Das Anti-inv-l-Rie

Ohne ZusammenfassungDirektor: Prof. Dr. Dr. H. Baitsch Mit 1 TextabbildungMit Unterstützung durch die Deutsche Forschungsgemeinschaft.Die vorliegende Arbeit bildet einen wesentlichen Teil der Dissertation von E. Schmidtmann.     

Orientierende Untersuchungen über die Einwirkung von gasförmigem Formaldehyd auf die grüne Pflanze

Ohne Zusammenfassung     

Transition of rhodopsin into the active metarhodopsin II state opens a new light-induced pathway linked to Schiff base isomerization

Rhodopsin bears 11-cis-retinal covalently bound by a protonated Schiff base linkage. 11-cis/all-trans isomerization, induced by absorption of green light, leads to active metarhodopsin II, in which the Schiff base is intact but deprotonated. The subsequent metabolic retinoid cycle starts with Schiff base hydrolysis and release of photolyzed all-trans-retinal from the active site and ends with the uptake of fresh 11-cis-retinal. To probe chromophore-protein interaction in the active state, we have studied the effects of blue light absorption on metarhodopsin II using infrared and time-resolved UV-visible spectroscopy. A light-induced shortcut of the retinoid cycle, as it occurs in other retinal proteins, is not observed. The predominantly formed illumination product contains all-trans-retinal, although the spectra reflect Schiff base reprotonation and protein deactivation. By its kinetics of formation and decay, its low temperature photointermediates, and its interaction with transducin, this illumination product is identified as metarhodopsin III. This species is known to bind all-trans-retinal via a reprotonated Schiff base and forms normally in parallel to retinal release. We find that its generation by light absorption is only achieved when starting from active metarhodopsin II and is not found with any of its precursors, including metarhodopsin I. Based on the finding of others that metarhodopsin III binds retinal in all-trans-C(15)-syn configuration, we can now conclude that light-induced formation of metarhodopsin III operates by Schiff base isomerization ("second switch"). Our reaction model assumes steric hindrance of the retinal polyene chain in the active conformation, thus preventing central double bond isomerization.     

Embryonic and larval development of jundiá (Rhamdia quelen, Quoy and Gaimard, 1824, Pisces, TeleosteI), a South American catfish.

The jundiá (Rhamdia quelen, Quoy and Gaimard) is an endemic South American fish species. Because this species supports cold winters and grows faster during warm months, it has begun to be viewed as an ideal species for fish production in southern South America. In the present study, jundiá oocytes used were obtained by extrusion from females after hormone injection. Soon after hydration, the eggs were transferred to 50 L conic glass incubators, with constant and controlled water influx. Samples of fertilized eggs were transferred to Petri dishes and, examined under a stereoscopic microscope, were spherical, demersal, and non-adhesive with defined perivitelline space and resistant chorion. Cleavage stages occurred during the first 3.5 h. After hatching, larvae were transferred to 200 L glass fiber incubators. First signs of embryo movement were observed 21 h after fertilization; larval eclosion occurred 30.5 h after fertilization. Present findings may provide a basis for studies aimed at determining the complete ontogeny of jundiá and may be useful in eco-toxicological studies.     

Minor folding defects trigger local modification of glycoproteins by the ER folding sensor GT

UDP-glucose:glycoprotein glucosyltransferase (GT) is a key component of the glycoprotein-specific folding and quality control system in the endoplasmic reticulum. By exclusively reglucosylating incompletely folded and assembled glycoproteins, it serves as a folding sensor that prolongs the association of newly synthesized glycoproteins with the chaperone-like lectins calnexin and calreticulin. Here, we address the mechanism by which GT recognizes and labels its substrates. Using an improved inhibitor assay based on soluble conformers of pancreatic ribonuclease in its glycosylated (RNase B) and unglycosylated (RNase A) forms, we found that the protein moiety of a misfolded conformer alone is sufficient for specific recognition by GT in vitro. To investigate the relationship between recognition and glucosylation, we tested a variety of glycosylation mutants of RNase S-Protein and an RNase mutant with a local folding defect [RNase C65S, C72S], as well as a series of loop insertion mutants. The results indicated that local folding defects in an otherwise correctly folded domain could be recognized by GT. Only glycans attached to the polypeptide within the misfolded sites were glucosylated.     

Definition of the consensus motif recognized by gamma-adaptin ear domains

The heterotetrameric adaptor complex 1 (AP-1) and the monomeric Golgi-localized, gamma ear-containing, Arf-binding (GGA) proteins are components of clathrin coats associated with the trans-Golgi network and endosomes. The carboxyl-terminal ear domains (or gamma-adaptin ear (GAE) domains) of two gamma-adaptin subunit isoforms of AP-1 and of the GGAs are structurally similar and bind to a common set of accessory proteins. In this study, we have systematically defined a core tetrapeptide motif PsiG(P/D/E)(Psi/L/M) (where Psi is an aromatic residue), which is responsible for the interactions of accessory proteins with GAE domains. The definition of this motif has allowed us to identify novel GAE-binding partners named NECAP and aftiphilin, which also contain clathrin-binding motifs. These findings shed light on the mechanism of accessory protein recruitment to trans-Golgi network and endosomal clathrin coats.     

Regiospecific phosphohydrolases from Dictyostelium as tools for the chemoenzymatic synthesis of the enantiomers D-myo-inositol 1,2,4-trisphosphate and D-myo-inositol 2,3,6-trisphosphate: non-physiological, potential analogues of biologically active D-myo-inositol 1,3,4-trisphosphate

A new de novo synthesis of the enantiomeric pair D-myo-inositol 1,2,4-trisphosphate and D-myo-inositol 2,3,6-trisphosphate is described. Starting from enantiopure dibromocyclohexenediol, several C2 symmetrical building blocks were synthesized which gave access to D-myo-inositol 1,2,4,5-tetrakisphosphate and D-myo-inositol 1,2,3,6-tetrakisphosphate. Exploiting the high regiospecificity of two partially purified phosphohydrolases from Dictyostelium, a 5-phosphatase and a phytase, the inositol tetrakisphosphates were converted enzymatically to the target compounds. Their potential to modulate the activity of Ins3,4,5,6P4 1-kinase was investigated and compared with the effects of D-myo-inositol 1,3,4-trisphosphate.