Effects of UV‐B radiation and nitrogen starvation on enzyme activities in isolated plasma membranes of Euglena gracilis

Abstract

Circadian rhythms are characteristic of many physiological and biochemical processes in the freshwater flagellate Euglena gracilis. Earlier, we found that the rhythms of photosynthesis, phototaxis and cell shape followed the same pattern in control organisms, but were differently affected by stress such as UV‐B irradiation and nitrogen deficiency. Here we extend our studies to use isolated plasma membranes to characterize the rhythms of some plasma membrane‐bound enzymes. Also, we wanted to see whether stress‐induced changes of these rhythms could be detected at the subcellular level and possibly be coupled to the changes seen in photosynthesis, phototaxis and cell shape. The isolation of plasma membranes using aqueous polymer two‐phase partitioning was successful, as judged by the large enrichment of the plasma membrane‐marker 5′‐nucleotidase, and the difference in the polypeptide pattern compared with the microsomal fraction from which it was prepared.Two other enzymes were analyzed, K+, Mg2+‐ATPase, and adenylyl cyclase. The specific activities of all three enzymes were decreased by UV‐B radiation by ca 30–50%, compared with the control cultures. On the other hand, nitrogen deficiency not only reduced the activity of the K+.Mg2+‐ATPase but also increased the activities of the 5′‐nucleotidase and adenylyl cyclase. The different treatments also resulted in differences in polypeptide pattern, e.g., a polypeptide around 30 kDa seemed to be specific to plasma membranes of nitrogen‐deficient cultures and one at 39 kDa for the UV‐B radiated ones.All three enzymes showed diurnal rhythms that were affected by UV‐B radiation. The peak in the rhythm of the ATPase was shifted by UV‐B radiation, the rhythm of the 5′‐nucleotidase nearly eliminated. The first peak of adenylyl cyclase activity was delayed, so that it looked more like a broad peak between 2 and 11 h after the onset of light. The rhythm of ATPase activity could be correlated with that of photosynthesis in both control and UV‐B irradiated cultures. Also, the rhythms of adenylyl cyclase activity and cell shape changes showed some similarities.