Abstract
Crassulacean acid metabolism (CAM) plants fix carbon dioxide at night by the carboxylation of phosphoenolpyruvate. If CO2 fixation is conducted with 13C18O2 , then in the absence of carbonic anhydrase, the malate formed by dark CO2 fixation should also contain high levels of carbon-13 and oxygen-18. Conversely, if carbonic anhydrase is present and highly active, oxygen exchange between CO2 and cellular H2O will occur more rapidly than carboxylation, and the [13C] malate formed will contain little or no oxygen-18 above the natural abundance level. The presence of oxygen-18 in these molecules can be detected either by nuclear magnetic resonance (using the oxygen-18 effect on the carbon-13 chemical shift of the carboxyl carbon) or by mass spectrometry (comparing the ions at three and five units above the molecular weight with that one unit above). Studies of phosphoenolpyruvate carboxylase in the presence and absence of carbonic anhydrase in vitro confirm the validity of the method. When CAM plants are studied by this method, we find that most species show incorporation of a significant amount of oxygen-18. Comparison of these results with results of isotope fractionation and gas exchange studies permits calculation of the in vivo activity of carbonic anhydrase toward HCO-3 compared with that of phosphoenolpyruvate carboxylase. The ratio (carbonic anhydrase activity/phosphoenolpyruvate carboxylase activity) is species dependent and varies from a low of about 7 for Ananas comosus to values near 20 for Hoya carnosa and Bryophyllum pinnatum , 40 for Kalancho ë daigremontiana , and 100 or greater for Bryophyllum tubiflorum , Kalancho ë serrata, and Kalancho ë tomentosa. Carbonic anhydrase activity increases relative to phosphoenolpyruvate carboxylase activity at higher temperature.
Highlights
Sample loading on the capillary column was adjusted to give optimum peak shapes and at least 10 scan cycles/peak were obtained in order that the mass cycling error should be kept below 1%.In contrast to theobservations of Murphy and Clay (25), extensive chromatographic separation of isotopic species was observed
We have chosen the second alternative. At this point we have reduced the problem to a system of two equations (2 and 3) in two unknowns (k5/k4 and k3/k,) and, it should be susceptible to analytical solution in closed form, we must acknowledgethat our experimental dataare imprecise and ourtheoretical treatment contains a numbeorf uncertainties
We show in the Miniprint that theconcentration ratio COz(ext)/ C02(i)for the three-step model is
Summary
Single operation.Thismethodisnotassensitive as the T o date we have assumed that the hydration of CO, is at carbon-14 method for studying compounds containinognly a equilibrium during nocturnalCOZ fixation inCAM plants (14, low level of label becauseof the natural abundancoef carbon- 15). This can only be true if carbonic anhydrase is present, 13 (1.1%), which provides a significant background against because therate of spontaneousinterchange of CO, and which isotopic enrichments must be detected.
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