Prediction of β-Turns

Kohonen's self-organization model, a neural network model, is applied to predict the -turns in proteins. There are 455 -turn tetrapeptides and 3807 non--turn tetrapeptides in the training database. The rates of correct prediction for the 110 -turn tetrapeptides and 30,229 non--turn tetrapeptides in the testing database are 81.8% and 90.7%, respectively. The high quality of prediction of neural network model implies that the residue-coupled effect along a polypeptide chain is important for the formation of reversal turns, such as -turns, during the process of protein folding.     

Transition-metal complexes as alternatives to proteolytic enzymes. Regioselective cleavage of myoglobin by palladium(II) aqua complexes

 The palladium(II) aqua complexes [Pd(H₂O)₄]²⁺, cis-[Pd(en)(H₂O)₂]²⁺, and cis-[Pd(dtco-OH)(H₂O)₂]²⁺ effect hydrolytic cleavage of horse myoglobin in aqueous solution. The conditions were optimized with the third complex. Its structure was determined by X-ray crystallographic analysis of its precursor, the square-planar complex cis-[Pd(dtco-OH)Cl₂], in which the chelating ligand adopts a boat-chair conformation. A weak interaction between the hydroxyl group and the palladium(II) atom seems to improve the stability of the reagent. The yield of cleavage after a 24-h incubation at 60  °C increases from 39% to 85% as the pH decreases from 6.2 to 3.2. The protein fragments are separated by SDS-PAGE electrophoresis and HPLC separation methods, and identified by ESIMS and MALDI-TOF mass spectrometric methods and by determination of terminal amino-acid sequences. Most of the 13 cleavage sites are clustered around the methionine, arginine, and some of the histidine residues, whose side chains can bind to palladium(II). Cleavage tends to occur at the peptide bonds one to three positions removed from the binding residues; the scissile bonds usually lie on the amino-terminal side, seldom on the carboxy-terminal side, of the binding residues. Removal of the heme and unfolding of the protein do not drastically alter the pattern of cleavage. The ability of palladium(II) aqua complexes to cleave proteins at relatively few sites, with explicable selectivity, with good to very good yield, and in weakly acidic and nearly neutral solutions, bodes well for their future use in biochemical and bioanalytical practice. Received: 23 December 1997 / Accepted: 14 April 1998     

Electrical currents generated by a partially purified Na/Ca exchanger from lobster muscle reconstituted into liposomes and adsorbed on black lipid membranes: Activation by photolysis of caged Ca2+

The Na/Ca exchanger from lobster muscle crossreacts specifically with antibodies raised against the dog heart Na/Ca exchanger. Immunoblots of the lobster muscle and mammalian heart exchangers, following SDS-PAGE, indicate that the invertebrate and mammalian exchangers have similar molecular weights: about 120 kDa. The exchanger from lobster muscle was partially purified and functionally reconstituted into asolectin vesicles which were loaded with 160 mm NaCl. ⁴⁵Ca uptake by these proteoliposomes was promoted by replacing 160 mm NaCl in the external medium with 160 mm KCl to produce an outwardly-directed Na⁺ concentration gradient. When the proteoliposomes were adsorbed onto black lipid membranes (BLM), and DMNitrophen-Ca²⁺ (caged Ca²⁺) was added to the KCl medium, photolytically-evoked Ca²⁺ concentration jumps elicited transient electric currents. These currents corresponded to positive charge exiting from the proteoliposomes, and were consistent with the Na/Ca exchanger-mediated exit of 3 Na⁺ in exchange for 1 entering Ca²⁺. The current was dependent upon the Ca²⁺ concentration jump, the protein integrity, and the outwardly directed Na⁺ gradient. KCl-loaded proteoliposomes did not produce any current. Low external Na⁺ concentrations augmented the current, whereas Na⁺ concentrations >25 mM reduced the current. The dependence of the current on free Ca²⁺ was Michaelis-Menten-like, with halfmaximal activation (K_M(Ca)) at <10 m Ca²⁺. Caged Sr²⁺ and Ba²⁺, but not Mg²⁺, also supported photolysisevoked outward current, as did Ni²⁺, but not Mn²⁺. However, Mg²⁺ and Mn²⁺ augmented the Cadependent current, perhaps by facilitating the adsorption of proteoliposomes to the BLM. The Ca-dependent current was irreversibly blocked by La³⁺ (added as 200 m DMN-La³⁺). The results indicate that the properties of the Na/Ca exchanger can be studied with these electrophysiological methods.The technical assistance of Verena Heiselpetz in some experiments is gratefully acknowledged. This work was partly supported by the Deutsche Forschungsgemeinschaft (SFB 169) and by National Institutes of Health grants HL30315 and GM39500 to JHK and HL45215 and NS16106 to MPB. MPB was the recipient of a Senior Scientist Award from the Alexander von Humboldt Stiftung.     

Metal ion binding affinities of gastrin and CCK in membrane mimetic environments

The fully active gastrin and CCK analogues [Nle¹⁵]-gastrin- 17 and [Nle, Thr]-CCK-9 were analysed for their Ca²⁺ and Tb³⁺ affinities in various membrane mimetic conditions. In TFE both gastrin and CCK exhibited high affinities for calcium and terbium. At saturation level identical metal ion/peptide ratios were determined with Ca²⁺ and Tb³⁺, i.e. R = 3 for gastrin and R = 1 for CCK, confirming the very similar coordination properties of the two metal ions. The conformational effects of both metal ions were found to be very similar with a disordering effect in the case of gastrin and a conformational transition to β-turn type structure in the case of CCK. In order to mimic more properly physiological conditions, similar experiments were performed in the prsence of phospholipid bilayers. No interaction of the peptides with the bilayers was observed even in the presence of phospholipid bilayers. No interaction of the peptides with the bilayers was observed even in the presence of mmolar Ca²⁺ concentrations. Induced lipid interaction via N-terminal lipodervatization of gastrin and CCK allowed to translocate quantitatively the two hormones into phospholipid bilayers and to examine the effect of extravesicular Ca²⁺ on the conformation of the peptide headgroups and on their display at the water/lipid interphase. The CCK moiety of the lipo-CCK inserted into phospholipid bilayers interacts with the lipid phase and addition of Ca²⁺ enhances the clustering of the peptide headgroups in a more β-sheet type conformation. Conversely, insertion of lipo-gastrin into the bilayers leads to full exposure of the gastrin headgroup to the bulk water in predominantly random coil structure. Again Ca²⁺ provokes aggregation. As the lipo-peptide/phospholipid system still represents only an artificial model, it remains hazardous to derive a biological relevance from these data. The significantly higher affinity of lanthanide ions than Ca²⁺ for the peptides could well play a role in the inhibibitory activity of lanthanum on the signal transduction of the CCK family of hormones.     

Immunological studies of an artificial antigen with specificity of a common polysaccharide antigen of Pseudomonas aeruginosa

Abstract Synthetic d -rhamnan, with the structure of Pseudomonas aeruginosa common polysaccharide antigen (CPA), was conjugated with BSA. The artificial antigen obtained, and the natural antigens, lipopolysaccharides (LPS) of P. aeruginosa and Pseudomonas cerasi with rhamnan chains of the same structure, were studied by ELISA with rabbit antibodies to the d -rhamnan-BSA conjugate and to the P. cerasi O-antigen. Immunological relations between the LPS of P. aeruginosa and P. cerasi determined by CPA as well as between these LPS and d -rhamnan-BSA were revealed by ELISA. O-antiserum to P. cerasi possesses protective activity in the mouse passive protection test when mice are challenged with some P. aeruginosa strains; the antiserum to the d -rhamnan-BSA does not possess protective activity in mice.     

Resolution of partition coefficients in the transverse plane of the lipid bilayer

The distribution of a small lipid soluble molecule across a lipid bilayer has been determined using fluorescence quenching techniques. The neutral form of the amine, N,N-dimethylaniline (DMA) quenches the fluorescence of a series of n-(9-anthroyloxy) fatty acids (n = 2,6,9,12,16) which place a fluorophore at a graded series of positions from the surface to the centre of the lipid bilayer. A method is described for determining the partition coefficient of a quencher at each transverse position. The results show that DMA is located at all depths within the bilayer leaflet but that it is concentrated at the bilayer centre and to a lesser extent at the bilayer surface.     

Sodium-dependent succinate uptake in purple bacterium Ectothiorhodospira shaposhnikovii

Succinate, malate and fumarate uptake in purple sulfur bacterium Ectothiorhodospira shaposhnikovii, strain 1 K MSU, obligatorily depends on the presence of Na⁺. Other monovalent cations such as K⁺, Li⁺, NH₄⁺ could not replace Na⁺. Experiments with energy-depleted cells have shown that succinate uptake against its concentration gradient can be energized by artificially imposed sodium gradients (ΔpNa).An artificial membrane potential (inside negative) inhibited ΔpNa-driven succinate uptake at pH 7.0 but stimulated it at pH 9.0.The results confirm the suggestion that succinate uptake in E. shaposhnikovii is carried out in symport with Na⁺.     

Determination of the fertilization rate of A.I. sires

The ovarian cycles of 160 beef heifers were controlled with two injections of PGF_2α and superovulation was induced by 2000 IU PMSG, administered 48 h prior to the second PGF_2α treatment. Heifers were inseminated with fresh or frozen semen from selected bulls that demonstrated consistent NR's above (n=5) or below (n=3) the stud average. Sixty-five percent of ovulated ova were recovered from the responding heifers. The mean fertilization rates for the above and below average fertility groups in this study were 89.0% (355 ova) and 69.7% (155 ova) respectively. This pilot study indicated the possibility of detecting differences in the fertilization rate between bulls using superovulated females to obtain large quantities of ova from relatively few experimental animals. Further research with more bulls would be required to accurately determine the correlation between fertilization rate and nonreturn rate but the results suggest the procedure may have application for selection of potential AI sires with above average fertility.     

Ionic permeation of lipid bilayer membranes mediated by a neutral,noncyclic Li+-selective carrier having imide and ether ligands. I. Selectivity among monovalent cations

Summary We have found that Simon's neutral, noncyclic, Li⁺-selective complexone, which has imide and ether ligands, renders lipid bilayer membranes selectively permeable to certain cations and anions. The present paper characterizes the ability of this molecule to carry monovalent cations; and we show it to be most selective for Li⁺ among the alkali cations, the first reconstitution of Li⁺-selective permeation in lipid bilayer membranes. This complexone acts as an equilibrium-domain carrier for Ag⁺> Li⁺>Tl⁺>Na⁺>NH ₄ ⁺ >Rb⁺>Cs⁺ over a wide range of experimental conditions. The major type of membrane-permeating species formed is a 21 carrier/cation complex dominant except at the lowest salt and carrier concentrations where a 11 carrier/cation, with a similar selectivity sequence, can be detected. Among the groupIa cations the selectivity sequence in bilayers, Li⁺>Na⁺>K⁺>Rb⁺>Cs⁺, is similar to that previously found for this molecule in thick solvent-polymer membrane electrodes. We find this carrier to be more selective to Ag⁺ than to any other monovalent cation yet studied. This high Ag⁺ selectivity is used, together with the dependence of the selectivity on the nature of the N-amide substitutents, to argue that the imide oxygens play a major role as ligands.     

Protozoan species accrual on artificial islands in differing lentic and wetland systems

In the recently broadened context of insular biogeographic theory, artificial substrates might well be considered as initially barren islands with the surrounding natural community acting as a source pool of potential colonists. In an attempt to relate protozoan communities forming on polyurethane foam (PF) substrates to their environments, a series of field experiments were performed in several aquatic and semi-aquatic (wetlands) systems in Michigan, Virginia, and Colorado. Results were considered in the context of island colonization theory.Given relatively stable environmental conditions and sufficient time to colonize, protozoa formed highly replicable communities on PF substrates; MacArthur & Wilson's equation for noninteractive island colonization, S = Seq ·(l-e^–Gt ), accuratelt described the process of species accrual in most cases. Sites were compared using non-linear regression estimates of the parameter, G, which reflects the rate at which substrates attained equilibrium species numbers. In general, the rate of equilibrium acquisition reflected differences in productivity and structure between the protozoan communities from the various habitats and systems studied.