Identification of protein side chains near the membrane-aqueous interface: a site-directed spin labeling study of KcsA.

This study presents an approach to identifying surface residues on membrane proteins that are exposed toward the membrane-aqueous interface. The method employs a lipid Ni(II) chelate that localizes the metal ion to a region near the membrane-aqueous interface. Lateral diffusion of the lipid chelate results in Heisenberg exchange (HE) with nitroxide side chains in the protein only if direct contact occurs between the paramagnetic species during a collision. Thus, HE serves as a signature for residues facing the bilayer in the neighborhood of the membrane-aqueous interface. To evaluate the method, 13 surface residues on the extracellular half of KcsA, a prokaryotic potassium channel of known structure, were examined for HE with the Ni(II) chelate. The HE rate between the two species is found to depend strongly on the vertical position of the nitroxide with respect to the membrane-aqueous interface. Nitroxides introduced near the interface experience relatively high HE rates, whereas nitroxides that are immersed in the bilayer interior or sterically sheltered from collision experience low or undetectable rates. The results indicate that residues near the interface can be identified on the basis of their high rates of collision with the headgroup region of the bilayer.     

Effect of adrenalin, insulin and contractions on the content of the free fatty acid fraction in skeletal muscle.

The fraction of free fatty acids (FFA) is present in skeletal muscles. However, there is almost no data regarding regulation in the content of this intramuscular lipid pool. We took advantage of the isolated muscle preparation to examine whether: a) increasing exogenous concentration of FFA (500microM or 700microM, 30min) b) insulin (10.00 I.U./L, 30min), c) adrenalin (4.4 nM, 30 min), or d) contractions (200ms, tetani, 1Hz, 30min), affect the FFA content inside myocytes. Incubation of soleus (S) and extensor digitorum longus (EDL) with increasing concentrations of exogenous FFA (from 500microM to 700microM) resulted in an increase in the total FFA fraction in both muscles studied (by 280.2% and 259.1%, respectively). In contracting muscles FFA pool was significantly reduced both in S (by 73.1%) and in EDL (by 31.1%). Neither stimulation by adrenalin nor insulin affected the total content of FFA fraction in the muscles examined. We conclude that a) increased availability of exogenous FFA at the sarcolemma level results in an increase in the size of intramuscular FFA fraction b) the intracellular FFA fraction is utilized by contracting muscles with regard to the fiber composition and to a greater extent in more oxidative muscles, c) FFA fraction remains stable upon stimulation by insulin or adrenalin.     

Rioarribasaurus,a new name for a Late Triassic dinosaur from New Mexico (USA)

The Late-Triassic-dinosaur generic nameCoelophysis Cope 1889 (type species C.bauri [Cope 1887]) is a nomen dubium because the lectotype of C.bauri, AMNH 2722, is four sacral vertebrae and a pubic process of the ilium that are not diagnostic. Dinosaur specimens from the famous Whitaker (“Coelophysis”) quarry in the Rock Point Member of the Chinle Formation at Ghost Ranch, New Mexico thus lack a valid name. We create a new genus and species name,Rioarribasaurus colberti, for these specimens.     

Rapid acquisition of three-dimensional triple-resonance experiments using pulsed field gradient techniques

Summary A rapid method for recording three-dimensional triple-resonance experiments utilising pulsed field gradient techniques is proposed, and applied to the HNCO experiment. In order to optimise the sensitivity of the method, a short phase cycle is used in conjunction with the pulsed field gradients to select the desired coherence transfer pathway. The method is demonstrated for the HU protein.     

Isolation of brush-border membranes from rat and rabbit colonocytes: Is alkaline phosphatase a marker enzyme?

Summary A method for the isolation of brush-border membranes of large intestinal epithelial cells was developed, which is based on the purification of intact brush-border caps by Percoll^® density-gradient centrifugation followed by separation of the vesiculated brush-border membranes on sucrose gradients. The procedure has two major advantages in comparison to known methods: 1) its first step does not depend on the determination of marker enzymes and 2) the method is applicable to rats as well as rabbits without major modifications. Due to the lack of an accepted marker for the colonic brush-border membrane the validity of the isolation procedure was tested by its application to the small intestine. Rat small intestinal brush-border membranes were enriched 21-fold when compared to the homogenate. The method was used to evaluate alkaline phosphatase as a marker enzyme for the colonic brush-border membrane. The results suggest that alkaline phosphatase is not exclusively localized in the brush-border membrane since this enzyme was also associated with membranes having different physical properties.     

Extracellular lipase from Pseudomonas aeruginosa is an amphiphilic protein.

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) secreted by Pseudomonas aeruginosa PAC1R was purified from cell-free growth medium by preparative isoelectric focusing. After blotting the N-terminal amino acid sequence and the amino acid composition were determined and compared to P. fragi and P. cepacia lipases yielding significant homology between all three species. Additionally, a consensus sequence K-Y-P-i-v-l-V-H-G was identified residing at the N-terminus of Pseudomonas lipases and in the central part of Staphylococcus lipases. Treatment of lipase with the serine-specific inhibitor diethyl p-nitrophenyl phosphate caused a rapid and complete inhibition of enzyme activity indicating the presence of a serine at the catalytic site as expected from lipase consensus sequences. Upon charge-shift electrophoresis the electrophoretic mobility of purified lipase was shifted either anodally or cathodally in the presence of sodium deoxycholate and cetyltrimethylammoniumbromide, respectively. This result demonstrates that extracellular lipase of P. aeruginosa exhibits an amphiphilic character like intrinsic membrane proteins.