] [PubMed] [Google Scholar]
  11. Konishi M., Baylor S. M. Myoplasmic calcium transients monitored with purpurate indicator dyes injected into intact frog skeletal muscle fibers. J Gen Physiol. 1991 Feb;97(2):245–270. doi: 10.1085/jgp.97.2.245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Konishi M., Olson A., Hollingworth S., Baylor S. M. Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements. Biophys J. 1988 Dec;54(6):1089–1104. doi: 10.1016/S0006-3495(88)83045-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  14. Lee H. C., Mohabir R., Smith N., Franz M. R., Clusin W. T. Effect of ischemia on calcium-dependent fluorescence transients in rabbit hearts containing indo 1. Correlation with monophasic action potentials and contraction. Circulation. 1988 Oct;78(4):1047–1059. doi: 10.1161/01.cir.78.4.1047. [DOI] [PubMed] [Google Scholar]
  15. Lorell B. H., Apstein C. S., Cunningham M. J., Schoen F. J., Weinberg E. O., Peeters G. A., Barry W. H. Contribution of endothelial cells to calcium-dependent fluorescence transients in rabbit hearts loaded with indo 1. Circ Res. 1990 Aug;67(2):415–425. doi: 10.1161/01.res.67.2.415. [DOI] [PubMed] [Google Scholar]
  16. Maylie J., Irving M., Sizto N. L., Boyarsky G., Chandler W. K. Calcium signals recorded from cut frog twitch fibers containing tetramethylmurexide. J Gen Physiol. 1987 Jan;89(1):145–176. doi: 10.1085/jgp.89.1.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maylie J., Irving M., Sizto N. L., Chandler W. K. Calcium signals recorded from cut frog twitch fibers containing antipyrylazo III. J Gen Physiol. 1987 Jan;89(1):83–143. doi: 10.1085/jgp.89.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Maylie J., Irving M., Sizto N. L., Chandler W. K. Comparison of arsenazo III optical signals in intact and cut frog twitch fibers. J Gen Physiol. 1987 Jan;89(1):41–81. doi: 10.1085/jgp.89.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Popov E. G., Gavrilov Y. Iu, Pozin EYa, Gabbasov Z. A. Multiwavelength method for measuring concentration of free cytosolic calcium using the fluorescent probe indo-1. Arch Biochem Biophys. 1988 Feb 15;261(1):91–96. doi: 10.1016/0003-9861(88)90107-5. [DOI] [PubMed] [Google Scholar]
  20. Sipido K. R., Wier W. G. Flux of Ca2+ across the sarcoplasmic reticulum of guinea-pig cardiac cells during excitation-contraction coupling. J Physiol. 1991 Apr;435:605–630. doi: 10.1113/jphysiol.1991.sp018528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Spurgeon H. A., Stern M. D., Baartz G., Raffaeli S., Hansford R. G., Talo A., Lakatta E. G., Capogrossi M. C. Simultaneous measurement of Ca2+, contraction, and potential in cardiac myocytes. Am J Physiol. 1990 Feb;258(2 Pt 2):H574–H586. doi: 10.1152/ajpheart.1990.258.2.H574. [DOI] [PubMed] [Google Scholar]
  22. Uto A., Arai H., Ogawa Y. Reassessment of Fura-2 and the ratio method for determination of intracellular Ca2+ concentrations. Cell Calcium. 1991 Jan;12(1):29–37. doi: 10.1016/0143-4160(91)90082-p. [DOI] [PubMed] [Google Scholar]
  23. Wier W. G., Cannell M. B., Berlin J. R., Marban E., Lederer W. J. Cellular and subcellular heterogeneity of [Ca2+]i in single heart cells revealed by fura-2. Science. 1987 Jan 16;235(4786):325–328. doi: 10.1126/science.3798114. [DOI] [PubMed] [Google Scholar]
  24. Williams D. A., Fay F. S. Intracellular calibration of the fluorescent calcium indicator Fura-2. Cell Calcium. 1990 Feb-Mar;11(2-3):75–83. doi: 10.1016/0143-4160(90)90061-x. [DOI] [PubMed] [Google Scholar]
  25. Williams D. A., Fogarty K. E., Tsien R. Y., Fay F. S. Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using Fura-2. Nature. 1985 Dec 12;318(6046):558–561. doi: 10.1038/318558a0. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES