1H-nmr studies of polyoxyethylene-bound homo-oligo-L-methionines

The use of ¹H-nmr spectroscopy is demonstrated to be a useful analytical method to characterize the structure of synthetic peptides attached to soluble, macromolecular polyoxyethylene (POE) supports in the liquid-phase method (LPM) of peptide synthesis. We report an extensive 360-MHz ¹H-nmr study of POE-bound homo-oligo-L -methionine peptides. A combination of high field and selective saturation or Redfield pulse methods allows resolution of individual backbone NH and α-CH resonances of dilute peptides in the presence of strong resonances from macromolecular POE and/or protonated solvents. The nmr spectra for the POE-bound peptides in CDCl₃ are qualitatively similar to those of the low-molecular-weight Boc-L -Met_n-OMe peptide esters. This corroborates other observations that POE has little effect on peptide stucture. The backbone α-CH region of peptides is overlapped by signals from the terminal oxyethylene group of POE, but the peptide side-chain and low-field backbone NH resonances are well resolved. In trifluoroethanol the Boc-(L -Met)_n-NH-POE heptamer and octamer adopt the right-handed α-helical structure, and the present nmr studies provide evidence for two strong intramolecular hydrogen bonds to stabilize the helices. In water, the N-deblocked derivatives, (L -Met)_n-NH-POE oligomers adopt β-sheet structure and manifest well-resolved nonequivalent NH resonances with 6–7 Hz ³J_NH-CH coupling constants.     

Annular and non-annular binding sites on the (Ca2+ + Mg2+)-ATPase

Quenching of the fluorescence of the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3β-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems.     

Veratridine-stimulated central synapses in culture: A quantitative ultrastructural analysis

Synapses in explant cultures of fetal rat neocortex at day 18 in vitro were stimulated by veratridine (10^?4M) for 20 min. The cultures were subsequently processed for electron microscopy and the synapses were analyzed by quantitative techniques, incorporating set mathematical treatment. The mean values of area, perimeter, and form factor of the presynaptic elements significantly increased following veratridine stimulation, compared to the values of control synapses. The length of the postsynaptic thickening also increased, while synaptic curvature did not change significantly in the veratridine group. A fivefold reduction was observed in the mean number of synaptic vesicles per presynaptic element and in the vesicle-terminal area ratio, following veratridine stimulation. The cytoplasm-terminal area ratio and the occurrence of vacuoles/cisternae significantly increased after veratridine application. Planar measurement of membranes (boundary length) of different presynaptic organelles revealed that the total membrane did not change significantly in the veratridine group. The data indicated an increase in volume and swelling of the pre- and postsynaptic elements, considerable depletion of synaptic vesicles, and preservation of the total presynaptic membrane following veratridine stimulation in nerve tissue culture.     

Lipid peroxidation of skeletal muscle: an in vitro study

The process of lipid peroxidation of skeletal muscle has been examined in vitro by monitoring the autoxidation of skeletal-muscle homogenates. Skeletal-muscle tissue has been shown to have considerable capacity for autoxidation and the process has been found to be initiated by a free-radical-mediated mechanism, critically dependent on the presence of free iron in the homogenate. The initiating radicals have not been firmly identified, but the results suggest that neither superoxide or hydroxyl radicals are involved. An in vitro technique for assessment of the antioxidant capacity of muscle tissue has also been developed which is capable of demonstrating differences between muscle tissues with differing vitamin E contents.     

A polymorphic variant of human erythrocyte carbonic anhydrase I with a widespread distribution in Australian aborigines,CAIAustralia-9 (8 Asp → Gly): Purification,properties, amino acid substitution,and possible physiological significance of the variant enzyme

Carbonic anhydrase I (EC 4.2.1.1) purified from the pooled packed red blood cells of 100 individuals typed as heterozygous for the common Australian Aboriginal carbonic anhydrase I variant CAI Australia-9 had a slightly higher specific CO2 hydratase or esterase (toward p-nitrophenyl acetate) activity than the normal component and a higher Km and Vmax using the esterase substrate. The variant enzyme was slightly more resistant to heat inactivation. The extent of inhibition of both enzymes by the specific inhibitor acetazolamide was identical, as was their immunological behavior and the lability of the active-site zinc ion. The variant enzyme was more resistant to chloride inhibition. The physiological importance of this observation is discussed in the context of a proposed adaptive advantage of the variant gene in the arid western and central regions of Australia. The amino acid substitution in the Aboriginal variant of a glycine for an aspartic acid residue has been located at residue 8 from the N terminus (i.e., 8 Asp leads to Gly), by proteolytic and partial acid hydrolyses. The possible effects of this substitution on the structure and function of the molecule are discussed.     

Determination of the chromosomal size of three different strains of Enterococcus faecalis and one strain of Enterococcus faecium.

Pulsed-field gel electrophoresis was used to determine the chromosomal size of three different strains of Enterococcus faecalis and one strain of Enterococcus faecium. The size determinations of OG1X, a strain of E. faecalis widely used in many laboratories for genetic studies, using Sma I, Not I, and Sfi I alone or in combination, ranged from 2,750 to 2,761 kb. Using the same enzymes as with OG1X, the size of HH-67, a plasmid-free clinical isolate of E. faecalis, was determined to be 2,170-2,288 kb and the size of JH2-2, an E. faecalis recipient strain, ranged from 2,008 to 2,135 kb. The size range generated for GE-1, a plasmid-free E. faecium strain, with the use of Sma I, Not I, and Apa I was 2,045-2,155 kb. Although OG1X differed in size from the other three enterococci, each individual enterococcal strain generated reproducible results in different experiments. However, for both E. faecalis OG1X and E. faecium GE-1, one of the enzymes used generated a considerably smaller molecular size than that generated by the other two enzymes. The discrepancy was due to visually undiscernible comigrating fragments, and serves to point out a potential source of error if fewer than two enzymes are used to size a genome. The size discrepancies were resolved by digesting individual fragments with a second enzyme. The molecular sizes of these enterococcal strains are larger than that recently reported for Campylobacter, smaller than that of Escherichia coli and Pseudomonas aeruginosa, and similar (OG1X) or smaller (JH2-2, HH67, and GE-1) than the 2,819-kb reported for Streptococcus mutans.     

Purification and characterization of yeast orotidine 5'-monophosphate decarboxylase overexpressed from plasmid PGU2

Orotidine 5'-monophosphate decarboxylase (ODCase) has been overexpressed in yeast 15C cells transformed with a plasmid carrying the URA3 gene that encodes ODCase. Twenty g of cells having ODCase activity equal to 30 mg of pure enzyme per liter of cell culture were obtained after 9 h of galactose induction. To remove yeast proteases, a 60-90% ammonium sulfate fractionation step plus the addition of EDTA as an inhibitor of metallopeptidases was necessary. The purification protocol yielded ODCase that was protease-free and stable to storage at 4 degrees C for 16 months. The pure enzyme had a specific activity of 40 units/mg in 50 mM phosphate buffer, pH 6, and could be stored at -20 degrees C in 20% glycerol with retention of full activity for more than 2 years. The enzyme had a Km for orotidine 5'-monophosphate of 0.7 microM at pH 6 and 25 degrees C. The molecular weight of the plasmid-derived ODCase monomer determined by electrophoresis on denaturing polyacrylamide gels was 29,500. ODCase sedimented through sucrose density gradients as a monomer of about 30 kDa at low protein concentration and in the absence of ligands that bind at the catalytic site. An increase in the sedimentation rate could be induced by increasing the ODCase concentration or by adding ligands that are competitive inhibitors. ODCase sedimented in a single band typical of a protein of 46 kDa at the highest protein concentration studied or in the presence of 50 mM phosphate or 933 microM substrate (orotidine 5'-monophosphate) or product (UMP). A dimer sedimenting as a protein of about 64 kDa occurred in the presence of 50 microM 6-azauridine 5'-monophosphate or 2 microM 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid, competitive inhibitors of ODCase. These results resemble the ligand-induced subunit association of the ODCase domain of bifunctional UMP synthase and support the use of yeast ODCase as a model for ODCases from other species.