Guanosine triphosphate-binding proteins on the plasmalemma of spinach leaf cells

The molecular mechanism of light perception through phytochrome is not well understood. This red-light photosensor has been implicated in various physiological processes, including the photoinduction of flowering. A few recent studies have shown that phytochrome initiates signal transduction chains via guanosine triphosphate (GTP)-binding proteins (G-proteins). We show here by different approaches that G-proteins exist in spinach (Spinacia oleracea L. cv. Nobel). Binding of GTP on the plasmalemma has been partially characterized and its possible regulation by red light examined by in-vitro assays. These experiments indicate a clear regulation of GTP binding by red light and also by Mastoparan. At least three G-proteins or protein subunits were found to be associated with the plasmalemma of leaf cells. The use of an antibody raised against an animal Gβ subunit confirmed the presence of heterotrimeric G-proteins. Separation of a crude membrane extract by free-flow electrophoresis also showed that some G-proteins could exist on the tonoplast.     

In vitro modification of Candida albicans invasiveness

Candida albicans produces germ-tubes (GT) when it is incubated in animal or human serum. This dimorphism is responsible for its invasive ability.The purpose of the present paper is (1) to evaluate the ability of rat peritoneal macrophages to inhibit GT production of ingested Candida albicans, obtained from immunized rats and then activated in vitro with Candida-induced lymphokines; (2) to determinate any possible alteration of phagocytic and candidacidal activities.The phagocytes were obtained from rats immunized with viable C. albicans. Some of them were exposed to Candida-induced lymphokines in order to activate the macrophages in vitro. The monolayers of activated, immune and normal macrophages were infected with a C. albicans suspension during 4 hr.Activated macrophages presented not only the highest phagocytic and candidacidal activities but a noticeable inhibition of GT formation and incremented candidacidal activity.     

In vitro plant regeneration of spinach from mature seed-derived callus

Summary A system for the regeneration of spinach (Spinacia oleracea L.) from mature dry seed explants has been established. The response of two commercial spinach cultivars, ‘Grandstand’ and ‘Baker’, was examined. Callus proliferation was most prominent on MS medium supplemented with 9.3 μM of 6-furfurylaminopurine (kinetin) and 3.39 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Adventitious shoot formation was observed within 8 wk after callus was transferred onto regeneration medium. Shoot regeneration was best from callus induced on 9.3 μM kinetin and 4.56 μM 2,4-D. The regeneration medium contained 9.3 μM kinetin, 0.045 μM 2,4-D, and 2.89 μM gibberellic acid (GA₃). Shoots were rooted on hormone-free medium, and plants grown in a greenhouse showed normal phenotype. This system is beneficial in rapid propagation of spinach plants, particularly when only a limited number of seeds are available.     

In vitro modification of bovine lens aldose reductase activity

Bovine lens aldose reductase can be activated in crude extracts upon incubation at 37 degrees C at relatively high ionic strength. This phenomenon shows a seasonal occurrence, the enzyme being susceptible to activation only in lenses of animals sacrified in summer. Systems generating oxygen activated species induce the enzyme activation, whereas scavengers of "oxygen radicals" preserve the activated state of the enzyme. Glutathione and other thiol compounds appear to prevent the enzyme activation.     

Light-dependent modification of Photosystem II in spinach leaves

In dark-adapted spinach leaves approximately one third of the Photosystem II (PS II) reaction centers are impaired in their ability to transfer electrons to Photosystem I. Although these inactive PS II centers are capable of reducing the primary quinone acceptor, Q_A, oxidation of Q_A ^– occurs approximately 1000 times more slowly than at active centers. Previous studies based on dark-adapted leaves show that minimal energy transfer occurs from inactive centers to active centers, indicating that the quantum yield of photosynthesis could be significantly impaired by the presence of inactive centers. The objective of the work described here was to determine the performance of inactive PS II centers in light-adapted leaves. Measurements of PS II activity within leaves did not indicate any increase in the concentration of active PS II centers during light treatments between 10 s and 5 min, showing that inactive centers are not converted to active centers during light treatment. Light-induced modification of inactive PS II centers did occur, however, such that 75% of these centers were unable to sustain stable charge separation. In addition, the maximum yield of chlorophyll fluorescence associated with inactive PS II centers decreased substantially, despite the lack of any overall quenching of the maximum fluorescence yield. The effect of light treatment on inactive centers was reversed in the dark within 10–20 mins. These results indicate that illumination changes inactive PS II centers into a form that quenches fluorescence, but does not allow stable charge separation across the photosynthetic membrane. One possibility is that inactive centers are converted into centers that quench fluorescence by formation of a radical, such as reduced pheophytin or oxidized P680. Alternatively, it is possible that inactive PS II centers are modified such that absorbed excitation energy is dissipated thermally, through electron cycling at the reaction center.Abbreviations A518 absorbance change at 518 nm, reflecting the formation of an electric field across the thylakoid membrane - A_FL1 amplitude of the fast (<100 ms) phase of A518 induced by the first of two saturating, single-turnover flashes spaced 30 ms apart - A_FL2 amplitude of the fast (<100 ms) phase of A518 induced by the second of two saturating, single-turnover flashes spaced 50 ms apart - DCBQ 2,6-dichloro-p-benzoquinone - Fo yield of chlorophyll fluorescence when Q_A is fully oxidized - Fm yield of chlorophyll fluorescence when Q_A is fully reduced - Fx yield of chlorophyll fluorescence when Q_A is fully reduced at inactive PS II centers, but fully oxidized at active PS II centers - Pheo pheophytin - P680 the primary donor of Photosystem II - PPFD photosynthetic photon flux density - Q_A Primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

In vitro high frequency plant regeneration from hypocotyl and root segments of spinach by organogenesis

An efficient protocol for spinach (Spinacia oleracea L.) plant regeneration from hypocotyl and root segments was established. When the sub-apical hypocotyl and tip-free root segments were cultured on Murashige & Skoog (1962)-based medium containing high concentrations of indole-3-acetic acid (85.62 M) and gibberellic acid (100 M), more than 75% and 90% of the hypocotyl and root explants, respectively, formed shoots. After elongation, more than 92% of the shoots rooted on medium supplemented with 2.85–5.71 M of indole-3-acetic acid. More than 70% of rooted plantlets survived in soil and were fertile. Significant interactions between growth regulator combinations, explant types and environmental conditions on shoot initiation, development and rooting were discussed.Abbreviations BA benzyladenine - BM Murashige & Skoog basal medium - B5 Gamborg et al. medium (1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - 2ip isopentenyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige & Skoog medium (1962) - NAA naphthaleneacetic acid - HS hypocotyl segments - RSS root segments of seedlings - RSV foot segments of in vitro plantlets     

In vitro phosphorylation and inactivation of spinach leaf sucrose-phosphate synthase by an endogenous protein kinase

(1) Partially purified preparations of spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) contain an endogenous protein kinase that phosphorylates and inactivates the enzyme with [gamma-32P]ATP. (2) The kinetic effect of phosphorylation is to alter affinities for substrates and the effector inorganic phosphate without affecting maximum velocity. (3) Two-dimensional peptide mapping of tryptic digests of in vitro labeled SPS yielded two phosphopeptides (designated sites 5 and 7). Labeling of the two sites occurred equally with time, and both correlated with inactivation. Maximum inactivation was associated with incorporation of 1.5 to 2.0 mol P/mol SPS tetramer, and about 70% of the phosphoryl groups were incorporated into one of the sites (phosphopeptide 7). (4) Phosphorylation and inactivation were strongly inhibited by NaCl, and the presence of salt alters some characteristics of the kinase reaction. In the absence of salt, the apparent Km for Mg.ATP was estimated to be 5 microM. (5) The dependence of the rate of phosphorylation on SPS concentration suggested that SPS and the protein kinase are distinct enzymes, but have some tendency to associate especially in the presence of ethylene glycol. (6) Ca2+/EGTA and polyamines have no effect on the rate of phosphorylation, whereas polycations (polylysine, polybrene and protamine) are inhibitory. (7) Of the metabolic intermediates tested, Glc 6-P inhibited phosphorylation and inactivation of the enzyme. The inhibition was not antagonized by inorganic phosphate, which suggests that Glc 6-P may be an effector of the kinase, rather than the target protein. Regulation by Glc 6-P may be of physiological significance.     

In vitro enzymatic modification of puerarin to puerarin glycosides by maltogenic amylase

Puerarin (daidzein 8-C-glucoside), the most abundant isoflavone in Puerariae radix, is prescribed to treat coronary heart disease, cardiac infarction, problems in ocular blood flow, sudden deafness, and alcoholism. However, puerarin cannot be given by injection due to its low solubility in water. To increase its solubility, puerarin was transglycosylated using various enzymes. Bacillus stearothermophilus maltogenic amylase (BSMA) was the most effective transferase used compared with Thermotoga maritima maltosyl transferase (TMMT), Thermus scotoductus 4-alpha-glucanotransferase (TS4alphaGTase), and Bacillus sp. I-5 cyclodextrin glucanotransferase (BSCGTase). TMMT and TS4alphaGTase lacked acceptor specificity for puerarin, which lacks an O-glucoside linkage between D-glucose and 7-OH-daidzein. The yield exceeded 70% when reacting 1% puerarin (acceptor), 3.0% soluble starch (donor), and 5U/100 microL BSMA at 55 degrees C for 45 min. The two major transfer products of the BSMA reaction were purified using C(18) and GPC chromatography. Their structures were identified as alpha-d-glucosyl-(1-->6)-puerarin and alpha-D-maltosyl-(1-->6)-puerarin using ESI+ TOF MS-MS and 13C NMR spectroscopy. The solubility of the transfer products was 14 and 168 times higher than that of puerarin, respectively.     

In vitro reconstruction of full thickness human skin on a composite collagen material

Rapid progress of in vitro techniques in the lastyears enabled the creation of organotypic skin cultures offering newpossibilities in wound treatment. Rebuilding of graft is one of the keyelementsof successful outcome of the procedure.In search for the best scaffold for organotypic skin culture, the novelcomposite xenogenic collagen based material with unique properties has beencreated and used to reconstitute full thickness human skin invitro. Based on our long established technology used for theproduction of collagen dressings for the treatment of burns, this novel,composite material offers excellent growth support of highly biodegradablespongy layer, combined with mechanical strength of collagen membrane. Themodulation of collagen properties was accomplished by consecutive treatmentwithhigh temperature and gamma irradiation. The use of the substrate enabled toobtain organotypic culture that resembles full thickness skin with fibroblastslayer and well-developed multilayer epithelium. Our new material offers easyhandling of obtained graft during surgery along with accelerated cell growth andcontrolled biodegradation of the culture support.     

In vitro activation of the Serratia marcescens hemolysin through modification and complementation.

The hemolytic activity of Serratia marcescens is determined by two polypeptides, termed ShlA and ShlB. ShlA is synthesized as an inactive precursor (ShlA*) and secreted with the help of ShlB, which is located in the outer membrane. In this study, it is shown that a cell lysate containing ShlB as well as partially purified ShlB converted ShlA* to the active ShlA hemolysin. ShlA remained active after removal of ShlB by column chromatography. In contrast to the stable modification of ShlA* by ShlB, a reversible activation was achieved by adding to ShlA* an N-terminal fragment of ShlA (ShlA16), consisting of 269 amino acid residues of ShlA and 18 residues of the vector. The nonhemolytic ShlA16 complemented ShlA* only when it was synthesized in an ShlB-producing cell. A deletion derivative of ShlA*, lacking residues 4 to 117, was complemented by ShlA16 but not activated by ShlB. Activation of ShlA* by ShlB at 4 degrees C proceeded at a much slower rate than complementation by ShlA16. It is concluded that ShlA* is modified by ShlB. ShlA16 modified by ShlB complements the missing modification of ShlA* in trans. Modification by ShlB occurs in the N-terminal part of ShlA*, which is also the reaction in vivo which results in active ShlA hemolysin in the culture supernatant. The HpmA hemolysin of Proteus mirabilis, which is very similar to ShlA, was also activated in vitro by ShlB and complemented by ShlA16.     

In vitro synthesis and modification of mRNA by exvectorial isolates of wound tumor virus

Nontransmissible (exvectorial) isolates of wound tumor virus retain the ability to catalyze in vitro synthesis of RNA. Furthermore, exvectorial virus particles exhibit mRNA-2′-O-methyltransferase activity even after long-term (30-year) passage in a host that lacks this enzyme activity.     

In vitro and in vivo age-related modification of human erythrocyte phosphoribosyl pyrophosphate synthetase.

Upon storage, human erythrocyte phosphoribosyl pyrophosphate synthetase (PRibPP synthetase, EC 2.7.6.1) from normal individuals was found to undergo a spontaneous dissociation into active enzyme components of much smaller molecular mass (60 000--90 000). These modified forms of enzyme exhibit kinetic properties different from the original large molecular weight enzyme (over 200 000). The small active components can be reversibly associated to form larger molecules in the presence of purine ribonucleotides as well as phosphoribosyl pyrophosphate (PRibPP). ATP was found to be most effective in associating PRibPP synthetase, while guanylate nucleotides seem to have no effect. The large molecular weight components, once separated from the milieu, were not able to undergo further dissociation. Fresh or stored human white cell tissue homogenates were found to lack the low-molecular-weight enzyme under all our experimental conditions. A characteristic enzyme modification similar to that observed in stored erythrocyte was also noted in erythrocytes of increasing ages. The physiological significance of these findings to the regulatory function of PRibPP synthetase in purine metabolism in vivo is discussed.     

In vivo and in vitro studies on the pathway of modification of mussel pyruvate kinase

1. On aerial exposure, pyruvate kinase is inactivated in various organs of M. edulis; the decrease of activity is slower in muscle than in non-muscular tissue. 2. Anoxic in vitro incubation of gills results in a rapid inactivation of pyruvate kinase. No change occurs in an aerated medium. 3. Enzyme inactivation is mimicked in part by oxic incubation in an acidified medium containing 5,5-dimethyloxazolidine-2,4-dione, and by the action of calcium ionophore A23187. 4. Incubation of supernatant of gill homogenate results in a slow inactivation of pyruvate kinase that is inhibited by trifluoperazine or EGTA and stimulated by exogenous calmodulin. 5. Addition of ATP plus cAMP stimulates pyruvate kinase inactivation in supernatant of homogenized muscle but not so in a high molecular weight fraction thereof.     

In vitro biosynthesis of a universal t6A tRNA modification in Archaea and Eukarya

N⁶-threonylcarbamoyladenosine (t⁶A) is a modified nucleotide found in all transfer RNAs (tRNAs) decoding codons starting with adenosine. Its role is to facilitate codon–anticodon pairing and to prevent frameshifting during protein synthesis. Genetic studies demonstrated that two universal proteins, Kae1/YgjD and Sua5/YrdC, are necessary for t⁶A synthesis in Saccharomyces cerevisiae and Escherichia coli. In Archaea and Eukarya, Kae1 is part of a conserved protein complex named kinase, endopeptidase and other proteins of small size (KEOPS), together with three proteins that have no bacterial homologues. Here, we reconstituted for the first time an in vitro system for t⁶A modification in Archaea and Eukarya, using purified KEOPS and Sua5. We demonstrated binding of tRNAs to archaeal KEOPS and detected two distinct adenosine triphosphate (ATP)-dependent steps occurring in the course of the synthesis. Our data, together with recent reconstitution of an in vitro bacterial system, indicated that t⁶A cannot be catalysed by Sua5/YrdC and Kae1/YgjD alone but requires accessory proteins that are not universal. Remarkably, we observed interdomain complementation when bacterial, archaeal and eukaryotic proteins were combined in vitro, suggesting a conserved catalytic mechanism for the biosynthesis of t⁶A in nature. These findings shed light on the reaction mechanism of t⁶A synthesis and evolution of molecular systems that promote translation fidelity in present-day cells.     

In vitro reconstitution of the spinach chloroplast cytochrome b6 protein from a fusion protein expressed in Escherichia coli

We have developed a strategy for overproduction of spinach apocytochrome b6 as a fusion protein to maltose-binding protein (MBP) in Escherichia coli, using the expression vector pMal-c2. The fusion protein was purified to virtual homogeneity by gel filtration chromatography and the method of insertion of hemes into fusion protein was elaborated. The ambient and low-temperature absorption spectra of the reconstituted cytochrome b6 were similar to those of cytochrome b6 spectra in isolated proteins or cytochrome b6f complexes and are typical for bis-histidine ligated b-type cytochromes. Optical circular dichroism (CD) spectra of the visible region further confirmed the appropriate binding of hemes by the apocytochrome b6 protein. We found that the incorporation of hemes was required for the refolding of the cytochrome b6 protein into the more compact structure found in the native cytochrome protein. Heme staining experiments suggested that the two hemes in the reconstituted cytochrome b6 protein are bound with different affinities. The reconstituted cytochrome b6 protein was cleaved by Xa factor proteolysis from fusion protein and separated for characterization. The procedure presented in this work for reconstitution of hemes into the cytochrome b6 protein should provide an important tool for structure/function studies of membrane-bound cytochrome proteins.     

Vitamin K-dependent carboxylation. In vitro modification of synthetic peptides containing the gamma-carboxylation recognition site

Synthetic peptides including the gamma-carboxylation recognition site and acidic amino acids were compared as substrates for vitamin K-dependent gamma-carboxylation by bovine liver carboxylase. The 28-residue proPT28 (proprothrombin -18 to +10) and proFIX28 (pro-Factor IX -18 to +10) were carboxylated with a Km of 3 microM. The Vmax of proPT28 was 2-3 times greater than that of proFIX28. An analog of proFIX28 that contained the prothrombin propeptide had a Vmax 2-3-fold greater than an analog of proPT28 that contained the Factor IX propeptide. proFIX28/RS-1, based upon Factor IX Cambridge, proFIX28/RQ-4, based upon Factor IX Oxford 3, and proFIX28 had equivalent Km and Vmax values. Analogs of proPT28 containing Ala6-Glu7 or Glu6-Ala7 were carboxylated at equivalent rates. A peptide containing Asp6-Asp7 was carboxylated at a rate of about 1% of that of Glu carboxylation. Carboxylation of peptides containing Asp6-Glu7 and Glu6-Asp7 yielded results identical with peptides containing Ala6-Glu7 and Glu6-Ala7. Carboxymethylcysteine was not carboxylated when substituted for Glu6 in a peptide containing Asp7. These results indicate that the prothrombin propeptide is more efficient in the carboxylation process than is the Factor IX propeptide, but that both propeptides direct carboxylation; the gamma-carboxylation recognition site does not include residues -4 and -1; aspartic acid and carboxymethylcysteine are poor substrates for the carboxylase, but aspartic acid does not inhibit the carboxylation of adjacent glutamic acids.