Marc Bonzon

Subchloroplastic localization of NAD kinase activity: evidence for a Ca2+, calmodulin‐dependent activity at the envelope and for a Ca2+, calmodulin‐independent activity in the stroma of pea chloroplasts

Chloroplasts were prepared from pea seedlings and tested for NAD kinase activity. More than half of a Ca2+, calmodulin‐dependent activity and most of a Ca2+, calmodulin‐independent activity of the homogenate were associated with chloroplasts. The Ca2+, calmodulin‐dependent activity could be detected by adding Ca2+ and calmodulin to the incubation medium containing intact chloroplasts. This activity could not be separated from the chloroplasts by successive washes or by phase partition in aqueous two‐polymer phase systems. After chloroplasts fractionation, the Ca2+, calmodulin‐dependent NAD kinase activity was localized at the envelope, and the Ca2+, calmodulin‐independent activity was recovered from the stroma. In view of these results and of a previous report [Simon (1982) Plant Cell Rep. 1, 119–122] the occurrence and presumed role of calmodulin in the chloroplast are discussed.

Calmodulin-dependent and independent NAD kinase activities from cytoplasmic and chloroplastic fractions of spinach (Spinacia oleracea L.).

NAD kinase activity has been found in a soluble, cytoplasmic fraction and in the chloroplasts prepared from green spinach leaves. A small amount of both the cytoplasmic and the chloroplastic NAD kinase activities was retained on a calmodulin-Sepharose affinity column. The cytoplasmic NAD kinase eluted from the affinity column was found to be enhanced by calmodulin in a Ca2+-dependent manner. The chloroplastic enzyme which is located exclusively in the stroma and not in the envelope and thylakoid fractions was not affected by Ca2+ and calmodulin. The stromal fraction of purified chloroplasts contained only a negligible amount of calmodulin, most probably due to cytoplasmic contamination. Based on these data, two different mechanisms for the light-dependent modulation of spinach NAD kinase activity are suggested.

Dynamically oriented biological water as studied by NMR

In spite of considerable effort, the significance of NMR line-pair spectra (doublets) due to “structured” water systems is not completely elucidated. We report here NMR doublet spectra of water in living systems interpreted in terms of the order parameter and of intermolecular chemical exchange, yielding exchange correlation times of about 10−4 s.

ADENYLATE KINASE IN TOBACCO CELL CULTURES. II: VARIABILITY AND REGULATION OF ISOFORM ACTIVITY PATTERNS IN DIFFERENT CELL LINES

Summary Specific activity and isoform patterns of adenylate kinase (AK) were investigated in tissue cultures of cloned tobacco cell lines that differed mainly in hormone requirement, proximate cause of hormoneautotrophy and neoplastic growth characteristics. Variations in the AK isoform pattern were related to growth and ageing processes, differentiation state of tissues, differences between culture cycles as well as the neoplastic characteristics of the cell lines. High specific AK activity and isoform patterns with dominating chloroplastic activities appeared as typical of green, photosynthetic and actively growing tissues (calli in growth phase, callus outer tissues). Lower specific AK activity and isoform patterns with dominating cytosolic activity were typical for senescent or non-photosynthetic tissue (calli in stationary phase, callus core tissues), but sometimes also occurred in entire young calli, apparently as a consequence of stress. This isoform pattern is therefore proposed as a stress marker in plant cell cultures. The isoform patterns of the different cell lines were analyzed in detail for the variations during different culture cycles and the differences appearing under optimal growth conditions. The observed characteristics were related to proximate cause of hormone-autotrophy and neoplastic state. The main differences appeared between normal and neoplastic, transformed tissues. Changes in compartmentation of AK activities under different metabolic situations are discussed with respect to the physiological characteristics of the cell lines, energy metabolism and ATP/ADP ratios of cellular compartments and the presumed role of AK in ageing and stress.

Chloroplast adenylate kinase from tobacco. Purification and partial characterization

Abstract A soluble isoform of adenylate kinase (AK, EC 2.7.4.3) from tobacco leaves ( Nicotiana tabacum L.) was purified about 60-fold by a protocol using ammonium sulphate fractionation, anion exchange chromatography, affinity chromatography and gel filtration. The purified protein was homogeneous, as judged by SDS-PAGE, IEF-PAGE and Mono Q ion exchange chromatography, and had a specific activity of 500 nkat mg −1 . Its M r was determined as 28 000 and 30 000 by SDS-PAGE and gel filtration, respectively. It is therefore monomeric and belongs to the long-variant-type adenylate kinases. The isoelectric point of ca 4.45, as measured by IEF-PAGE and the elution profile of the Mono Q column, is characteristic for a chloroplast AK isoform. Like the chloroplast AK of maize, the activity with ATP/AMP as substrates was about two times higher than with ADP and the apparent K m was about 10-times higher for ATP/AMP than for ADP. In contrast to the maize enzyme and many other eukaryotic AKs, both substrate binding sites showed an exceptionally high specificity for all three adenylate substrates, together with a rather low affinity, as judged by the apparent K m -values. These differences at the substrate-binding sites are confirmed by a low sensitivity of the enzyme to the competitive AK inhibitor diadenosine pentaphosphate, i.e. high K i -values.

Membrane-Oscillator Hypothesis of Metabolic Control in Photoperiodic Time Measurement and the Temporal Organization of Development and Behaviour in Plants

As a working hypothesis we proposed that the oscillatory feedback system of cellular energy metabolism should be the basis for the endogenous timing of growth, development and behaviour in eukaryotic systems. The interaction of environmental signals with an endogenous physiological rhythm or clock was assumed to occur at membrane-organized receptors which modulate membrane-bound energy transduction. The energy-dependent state of membranes was in turn considered to determine the sensitivity of membrane-bound receptors. The structural and functional principles for the physiological oscillators were supposed to be the same as those underlaying the theory of membrane-bound energy transduction (1,2). The circadian system is genetically fixed and provides the temporal frame for physiological and behavioural patterns that are necessary for survival of organisms and populations. In photoperiodic acclimation of organisms photoredox systems most likely function in signal transduction as modulators of vectorial metabolism in general and of co-translational and post-translational protein translocation in particular (1,3,4).

Irreversible Thermodynamics and Biological Evolution in Spinach Leaves as Studied by NMR

Summary Changes in molecular dynamics, which can be measured by nuclear magnetic resonance (NMR) methods, are closely related to internal energy and entropy changes and can give a valuable approximation of the total entropy evolution of a system. It is thus possible to analyse the different physiological states of a living organism in terms of its global thermodynamical properties. As an experimental system, leaves of vegetative or floral spinach plants were used, as well as leaves subjected to changes in day length or to chemical treatments which promote or inhibit the induction of the flowering process. The dynamics of the hexafluorobenzene spin-labels in lyophilized leaves indicate that the molecular movements are partially blocked in the floral state, which can thus be considered as more structured and ordered than the vegetative state. The time-dependent changes in NMR line widths of living leaves show a nearly circadian rhythm in entropy production in both the vegetative and the floral states, this latter state being characterized by a lower order of magnitude and a downward trend. This oscillatory behaviour can be interpreted in terms of non-equilibrium thermodynamics and of theory of irreversibility. The biophysical and biochemical changes occurring during the floral induction in the leaves seem to be closely related to fast energy and entropy changes, which may result in a rapid modification of the membrane permeability properties. It is supposed that a new balance in the fluxes of matter and energy may result from these changes and thus give rise to a thermodynamically more stable floral state.

Migration of adult spinach chloroplasts in the S-rho space, before and after photoperiodic floral induction

Summary The migration of adult chloroplasts isolated from spinach leaves submitted to different photoperiodic treatments was followed in time series through the S-rho space (sedimentation coefficient and density measured in zonal rotors). It was shown that a large S-rho surface is the same for plants in short days of 8 hours (vegetative state), in continuous days (fully induced and floral states) and during the mimicked inductive treatment (see Material and methods). In sharp contrast to this, the S-rho surface occupied by the chloroplasts isolated from plants transferred from short days to continuous illumination (floral inductive treatment) is quite specific and significantly different from the other light treatments. The acquisition of the induced state is characterized by a very sharp increase in the sedimentation coefficients of the two chloroplastic subpopulations differing by their densities. After a comparison of these results with the in vivo ultrastructural observations, an interpretation of the events accompanying the acquisition of the floral state in the leaf is proposed. These events can be considered as a transitory change in the regulation control systems of the chloroplasts. If it is assumed that the logics of the vegetative state (steady-state) is different from that of the floral state (another steady-state), then this transition must be accompanied by important disturbances in the system, especially in the energy transducers which are responsible for the maintenance of these steady-states. This transitory period appears as a particular moment in which some negative feedbacks become positive, permitting thus the new floral steady-state and a new equilibrium in the structuro-functional relationships to take place.

Molecular dynamics and entropy changes in the agar-water system

Abstract Molecular dynamics in the agar-water systems was studied using proton spin relaxation and rotary viscosimetry techniques. The decrease of spin-lattice relaxation during biological evolution in the agar medium for the tissue culture contributes to the problem of the entropy changes in the open systems.