NEST is a hydrophobic recombinant polypeptide comprising the catalytic domain (residues 727–1216) of neuropathy target esterase. NEST in bacterial lysates has potent esterase activity, which is lost after its solubilization and purification in detergent-containing solutions. Activity in purified NEST preparations was restored by the addition of phospholipids before the removal of detergent by dialysis. The pattern of digestion by proteinase K of NEST–phospholipid complexes suggested that NEST might incorporate in a topologically random fashion into nascent liposomes and that the bulk of each NEST molecule might be exposed either to the liposome lumen or the external medium. Significant quantities of NEST were liberated from NEST–phospholipid complexes by treatment with dilute acid or alkali, suggesting that charge interactions might contribute to the association; however, NEST was irreversibly denatured at these pH values. Treatment of NEST–phospholipid complexes with glutaraldehyde afforded some protection against the inactivation of esterase activity by detergent but the pattern of cross-linked forms of NEST generated did not indicate pre-existing oligomers. Similarly, the inactivation of esterase activity in NEST–phospholipid complexes by radiation indicated that NEST monomers are catalytically active. The foregoing observations are not compatible with structural algorithms predicting that the catalytic serine residue lies at the centre of one of three transmembrane helices in NEST.
The high water permeability of kidney proximal tubules is of paramount importance for isotonic reabsorption of 70% of the glomerular filtrate, and water channels have been postulated to account for the high water permeability. Target analysis following radiation inactivation was used to probe the molecular size of the water channel. Samples of brush border membranes from rat renal cortex were subjected to 3-MeV electron pulses from the Van de Graaff accelerator at a temperature of -130 degrees C. The inactivation of the renal brush border enzymes, alkaline phosphatase, and maltase was used for internal standardization of accumulated dose measurements in target analysis of the water channel. Osmotic water permeability was measured by following the change in scattered light intensity upon rapid mixing of vesicles with a hypertonic solution using stopped-flow spectrophotometry. The vesicle shrinkage response was biphasic and the rate of the fast phase decreased dose dependently by irradiation corresponding to a target size of 30 +/- 3.5 kDa. The total change in scattered light intensity was unaltered, indicating that irradiation did not destroy the lipid barrier. Our results provide strong support for the hypothesis that the high osmotic water permeability of renal proximal tubules results from a water channel-specific protein with a functional unit of 30 kDa.
An electronic system is described that subtracts the analyzing light signal level from the analyzing light signal modulated by a transient absorption signal at the output of a photodetector in a transient absorption spectrophotometer. The system enables sensitive measurement of the transient with simultaneous measurement of the analyzing light level immediately before the transient. The shortest rise time is 50 ns. The time window for the measurement can be selected to a maximum of 20 ms with a droop of 20 μV. Input signals of 10 V of either polarity are permitted. The minimum detectable absorption signal is determined by the noise of the photomultiplier used and can be about 0.2% for an experiment without signal averaging.
Presented in detail are the construction and relevant properties of a high-pass wide band filter with a low frequency cutoff of several kilohertz and a high frequency cutoff of about 1 GHz to be used with 50 Ω characteristic impedance systems. The application to absorption spectrophotometry makes it possible to bring small transient signals on very high levels of analyzing light automatically on the screen of a fast oscilloscope with negligible offset at high gain settings.
SummarySuspensions of bacteriophage T4 in 25 mM phosphate buffer, pH 7·1, were irradiated with 60Co γ-rays. Measurements of the viscosity of irradiated samples before and after treatment with DNA-ase and trypsin showed that DNA is released from damaged phage particles. The phage debris acts as an effective radical scavenger and at higher doses protects the residual phage, thereby decreasing the inactivation rate. In irradiated suspensions of T4 and ΦX174 phage particles biologically-active subviral particles able to infect bacterial spheroplasts were found. Sedimentation in a sucrose gradient showed that the subviral particles of T4 have nearly the same sedimentation coefficient as unirradiated particles. Examination of irradiated samples with the electron microscope revealed that they contained many particles with the sheath contracted. The subviral particles produced by irradiation resemble those obtained with urea but do not show the tail clusters found in samples treated with urea.
The polymerization of styrene in latex particles by means of pulsed high-energy electrons has been studied. Using a sequence of electron beam pulses, periodic initiation is accomplished, similar to that used in pulsed laser polymerization. The larger penetration depth of electron beams offers advantages as compared to laser light in heterogeneous systems since samples do not need to be optically transparent. Therefore pulsed electron beam irradiations are more suitable to study polymerizations homogeneously irradiated in emulsion. From the molar mass distribution of the formed polymer, the monomer concentration in the particles can be determined. Polystyrene particles swollen with styrene were used. A typical dose per pulse was 1.5 Gy. The monomer concentration in 46 nm diameter particles was found to be 6 mol dm-3. Cationic and anionic polymerization of styrene was suppressed by the presence of water.
NEST is a hydrophobic recombinant polypeptide comprising the catalytic domain (residues 727–1216) of neuropathy target esterase. NEST in bacterial lysates has potent esterase activity, which is lost after its solubilization and purification in detergent-containing solutions. Activity in purified NEST preparations was restored by the addition of phospholipids before the removal of detergent by dialysis. The pattern of digestion by proteinase K of NEST–phospholipid complexes suggested that NEST might incorporate in a topologically random fashion into nascent liposomes and that the bulk of each NEST molecule might be exposed either to the liposome lumen or the external medium. Significant quantities of NEST were liberated from NEST–phospholipid complexes by treatment with dilute acid or alkali, suggesting that charge interactions might contribute to the association; however, NEST was irreversibly denatured at these pH values. Treatment of NEST–phospholipid complexes with glutaraldehyde afforded some protection against the inactivation of esterase activity by detergent but the pattern of cross-linked forms of NEST generated did not indicate pre-existing oligomers. Similarly, the inactivation of esterase activity in NEST–phospholipid complexes by radiation indicated that NEST monomers are catalytically active. The foregoing observations are not compatible with structural algorithms predicting that the catalytic serine residue lies at the centre of one of three transmembrane helices in NEST.
SummaryProtection of purified biologically-active bacteriophage DNA by cysteamine against radiation damage was studied in rapid-mixing experiments, under conditions where protection is only due to reactions between irradiated DNA and unirradiated protector.It is shown that the formation of single-strand breaks, a few msec after irradiation in the absence of oxygen, can be influenced by the addition of cysteamine, leading to a reduction of the number of breaks. The amount of lethal nucleotide damage was shown not to be affected.
