An optimized procedure for sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of hydrophobic peptides from an integral membrane protein

A procedure for successful analysis of the hydrophobic tryptic peptides of the Neurospora crassa plasma membrane H+-ATPase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is described. The features of this procedure that are essential for the best results include (i) treatment of the hydrophobic peptide samples with neat trifluoroacetic acid, (ii) dissolution and disaggregation of the hydrophobic peptide samples with SDS at 0 degrees C, (iii) SDS-PAGE of the hydrophobic peptide samples in gels containing a 200:1 ratio of acrylamide to bisacrylamide and a 5-20% convex acrylamide gradient, and (iv) silver-staining of the gels after electrophoresis. This method results in the reproducible resolution and visualization of the H+-ATPase hydrophobic tryptic peptides, which range in size from ca. 5 to 21 kDa, as well as other peptides and proteins ranging in size from ca. 2.5 to 150 kDa. The methods described should also prove useful in other studies where resolution and visualization of hydrophobic peptides of integral membrane proteins are required.     

Cholesterol heterogeneity in bovine rod outer segment disk membranes

Rod outer segment disk membranes have been used to study visual transduction events. Numerous studies have also focused on protein-lipid interactions in these membranes. The possible heterogeneity of the disk membrane composition has not been addressed in such studies. Freeze fracture studies (Andrews, L. D., and Cohn, A. I. (1979) J. Cell Biol. 81, 215-220; Caldwell, R., and McLaughlin, B. (1985) J. Comp. Neurol. 236, 523-537) suggest a difference in cholesterol content between newly formed and old disks. This potential heterogeneity in disk membrane composition was investigated using digitonin. Osmotically intact bovine rod outer segment disk membranes prepared by Ficoll flotation were separated based on the cholesterol content of the disks. The addition of digitonin to disk membrane suspensions in a one-to-one molar ratio with respect to cholesterol produced an increase in the density of the membranes in proportion to the amount of cholesterol present. The digitonin-treated disks were separated into subpopulations using a sucrose density gradient. Disks were shown to vary in cholesterol to phospholipid ratio from 0.30 to 0.05. The ratio of phospholipid to protein remained constant in all disk subpopulations at approximately 65 phospholipids per protein. No significant change in the fatty acid composition of the disks was observed as a function of change in cholesterol content. This work demonstrates compositional heterogeneity in disk membranes which may ultimately affect function.     

Carbohydrate composition analysis of bacterial polysaccharides: optimized acid hydrolysis conditions for HPAEC-PAD analysis.

The capsular polysaccharide from Haemophilus influenzae type b (polyribosyl ribitol-phosphate; PRP) and the capsular polysaccharides from Streptococcus pneumoniae types 6B, 14, 18C, and 23F (Pn6B, Pn14, Pn18C, and Pn23F) were subjected to acid hydrolysis using hydrofluoric (HF) and/or trifluoroacetic acid (TFA) and high-pH anion-exchange chromatography with pulsed amperometric detection in an effort to identify optimum hydrolysis conditions for composition analysis of their carbohydrate components. With the exception of PRP, composition analyses of polysaccharides containing a phosphate moiety in the repeating unit structure (Pn6B, Pn18C, and Pn23F) are significantly improved by subjecting the sample to HF hydrolysis (65 degrees C, 1 h) followed by TFA hydrolysis (98 degrees C, 16 h). This results in essentially quantitative hydrolysis of the phosphodiester bond to the carbohydrate components, which otherwise remained predominantly phosphorylated and poorly accounted for in the analysis. Optimum analysis of PRP was achieved following a 2-h hydrolysis with TFA at 80 degrees C, whereas Pn14 showed optimum results after a 16-h hydrolysis with TFA at 98 degrees C. These analyses also provide information about the relative susceptibility to acid hydrolysis of the various glycosidic and phosphodiester bonds in these polysaccharides, with evidence to suggest that the acid lability of a given bond can be dramatically different from one polysaccharide to another.     

Cell-intrinsic melanin fails to protect melanocytes from ultraviolet-mutagenesis in the absence of epidermal melanin

Melanin is a free-radical scavenger, antioxidant, and broadband absorber of ultraviolet (UV) radiation which protects the skin from environmental carcinogenesis. However, melanin synthesis and UV-induced reactive melanin species are also implicated in melanocyte genotoxicity. Here, we attempted to reconcile these disparate functions of melanin using a UVB-sensitive, NRAS-mutant mouse model, TpN. We crossed TpN mice heterozygous for an inactivating mutation in Tyrosinase to produce albino and black littermates on a C57BL/6J background. These animals were then exposed to a single UVB dose on postnatal day three when keratinocytes in the skin have yet to be melanized. Approximately one-third (35%) of black mice were protected from UVB-accelerated tumor formation. However, melanoma growth rates, tumor mutational burdens, and gene expression profiles were similar in melanomas from black and albino mice. Skin from albino mice contained more cyclobutane pyrimidine dimer (CPD) positive cells than black mice 1-h post-irradiation. However, this trend gradually reversed over time with CPDs becoming more prominent in black than albino melanocytes at 48 h. These results show that in the absence of epidermal pigmentation, melanocytic melanin limits the tumorigenic effects of acute UV exposure but fails to protect melanocytes from UVB-induced mutagenesis.     

Mutations in genes encoding inner arm dynein heavy chains in Tetrahymena thermophila lead to axonemal hypersensitivity to Ca2+

Calcium-dependent ciliary reversals are seen in ciliated protozoans such as Tetrahymena in response to depolarizing stimuli, but the axonemal mechanisms responsible for this response are not well understood. The model is that the outer arm dyneins (OADs) control the beating frequency while the inner arm dyneins (IADs) regulate ciliary waveform. Since ciliary reversal is a type of waveform change, the model would predict that IAD mutations could affect ciliary reversal. We have used gene disruption techniques to generate several behavioral mutants of Tetrahymena with functional disruptions of various IADs. One such mutant, called KO-6, is missing I1 (the two-headed IAD) and is unable to show ciliary reversals in response to any stimuli due to a loss of axonemal Ca2+ sensitivity [Eur J Cell Biol 80 (2001) 486-497; Cell Motil Cytoskeleton 53 (2002) 281-288.]. In contrast, disruption of 3 one-headed IADs [Liu et al., Cell Motil Cytoskeleton 59 (2004), 201-214] produced mutants, which showed over-responsiveness in bioassays measuring either their depolarization-induced avoiding reactions (AR) in Na+ and Ba2+ solutions or their duration of backward swimming (continuous ciliary reversal or CCR) in K+ solutions. Detergent-extracted and reactivated mutants also showed increased probabilities of CCR at lower Ca2+ concentrations suggesting that the behavioral over-responsiveness of these three mutants in vivo is due to increased axonemal Ca2+ sensitivity. Our data suggest the possibility that the one-headed IADs and the two-headed IAD act antagonistically in vivo and that loss of any one of the one-headed IADs leads to behavioral over-responsiveness due to less resistance to I1-induced reversals.     

Development and validation of an NMR-based identity assay for bacterial polysaccharides

A method utilizing NMR spectroscopy has been developed to confirm the identity of bacterial polysaccharides used to formulate a polyvalent pneumococcal polysaccharide vaccine. The method is based on 600 MHz proton NMR spectra of individual serotype-specific polysaccharides. A portion of the anomeric region of each spectrum (5.89 to 4.64 ppm) is compared to spectra generated for designated reference samples for each polysaccharide of interest. The selected region offers a spectral window that is unique to a given polysaccharide and is sensitive to any structural alteration of the repeating units. The similarity of any two spectral profiles is evaluated using a correlation coefficient (rho), where rho >/= 0.95 between a sample and reference profile indicates a positive identification of the sample polysaccharide. This method has been shown to be extremely selective in its ability to discriminate between serotype-specific polysaccharides, some of which differ by no more than a single glycosidic linkage. Furthermore, the method is rapid and does not require extensive sample manipulations or pretreatments. The method was validated as a qualitative identity assay and will be incorporated into routine quality control testing of polysaccharide powders to be used in preparation of the polyvalent pneumococcal vaccine PNEUMOVAX 23. The specificity and reproducibility of the NMR-based identity assay is superior to the currently used colorimetric assays and can be readily adapted for use with other bacterial polysaccharide preparations as well.     

Variation in response among threeAlnus spp. clones to progressive water stress

Summary Experiments to quantify the response of unnodulated, fertilizedAlnus species from disparate habitats to progressive water stress showed contrasting xerotolerance amongspp. clones, using stomatal diffusive resistance, leaf area, and height as indices of drought sensitivity. At moderate or severe levels of water stress, stomatal resistance values forA. glutinosa were consistently higher than values forA. serrulata orA. maritima. In addition, leaf area and height ofA. glutinosa were significantly reduced at moderate water stress as compared to well-watered controls, whereas control and moderately-stressedA. maritimia exhibited similar leaf area and height.Journal Article J-4612 of the Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma, USA     

Secondary structure of the Neurospora crassa plasma membrane H+-ATPase as estimated by circular dichroism

In a previous communication, a water-soluble, hexameric form of the Neurospora crassa plasma membrane H+-ATPase was described (Chadwick, C. C., Goormaghtigh, E., and Scarborough, G. A. (1987) Arch. Biochem. Biophys. 252, 348-356). To facilitate physical studies of the hexamers, the H+-ATPase isolation procedure has been improved, resulting in a structurally and functionally stable hexamer preparation that contains only 5 to 10% non-ATPase protein, approximately 12 mol of enzyme-bound lysophosphatidylcholine/mol of H+-ATPase monomer, and little or no residual plasma membrane phospholipid. Importantly, when activated by lysophosphatidylglycerol, which satisfies the acidic phospholipid requirement of the enzyme, the hexameric quaternary structure of the enzyme is retained, indicating that the functional properties of the water-soluble hexamers are relevant to those of the native, membrane-bound enzyme. The circular dichroism (CD) spectrum of this H+-ATPase preparation has been measured from 184 to 260 nm and used to estimate the secondary structure of the enzyme. The H+-ATPase is estimated to consist of approximately 36% helix, 12% antiparallel beta-sheet, 8% parallel beta-sheet, 11% beta-turn, and 26% other (irregular) structure. There is no change in the CD spectrum when known enzyme ligands are added to the H+-ATPase solution, suggesting that any changes in secondary structure that might occur during ligand binding and/or catalytic cycling are either minor or result in compensatory changes in secondary structure. The CD spectrum of the H+-ATPase is also compared to published spectra of the animal cell Na+/K+- and Ca2+-ATPases and is shown to be quite similar in shape and intensity, suggesting that all of these ATPases, which have significant sequence homology and are mechanistically similar, may have similar secondary structure composition as well.