CYTOPLASMIC CA2-H+-EXCHANGE BUFFERS IN GREEN-ALGAE()

Fluorescence ratio imaging was used for simultaneous measurement of cy tosolic pH(c) and pCa(c) in Chara corallina, Nitella flexilis, and Ere mosphaera viridis. In some experiments the electrical membrane potenti al was also recorded. The first hint of coupling between changes in pH (c) and pCa(c) was found in characean cells when the influence of buty rate on cytosolic streaming was studied by laser-Doppler-anemometry (L DA). The observed butyrate-induced cessation of cytosolic streaming su pports the assumption that changes in pH(c) cause changes in pCa(c). T his hypothesis was tested by simultaneously loading cells with Fura-2- dextran and BCECF-dextran. The addition of butyrate revealed strong co upling between pCa(c) and pH(c) although this only occurred when the d ifference between pH(c) and pCa(c) was less than 0.4 units (+/- 0.24, n = 7). The measured relationship between the changes in pCa(c) and pH (c) could be fitted by a cytoplasmic buffer exchange process. Protons imported by butyrate into the cytoplasm are able to displace Ca++ ions from cytoplasmic buffer sites. Three dissociation constants of the cy toplasmic buffer were pK(1) = 6.2, pK(2) = 7.1 for proton buffering, a nd pK(Ca) = 6.7 for Ca++ ion buffering. Other possible mechanisms, suc h as butyrate-induced Ca++ influx through the plasmalemma and Ca++ rel ease from internal stores are discussed. They are not necessary to exp lain the observed coupling but cannot be excluded from the process. Us ing the butyrate technique, the cytosolic in vivo proton buffer capaci ties of N. flexilis, C. corallina, and E. viridis were determined as b eta(i) = 30 mM . H+/pH-unit, beta(i) = 46 mM . H+/pH-unit, and beta(i) = 90 mM . H+/pH-unit, respectively. The values obtained in vivo are g reater than those found previously using extraction methods. This can be explained in terms of pump activity and exchange with cell organell es, i.e., the vacuole. The high value of beta(i) found in Eremosphaera reflects adaptation to its habitat.