Structure-wise discrimination of adenine and guanine by proteins on the basis of their nonbonded interactions

We have analyzed the nonbonded interactions of the structurally similar moieties, adenine and guanine forming complexes with proteins. The results comprise (a) the amino acid–ligand atom preferences, (b) solvent accessibility of ligand atoms before and after complex formation with proteins, and (c) preferred amino acid residue atoms involved in the interactions. We have observed that the amino acid preferences involved in the hydrogen bonding interactions vary for adenine and guanine. The structural variation between the purine atoms is clearly reflected by their burial tendency in the solvent environment. Correlation of the mean amino acid preference values show the variation that exists between adenine and guanine preferences of all the amino acid residues. All our observations provide evidence for the discriminating nature of the proteins in recognizing adenine and guanine.     

Orientational ordering and dynamics of the hydrate and exchangeable hydrogen atoms in crystalline crambin

Deuterium nuclear magnetic resonance studies of crambin crystals grown from deuterated solvent (2H2O/CH3CH2O2H or H2O/C2H3CH2OH) are reported. The extent to which the hydrate and exchangeable hydrogen atoms are dynamically disordered are then determined from the size of the residual deuterium quadrupole couplings, qcc. Rapid molecular reorientation (tau c-1 greater than 10(5) s-1) reduces the magnitude of the quadrupole coupling from its static value (216 kHz for solid water). We find that the room temperature spectrum of crambin is dominated by two features: a sharp line with very small residual quadrupolar coupling less than 3 kHz, and a broad pattern with a quadrupolar coupling in the range 185 to 195 kHz. The former is indicative of very nearly isotropically reorienting deuterons, whereas the latter is somewhat narrower than that observed for the amide deuterons of poly-gamma-benzyl-L-glutamate and thus indicative of deuterons that are almost but not completely stationary. By considering the nuclear magnetic resonance spectrum intensities along with the amino acid sequence, X-ray structure and the manner in which quadrupole couplings are reduced by dynamics, we conclude that the nuclear magnetic resonance signal from most of the water molecules of hydration are contained in the sharp line, i.e. reorient nearly isotropically in the crystalline protein. Unlike bulk water, which freezes abruptly in the manner of a phase transition, the water of hydration in crambin has a broad freezing range from 180 to 250K, as evidenced by the decreasing intensity of the sharp line that disappears at 180K. At temperatures between 150 and 200K, a typical hydrate molecule reorients at a rate comparable to the quadrupole coupling, 10(4) s-1 to 10(5) s-1, a process that occurs in hexagonal ice in the range of 240 to 270K. At 140K, the hydrate is stationary, tau c-1 less than 10(3) s-1. Studies of the protein crystallized from solvent deuterated only at the non-exchangeable methyl group of ethanol confirm that ethanol is in the lattice and show that this solvate behaves in much the same way as the hydrate. The refined X-ray structure has identified four ethanol solvate molecules. The deuterium spectrum at room temperature has a well-defined residual pattern with qcc = 2.2 kHz, i.e. a small-order parameter consistent with nearly isotropically reorienting molecules. The spectrum width broadens substantially only at temperatures below 200K and achieves the characteristic spectrum of a rotating methyl group with stationary C-C axis at 140K.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of isovaleroyl lipids in channeling of sound in the porpoise melon

Physical properties (e.g. specific gravity, adiabatic compressibility and sound velocity) of lipids isolated from tissues from contiguous areas of the fatty melon of an echo-locating porpoise (Delphinus delphis) were determined to elucidate relations between lipid composition and structure, and sound transmission in the head. Lipid content varied greatly within the melon (13.6–77.6% of the tissue weight) and triacylglycerols (80–100%) were the major lipid components. This lipid class was composed of diisovaleroylglycerides (triacylglycerols containing two isovaleroyl moieties and a long-chain acyl moiety), monoisovaleroyldiacylglycerols and triacylglycerols consisting of long-chain acids. The lipid-rich (>45%) areas in the melon contained a high proportion (>45% of total triacylglycerols) of diisovaleroylglycerides. There were gradations of sound velocities within the melon; the lowest sound velocities were associated with high concentrations of diisovaleroylglycerides (<1400 m/s) and the highest with high concentrations of long-chain triacylglycerols. Assuming an average sound frequency of 75 kHz, and considering dimensions of melon (path length and width of 12–14 cm and 5 cm, respectively), a forward radiating lobe of 15–25 degrees is produced. Thus, the deposition of lipids of different acoustic properties in a three-dimensional matrix within the porpoise melon results in a lens for the projection of sound into the marine environment.     

The energy utilized in protein breakdown by the ATP-dependent protease (La) from Escherichia coli

A crucial enzyme in the pathway for protein degradation in Escherichia coli is protease La, an ATP-hydrolyzing protease encoded by the lon gene. This enzyme degrades various proteins to small polypeptides containing 10-20 amino acid residues. To learn more about its energy requirement, we determined the number of ATP molecules hydrolyzed by the purified protease for each peptide bond cleaved. The enzyme hydrolyzed about 2 molecules of ATP for each new amino group generated with casein, bovine serum albumin, glucagon, or guanidinated casein as substrates, even though these proteins differ up to 20-fold in size and 3-4 fold in rates of hydrolysis of peptide bonds. Similar values for the stoichiometry (from 1.9 to 2.4) were obtained using fluorescamine or 2,4,6-trinitrobenzene sulfonic acid to estimate the appearance of new amino groups. These values appeared lower at 1 mM than at 10 mM Mg2+. The coupling between ATP and peptide bond hydrolysis appeared very tight. However, when the protease was assayed under suboptimal conditions (e.g. at lower pH or with ADP present), many more ATP molecules (from 3.5 to 12) were consumed per peptide bond cleaved. Our data would indicate that the early steps in protein degradation consume almost as much energy (2 ATPs for each cleavage) as does the formation of peptide bonds during protein synthesis.     

Metabolism of high-density lipoproteins in cultured rat luteal cells

The uptake of cholesterol from high-density lipoproteins (HDL) labeled with 125I and [3H]cholesterol was examined in cultured rat luteal cells. Luteal cells were incubated with labeled HDL, following which the metabolic fate of the apolipoproteins and cholesterol moieties of the receptor-bound HDL were examined. About 50% of the originally bound HDL apolipoproteins were released into the medium in 24 h by a temperature-dependent process while only 5% of the HDL cholesterol was released unmetabolized. Inclusion of unlabeled HDL in the chase incubation resulted in increased release of apolipoprotein-derived radioactive products without significant change in the release of unmetabolized cholesterol. 60% of the apolipoprotein-derived radioactivity could be precipitated with trichloroacetic acid; the remaining trichloroacetic acid-soluble radioactive fraction was identified as [125I]iodotyrosine. Gel filtration chromatography of the chase-released material showed that the trichloroacetic acid-precipitable products, which contained no detectable amounts of cholesterol, eluted over a range of molecular sizes (9-80 kDa). No intact HDL was retroendocytosed. About 80% of trichloroacetic acid-precipitable products could be immunoadsorbed on anti-apolipoprotein A-I antibody immobilized on CNBr-activated Sepharose, suggesting the presence of fragments containing apolipoprotein A-I. This material was also capable of reassociating with native HDL. Lysosomal inhibitors were partially effective in inhibiting the amount of trichloroacetic acid-soluble products formed. The lysosomal degradation appeared to have no role in the uptake of HDL-derived cholesterol. These studies demonstrate preferential and total uptake of HDL cholesterol by luteal cells, with concomitant degradation of the lipoprotein.     

Metallothionein mRNA and protein induction by cadmium in peripheral-blood leucocytes.

To help to evaluate the role of metallothionein (MT) in peripheral-blood leucocytes, we examined MT protein and mRNA levels in these cells before and after exposure to CdCl2 in culture. Protein was assayed by 109Cd2+ binding, and RNA by dot-blot hybridization. MT was induced in both lymphocytes and adherent monocytes about 10-fold with a 12 h exposure to 10 microM-CdCl2, but absolute levels were 3-fold higher in monocytes: 57 x 10(5) (+Cd2+) versus 6 x 10(5) (-Cd2+) molecules/cell for monocytes; 18 x 10(5) (+Cd2+) versus 2 x 10(5) (-Cd2+) for lymphocytes. Polymorphonuclear cells expressed relatively little MT (0.6 x 10(5) molecules/cell), and this did not change with phorbol ester stimulation or exposure to Cd2+, arguing against a direct protective role for MT in activated neutrophils. MT mRNA levels corresponded qualitatively to expression of protein in these cells. Our data provide quantitative comparisons of leucocyte MT expression and regulation in the human population. Variation in both basal and induced MT mRNA levels reflects environmental or experimental (intra-individual) and possibly genetic (inter-individual) differences.     

Developmental and Dimensional Changes in the Cambial Fusiform Initials ofHoloptelea integrifolia (Roxb.) Planch.

Divisional activity, intrusive growth of the cell wall and loss of fusiform initials have been studied in Holoptelea integrifolia. The dimensional changes in relation to mean length, length frequency, mean width and length variation in relation to fibre length have also been analysed.