To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human ...serum pseudocholinesterase (BtChoEase; EC 3.1.1.8) and Torpedo electric organ ``true'' acetylcholinesterase (AcChoEase; EC 3.1.1.7). Using these probes, we isolated several cDNA clones from λ gt10 libraries of fetal brain and liver origins. These include 2.4-kilobase cDNA clones that code for a polypeptide containing a putative signal peptide and the N-terminal, active site, and C-terminal peptides of human BtChoEase, suggesting that they code either for BtChoEase itself or for a very similar but distinct fetal form of cholinesterase. In RNA blots of poly(A)+RNA from the cholinesterase-producing fetal brain and liver, these cDNAs hybridized with a single 2.5-kilobase band. Blot hybridization to human genomic DNA revealed that these fetal BtChoEase cDNA clones hybridize with DNA fragments of the total length of 17.5 kilobases, and signal intensities indicated that these sequences are not present in many copies. Both the cDNA-encoded protein and its nucleotide sequence display striking homology to parallel sequences published for Torpedo AcChoEase. These findings demonstrate extensive homologies between the fetal BtChoEase encoded by these clones and other cholinesterases of various forms and species.
Factors affecting the C = N stretching frequency of protonated retinal Schiff base (RSBH+) were studied with a series of synthetic chromophores and measured under different conditions. Interaction of ...RSBH+ with nonconjugated positive charges in the vicinity of the ring moiety or a planar polyene conformation (in contrast to the twisted retinal conformation in solution) shifted the absorption maxima but did not affect the C = N stretching frequency. The latter, however, was affected by environmental perturbations in the vicinity of the Schiff base linkage. Diminished ion pairing (i.e., of the positively charged nitrogen to its anion) achieved either by substituting a more bulky counteranion or by designing models with a homoconjugation effect lowered the C = N stretch energy. Decreasing solvation of the positively charged nitrogen leads to a similar trend. These effects in the vicinity of the Schiff base linkage also perturb the deuterium isotope effect observed upon deuteriation of the Schiff base. The results are interpreted by considering the mixing of the C = N stretching and C = N-H bending vibration. The C = N mode is shifted due to electrostatic interaction with nonconjugated positive charges in the vicinity of the Schiff base linkage, an interaction that does not influence the isotope effect. Weak hydrogen bonding between the Schiff base linkage in bacteriorhodopsin (bR) and its counteranion or, alternatively, poor solvation of the positively charged Schiff base nitrogen can account for the C = N stretching frequency of 1640 cm-1 and the deuterium isotope effect of 17 cm-1 observed in this pigment.
The incorporation of Mn(II) into framework sites in the aluminophosphate zeotype AlPO{sub 4}-20, an analog of sodalite, has been investigated using pulsed electron nuclear double resonance (ENDOR) ...spectroscopy at 95 GHz. The field sweep echo-detected EPR spectrum showed the presence of a single Mn(II) site with a {sup 55}Mn hyperfine coupling of 8.7 mT. ENDOR spectra, recorded using the Mims and Davies sequences, consist of an {sup 27}Al signal at the Larmor frequency and a {sup 31}P Larmor frequency indicates that it originates from the M{sub s} = {+-}1/2 manifolds and that the interaction is primarily isotropic. The relatively large {sup 31}P hyperfine interaction and the weak interaction with {sup 27}Al provide unique and direct evidence for Mn(II) substitution of framework Al. X-band electron spin echo envelope modulation (ESEEM) measurements showed only signals at the {sup 1}H, {sup 14}N, {sup 27}Al, and {sup 31}P Larmor frequencies. The first two are due to weak dipolar interaction with the template molecules, while the others reflect interactions with the framework.
Artificial bacteriorhodopsin pigments based on synthetic retinal analogues carrying an electron-withdrawing CF3 substituent group were prepared. The effects of CF3 on the spectra, photocycles, and ...Schiff base pKa values of the pigments were analyzed. A reduction of 5 units in the pKa of the Schiff base is observed when the CF3 substituent is located at the C-13 polyene position, in the vicinity of the protonated Schiff base nitrogen. The results lead (i) to the unambiguous characterization of the (direct) titration of the Schiff base in bacteriorhodopsin and (ii) to the conclusion that the deprotonation rate of the Schiff base during the photocycle (i.e., the generation of the M412 intermediate) is determined by a structural change in the protein.
Communication between cells is a fundamental component of development and morphogenesis. Identification of the molecules mediating cell-cell communication is crucial for elucidation of the molecular ...basis of these processes. Receptor tyrosine kinases (RTKs) appear to play a central role in this context by transmitting into cells information dictating their fate. The functions of RTKs in Drosophila are extremely diverse, and include maternal determination of embryonic polarity (torso and torpedo), determination of neuroblast identity (faint little ball), and guidance of tracheal cell migration in the embryo (breathless). During compound eye development, RTKs affect the number of photoreceptor clusters (Ellipse) and the determination of photoreceptor R7 identity (sevenless). The phenotypes of mutations in RTK loci serve as a starting point for understanding processes dictating cell identity at the level of the whole organism. Recently, they have also begun to provide a basis for selection of second-site suppressor mutations, encoding additional elements in their signal transduction pathway. Common themes between the functions, regulation, and signal transduction pathways of Drosophila RTKs are drawn.
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