Detergent effects on a reverse transcriptase activity and on inhibition by rifamycin derivatives.

A reverse transcriptase activity, extracted from virus-transformed cells, is activated by very low concentrations of nonionic detergents. These same detergents also significantly reduce the effectiveness of certain rifamycin derivatives as inhibitors of the polymerase activity when the detergents are present at micelle-forming concentrations.

Medium chain fatty acid synthesis in rodent mammary adenocarcinomas in vitro.

The ability or inability of certain rodent mammary adenocarcinomas to synthesize medium chain fatty acids in vitro, correlates with the presence or absence of the specific mammary chain-terminating enzyme, thioesterase II.

Application of method of moments analysis to fluorescence decay lifetime distributions.

In fluorescence decay work, distributions of exponential decay lifetimes are anticipated where complex systems are examined. We describe here methods of gaining information on such distributions using the method of moments analysis approach. The information obtained may be as simple as the average and deviation of the lifetime distribution, quantities which we show may be estimated directly from the results of a multiexponential analysis. An approximation to the actual distribution shape may also be obtained using a procedure we call the variable filter analysis (VFA) method without making any assumptions about the shape of the distribution. Tests of VFA using both simulated and experimental data are described. Limitations of this method and of distribution analysis methods in general are discussed. Results of analyses on experimental decays for ethidium intercalated in core particles and in free DNA are reported.

Analysis of photoacoustic waveforms using the nonlinear least squares method.

Pulsed-laser photoacoustics is a technique which measures photoinduced enthalpic and volumetric changes on the nano- and microsecond timescales. Analysis of photoacoustic data generally requires deconvolution for a sum of exponentials, a procedure which has been developed extensively in the field of time-resolved fluorescence decay. Initial efforts to adapt an iterative nonlinear least squares computer program, utilizing the Marquardt algorithm, from the fluorescence field to photoacoustics indicated that significant modifications were needed. The major problem arises from the wide range of transient decay times which must be addressed by the photoacoustic technique. We describe an alternative approach to numerical convolution with exponential decays, developed to overcome the problems. Instead of using an approximation method (Simpson's rule) for evaluating the convolution integral, we construct a continuous instrumental response function by quadratic fitting of the discrete data and evaluate the convolution integral directly, without approximations. The success and limitations of this quadratic-fit convolution program are then demonstrated using simulated data. Finally, the program is applied to the analysis of experimental data to compare the resolution capabilities of two commercially available transducers. The advantages of a broadband, heavily damped transducer are shown for a standard organic photochemical system, the quenching of the triplet state of benzophenone by 2,5-dimethyl-2,4-hexadiene.

An experimental methodology for measuring volume changes in proton transfer reactions in aqueous solutions.

A fast perturbation in proton concentration can be induced in aqueous solution using a pulsed ultraviolet laser and suitable photolabile compounds which, upon photoexcitation, irreversibly release protons. The volume change and the rate constant for the reaction of the photodetached protons with proton-accepting groups in solution can be monitored using time resolved photoacoustics. A typical proton concentration jump of 1 microM can be obtained with a 200-microJ laser pulse at 308 nm. Reaction dynamics from 20 ns to 5 micros can be easily followed. The methodology we establish represents a direct, time-resolved measurement of the reaction volume in proton transfer processes and an extension to the nanosecond-microsecond range of traditional relaxation techniques, such as stopped-flow. We report example applications to reactions involving simple molecules and polypeptides.

On the choice of laser dyes for use in exciting tyrosine fluorescence decays.

The choice of laser dyes for exciting tyrosine fluorescence using synchronously pumped cavity-dumped dye laser systems is discussed. Rhodamine 560 was found to be optimal for a system based on an argon-ion pumping laser, whereas rhodamine 575 was preferred using a frequency-doubled Nd:YAG laser. Modifications of our fluorescence decay instrument to permit rejection of multiphoton events using a microchannel plate photomultiplier are described. An example of a four-component resolution of tyrosine decays illustrates the dramatic resolution capabilities attainable.

Reversibility of the low-salt transition of chromatin core particles.

The low-salt transition of chromatin core particles is reversible if the monovalent cation concentration is kept above 0.2 mM. Exposure of the particles to salt concentrations below this value results in a nonreversible secondary transition. The nonreversible changes are relatively slow with a half-time of about 15 minutes. Once exposed to such low ionic strength, the particles then begin to refold with increasing salt in at least two steps over a much higher ionic strength range than is required for the usual low-salt transition. The refolding is very fast, with a half-time less than a minute. Small differences between particles which had or had not been exposed to very low salt persist even when the particles are returned to near physiological ionic strengths.

Salt induced transitions of chromatin core particles studied by tyrosine fluorescence anisotropy.

Chromatin core particles containing 146 base pairs of DNA have been found to undergo a single defined transition below 10 mM ionic strength as studied by both sedimentation velocity and tyrosine fluorescence anisotropy. A method is described for the preparation of such core particles from chicken erythrocytes with greater than 50% yield.

Effects of pH on the stability of chromatin core particles.

Chromatin core particles near physiological ionic strength undergo a reversible transition induced by changes in pH near neutrality. While sedimentation studies indicate no significant effect on size or shape, changes in tyrosine fluorescence anisotropy and in circular dichroism suggest a somewhat looser structure at high pH. Further support of this suggestion is given by high salt dissociation experiments; at pH 8 core particles begin to show changes at lower salt concentration than at pH 6. The pH transition appears unaffected by the presence of Mg2+ but can be blocked by crosslinking of the histones. A possible relationship is suggested between this transition and increases in intracellular pH which correlate with enhancement in several aspects of cellular activity including DNA replication.

F/F deconvolution of fluorescence decay data.

An approach for the deconvolution of multiexponential fluorescence decay data in which a single exponential decay is used in place of the usual excitation profile is described. For analysis by the method of moments, the resulting decay lifetimes are identical to those in the multiexponential decay, while the pre-exponential factors are a simple function of the true values and the parameters of the single exponential decay. This approach, which we call the F/F deconvolution method, is capable of eliminating the errors in decay analyses which arise from the wavelength dependence of the instrument response function.

Effects of pH on low-salt transition of chromatin core particles.

The low-salt transition of chicken erythrocyte core particles containing uniform 145 base pair DNA was studied as a function of pH and of salt concentration. Intrinsic tyrosine fluorescence was used to follow the changes. Potassium salts of the anions C1-, H2PO4-, and SO4(2-) were indistinguishable in their ability to affect the transition. Divalent cations (Mg2+, Mn2+, Ca2+) were effective at 36-fold lower total concentration than monovalent cations (Li+, Na+, K+, Tris+), but no significant differences were observed within the two classes of cations. These results indicate that cation binding to the core particle is involved in the transition. At pH 9 the transition was broadened and shifted to higher monovalent cation concentration as compared to that at pH 6. At both pHs the fluorescence changes could be resolved into two steps by numerical least-squares analysis. On the basis of what is known about histone--histone interactions, a two-step mechanism is suggested, involving changes in the interactions between dimers of histones 2a and 2b with a tetramer of histones 3 and 4. The pH-induced changes appear to be correlated with a structural transition, which was detected as a function of pH at near physiological ionic strength (0.1 M). This structural change was accompanied by a small decrease in the tyrosine fluorescence anisotropy. An apparent pKa value near 7 is indicated, suggesting that the structural changes involved may be of physiological significance.

Purification and properties of a thioesterase from lactating rat mammary gland which modifies the product specificity of fatty acid synthetase.

An acyl coenzyme A hydrolase (thioesterase II) has been purified to near homogeneity from lactating rat mammary gland. The enzyme is a monomer of molecular weight 33,000 and contains a single active site residue. The enzyme is specific for acyl groups, as acyl-CoA thioesters, containing eight or more carbon atoms and can also hydrolyze oxygen esters. Thioesterase II is capable of shifting the product specificity of rat mammary gland fatty acid synthetase from predominately long chain fatty acids (C14, C16, and C18) to mainly medium chain fatty acids (C8, C10, and C12). Thioesterase II can restore the capacity for fatty acid synthesis to fatty acid synthetase in which the thioesterase component (thioesterase I) has been inactivated with phenylmethanesulfonyl fluoride or removed by trypsinization. No evidence was found of significant levels of thioesterase II in lactating rat liver. The presence of thioesterase II in the lactating mammary gland and the ability of the enzyme to hydrolyze acyl-fatty acid synthetase thioesters of intermediate chain length, are indicative of a major role for this enzyme in the synthesis of the medium chain fatty acids characteristic of milk fat.

The intrinsic tyrosine fluorescence of histone H1. Steady state and fluorescence decay studies reveal heterogeneous emission.

In wavelength-resolved steady state spectra we observe three different kinds of emission from histone H1, a class A protein with only a single tyrosine residue. Unfolded H1 emissions that peak at approximately 300 and 340 nm can both be excited maximally at approximately 280 nm. Another, peaking much further to the red at approximately 400 nm, can be excited maximally at approximately 320 nm. The 300-nm fluorescence can be resolved by lifetime measurements into three components with decay times of approximately 1, 2, and 4 ns. On sodium-chloride-induced refolding of H1, simplification of the emission properties occurs. The 340 and 400-nm components disappear while the two shorter lifetime components of the 300-nm band diminish in amplitude and are replaced by the 4-ns decay. We believe that the 340-nm emission is tyrosinate fluorescence resulting from excited-state proton transfer. The origin of the 400-nm emission remains uncertain. We assign the 1 and 2-ns components of the 300-nm emission to two states of tyrosine in denatured H1 and the 4-ns decay to fluorescence of the single tyrosine residue in the globular region of refolded H1. Our results support the contention that salt induced folding of H1 is a cooperative two state process, and permit us to better understand the previously reported increases in fluorescence intensity and anisotropy on salt-induced folding.

Fluorescence anisotropy decay of ethidium bound to nucleosome core particles. 1. Rotational diffusion indicates an extended structure at low ionic strength.

The fluorescence decay of ethidium intercalated into the DNA of nucleosome core particles increases in average lifetime from about 22 ns in H2O to about 39 ns in D2O. This increase, combined with the acquisition of large amounts of data (on the order of 10(8) counts per decay), allows measurement of anisotropy decays out to more than 350 ns. The overall slow rotational motions of the core particle may thereby be more clearly distinguished from the faster torsional motions of the DNA. In 10 mM NaCl at 20 degrees C, we recover a long correlation time of 198 ns in D2O (159 ns when corrected to a viscosity of 1.002 cP), in agreement with the value of 164 ns obtained in H2O. These values are consistent with hydrodynamic calculations based on the expected size and shape of the hydrated particle. To support our conclusion that this long correlation time derives from Brownian rotational diffusion, we show that the value is directly proportional to the viscosity and inversely proportional to the temperature. No significant changes in the rotational correlation time are observed between 1 and 500 mM ionic strength. Below 1 mM, the particle undergoes the "low-salt transition" as measured by steady-state tyrosine fluorescence anisotropy. However, we observe little change in shape until the ionic strength is decreased below approximately 0.2 mM, where the correlation time increases nearly 2-fold, indicating that the particle has opened up into an extended form. We have previously shown that the transition becomes nonreversible below 0.2 mM salt.

Kinetics of local helix formation in poly-L-glutamic acid studied by time-resolved photoacoustics: neutralization reactions of carboxylates in aqueous solutions and their relevance to the problem of protein folding.

Photoactivatable caged protons have been used to trigger proton transfer reactions in aqueous solutions of acetate, glutamate, and poly-L-glutamic acid, and the volumetric and enthalpic changes have been detected and characterized by means of time-resolved photoacoustics. Neutralization of carboxylates in aqueous solutions invariably results in an expansion of the solution due to the disappearance of two charges and is accompanied by little enthalpic change. The reactions occur with thermally activated, apparent bimolecular rates on the order of 10(10) M(-1)s(-1). In the case of aqueous solutions of poly-L-glutamic acid at pH around the pK(a) of the coil-to-helix transition, diffusional binding of a proton by carboxylates is followed by a sequential reaction with rate 1.06 (+/- 0.05) x 10(7)s(-1). This step is not thermally activated in the temperature range we have investigated and is likely related to local formation of hydrogen bonds near the protonation site. This structural event may constitute a rate-limiting step in helix propagation.

Unfolding of nucleosome cores dramatically changes the distribution of ultraviolet photoproducts in DNA.

Nucleosome core particles undergo a conformational change at ionic strengths below 0.2 mM; the fluorescence anisotropy decay of bound ethidium indicates that under these conditions the particle adopts a highly extended structure. We have measured the distribution of UV-induced DNA damage (primarily cyclobutane-pyrimidine dimers) through a process termed photofootprinting. As the core particle is exposed to ionic strengths below 0.2 mM, the photofootprint pattern changes from that observed for native cores, with a characteristic 10.3 base repeat pattern presumably derived largely from the bending of DNA around the histone octamer, to a more evenly distributed pattern resembling that of free DNA. These results provide clear evidence that the DNA in the core particle at these very low ionic strengths, although still tightly bound to histones, is no longer bent to a significant degree.

Histone hyperacetylation. Its effects on nucleosome core particle transitions.

Effects of histone hyperacetylation on transitions of HeLa cell nucleosome core particles were studied. The transitions examined were induced by low salt concentrations, pH, temperature, and nondissociating high salt. Effects of salt dissociation were also examined. The low-salt transition was found to shift to higher ionic strength by approximately three fold for hyperacetylated particles, a change which may be due simply to the increased overall negative charge on the particles caused by acetylation of lysine residues. Some differences were also seen in the way in which core particles refold after exposure to very low salt (which induces a nonreversible change in the particles). Otherwise no significant effects of hyperacetylation were observed.