Simultaneous coupling of alpha 2-adrenergic receptors to two G-proteins with opposing effects. Subtype-selective coupling of alpha 2C10, alpha 2C4, and alpha 2C2 adrenergic receptors to Gi and Gs.

Coupling of the three alpha 2-adrenergic receptor (alpha 2AR) subtypes to Gi and Gs was studied in membranes from transfected CHO cells. We observed that in the presence of low concentrations of the alpha 2AR agonist UK-14304, alpha 2C10 mediated inhibition of adenylyl cyclase activity, whereas at high concentrations of agonist, alpha 2C10 mediated stimulation of adenylyl cyclase activity. We considered that this biphasic response was due to the coupling of alpha 2C10 to both Gi and Gs. To isolate functional Gs and Gi coupling, cells were treated with pertussis toxin or cholera toxin in doses sufficient to fully ADP-ribosylate the respective G-proteins. Following treatment with cholera toxin, agonists elicited only alpha 2C10-mediated inhibition (approximately 50%) of adenylyl cyclase while after pertussis toxin treatment, agonists elicited only alpha 2C10-mediated stimulation (approximately 60%) of adenylyl cyclase. Incubation of membranes with antisera directed against the carboxyl-terminal portion of Gs alpha blocked this functional alpha 2AR.Gs coupling to the same extent as that found for beta 2AR.Gs coupling. In addition to functional Gs coupling, we also verified direct, agonist-dependent, physical coupling of alpha 2AR to Gs alpha. In agonist-treated membranes, an agonist-receptor-Gs alpha complex was immunoprecipitated with a specific alpha 2C10 antibody, and the Gs component identified by both western blots using Gs alpha antibody, and cholera toxin mediated ADP-ribosylation. Due to the differences in primary amino acid structure in a number of regions of the alpha 2AR subtypes, we investigated whether G-protein coupling was subtype-selective, using UK-14304 and cells with the same alpha 2AR expression levels (approximately 5 pmol/mg). Coupling to Gi was equivalent for alpha 2C10, alpha 2C4, and alpha 2C2: 53.4 +/- 8.8% versus 54.9 +/- 1.0% versus 47.6 +/- 3.5% inhibition of adenylyl cyclase, respectively. In marked contrast, distinct differences in coupling to Gs were found between the three alpha 2AR subtypes: stimulation of adenylyl cyclase was 57.9 +/- 6.3% versus 30.7 +/- 1.1% versus 21.8 +/- 1.7% for alpha 2C10, alpha 2C4, and alpha 2C2, respectively. Thus, alpha 2AR have the potential to couple physically and functionally to both Gi and Gs; for Gi coupling we found a rank order of alpha 2C10 = alpha 2C4 = alpha 2C2, while for Gs coupling, alpha 2C10 greater than alpha 2C4 greater than alpha 2C2.     

Purification and Characterization of an Autolysin from Clostridium acetobutylicum

A proteinaceous substance with antibiotic-like activity, resembling that of a bacteriocin, was isolated from an industrial-scale acetone-butanol fermentation of Clostridium acetobutylicum. The substance, purified by acetone precipitation, diethylaminoethyl cellulose chromatography, and polyacrylamide gel electrophoresis, was characterized as a glycoprotein with a molecular weight of 28,000. The glycoprotein was partially inactivated by certain protease enzymes. It had no effect on deoxyribonucleic acid, ribonucleic acid, or protein synthesis, and it did not result in the loss of intracellular adenosine triphosphate. The glycoprotein lysed sodium dodecyl sulfate-treated cells and cell wall preparations, and therefore it is referred to as an autolysin. The autolysin gene appeared to be chromosomal since plasmid deoxyribonucleic acid was not detected in the C. acetobutylicum strain.     

Molecular weight and amino acid composition of purified spinach beet phenolase

The purification of spinach beet phenolase has been modified to include equilibration of the crude macerate with 0.5% cetyl-trimethylammonium bromide a     

《基因Ⅺ》

<正>出版日期:2014年5月定价:398.00元字数:180万内容简介分子生物学是一门发展迅速的学科,其知识更新之快及前沿的蓬勃发展,对人类生活产生了持久而深入的影响。Lewin的《基因》自问世以来,一     

Mutations in the chloroplast inner envelope protein TIC100 impair and repair chloroplast protein import and impact retrograde signaling

Chloroplast biogenesis requires synthesis of proteins in the nucleocytoplasm and the chloroplast itself. Nucleus-encoded chloroplast proteins are imported via multiprotein translocons in the organelle’s envelope membranes. Controversy exists around whether a 1-MDa complex comprising TIC20, TIC100, and other proteins constitutes the inner membrane TIC translocon. The Arabidopsis thaliana cue8 virescent mutant is broadly defective in plastid development. We identify CUE8 as TIC100. The tic100^cue8 mutant accumulates reduced levels of 1-MDa complex components and exhibits reduced import of two nucleus-encoded chloroplast proteins of different import profiles. A search for suppressors of tic100^cue8 identified a second mutation within the same gene, tic100^soh1, which rescues the visible, 1 MDa complex-subunit abundance, and chloroplast protein import phenotypes. tic100^soh1 retains but rapidly exits virescence and rescues the synthetic lethality of tic100^cue8 when retrograde signaling is impaired by a mutation in the GENOMES UNCOUPLED 1 gene. Alongside the strong virescence, changes in RNA editing and the presence of unimported precursor proteins show that a strong signaling response is triggered when TIC100 function is altered. Our results are consistent with a role for TIC100, and by extension the 1-MDa complex, in the chloroplast import of photosynthetic and nonphotosynthetic proteins, a process which initiates retrograde signaling.

Complementary mutations in TIC100 of the chloroplast inner envelope membrane cause reductions or corrective improvements in chloroplast protein import, and highlight a signaling role.

IN A NUTSHELLBackground: Plants harvest energy from the sun and CO₂ from the air and convert them into the energy-rich molecules they, and eventually us, are made of. Plants do this, photosynthesis, in bodies called chloroplasts inside their cells. Chloroplasts, made of protein and membrane material, were, before plants evolved, free-living bacteria, but the synthesis of most of their proteins occurs outside them, using information carried by the cell’s nuclear DNA, so most proteins have to be brought into developing chloroplasts, across the double membrane surrounding them, through dedicated, selective channels, formed by TOC (outer) and TIC (inner envelope) proteins. The identity of those channels matters as it helps determine versions of chloroplasts suited for particular environments. Which TIC proteins constitute the inner envelope channel has been a matter of controversy.Question: A mutant Arabidopsis plant called cue8 is slow-to-green (young leaves begin almost white) and shows delayed chloroplast and plant development. We looked for the molecular identity of the CUE8 gene. We also caused further mutations in this mutant and searched whether any corrected the defects in cue8.Findings: We found the mutated gene causing the cue8 defects is the TIC100 gene. This is one essential component of the “TIC 1-MDa complex,” one of the two versions of the TIC import complex under debate. That complex is made of several proteins, all present at reduced levels in cue8. In laboratory assays in which proteins are imported into isolated chloroplasts, cue8 performed worse than normal plants for a photosynthetic and a housekeeping chloroplast protein. A corrective, “suppressor” mutant was identified, and it carried a second mutation in TIC100, one physically complementary to the first one. Both the single and the double (suppressed) mutant still were slow-to-green, which evidences a signaling role for import defects to the nucleus, making photosynthetic genes active or not.Next steps: Surprisingly the grasses, including the cereals, have one core protein of the TIC 1 MDa complex but not the rest (including TIC100). We don’t know how their TIC channels operate. We also need to learn how the information on the defect in protein import, which occurs at the chloroplast envelope, is relayed to the cell’s nucleus (but we do have some clues).     

Hormone-regulated expression and distribution of versican in mouse uterine tissues

Background  

Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination.