Pr-specific phytochrome phosphorylation in vitro by a protein kinase present in anti-phytochrome maize immunoprecipitates

Protein kinase activity has repeatedly been found to co-purify with the plant photoreceptor phytochrome, suggesting that light signals received by phytochrome may be transduced or modulated through protein phosphorylation. In this study immunoprecipitation techniques were used to characterize protein kinase activity associated with phytochrome from maize (Zea mays L.). A protein kinase that specifically phosphorylated phytochrome was present in washed anti-phytochrome immunoprecipitates of etiolated coleoptile proteins. No other substrate tested was phosphorylated by this kinase. Adding salts or detergents to disrupt low-affinity protein interactions reduced background phosphorylation in immunoprecipitates without affecting phytochrome phosphorylation, indicating that the protein kinase catalytic activity is either intrinsic to the phytochrome molecule or associated with it by high-affinity interactions. Red irradiation (of coleoptiles or extracts) sufficient to approach photoconversion saturation reduced phosphorylation of immunoprecipitated phytochrome. Subsequent far-red irradiation reversed the red-light effect. Phytochrome phosphorylation was stimulated about 10-fold by a co-immunoprecipitated factor. The stimulatory factor was highest in immunoprecipitates when Mg2+ was present in immunoprecipitation reactions but remained in the supernatant in the absence of Mg2+. These observations provide strong support for the hypothesis that phytochrome-associated protein kinase modulates light responses in vivo. Since only phytochrome was found to be phosphorylated, the co-immunoprecipitated protein kinase may function to regulate receptor activity.     

Identification and isoprenylation of plant GTP-binding proteins

To identify isoprenylated plant GTP-binding proteins,Arabidopsis thaliana andNicotiana tabacum cDNA expression libraries were screened for cDNA-encoded proteins capable of binding [³²P]GTPin vitro. ATGB2, anArabidopsis homologue of the GTP-binding protein Rab2, was found to bind GTPin vitro and to be a substrate for a geranylgeranyl:protein transferase (GGTase) present in plant extracts. The carboxyl terminus of this protein contains a-GCCG sequence, which has not previously been shown to be recognized by any prenyl:protein transferase (PTase), but which most closely resembles that isoprenylated by the type II GGTase (-XXCC,-XCXC, or-CCXX).In vitro geranylgeranylation of anArabidopsis Rab1 protein containing a carboxyl-terminal-CCGQ sequence contirmed the presence of a type II GGTase-like activity in plant extracts. Several other proteins were also identified byin vitro GTP binding, includingArabidopsis and tobacco homologues of Rab11, ARF (ADP-ribosylation factor) and Sar proteins, as well as a novel 22 kDaArabidopsis protein (ATG81). This 22 kDa protein had consensus GTP-binding motifs and bound GTP with high specificity, but its structure was not closely related to that of any known GTP-binding protein (it most resembled proteins within the ARF/Sar and G protein -subunit superfamilies).     

PHA and endotoxin stimulation of human lymphocytes separated by albumin density gradient centrifugation.

Venous blood from eight healthy subjects was divided into four fractions on a discontinuous albumin density gradient. The percentage recovery of lymphocytes was 82.3%; the purity of the lymphocyte fractions was 83.6%. The lymphocytes were cultured with PHA and Endotoxin, and the samples were analysed after 24, 48, and 72 hours. After PHA stimulation immunoblasts appeared up to 59.3% in the cultures from the 19-21% albumin fraction. After Endotoxin stimulation the maximum (75.8%) was reached in the heavy (25-27% albumin) fraction. Thus, it is concluded that the lymphocytes which can be stimulated with both the mitogens have different densities, the PHA-stimulable T lymphocytes being ligther than the Endotoxin-stimulable B lymphocytes. It is also concluded that as a mitogen Endotoxin is equal to PHA.     

Elucidation of active site residues of Arabidopsis thaliana flavonol synthase provides a molecular platform for engineering flavonols

Arabidopsis thaliana flavonol synthase (aFLS) catalyzes the production of quercetin, which is known to possess multiple medicinal properties. aFLS is classified as a 2-oxoglutarate dependent dioxygenase as it requires ferrous iron and 2-oxoglutarate for catalysis. In this study, the putative residues for binding ferrous iron (H221, D223 and H277), 2-oxoglutarate (R287 and S289) and dihydroquercetin (H132, F134, K202, F293 and E295) were identified via computational analyses. To verify the proposed roles of the identified residues, 15 aFLS mutants were constructed and their activities were examined via a spectroscopic assay designed in this study. Mutations at H221, D223, H277 and R287 completely abolished enzymes activities, supporting their importance in binding ferrous iron and 2-oxoglutarate. However, mutations at the proposed substrate binding residues affected the enzyme catalysis differently such that the activities of K202 and F293 mutants drastically decreased to approximately 10% of the wild-type whereas the H132F mutant exhibited approximately 20% higher activity than the wild-type. Kinetic analyses established an improved substrate binding affinity in H132F mutant (Km: 0.027+/-0.0028 mM) compared to wild-type (Km: 0.059+/-0.0063 mM). These observations support the notion that aFLS can be selectively mutated to improve the catalytic activity of the enzyme for quercetin production.     

Structural comparison of crystal and solution states of the 138 kDa complex of methylamine dehydrogenase and amicyanin from Paracoccus versutus

Methylamine can be used as the sole carbon source of certain methylotrophic bacteria. Methylamine dehydrogenase catalyzes the conversion of methylamine into formaldehyde and donates electrons to the electron transfer protein amicyanin. The crystal structure of the complex of methylamine dehydrogenase and amicyanin from Paracoccus versutus has been determined, and the rate of electron transfer from the tryptophan tryptophylquinone cofactor of methylamine dehydrogenase to the copper ion of amicyanin in solution has been determined. In the presence of monovalent ions, the rate of electron transfer from the methylamine-reduced TTQ is much higher than in their absence. In general, the kinetics are similar to those observed for the system from Paracoccus denitrificans. The complex in solution has been studied using nuclear magnetic resonance. Signals of perdeuterated, (15)N-enriched amicyanin bound to methylamine dehydrogenase are observed. Chemical shift perturbation analysis indicates that the dissociation rate constant is approximately 250 s(-1) and that amicyanin assumes a well-defined position in the complex in solution. The most affected residues are in the interface observed in the crystal structure, whereas smaller chemical shift changes extend to deep inside the protein. These perturbations can be correlated to small differences in the hydrogen bond network observed in the crystal structures of free and bound amicyanin. This study indicates that chemical shift changes can be used as reliable indicators of subtle structural changes even in a complex larger than 100 kDa.     

Carbohydrate‐based species recognition in sea urchin fertilization: another avenue for speciation?

Spawning marine invertebrates are excellent models for studying fertilization and reproductive isolating mechanisms. To identify variation in the major steps in sea urchin gamete recognition, we studied sperm activation in three closely related sympatric Strongylocentrotus species. Sperm undergo acrosomal exocytosis upon contact with sulfated polysaccharides in the egg-jelly coat. This acrosome reaction exposes the protein bindin and is therefore a precondition for sperm binding to the egg. We found that sulfated carbohydrates from egg jelly induce the acrosome reaction species specifically in S. droebachiensis and S. pallidus. There appear to be no other significant barriers to interspecific fertilization between these two species. Other species pairs in the same genus acrosome react nonspecifically to egg jelly but exhibit species-specific sperm binding. We thus show that different cell-cell communication systems mediate mate recognition among very closely related species. By comparing sperm reactions to egg-jelly compounds from different species and genera, we identify the major structural feature of the polysaccharides required for the specific recognition by sperm: the position of the glycosidic bond of the sulfated alpha-L-fucans. We present here one of the few examples of highly specific pure-carbohydrate signal transduction. In this system, a structural change in a polysaccharide has far-reaching ecological and evolutionary consequences.