Biophysical Journal 63: 89-97 (1992)
© 1992 the Biophysical Society

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hove-Madsen, L Articles by Bers, D M Search for Related Content PubMed PubMed Citation Articles by Hove-Madsen, L Articles by Bers, D M

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

Division of Biomedical Sciences, University of California, Riverside 92521-0121.

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.

This article has been cited by other articles:

				J. Gomez, P. Neco, M. DiFranco, and J. L. Vergara Calcium Release Domains in Mammalian Skeletal Muscle Studied with Two-photon Imaging and Spot Detection Techniques J. Gen. Physiol., May 30, 2006; 127(6): 623 - 637. [Abstract] [Full Text] [PDF]

				H. A. Shiels and E. White Temporal and spatial properties of cellular Ca2+ flux in trout ventricular myocytes Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2005; 288(6): R1756 - R1766. [Abstract] [Full Text] [PDF]

				T. R. Shannon, T. Guo, and D. M. Bers Ca2+ Scraps: Local Depletions of Free [Ca2+] in Cardiac Sarcoplasmic Reticulum During Contractions Leave Substantial Ca2+ Reserve Circ. Res., July 11, 2003; 93(1): 40 - 45. [Abstract] [Full Text] [PDF]

				R. Brandes and D. M. Bers Simultaneous Measurements of Mitochondrial NADH and Ca2+ during Increased Work in Intact Rat Heart Trabeculae Biophys. J., August 1, 2002; 83(2): 587 - 604. [Abstract] [Full Text] [PDF]

				A. Takahashi, P. Camacho, J. D. Lechleiter, and B. Herman Measurement of Intracellular Calcium Physiol Rev, October 1, 1999; 79(4): 1089 - 1125. [Abstract] [Full Text] [PDF]

				G.A. Ng, S. M Cobbe, and G. L Smith Non-uniform prolongation of intracellular Ca2+ transients recorded from the epicardial surface of isolated hearts from rabbits with heart failure Cardiovasc Res, February 1, 1998; 37(2): 489 - 502. [Abstract] [Full Text] [PDF]