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Determination of the intracellular dissociation constant, K D , of the Fluo‐3 · Ca 2+ complex in mouse sperm for use in estimating intracellular Ca 2+ concentrations
Author(s) -
Rockwell Pamela L.,
Storey Bayard T.
Publication year - 1999
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/(sici)1098-2795(199912)54:4<418::aid-mrd13>3.0.co;2-i
Subject(s) - nigericin , egta , ionomycin , intracellular , dissociation constant , sperm , biophysics , gramicidin , intracellular ph , fluorescence , ionophore , biology , valinomycin , analytical chemistry (journal) , calcium , membrane potential , membrane , biochemistry , chemistry , chromatography , botany , receptor , physics , organic chemistry , quantum mechanics
In order to calculate the actual, rather than the relative, intracellular Ca 2+ concentration (Ca 2+ ) i in mammalian sperm cells, using fluorescent probes whose fluorescence emission differs between the probe · Ca 2+ complex and free probe, the value of the dissociation constant for the probe · Ca 2+ complex, K D , is required. Interaction of the probe with cellular components may change the intracellular value of K D from that determined in buffered solution. We had previously shown that fluo‐3, whose Ca 2+ complex is highly fluorescent whereas free fluo‐3 is not, could be used to monitor changes of (Ca 2+ ) i in mouse sperm. In this report, we describe a method for determining K D for the fluo‐3 · Ca 2+ complex in mouse sperm suspended in medium MJB, a medium in which the sperm remain viable, but which contains high Ca 2+ . The method involved treating the sperm with ionomycin to provide a plasma membrane Ca 2+ carrier, with nigericin to eliminate pH gradient, and with gramicidin D to eliminate membrane potential, such that (Ca 2+ ) i equilibrates with medium Ca 2+ concentration (Ca 2+ ) e , then titrating (Ca 2+ ) e with EGTA in added aliquots to near nil concentration. At EGTA concentrations in excess of total medium Ca 2+ , an approximation algorithm was used to calculate (Ca 2+ ) e , based on the known K D for the EGTA · Ca 2+ complex. The fluorescence of the intracellular fluo‐3 · Ca 2+ complex, F, decreased with increasing additions of EGTA; (Ca 2+ ) i = (Ca 2+ ) e was plotted as a linear function of F/[F max − F]; the slope gives K D . At 37°C, intracellular K D was calculated to be 0.636 ± 0.018 μM (±SEM, n = 8). At 37°C and 20°C, K D values in MJB were calculated to be 0.502 ± 0.022 and 0.578 ± 0.029 (±SEM, n =8 and n = 6), respectively. The higher intracellular K D value implies probe interaction with cytosol components, primarily those in the head, as this compartment is the major contributor to sperm fluorescence. Changes in (Ca 2+ ) i , monitored with fluo‐3 fluorescence, that occur on interaction of capacitated mouse sperm with the zona pellucida and may now be quantified, using 0.636 μM for K D of the intracellular fluo‐3 · Ca 2+ complex. Mol. Reprod. Dev. 54:418–428, 1999. © 1999 Wiley‐Liss, Inc.