Photoinhibition and light-dependent turnover of the D1 reaction-centre polypeptide of photosystem II are enhanced by mineral-stress conditions

The function of photosystem II (PSII) and the turnover of its D1 reaction-center protein were studied in spinach (Spinacia oleracea L.) plants set under mineral stress. The mineral deficiencies were induced either by supplying the plants with an acidic nutrient solution or by strongly reducing the supply of magnesium alone or together with sulfur. After exposure for 8–10 weeks to the different media, the plants were characterized by a loss of chlorophyll and an increase in starch content, indicating a disturbance in the allocation of assimilates. Depending on the severity of the mineral deficiencies the plants lost their ability to adapt even to moderate iradiances of 400 mol photons·m^–2·s^–1 and became photoinhibited, as indicated by the decrease in F_v/F_m (the ratio of yield of variable fluorescence to yield of maximal fluorescence when all reaction centers are closed). The loss of PSII function was induced by changes on the acceptor side of PSII. Fast fluorescence decay showed a loss of PSII centers with bound Q_B, the secondary quinone acceptor of PSII, and a fast reoxidation kinetic of q _a ^- , the primary quinone acceptor of PSII, in the photoinactivated plants. No appreciable change could be observed in the amount of PSII centers with unbound Q_B and in Q_B-nonreducing PSII centers. Immunological studies showed that the contents of the D1 and D2 proteins of the PSII reaction center and of the 33-kDa protein of the water-splitting complex were diminished in the photoinhibited plants, and the occurrance of a new polypetide of 14 kDa that reacted with an antibody against the C-termius of the D1 protein. As shown by pulse-labelling experiments with [¹⁴C]leucine both degradation and synthesis of the D1 protein were enhanced in the mineral-deficient plants when compared to non-deficient plants. A stimulation of D1-protein turnover was also observed in pH 3-grown plants, which were not inhibited at growth-light conditions. Obviously, stimulation of D1-protein turnover prevented photoinhibition in these plants. However, in the Mg- and Mg/S-deficient plants even a further stimulation of D1-protein turnover could not counteract the increased rate of photoinactivation.Abbreviations amp_(f,m,s) amplitude of the fast, (medium and slow) exponential component of fluorescence decay - F_m yield of maximum fluorescenc when all reaction centers are closed - F_o yield of intrinsic fluorescence at open PSII reaction centers in the dark - F_v yield of variable fluorescence, (difference between F_m and F_o) - LHC light-harvesting complex - PFD photon flux density - Q_A primary quinone acceptor of PSII - Q_B secondary quinone acceptor of PSII Dedicated to Professor Dr. Dres. hc. Achim Trebst on the occasion of his 65th birthdayThis work was supported by grants from the BMFT and the Ministerium für Umwelt, Raumordnung and Landwirtschaft, Nordrhein-Westfalen. The authors thank H. Wietoska and M. Bronzel for skilful technical assistance.     

Similarity between pyridoxal/pyridoxamine phosphate-dependent enzymes involved in dideoxy and deoxyaminosugar biosynthesis and other pyridoxal phosphate enzymes.

A multiple sequence alignment among aspartate aminotransferase, dialkylglycine decarboxylase, and serine hydroxymethyltransferase (DAS) was used for profile databank search. The DAS profile could detect similarities to other pyridoxal or pyridoxamine phosphate-dependent enzymes, like several gene products involved in dideoxysugar and deoxyaminosugar synthesis. The alignment among DAS and such gene products shows the conservation of aspartate 222 and lysine 258, which, in aspartate aminotransferase, interacts with the N1 of the coenzyme pyridine ring and forms the internal Schiff base, respectively. The lysine is replaced by histidine in the pyridoxamine phosphate-dependent gene products. The alignment indicates also that the region encompassing the coenzyme binding site is the most conserved.     

质体醌重组的D1／D2／Cytb559复合物的荧光衰减动力学和光破坏作用

光系统Ⅱ反应中心Ｄ１／Ｄ２／Ｃｙｔｂ５５９ 在分离纯化过程中失去了电子受体ＱＡ 和ＱＢ,人工合成的质体醌可以与Ｄ１／Ｄ２／Ｃｙｔｂ５５９ 复合物发生重组。癸基质体醌（ＤＰＱ）与Ｄ１／Ｄ２／Ｃｙｔｂ５９９ 复合物的重组导致该复合物的荧光强度下降及发射光谱蓝移,同时两个与光化学活性相关的长寿命（２４ ｎｓ和７３ ｎｓ）荧光衰减组分占整个荧光的百分数下降,这些结果表明ＤＰＱ作为Ｐｈｅｏ－ 的电子受体,限制了Ｐ６８０＋ ·Ｐｈｅｏ－ 的电荷重组。ＤＰＱ 的加入对Ｄ１／Ｄ２／Ｃｙｔｂ５５９复合物中Ｃｈｌａ 分子的光破坏敏感性影响不大,但β－胡萝卜素在加入ＤＰＱ 之后可以被光照破坏,这个过程可能与β－胡萝卜素的生理功能相关。     

Inhibition of G1 phase cyclin dependent kinases by transforming growth factor β1

Transforming growth factor β1 (TGFβ1) inhibits epithelial cell proliferation late in the G1 phase of the cell cycle. We examined the effect of TGFβ1 on known late G1 cell cycle regulators in an attempt to determine the molecular mechanism of growth inhibition by this physiological inhibitor. The results demonstrate the TGFβ1 inhibits the late G1 and S phase specific histone H1 kinase activity of p33^cdk2. This inhibitiion is not dur to TGFβ1's effect on p33^cdk2 synthesis, but rather due to its negative effect on the late G1 phosphorylation of p33^cdk2. It is also shown that TGFβ1 inhibits both late G1 cyclin A and cyclin E associated histon H1 kinase activities. The inhibitor has no effects on the synthesis of cyclin E but to inhibit the synthesis of cyclin A protein in a cell cycle dependent manner. If TGFβ1 is added to cells which have progressed futher than 8 hours into G1, then it is without inhibitory effect on cyclin A synthesis. These effect on TGFβ1 on late G1 cell cycle regulators correlate well with its inhibitory effects on cellular growth and suggest that these G1 cyclin dependent kinases might serve as targets for TGFβ1-mediated growth arrest.     

Identification of type-2 phosphatidic acid phosphohydrolase (PAPH-2) in neutrophil plasma membranes

Plasma membrane phosphatidic acid phosphohydrolase (PAPH) plays an important role in signal transduction by converting phosphatidic acid to diacylglycerol. PAPH-2, a Mg²⁺-independent, detergent-dependent enzyme involved in cellular signal transduction, is reportedly absent from the plasma membranes of neutrophilic leukocytes, a cell that responds to metabolic stimulation with abundant phospholipase -dependent diacylglycerol generation. The present study was designed to resolve this discrepancy, focusing on the influence of cellular disruption techniques, detergenta availability and cation sensitivity on the apparent distribution of PAPH in neutrophil sub-cellular fractions. The results clearly indicate the presence of two distinct types of PAPH within the particulate and cytosolic fractions of disrupted cells. Unlike the cytosolic enzyme, the particulate enzyme was not potentiated by magnesium and was strongly detergent-dependent. The soluble and particulate enzymes displayed dissimilar pH profiles. Separation of neutrophil particulate material into fractions rich in plasma membranes, specific granules and azurophilic granules by high speed discontinuous density gradient centrifugation revealed that the majority of the particulate activity was confined to plasma membranes. This activity was not inhibited by pretreatment with n-ethyl-maleimide in concentrations as high as 25 mM. PAPH activity recovered in the cytosolic fraction of disrupted neutrophils was almost completely inhibited by 5.0 mM n-ethylmaleimide. We conclude that resting neutrophils possess n-ethylmaleimide-resistant PAPH (type 2) within their plasma membranes. This enzyme may markedly influence the kinetics of cell activation by metabolizing second messengers generated as a result of activation of plasma membrane phospholipase D.     

Bayesian signal extraction from noisy FT NMR spectra

Summary The statistical interpretation of the histogram representation of NMR spectra is described, leading to an estimation of the probability density function of the noise. The white-noise and Gaussian hypotheses are discussed, and a new estimator of the noise standard deviation is derived from the histogram strategy. The Bayesian approach to NMR signal detection is presented. This approach homogeneously combines prior knowledge, obtained from the histogram strategy, together with the posterior information resulting from the test of presence of a set of reference shapes in the neighbourhood of each data point. This scheme leads to a new strategy in the local detection of NMR signals in 2D and 3D spectra, which is illustrated by a complete peak-picking algorithm.     

HNCCH-TOCSY,a triple resonance experiment for the correlation of backbone 13Cα and 15N resonances with aliphatic side-chain proton resonances and for measuring vicinal 13CO, 1Hβ coupling constants in proteins

Summary A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and -carbons in uniformly ¹³C¹⁵N-labeled proteins. In-phase ¹³C magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from ¹³C to ¹H leads to E.COSY-type cross peaks, from which the ³J_{H co} coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.     

Carotenoids in photosynthesis: Protection of D1 degradation in the light

Photosynthesis has been determined with mutants of Anacystis which form different amounts of carotenoids. With these cultures a highly significant correlation between photosynthetic oxygen evolution and the amounts of synthesized carotenoids was observed. In addition, the influence of carotenoids on light-dependent degradation of thylakoid proteins was investigated with Scenedesmus cultures grown in darkness in the presence of norflurazon, an inhibitor of carotenoid biosynthesis. Pre-illumination of cells resulted in decrease of photosynthetic activity accompanied by loss of the D1 protein. This effect is dependent on the length of illumination, and the light intensity, and increased when carotenoid content was lowered during previous growth of the norflurazon-treated cultures.Abbreviations BSA bovine serum albumin - D1 32 kDa Q_B-binding protein - EDTA ethylenediaminetetraacetic acid - LHCII light-harvesting complex II - PMSF phenylmethylsulfonyl fluoride - PS photosystem - tricine N-[tris(hydroxymethyl)methyl] glycine