Faculty Opinions recommendation of Cyanobacterial circadian pacemaker: Kai protein complex dynamics in the KaiC phosphorylation cycle in vitro.

SUMMARYThe expression of a protein complex designated gp15/400, previously identified via extrinsic iodination of adultBrugia malayi, was examined by labelling all stages found in the mammalian host and immunoprecipitation with a specific antibody raised to a recombinant protein. In this way, gp15/400 could be detected in L3, L4, adult worms and microfilariae recovered from jirds and labelled with Bolton–Hunter reagent. Metabolic labelling indicated that gp15/400 was released into culture medium when adult worms were maintainedin vitro, but at a rate slower than that of gp29, the major soluble cuticular glycoprotein. Immuno-electron microscopy showed that the protein complex was broadly distributed in different tissues, although it was not detectable in the cuticle of adult worms. Dense labelling was observed in the matrix of the basal laminae bordering the hypodermis, somatic musculature and oesophagus, and lower but significant labelling was seen in the cells overlying these extracellular matrices. Hybridization of genomic DNA with a cDNA probe encoding gp15/400 indicated that homologous genes were present inDirofilaria immitisandAcanthocheilonema viteae. The failure to detect related genes in non-filarial nematodes was presumed to be due to divergence beyond the practical limits of detection by nucleic acid probes, as antibody reagents showed that the protein cross-reacted immunologically with ABA-1, a major protein allergen from the body fluid ofAscaris.

Download Full-text

In Vitro Synthesis, Transport, and Assembly of the Constituent Polypeptides of the Light-Harvesting Chlorophyll a/b Protein Complex

Genome Organization and Expression in Plants ◽

10.1007/978-1-4613-3051-6_29 ◽

1980 ◽

pp. 337-351 ◽

Cited By ~ 26

Author(s):

Gregory W. Schmidt ◽

Sue Bartlett ◽

Arthur R. Grossman ◽

Anthony R. Cashmore ◽

Nam-Hai Chua

Keyword(s):

Chlorophyll A ◽

Protein Complex ◽

Light Harvesting ◽

In Vitro Synthesis

Download Full-text

Bacterial Microtubules Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis

10.1101/112987 ◽

2017 ◽

Author(s):

César Díaz-Celis ◽

Viviana I. Risca ◽

Felipe Hurtado ◽

Jessica K. Polka ◽

Scott D. Hansen ◽

...

Keyword(s):

Molecular Motors ◽

Intracellular Transport ◽

Complex Dynamics ◽

Critical Concentration ◽

Polarized Growth ◽

Gtp Hydrolysis ◽

Filament Dynamics ◽

Mixed Polarity ◽

Self Association

AbstractBacteria of the genusProsthecobacterexpress homologs of eukaryotic α-and β-tubulin, called BtubA and BtubB, that have been observed to assemble into bacterial microtubules (bMTs). ThebtubABgenes likely entered theProsthecobacterlineage via horizontal gene transfer and may derive from an early ancestor of the modern eukaryotic microtubule (MT). Previous biochemical studies revealed that BtubA/B polymerization is GTP-dependent and reversible and that BtubA/B folding does not require chaperones. To better understand bMT behavior and gain insight into the evolution of microtubule dynamics, we characterizedin vitrobMT assembly using a combination of polymerization kinetics assays, and microscopy. Like eukaryotic microtubules, bMTs exhibit polarized growth with different assembly rates at each end. GTP hydrolysis stimulated by bMT polymerization drives a stochastic mechanism of bMT disassembly that occurs via polymer breakage. We also observed treadmilling (continuous addition and loss of subunits at opposite ends) of bMT fragments. Unlike MTs, polymerization of bMTs requires KCl, which reduces the critical concentration for BtubA/B assembly and induces bMTs to form stable mixed-orientation bundles in the absence of any additional bMT-binding proteins. Our results suggest that at potassium concentrations resembling that inside the cytoplasm ofProsthecobacter, bMT stabilization through self-association may be a default behavior. The complex dynamics we observe in both stabilized and unstabilized bMTs may reflect common properties of an ancestral eukaryotic tubulin polymer.ImportanceMicrotubules are polymers within all eukaryotic cells that perform critical functions: they segregate chromosomes in cell division, organize intracellular transport by serving as tracks for molecular motors, and support the flagella that allow sperm to swim. These functions rely on microtubules remarkable range of tunable dynamic behaviors. Recently discovered bacterial microtubules composed of an evolutionarily related protein are evolved from a missing link in microtubule evolution, the ancestral eukaryotic tubulin polymer. Using microscopy and biochemical approaches to characterize bacterial microtubules, we observed that they exhibit complex and structurally polarized dynamic behavior like eukaryotic microtubules, but differ in how they self-associate into bundles and become destabilized. Our results demonstrate the diversity of mechanisms that microtubule-like filaments employ to promote filament dynamics and monomer turnover.

Download Full-text

Identification of Potential Plants Producing Tannin-protein Complex for a-amylase as Botanical Pesticide

Pelita Perkebunan (a Coffee and Cocoa Research Journal) ◽

10.22302/iccri.jur.pelitaperkebunan.v29i1.189 ◽

2013 ◽

Vol 29 (1) ◽

Author(s):

Asriyah Firdausi ◽

Tri Agus Siswoyo ◽

Soekadar Wiryadiputra

Keyword(s):

Protein Complex ◽

Protein Complexes ◽

Gliricidia Sepium ◽

In Vitro Test ◽

Betel Nut ◽

Botanical Pesticides ◽

Botanical Pesticide ◽

Areca Catechu ◽

Gnetum Gnemon

Research on the development of botanical pesticides should be developed through new methods, such as by inhibiting the activity of digestive enzymes by secondary metabolites. The aim of this study was to identify some of potential plants as a source of tannin-protein complexes to inhibitthe activity of - amylase. The study of identification of potential plants producing the active ingredient tannin-protein complex was divided into three stages, 1) identification of potential plants producing tannin, 2) isolation of tannin-protein complexes, and 3) in vitro test of tannin-protein complexes effect of the -amylase activity. Some of the observed plants were sidaguri leaf (Sida rhombifolia), melinjo leaf (Gnetum gnemon), gamal leaf (Gliricidia sepium),lamtoro leaf (Leucaena leucocephala) , betel nut (Areca catechu) , and crude gambier (Uncaria gambir) a s a source of tannins and melinjo seed was used asprotein source. Betel nut and melinjo seed were the best source of tannin-protein complex, tannin content 1.77 mg TAE/mL with antioxidant activity of 90%,the ability to inhibit the activity of -amylase by 95% with IC 50 values of 10 mg/mL.Key words: Tannin, protein, -amylase, botanical pesticides,Areca catechu, Gnetum gnemon.

Download Full-text

Disulfide Bond Formation at the C Termini of Vaccinia Virus A26 and A27 Proteins Does Not Require Viral Redox Enzymes and Suppresses Glycosaminoglycan-Mediated Cell Fusion

Journal of Virology ◽

10.1128/jvi.02295-08 ◽

2009 ◽

Vol 83 (13) ◽

pp. 6464-6476 ◽

Cited By ~ 18

Author(s):

Yao-Cheng Ching ◽

Che-Sheng Chung ◽

Cheng-Yen Huang ◽

Yu Hsia ◽

Yin-Liang Tang ◽

...

Keyword(s):

Vaccinia Virus ◽

Cell Fusion ◽

Disulfide Bond ◽

Protein Complex ◽

Disulfide Bonds ◽

Bond Formation ◽

In Vitro Mutagenesis ◽

Disulfide Bond Formation ◽

Infected Cells

ABSTRACT Vaccinia virus A26 protein is an envelope protein of the intracellular mature virus (IMV) of vaccinia virus. A mutant A26 protein with a truncation of the 74 C-terminal amino acids was expressed in infected cells but failed to be incorporated into IMV (W. L. Chiu, C. L. Lin, M. H. Yang, D. L. Tzou, and W. Chang, J. Virol 81:2149-2157, 2007). Here, we demonstrate that A27 protein formed a protein complex with the full-length form but not with the truncated form of A26 protein in infected cells as well as in IMV. The formation of the A26-A27 protein complex occurred prior to virion assembly and did not require another A27-binding protein, A17 protein, in the infected cells. A26 protein contains six cysteine residues, and in vitro mutagenesis showed that Cys441 and Cys442 mediated intermolecular disulfide bonds with Cys71 and Cys72 of viral A27 protein, whereas Cys43 and Cys342 mediated intramolecular disulfide bonds. A26 and A27 proteins formed disulfide-linked complexes in transfected 293T cells, showing that the intermolecular disulfide bond formation did not depend on viral redox pathways. Finally, using cell fusion from within and fusion from without, we demonstrate that cell surface glycosaminoglycan is important for virus-cell fusion and that A26 protein, by forming complexes with A27 protein, partially suppresses fusion.

Download Full-text

The relationship between in vivo generated hemoglobin skeletal protein complex and increased red cell membrane rigidity

Blood ◽

10.1182/blood.v71.5.1427.1427 ◽

1988 ◽

Vol 71 (5) ◽

pp. 1427-1431 ◽

Cited By ~ 44

Author(s):

N Fortier ◽

LM Snyder ◽

F Garver ◽

C Kiefer ◽

J McKenney ◽

...

Keyword(s):

Protein Complex ◽

Sodium Dodecyl ◽

Red Cells ◽

Red Cell ◽

Red Cell Membrane ◽

Cellular Dehydration ◽

Thalassemia Minor ◽

Membrane Rigidity

Abstract In vitro induced oxidative damage to normal human RBCs has previously been shown to result in increased membrane rigidity as a consequence of the generation of a protein complex between hemoglobin and spectrin. In order to determine if in vivo generated hemoglobin-spectrin complexes may play a role in increased membrane rigidity of certain pathologic red cells, we measured both these parameters in membranes prepared from hereditary xerocytosis (Hx), sickle cell disease (Sc), and red cells from thalassemia minor (beta thal). Membranes were prepared from density-fractionated red cells, and membrane deformability was measured using an ektacytometer. Hemoglobin-spectrin complex was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis, as well as by Western blot analysis using a monoclonal antibody against the beta- subunit of hemoglobin. For these three types of pathologic red cells, progressive cellular dehydration was associated with increased membrane rigidity and increased content of hemoglobin-spectrin complex. Moreover, the increase in membrane rigidity appeared to be directly related to the quantity of hemoglobin-spectrin complex associated with the membrane. Our findings imply that hemoglobin-spectrin complex is generated in vivo, and this in turn results in increased membrane rigidity of certain pathologic red cells. The data further suggest that oxidative crosslinking may play an important role in the pathophysiology of certain red cell disorders.

Download Full-text