Biomedical subjects
Publications and source records attributed to D B Millar.
Exposure of microtubular protein to ultraviolet light inhibits its assembly into morphologically normal microtubules. This effect appeared to result primarily from damage to the tubulin dimers. The damage consisted of a conformational change, a loss of two free sulfhydryl groups, a production of higher molecular weight cross-linked species, and the formation of aggregated amorphous material upon polymerization.
The effect of 2,450-MHz pulsed microwave radiation on the enzyme activity of membrane-free acetylcholinesterase was studied while the enzyme was in the microwave field. We found no significant effect of microwave radiation on enzyme activity using a wide variety of power densities, pulse widths, repetition rates, and duty cycles. This suggests that simple, direct modification by microwave energy of acetylcholinesterase structure and enzymic activity is not related to microwave alteration of acetylcholinesterase central nervous system levels.
The dimensions of the small intestinal diffusion barrier interposed between luminal nutrients and their membrane receptors were determined from kinetic analysis of substrate hydrolysis by integral surface membrane enzymes. The calculated equivalent thickness of the unstirred water layer was too large to be compatible with the known dimensions of rat intestine. The discrepancy could be reconciled by consideration of the mucous coat overlying the intestinal surface membrane. Integral surface membrane proteins could not be labeled by an iodine-125 probe unless the surface coat was first removed. The mucoprotein surface coat appears to constitute an important diffusion barrier for nutrients seeking their digestive and transport sites on the outer intestinal membrane.
Ultraviolet irradiation of 11S acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) produces a loss of tryptophan fluorescence which is best described as the sum of two separable first-order processes, one much more rapid than the other. In addition, the enzyme undergoes an all-or-none inactivation that is monotonically first order. Simultaneous with activity loss, photoscission takes place and results in a molecular weight drop of 1 x 10(5); this decrease is first order with a rate constant identical to that for enzymatic inactivation. These processes are accompanied by apparent conformational changes, as shown by circular dichroic and difference absorption spectra. The relative photochemical inactivation efficiency of incident light is unity when corrected for the wavelength dependence of fluorescence excitation, which is consistent with an efficient Förster resonance transfer of energy among the aromatic chromophores. The extreme sensitivity of acetylcholinesterase to photodestruction upon photon absorption and the several events that follow it not only suggest that these findings might be a basis for a useful molecular probe of the structure of this enzyme, but also indicate that additional care should be taken when conducting spectroscopic studies in the UV region.
The bis-acridine ring system forms the basis for new biophysical probes of novel stereochemistry. Spectral data indicate that certain alkylene bridged bis-9-aminoacridines have a parallel plane conformation of predictable interplane distance. The parallel plane conformation is independent of solvent and thus is different from nucleic acid systems. This stable conformation allows these compounds to be used as sensitive "rulers" for describing binding site geometry in cholinergic enzymes and in the delineation of the mechanism of allosteric control in acetylcholinesterase.
At high detergent concentrations, approximately the equivalent of 2 micelles of TX-100 reversibly bind to acetylcholinesterase and fully inhibit the enzyme. This result suggests that the appropriate lipid environment might regulate this neuronal enzyme's function.
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11-S acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) purified by affinity chromatography of trypsin-digested homogenates was shown to be contaminated with three other active forms of enzyme. The initial purification used an affinity column of the inhibitor, N-methylacridinium ion. Chromatography of the "affinity-pure" sample on hydroxyapatite resulted in two peaks of acetylcholinesterase activity. One peak contained only a form sedimenting at 11-S (approx. 85% of the recovered activity). The other peak consisted of a 9.5-S form, in addition to 14-S and 18-S forms. The 9.5-S form (approx. 7% of the activity) co-electrophoresed with 11-S in 6% polyacrylamide gels and co-sedimented with the same form in sucrose density gradients containing 0.1 M NaCl. The purified 11-S enzyme was shown to be homogeneous by sucrose density gradient centrifugation and electrophoresis. These results indicate that 11-S acetylcholinesterase may be unsuitable for some characterization studies due to undetected contamination by the 9.5-S form.
Chloroform/methanol rechromatography on Sephadex LH-20 of fractions isolated from organic solvent extracts of E. electricus electric organs exhibited alterations in elution point compared to the original chromatography. Additionally, 3H decamethonium bromide did not coelute with any of the observed fractions. It is concluded that as presently described the organic solvent isolation procedure for the cholinergic receptor exhibits undesirable characteristics.
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Detergent binding studies indicated that the neural enzyme, acetylcholinesterase, did not exhibit the properties of an integral membrane protein. The 11S form was isolated by affinity chromatography from a tryptic digest and the 14S and 18S forms in like manner from an undigested preparation. Studies were performed with [3H]TX-100 to determine the extent of binding by these forms and with catalase and human low density lipoprotein as reference proteins. All forms of the enzyme bound less than 0.04 mg TX-100/mg protein which is only slightly higher than binding by catalase and about 25 fold lower than the binding exhibited by low density lipoprotein.
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Five spin-labeled 9-aminoacridines, each bearing either a 4-(2,2,6,6-tetramethyl-1-piperidinyloxy) or a 3-(2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety in the 9 position, have been synthesized and assayed for biological activity in three different test systems. Sedimentation velocity measurements indicated that the labels caused unwinding of calf thymus DNA. Those acridines which contained both 6-chloro and 2-methoxy substituents were less toxic to leukemia L1210 in static culture than the corresponding unsubstituted analogues. While the unsubstituted aminoacridines were quite good inhibitors of Escherichia coli DNA-primed RNA polymerase, the 6-chloro-2-methoxy-substituted compounds stimulated this enzyme system. In the presence of E.coliDNA, the ESR spectrum of 4-[(6-chloro-2-methoxy-9-acridinyl)amino]-2,2,6,6-tetramethyl-1-piperidinyloxyl (12) became broad and highly asymmteric with a maximal hyperfine splitting of 57.5 G. This observation suggests that when 12 intercalates into DNA the piperidinyl moiety that bears the nitroxide group becomes highly immobilized. These results suggest that the spin-labeled 9-aminoacridines will be useful probes for nucleic acids.
The effect of adrenochrome semicarbazide on the conformation of erythrocyte ghost membranes has been studied by ANS fluorescence, lipid and sulfhydryl spin labels and circular dichroism. No large conformational alterations in the membrane were detected by these techniques. Noncompetitive quenching of ANS fluorescence by ADCS suggests ADCS to interact with the membrane at sites close to the ANS binding domain.
Triton X-100 micelle formation at 25 degrees C was studied by use of sedimentation equilibrium and fluorescence spectroscopic techniques. The apparent molecular weight of the major Triton X-100 micelle was found to be 81250, indicating a micelle number of 125. A micelle number of 121 was obtained with fluorescence titration experiments, which showed one molecule of 1-anilino-8-naphthalene sulfonate binding per micelle with an apparent association constant of 0.9 x 10(5) M. The fluorescent titration experiments also indicated the presence of another TX-100 binding species of variable size.
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