General and local anaesthetics perturb the fusion of phospholipid vesicles.

The effects of general and local anaesthetics on Ca2+-induced fusion of negatively charged lipid vesicles have been investigated. Vesicles composed of phosphatidylcholine and phosphatidic acid (2:1 molar ratio) were induced to fuse using 5 mM free Ca2+. Fusion, assessed by an increase in size using gel filtration techniques and confirmed by electron microscopy, displayed a dependence on Ca2+ and Mg2+ concentration and on temperature. The inhalational anaesthetics halothane, methoxyflurane and diethyl ether enhanced fusion as did the uncharged local anaesthetic benzocaine. In contrast, the charged local anaesthetics lignocaine and bupivacaine inhibited the fusion process. It is suggested that the enhancement observed with the inhalational anaesthetics and benzocaine was mediated by an effect on lipid fluidity and the inhibition observed with the charged tertiary amine anaesthetics was due to an antagonism towards Ca2+.

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

Quenching of the fluorescence of the (Ca2+ + Mg2+)-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-3 beta-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.

Binding of dansyl propranolol to the (Ca2+ + Mg2+)-ATPase.

We have studied the binding of dansyl propranolol to lipid bilayers and to the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum. The fluorescence emission spectra for dansyl propranolol bound to the ATPase system can be fitted to the sum of three peaks, characteristic of probe bound to lipid and to protein and free in solution, respectively. Titrations show that binding to the lipid component of the ATPase system is comparable to binding to simple lipid bilayers. Binding constants obtained using fluorescence spectroscopy for binding to lipid bilayers agree with constants obtained from microelectrophoresis measurements. Binding to sites on the ATPase can be described either in terms of the aqueous concentration of dansyl propranolol or in terms of the mole fraction of dansyl propranolol in the lipid phase of the membrane. Both descriptions suggest extensive binding to annular sites at the lipid/protein interface of the ATPase. Binding at other sites on the ATPase might also be present. Binding of dansyl propranolol to the ATPase results in a marked inhibition of activity. At high Ca2+ concentrations, inhibition fits to a non-competitive model of inhibition, described by a Ki of 5 microM. We attribute this effect to binding at annular sites. At lower Ca2+ concentration, a decrease is observed in the apparent affinity of the ATPase for Ca2+ which can be attributed to a build-up of positive charge on the membrane as a result of binding.

Factors important to students in selecting a residency program.

A 22-item questionnaire, designed to assess the factors students considered important when they ranked residency programs, was distributed to the 1988 senior class of Tulane University School of Medicine just before the submission deadline of the National Residency Matching Program. Completed surveys were obtained from 111 of the 157 graduating students (approximately 71%) and were representative of the entire class in terms of sex, age, race, marital status, and anticipated field of specialization. Results of this investigation suggest that the satisfaction of a program's house officers and the seniors' general impression at the interview were the most important selection factors of the matriculating seniors surveyed. Diversity of the training experience and geographic location were also important selection factors. House officer benefits and salary were low-priority factors in the seniors' program selections.

Interactions of cholesterol hemisuccinate with phospholipids and (Ca2+-Mg2+)-ATPase.

Cholesterol hemisuccinate has been shown to equilibrate readily with liposomes and with the (Ca2+-Mg2+)-ATPase from sarcoplasmic reticulum and has been used to modify the sterol content of these membranes. Cholesterol hemisuccinate incorporates into dioleoylphosphatidylcholine (DOPC) up to a molar ratio of 3:1 sterol to DOPC. Effects on lipid order as detected by electron spin resonance and fluorescence polarization are comparable to those of cholesterol. Binding constants have been determined, and the uncharged form of the sterol binds more strongly than the anionic form. Binding to DOPC and to the lipid component of the ATPase system is comparable. From use of the fluorescence quenching properties of 1,2-bis(9,10- dibromooleoyl )phosphatidylcholine and dibromocholesterol hemisuccinate, two classes of binding sites on the ATPase have been deduced. At the lipid/protein interface, the binding constant for cholesterol hemisuccinate is considerably less than that for DOPC. At the second set of sites ( nonannular sites), binding occurs with Kd = 0.55 in molar ratio units. The effect of cholesterol hemisuccinate on the activity of the ATPase depends on the phospholipid present in the system: ATPase reconstituted with DOPC is inhibited whereas ATPase reconstituted with dimyristoleoylphosphatidylcholine is activated. We conclude that changes in membrane fluidity are not important in determining ATPase activity in these systems.

Membrane fluidity is not an important physiological regulator of the (Ca2+-Mg2+)-dependent ATPase of sarcoplasmic reticulum.

The (Ca2+-Mg2+)-ATPase from sarcoplasmic reticulum has been reconstituted into phospholipid bilayers of different fatty acyl chain length and degree of unsaturation, and ATPase activity was found to vary about 10-fold. Order parameters for bilayers of these lipids measured using the ESR probe 5-doxylstearic acid varied from 0.5 to 0.6 at 37 degrees C. There was no correlation between phospholipid order parameter and ATPase activity, and we conclude that phospholipid structure is a more important determinant of protein activity than is membrane fluidity.

Membrane-permeable luciferin esters for assay of firefly luciferase in live intact cells.

Five esters of luciferin were synthesized and compared with native luciferin as substrates for firefly luciferase expressed in live intact mammalian cells. The esters themselves were not substrates for purified luciferase, but four were substrates for a purified esterase and all appeared to be hydrolysed to luciferin within mammalian cells. At a substrate concentration of 0.01 mM, the peak luminescence from the cos cells expressing luciferase was up to 6-fold greater with the esters than with unmodified luciferin. At 0.1 mM, the difference between luciferin and the esters was decreased. The kinetics of the luminescent signal with the different luciferin esters varied significantly, indicating possible differences in the rates of uptake, breakdown and enzyme inhibition. The esters did not support luminescence from Escherichia coli cells expressing firefly luciferase, suggesting a lack of appropriate esterase activity in this particular strain. The esters could be useful for the assay of luciferase expression in intact mammalian cells when luciferin levels are limiting, for example in tissues, and in plants. Alternative luciferin derivatives may allow further improvements in sensitivity.

Nonradioactive DNA detection on Southern blots by enzymatically triggered chemiluminescence.

A chemiluminescent reaction based on the deprotection of a phosphorylated phenyl dioxetane by alkaline phosphatase has recently been described (Schaap, A.P., 1988, J. Biolumin. Chemilumin. 2, 253). Light output is enhanced by intermolecular energy transfer to a micelle-solubilized fluorophore. This system is applied here to the detection of DNA probes on Southern blots. Enzyme solution assays which give an indication of sensitivity show that using this substrate 100 fg (0.7 amol) alkaline phosphatase can be detected on a luminescence plate reader (200 ms reading time). In a model Southern blotting system 180 fg HindIII digested lambda DNA was detected on film with homologous biotinylated DNA and a streptavidin-alkaline phosphatase complex. The single copy genes mos and raf-1, representing targets of 4.2 and 2.4 pg target DNA respectively, have also been detected in Southern-blotted human genomic DNA. A delay in reaching a plateau level of light output which is dependent on pH is observed but signal continues for at least 7 days. Typically, 12-h exposures to X-ray film were performed but once a steady-state light output had been achieved this time could be reduced to 2 h by preflashing film. This detection system represents a sensitive nonradioactive method, which is applicable not only to Southern blots but also to Northern and Western blots and any assay in which alkaline phosphatase is the label.

Interaction of fatty acids with the calcium-magnesium ion dependent adenosinetriphosphatase from sarcoplasmic reticulum.

The fluorescence emission spectrum of dansylundecanoic acid is sensitive to the environment and appears at a lower wavelength when the fatty acid is bound to protein than when it is bound to phospholipid. When bound to the (Ca2+-Mg2+)-ATPase of sarcoplasmic reticulum, the emission spectrum can be resolved into separate components assigned to fatty acid bound to protein and to lipid. Efficiency of energy transfer from the tryptophan residues of the ATPase to dansylundecanoic is higher for protein-bound probe than for lipid-bound probe. Fluorescence titrations are consistent with three fatty acid binding sites per ATPase with a Kd of 7 microM, and these sites are postulated to occur at the protein-protein interface in ATPase oligomers. Fatty acid incorporated into the lipid component of the membrane appears to be bound outside the lipid annulus around the protein.

Use of 5-nitroindole-2'-deoxyribose-5'-triphosphate for labelling and detection of oligonucleotides.

The 5'-triphosphate of 5-nitroindole-2'-deoxyriboside has been shown to be a good substrate for terminal deoxynucleotidyl transferase (TdT). An antibody has been prepared for the detection of 5-nitroindole and has been used for the detection of 5-nitroindole tailed DNA both in single-stranded form and after hybridisation to a template. This is therefore a new method for the detection of nucleic acid probes.