Ruud Kraayenhof

Studies on well coupled Photosystem I-enriched subchloroplast vesicles. Content and redox properties of electron-transfer components

Abstract Stable and well coupled Photosystem (PS) I-enriched vesicles, mainly derived from the chloroplast stroma lamellae, have been obtained by mild digitonin treatment of spinach chloroplasts. Optimal conditions for chloroplast solubilization are established at a digitonin/chlorophyll ratio of 1 ( w w ) and a chlorophyll concentration of 0.2 mM, resulting in little loss of native components. In particular, plastocyanin is easily released at higher digitonin/chlorophyll ratios. On the basis of chlorophyll content, the vesicles show a 2-fold enrichment in ATPase, chlorophyll-protein Complex I, P-700, plastocyanin and ribulose-1,5-bisphosphate carboxylase as compared to chloroplasts, in line with the increased activities of cyclic photophosphorylation and PS I-associated electron transfer as shown previously (Peters, A.L.J., Dokter, P., Kooij, T. and Kraayenhof, R. (1981) in Photosynthesis I (Akoyunoglou, G., ed.), pp. 691–700, Balaban International Science Services, Philadelphia). The vesicles have a low content of the light-harvesting chlorophyll-protein complex and show no PS II-associated electron transfer. Characterization of cytochromes in PS I-enriched vesicles and chloroplasts at 25°C and 77 K is performed using an analytical method combining potentiometric analysis and spectrum deconvolution. In PS I-enriched vesicles three cytochromes are distinguished: c -554 ( E ′ 0 = 335 mV), b -559 LP ( E ′ 0 = 32 mV) and b -563 ( E ′ 0 = − 123 mV); no b -559 HP is present (LP, low-potential; HP, high-potential). Comparative data from PS I vesicles and chloroplasts are consistent with an even distribution of the cytochrome b -563- cytochrome c -554 redox complex in the lateral plane of exposed and appressed thylakoid membranes, an exclusive location of plastocyanin in the exposed membranes and a dominant location of plastoquinone in the appressed membranes. The results are discussed in view of the lateral heterogeneity of redox components in chloroplast membranes.

A thermoelectrically regulated multipurpose cuvette for simultaneous time-dependent measurements

Abstract An improved design of a thermostatically controlled reaction cuvette for time-dependent biochemical measurements is described. The design is such that a multiple choice of single and simultaneous spectroscopic and electrode analyses can be performed in a sample of about 1.8 ml. This choice is quite flexible due to the use of exchangeable tapered plugs suited with either optical quartz-rod windows for absorption or fluorescence measurements or selective electrodes for changes of O 2 , H 2 , H + , etc. Temperature is accurately controlled by a thermoelectric (Peltier) module. An overhead constant-stirring device includes solute addition and gasflow ports. A bottom window allows actinic illumination for photobiological and photochemical experiments. Some examples of application in combination with commercial or laboratorymade instruments are presented.

Interactions of histatin 5 and histatin 5-derived peptides with liposome membranes: surface effects, translocation and permeabilization.

A number of cationic antimicrobial peptides, among which are histatin 5 and the derived peptides dhvar4 and dhvar5, enter their target cells and interact with internal organelles. There still are questions about the mechanisms by which antimicrobial peptides translocate across the membrane. We used a liposome model to study membrane binding, translocation and membrane-perturbing capacities of histatin 5, dhvar4 and dhvar5. Despite the differences in amphipathic characters of these peptides, they bound equally well to liposomes, whereas their membrane activities differed remarkably: dhvar4 translocated at the fastest rate, followed by dhvar5, whereas the histatin 5 translocation rate was much lower. The same pattern was seen for the extent of calcein release: highest with dhvar4, less with dhvar5 and almost none with histatin 5. The translocation and disruptive actions of dhvar5 did not seem to be coupled, because translocation occurred on a much longer timescale than calcein release, which ended within a few minutes. We conclude that peptide translocation can occur through peptide-phospholipid interactions, and that this is a possible mechanism by which antimicrobial peptides enter cells. However, the translocation rate was much lower in this model membrane system than that seen in yeast cells. Thus it is likely that, at least for some peptides, additional features promoting the translocation across biological membranes are involved as well.

Transmembrane electrical potential formation in spinach chloroplasts: Investigation using a rapidly-responding extrinsic probe

It is well established that an electrochemical proton gradient (AT,+) can drive ATP synthesis [l-3] and that a ArH+ is formed during photosynthetic phosphorylation [4], consistent with the chemiosmotic hypothesis of Mitchell [5,6] . Whilst, in the steady state, the bulk of this ApH+ is in the form of a ApH [7,8] and less in the form of its electrical component (A

Monovalent cations differentially affect membrane surface properties and membrane curvature, as revealed by fluorescent probes and dynamic light scattering

), considerable evidence indicates that there is a ‘burst’ of A

Fluorescent probes used to monitor membrane interfacial polarity.

J on illumination of chloroplasts, which decays rapidly to its steady state level [4,9] . Furthermore this initial A J/ appears to play a fundamental role in the regulation of photosynthetic phosphorylation [lo] . Investigations of the initial A

Studies on well coupled Photosystem I-enriched subchloroplast vesicles. Optimization of ferredoxin-mediated cyclic photophosphorylation and electric potential generation

formation have employed chloroplasts activated by flashes of saturating light, which induce a single turnover of the photosynthetic apparatus [ 1 l] . The rapid formation and decay of A

Energy metabolism in the cyanobacterium Plectonema boryanum. Participation of the thylakoid photosynthetic electron transfer chain in the dark respiration of NADPH and NADH

in this system have been observed, in the main, using two techniques. A shift in the absorbance spectrum of the carotenoids in the chloroplastthylakoid membrane occur on illumination [9] and this shift is A

Fluorescent probes of membrane surface properties.

-linked [ 12,131 . Micro-electrodes, implanted in the giant chloroplasts of Peperomia metallica, also detect a light-induced A

CTP:phosphocholine cytidylyltransferase and protein kinase C recognize different physical features of membranes: differential responses to an oxidized phosphatidylcholine

[ 141 . There are considerable differences, however, between the magnitude and kinetics of formation of