Structure of the KcsA potassium channel from Streptomyces lividans: a site-directed spin labeling study of the second transmembrane segment.

KcsA is a prokaryotic potassium channel. The present study employs cysteine scanning mutagenesis and site-directed spin labeling to investigate the structure of the second transmembrane segment (residues 82-120) in functional tetrameric channels reconstituted in lipid bilayers. Spin-spin interactions are observed between nitroxide side chains at symmetry-related sites close to the 4-fold axis of symmetry. To aid in quantitative analysis of these interactions, a new diamagnetic analogue of the nitroxide side chain is used to prepare magnetically dilute samples with constant structure. Using constraints imposed by the spin-spin interactions, a packing model for this segment is deduced that is in excellent agreement with the recently reported crystal structure [Doyle, D., et al. (1998) Science 280, 69-77]. The relatively immobilized state of the nitroxide side chains suggests that the channel is rigid on the electron paramagnetic resonance time scale. Moreover, the poor sulfhydryl reactivity of the cysteine at many locations indicates that the channel is not subject to the low-frequency fluctuations that permit reaction of buried cysteines. At sites expected to be located in the pore, the accessibility of the side chains to collision with O(2) or nickel(II) ethylenediaminediacetate is low. This inaccessibility, together with the generally low mobility of the side chains throughout the sequence, makes it difficult to detect the presence of the pore based on these measurements. However, the presence of a solvated pore can be directly demonstrated using a polarity parameter deduced from the EPR spectra recorded at low temperature. These measurements also reveal the presence of a polarity gradient in the phospholipid bilayer.     

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.     

Fractionation and molecular-weight determination of large polypeptides by gel filtration in guanidiniun chloride

The interaction of several N-acetyl-d-glucosamine analogs and of sialyl lactose with the lectin wheat germ agglutinin was studied by nuclear magnetic resonance. N-²H₃-acetyl-d-gluocosamine was synthesized and found to displace the N-acetyl methyl signal toward its free chemical shift in N-acetylglucosamine and N-acetylneuraminic acid demonstrating common binding sites for the latter two compounds. The N-acetyl methyl signal of the α-methylglucoside of N-acetylglucosamine could be titrated but a 3-deoxy analog could not, the latter exhibiting very weak binding and demonstrating the importance of the 3-OH group in the binding process. Sialyl lactose (an N-acetylneuraminic acid analog) was rather tightly bound to the lectin. N-F₃-acetyl-d-glucosamine was synthesized and its binding to the lectin was studied at pH 4, 4.5, 5.1 by ¹⁹F NMR. The two anomers were found to bind with nearly equal K_d′s but exhibited a pH and anomer dependent Δ (total bound chemical shift). The -CF₃ analog was found to bind considerably stronger to the lectin than the -CH₃ compound. The clear resolution of the α and β anomers of this molecule make it a very useful probe of the lectin binding site.     

Plant Cell Wall Carbohydrates as Substrates for Azospirillum brasiliense

Carbohydrate components (simple sugars and polysaccharides) of cell walls of pearl millet (Pennisetum americanum L., cv. Gahi) were studied as potential substrates for the root-associated diazotroph Azospirillum brasiliense Sp. 7. Simple sugars were utilized, but no evidence was obtained to support the suggestion that the polysaccharide components tested might serve as substrates for growth following hydrolysis by the associated azospirilla.     

Potentiated lymphokine-activated killer cell activity generated by low-dose interleukin-2 and mismatched double-stranded RNA

Summary Lymphokine-activated killer (LAK) cell activity was measured in human peripheral blood mononuclear cells (PBMC) treated in vitro for 3 days with recombinant interleukin-2 (rIL-2) and mismatched double-stranded RNA (dsRNA). Lytic activity was measured utilizing K562 (NK-sensitive) and 786-0 (NK-resistant) target cells. PBMC cultured with rIL-2 (10–1000 BRMP U/ml) alone showed concentration-dependent lytic activity against the 786-0 target cells, while cells cultured in unsupplemented medium or medium supplemented with mismatched dsRNA (200 µg/ml) alone could not lyse the 786-0 targets. The combination of mismatched dsRNA with suboptimal concentrations of rIL-2 (10–30 U/ml) showed enhancement of both natural killer (NK) and LAK cell activities. The uptake of [³H]thymidine by treated effector cells was dependent on time and rIL-2 concentration and was not increased in the cells treated with low-dose rIL-2/mismatched dsRNA, compared to those treated with low-dose rIL-2 or mismatched dsRNA alone. Similarly, changes in the expression of CD3, CD4, CD8, CD57, CD16 and CD25 cell surface antigens were dependent on rIL-2 concentration and not altered by the presence of mismatched dsRNA. These results indicate that mismatched dsRNA can potentiate rIL-2-induced LAK cell activity by increasing the functional activity per cell, rather than by increasing the number of activated cells.     

The intrinsic pKa values for phosphatidylserine and phosphatidylethanolamine in phosphatidylcholine host bilayers.

Potentiometric titrations and surface potential measurements have been used to determine the intrinsic pKa values of both the carboxyl and amino groups of phosphatidylserine (PS) in mixed vesicles of PS and phosphatidylcholine (PC), and also of the amino group of phosphatidylethanolamine (PE) in mixed PE-PC vesicles. The pKa of the carboxyl group of PS in liposomes with different PS/PC lipid ratios measured by the two different methods is 3.6 +/- 0.1, and the pKa of its amino group is 9.8 +/- 0.1. The pKa of the amino group of PE in PE-PC vesicles, determined solely by surface potential measurements, is 9.6 +/- 0.1. These pKa values are independent of the aqueous phase ionic strength and of the effect of the liposome's surface potential due to the presence of these partially charged lipids.     

Expression of rat L-FABP in mouse fibroblasts: role in fat absorption

Fatty acid-binding proteins (FABP) are abundant cytosolic proteins whose level is responsive to nutritional, endocrine, and a variety of pathological states. Although FABPs have been investigatedin vitro for several decades, little is known of their physiological function. Liver L-FABP binds both fatty acids and cholesterol. Competitive binding analysis and molecular modeling studies of L-FABP indicate the presence of two ligand binding pockets that accomodate one fatty acid each. One fatty acid binding site is identical to the cholesterol binding site. To test whether these observations obtainedin vitro were physiologically relevant, the cDNA encoding L-FABP was transfected into L-cells, a cell line with very low endogenous FABP and sterol carrier proteins. Uptake of both ligands did not differ between control cells and low expression clones. In contrast, both fatty acid uptake and cholesterol uptake were stimulated in the high expression cells. In high expression cells, uptake of fluorescent cis-parinaric acid was enhanced more than that of trans-parinaric acid. This is consistent with the preferential binding of cis-fatty acids to L-FABP but in contrast to the preferential binding of trans-parinaric acid to the L-cell plasma membrane fatty acid transporter (PMFABP). These data show that the level of cytosolic fatty acids in intact cells can regulate both the extent and specificity of fatty acid uptake. Last, sphingomyelinase treatment of L-cells released cholesterol from the plasma membrane to the cytoplasm and stimulated microsomal acyl-CoA: cholesteryl acyl transferase (ACAT). This process was accelerated in high expression cells. These observations show for the first time in intact cells that L-FABP, a protein most prevalent in liver and intestine where much fat absorption takes place, may have a role in fatty acid and cholesterol absorption.Abbreviations FABP fatty acid-binding protein - L-FABP liver fatty acid-binding protein - I-FABP intestinal fatty acid-binding protein - H-FABP heart fatty acid-binding protein - A-FABP adipocyte fatty acid-binding protein - PMFABP plasma membrane fatty acid-binding protein - SCP-2 sterol carrier protein-2 - Dehydroergosterol (DHE) d-5,7,9(11),22-ergostatetraene-3b-ol - cis-parinaric acid-9Z, 11E, 13E, 15Z-octatetraenoic acid - trans parinaric acid, 9E, 11E, 13E, 14E-octatetraenoic acid - BSA bovine serum albumin - KRH Krebs-Ringer-Henseleit buffer     

An Ustilago maydis Mutant Partially Blocked in P45014DM Activity Is Hypersensitive to Azole Fungicides

An Ustilago maydis ergosterol biosynthesis mutant (A14) which is partially blocked in sterol 14alpha-demethylase (P45014DM) activity is described. This mutant accumulated the abnormal 14alpha-methyl sterols, eburicol, 14alpha-methylfecosterol, and obtusifoliol, along with significant amounts of ergosterol. Although the A14 mutant grew nearly as well as the wild type, it was impaired in cell extension growth, which indicated a dysfunction in apical cell wall synthesis. The mutant was also found to be hypersensitive to the azole fungicides penconazole and tebuconazole.     

Cellulase production by Rhizobium

Summary The production of cellulase byRhizobium species was studied.Rhizobium trifolii cellulase was induced by a variety of polysaccharides, including celluloses and hemicelluloses. Cellobiose and myo-inositol also allowed enzyme expression but mannitol prevented it at concentrations higher than 0.25%. Both soluble and insoluble plant root substances moderately stimulated cellulase production byRhizobium trifolii.Most substances tested did not induce the production of cellulases by the slow-growing, cowpea type rhizobia strain CIAT 79. Effective inducers were carboxymethylcellulose, gluconate and myo-inositol.Cellulase production was very low under all conditions tested. In most cases the enzyme activity was loosely bound to the capsular material. The enzyme in fast-growers is an 1,4--D-glucan-4-glucanohydrolase (endo-glucanase EC 3.2.1.4) with specificity for high molecular weight polysaccharides.There was no correlation between infectiveness ofRhizobium trifolii strains and cellulase production. One strain, which lacks the nodulation plasmid, produced cellulase at the same rate as its parental infective strain.