Characterization of Nitrate Reductase from Light- and Dark-Exposed Leaves (Comparison of Different Species and Effects of 14-3-3 Inhibitor Proteins)

Nitrate reductase (NR) was extracted and partially purified from leaves of squash (Curcurbita maxima), spinach (Spinacia oleracea), and three transgenic Nicotiana plumbaginifolia leaves in the presence of phosphatase inhibitors to preserve its phosphorylation state. Purified squash NR showed activation by substrates (hysteresis) when prepared from leaves in the light as well as in darkness. A 14-3-3 protein known to inhibit phosphorylated spinach NR in the presence of Mg2+ decreased by 70 to 85% the activity of purified NR from dark-exposed leaves, whereas NR from light-exposed leaves decreased by 10 to 25%. Apparent lack of posttranslational NR regulation in a transgenic N. plumbaginifolia expressing an NR construct with an N-terminal deletion ([delta]NR) may be explained by more easy dissociation of 14-3-3 proteins from [delta]NR. Partially purified [delta]NR was, however, inhibited by 14-3-3 protein, and the binding constant of 14-3-3 protein (4 x 108 M-1) and the NR-inhibiting protein concentration that results in a 50% reduction of free NR (2.5 nM) were the same for NR and [delta]NR. Regulation of NR activity by phosphorylation and binding of 14-3-3 protein was a general feature for all plants tested, whereas activation by substrates as a possible regulation mechanism was verified only for squash.     

Biological timing and the clock metaphor: oscillatory and hourglass mechanisms

Living organisms have developed a multitude of timing mechanisms--"biological clocks." Their mechanisms are based on either oscillations (oscillatory clocks) or unidirectional processes (hourglass clocks). Oscillatory clocks comprise circatidal, circalunidian, circadian, circalunar, and circannual oscillations--which keep time with environmental periodicities--as well as ultradian oscillations, ovarian cycles, and oscillations in development and in the brain, which keep time with biological timescales. These clocks mainly determine time points at specific phases of their oscillations. Hourglass clocks are predominantly found in development and aging and also in the brain. They determine time intervals (duration). More complex timing systems combine oscillatory and hourglass mechanisms, such as the case for cell cycle, sleep initiation, or brain clocks, whereas others combine external and internal periodicities (photoperiodism, seasonal reproduction). A definition of a biological clock may be derived from its control of functions external to its own processes and its use in determining temporal order (sequences of events) or durations. Biological and chemical oscillators are characterized by positive and negative feedback (or feedforward) mechanisms. During evolution, living organisms made use of the many existing oscillations for signal transmission, movement, and pump mechanisms, as well as for clocks. Some clocks, such as the circadian clock, that time with environmental periodicities are usually compensated (stabilized) against temperature, whereas other clocks, such as the cell cycle, that keep time with an organismic timescale are not compensated. This difference may be related to the predominance of negative feedback in the first class of clocks and a predominance of positive feedback (autocatalytic amplification) in the second class. The present knowledge of a compensated clock (the circadian oscillator) and an uncompensated clock (the cell cycle), as well as relevant models, are briefly re viewed. Hourglass clocks are based on linear or exponential unidirectional processes that trigger events mainly in the course of development and aging. An important hourglass mechanism within the aging process is the limitation of cell division capacity by the length of telomeres. The mechanism of this clock is briefly reviewed. In all clock mechanisms, thresholds at which "dependent variables" are triggered play an important role.     

The Goodwin model: simulating the effect of cycloheximide and heat shock on the sporulation rhythm of Neurospora crassa

The Goodwin model is a negative feedback oscillator which describes rather closely the putative molecular mechanism of the circadian clock of Neurospora and Drosophila. An essential feature is that one or two clock proteins are synthesized and degraded in a rhythmic fashion. When protein synthesis in N. crassa (wild-type frq+and long-period mutant frq7) was inhibited by continuous incubation with increasing concentrations of cycloheximide (CHX) the period of the circadian sporulation rhythmicity is only slightly increased. The explanation of this effect may be seen in the inhibition of protein synthesis and protein degradation. In the model, increasing inhibition of both processes led to very similar results with respect to period length. That protein degradation is, in fact, inhibited by CHX is shown by determining protein degradation in N. crassa by means of pulse chase experiments. Phase response curves (PRCs) of the N. crassa sporulation rhythm toward CHX which were reported in the literature and investigated in this paper revealed significant differences between frq+and the long period mutants frq7and csp -1 frq7. These PRCs were also convincingly simulated by the model, if a transient inhibition of protein degradation by CHX is assumed as well as a lower constitutive degradation rate of FRQ-protein in the frq7/ csp -1 frq7mutants. The lower sensitivities of frq7and csp -1 frq7towards CHX may thus be explained by a lower degradation rate of clock protein FRQ7. The phase shifting by moderate temperature pulses (from 25 to 30 degrees C) can also be simulated by the Goodwin model and shows large phase advances at about CT 16-20 as observed in experiments. In case of higher temperature pulses (from 35 to 42 or 45 degrees C=heat shock) the phase position and form of the PRC changes as protein synthesis is increasingly inhibited. It is known from earlier experiments that heat shock not only inhibits the synthesis of many proteins but also inhibits protein degradation. Taking this into account, the Goodwin model also simulates the PRCs of high temperature (heat shock) pulses.     

Robust Concentration and Frequency Control in Oscillatory Homeostats

Homeostatic and adaptive control mechanisms are essential for keeping organisms structurally and functionally stable. Integral feedback is a control theoretic concept which has long been known to keep a controlled variable robustly (i.e. perturbation-independent) at a given set-point by feeding the integrated error back into the process that generates . The classical concept of homeostasis as robust regulation within narrow limits is often considered as unsatisfactory and even incompatible with many biological systems which show sustained oscillations, such as circadian rhythms and oscillatory calcium signaling. Nevertheless, there are many similarities between the biological processes which participate in oscillatory mechanisms and classical homeostatic (non-oscillatory) mechanisms. We have investigated whether biological oscillators can show robust homeostatic and adaptive behaviors, and this paper is an attempt to extend the homeostatic concept to include oscillatory conditions. Based on our previously published kinetic conditions on how to generate biochemical models with robust homeostasis we found two properties, which appear to be of general interest concerning oscillatory and homeostatic controlled biological systems. The first one is the ability of these oscillators (“oscillatory homeostats”) to keep the average level of a controlled variable at a defined set-point by involving compensatory changes in frequency and/or amplitude. The second property is the ability to keep the period/frequency of the oscillator tuned within a certain well-defined range. In this paper we highlight mechanisms that lead to these two properties. The biological applications of these findings are discussed using three examples, the homeostatic aspects during oscillatory calcium and p53 signaling, and the involvement of circadian rhythms in homeostatic regulation.     

Evidence for increased proton dissociation in low-activity forms of dephosphorylated squash-leaf nitrate reductase

The pH dependence of squash-leaf nitrate reductase has been studied. It has been found that high- and low-activity forms of purified nitrate reductase (both forms dephosphorylated) have different optimum pH values. A high-activity form has always a higher pH optimum compared with a low-activity form. Model computations show that the decrease in activity and the corresponding change of the pH optimum is apparently due to a conformation-dependent increase of proton dissociation of the enzyme. As previously shown, this behavior is also observed in leaf extracts during the conversion (and probably phosphorylation of nitrate reductase) from a high-active form to a low-active form when plants are transferred from light to darkness.