Resolution of end-to-end distance distributions of flexible molecules using quenching-induced variations of the Forster distance for fluorescence energy transfer.

We describe a new method to recover the distribution of donor-to-acceptor (D-A) distances in flexible molecules using steady-state measurements of the efficiency of fluorescence energy transfer. The method depends upon changes in the Forster distance (Ro) induced by collisional quenching of the donor emission. The Ro-dependent transfer efficiencies are analyzed using nonlinear least squares to recover the mean D-A distance and the width of the distribution. The method was developed and tested using three synthetic D-A pairs, in which the chromophores were separated by alkyl chains of varying lengths. As an example application we also recovered the distribution of distances from the single tryptophan residue in troponin I (trp 158) to acceptor-labeled cysteine 133. The half-width of the distribution increases from 12 A in the native state to 53 A when unfolded by guanidine hydrochloride. For both TnI and the three model compounds the distance distributions recovered from the steady-state transfer efficiencies were in excellent agreement with the distributions recovered using the more sophisticated frequency-domain method (Lakowicz, J.R., M.L. Johnson, W. Wiczk, A. Bhat, and R.F. Steiner. 1987. Chem. Phys. Lett. 138:587-593). The method was found to be reliable and should be generally useful for studies of conformational distributions of macromolecules.     

Polarized Raman scattering from oriented single microcrystals of d(A5T5)2 and d(pTpT)

Polarized Raman spectra have been obtained from single microcrystals of the duplex of the decamer d(A₅T₅)₂ using a Raman microscope. This is the first report of Raman spectra from a crystal of a deoxyoligomer that contains only long, nonalternating sequences of adenine and thymine. Sequences containing d(A)_n and d(T)_n are of interest in view of recent suggestions that they induce bends in DNA and that they might exist in a nonstandard B-conformation. Polarized Raman spectra of a crystal of d(pTpT) have also been obtained. Both crystals display Raman bands whose intensities are very sensitive to the orientation of the crystal with respect to the direction of polarization of the incident laser beam. These spectra indicate that the helical axes of the oligonucleotides are parallel to the long axes of the crystals and that the d(A₅T₅)₂ is not appreciably bent in the crystal. The Raman spectrum from the d(pTpT) crystal indicates that all of the furanose ring puckers are in a C2′-endo configuration since only the C2′-endo marker band at 835 ± 5 cm^?1 is present. Crystals of d(A₅T₅)₂ show measurable Raman intensities in both the 838- and 816-cm^?1 bands. This indicates the presence of both the C2′-endo and C3′-endo, or possibly other non-C2′-endo, furanose conformations. The 816-cm^?1 band is weak so that only a small fraction of the residues are estimated to be in the non-C2′-endo conformation. In both the d(pTpT) and d(A₅T₅)₂ crystals the intensity of the bands due to vibrations of the backbone show only a small dependence on orientation of the crystals. This result is explained by the low symmetry of the puckered sugar rings. It is concluded that Raman spectra obtained from oligonucleotide crystals in which the orientation of the crystal axes to the laser polarization is not carefully controlled may contain intensity artifacts that are due to polarization effects.     

Delivery of folates to the cytoplasm of MA104 cells is mediated by a surface membrane receptor that recycles

MA104 cells, as well as several other rapidly dividing tissue culture cells, have a folate-binding protein associated with their cell surface. The protein has the properties of a membrane receptor: (a) 5-methyl[3H]tetrahydrofolic acid binds with high affinity (Kd approximately equal to 3 nM); (b) the protein is an integral membrane protein; (c) it appears to deliver physiological concentrations of 5-methyl[3H]tetrahydrofolic acid to the inside of the cell; (d) binding activity is regulated by the concentration of folate within the cell. To better understand the mechanism of action of this receptor, we have studied the pathway of folate internalization. We present evidence that during internalization: (a) folate binds to the membrane receptor; (b) the ligand-receptor complex moves into the cell; (c) the ligand is released from the receptor in an acidic intracellular compartment and moves into the cytoplasm; and (d) the unoccupied receptor returns to the cell surface.     

Multiple phosphorylation sites of rat liver glycogen synthase

Rat liver glycogen synthase was purified to homogeneity by an improved procedure that yielded enzyme almost exclusively as a polypeptide of Mr 85,000. The phosphorylation of this enzyme by eight protein kinases was analyzed by cleavage of the enzyme subunit followed by mapping of the phosphopeptides using polyacrylamide gel electrophoresis in the presence of SDS, reverse-phase high-performance liquid chromatography and thin-layer electrophoresis. Cyclic AMP-dependent protein kinase, phosphorylase kinase, protein kinase C and the calmodulin-dependent protein kinase all phosphorylated the same small peptide (approx. 20 amino acids) located in a 14 kDa CNBr-fragment (CB-1). Calmodulin-dependent protein kinase and protein kinase C also modified second sites in CB-1. A larger CNBr-fragment (CB-2) of approx. 28 kDa was the dominant site of action for casein kinases I and II, FA/GSK-3 and the heparin-activated protein kinase. The sites modified were all localized in a 14 kDa species generated by trypsin digestion. Further proteolysis with V8 proteinase indicated that FA/GSK-3 and the heparin-activated enzyme recognized the same smaller peptide within CB-2, which may also be phosphorylated by casein kinase 1. Casein kinase 1 also modified a distinct peptide, as did casein kinase II. The results lead us to suggest homology to the muscle enzyme with regard to CB-1 phosphorylation and the region recognized by FA/GSK-3, which in rabbit muscle is characterized by a high density of proline and serine residues. A striking difference with the muscle isozyme is the apparent lack of phosphorylations corresponding to the muscle sites 1a and 1b. These results provide further evidence for the presence of liver- and muscle-specific glycogen synthase isozymes in the rat. That the isozymes differ subtly as to phosphorylation sites may provide a clue to the functional differences between the isozymes.     

A comparison of fragile X expression in lymphocyte and lymphoblastoid cultures

This study compares fragile X expression in peripheral blood lymphocyte cultures with expression in lymphoblastoid cell lines established from 23 individuals from families in which the fragile X is segregating. Most patients expressed the fragile X in lymphoblastoid cell lines treated with FUdR under optimal conditions at approximately the same frequency as in peripheral blood cultures from the same individual. No fragile X cells were seen in the lymphoblastoid cell lines from three phenotypically normal males who had transmitted the fragile X gene to offspring or in the lines from three phenotypically normal obligate-carrier females, all of whom were also negative in peripheral blood cultures. Two individuals, however, who expressed at high levels in peripheral blood lymphocytes expressed in lymphoblastoid cells only at low levels or not at all. We describe the considerations needed for the consistent demonstration of the fragile X in lymphoblastoid cell lines.     

Temperature dependence of the reduction potential of CuA in carbon monoxide inhibited cytochrome c oxidase

The temperature dependence of the reduction potential of the CuA site in carbon monoxide inhibited cytochrome c oxidase has been measured with a spectroelectrochemical method adapted to the relatively weak near-infrared absorption of this copper ion. These measurements, together with parallel measurements on the 604-nm absorption due to Fea, indicate that an interaction between CuA and Fea causes the reduction potential for one of these sites to be decreased by approximately 40 mV upon reduction of the other. The temperature dependence of the CuA reduction potential indicates a relatively large and negative standard entropy of reduction of CuA (delta So' = -48.7 +/- 2.3 eu). Possible implications of the intersite redox interaction and the large standard entropy of reduction of the CuA site are discussed.     

Interaction and reconstitution of carboxyl-terminal-shortened B chains with the intact A chain of insulin

With the S-(thiomethyl)-A chain and despentapeptide (26-30) and desoctapeptide (23-30) S-(thiomethyl)-B chains of insulin at pH 10.8 and a molar ratio of A/B = 1.5, difference spectra of the mixed against the separated chains with negative peaks at 245 and 295 nm and a weak positive peak at 278 nm indicate interaction of the chains leading to Tyr environmental changes as in the case for the intact chains. With the shortened B chains, freshly dissolved from lyophilized powders, it takes some 2 h for the difference spectra to approach completion whereas with the solutions of the shortened B chains left standing overnight at pH 10.8 and 4 degrees C the difference spectra, similar in shape to that described above, appear almost immediately after mixing. Solvent perturbation with 20% ethylene glycol suggests some ordered structure for the despentapeptide but not for the desoctapeptide B chain. The interactions of the A chain with the shortened B chains appear to be weaker as compared to that with the intact B chain as shown by decreasing reconstitution yields for the intact, despentapeptide, and desoctapeptide B chains respectively with the A chain. The above results indicate that the C-terminal portion of the B chain is important not only for the activity of insulin but also for the correct pairing of the chains.     

Rapid voltage-dependent dissociation of scorpion alpha-toxins coupled to Na channel inactivation in amphibian myelinated nerves

The voltage-dependent action of several scorpion alpha-toxins on Na channels was studied in toad myelinated nerve under voltage clamp. These toxins slow the declining phase of macroscopic Na current, apparently by inhibiting an irreversible channel inactivation step and thus permitting channels to reopen from a closed state in depolarized membranes. In this article, we describe the rapid reversal of alpha-toxin action by membrane depolarizations more positive than +20 mV, an effect not achieved by extensive washing. Depolarizations that were increasingly positive and of longer duration caused the toxin to dissociate faster and more completely, but only up to a limiting extent. Repetitive pulses had a cumulative effect equal to that of a single pulse lasting as long as their combined duration. When the membrane of a nonperfused fiber was repolarized, the effects of the toxin returned completely, but if the fiber was perfused during the conditioning procedure, recovery was incomplete and occurred more slowly, as it did at lower applied toxin concentrations. Other alpha-type toxins, from the scorpion Centruroides sculpturatus (IVa) and the sea anemone Anemonia sulcata (ATXII), exhibited similar voltage-dependent binding, though each had its own voltage range and dissociation rate. We suggest that the dissociation of the toxin molecule from the Na channel is coupled to the inactivation process. An equivalent valence for inactivation gating, of less than 1 e per channel, is calculated from the voltage-dependent change in toxin affinity.     

Chronic measurement, using a Doppler probe, of uterine artery flow in the gravid guinea-pig

A chronic animal model is described which permits for the first time the continuous measurement of uterine artery blood flow velocity in the pregnant guinea-pig by using a miniaturized Doppler flow probe. Preliminary validation revealed that alterations in actual blood flow are directly and proportionally related to the change in the Doppler shift (r = 0.984) from 0 to 100 ml/h. The velocity signal baseline was as stable as that of systemic blood pressure. Depending upon the individual animal's flow velocity, a deviation of 2-5% from baseline was statistically significant. With experience, greater than 90% of preparations were successful and a 30-day interval was often available for study. Uterine artery flow velocity increased steadily between 45 and 55 days of gestation. Instrumentation did not result in fetal growth retardation. A reduction in flow velocity occurred during general anaesthesia using ketamine and the antianxietal xylazine. In agreement with the reports of other investigators using a different model, both hydralazine and angiotensin II increased uterine blood velocity and adrenaline reduced it.     

Structure-activity relationships of nitro and methyl-nitro derivatives of indoline, indole, indazole and benzimidazole in Salmonella typhimurium

The mutagenic activities of eleven nitro derivatives and eleven N-methyl-nitro derivatives of indoline, indole, indazole and benzimidazole were investigated in Salmonella TA98 and TA100. The presence of a nitro group at C4 or C7 resulted in only weakly or nonmutagenic compounds, while a nitro group at C2, C5 or C6 usually resulted in measurable mutagenic activity in the non-N-methylated compounds. Methylation of a ring nitrogen usually reduced the mutagenic activity of these nitroheterocyclics except 2-nitro-benzimidazole, which resulted in a better than 300-fold increase in mutagenic activity. A proposed mechanism for the increased mutagenic activity obtained by methylation of imidazole nitrogens may provide insights into the reasons for the potent mutagenicities observed for several similarly methylated cooked-food mutagens.