Inactivation of taste receptor cell function by two cationic protein modification reagents

Two cationic protein modification reagents, 1‐cyclohexyl‐3‐(2‐morpholinylethyl) carbodiimide (CMCD) and dimethyl (2‐hydroxy‐5‐nitrobenzyl) sulfonium bromide (HNB‐dmS), inhibit taste receptor cell stimulation by NaCl, sucrose, and HCl. Modified inactive derivatives of the reagents under the same conditions are ineffective. Inactivation by HNB‐dmS is essentially irreversible. The effects of inactivation by CMCD are reversible after about 10–15 minutes of a water rinse, however, when applied in the presence of glycine methyl ester, the inhibited response is stabilized and only recovers after about 1.5–3 hours. Glycine methyl ester alone has no inhibitory properties. The kinetics of inactivation by both HNB‐dmS and CMCD are consistent with a second‐order reaction with rate constants of 0.041 ± 0.001 M−1 sec−1 and 0.121 ± 0.012 M−1 sec−1, respectively. The rate of inactivation by both compounds is independent of NaCl concentration as well as degree of receptor stimulation. This, together with the observation that the response to stimulation by all effectors examined is altered, suggests the inactivation occurs at an event which is common to the transduction of the response from all three stimuli. The ether:water partition coefficients, as well as previous results from inactivation by N–substituted maleimides, indicate that hydrophilic reagents do not cross the cell membrane in significant concentrations within the time period of application. This suggests the site of modification by the cationic protein modification reagents is at the surface of the cell membrane. Significant residual NaCl, sucrose, and HCl activity remains after total inactivation. To account for this, a two‐state membrane receptor system is postulated.