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Biomedical subjects

P Woolley

Publications and source records attributed to P Woolley.

At least 55 records · Page 3Linked to original sources
PubMed1983

Effect of codon shortening and the antibiotics viomycin and sparsomycin upon the behaviour of bound aminoacyl-tRNA. Decoding at the ribosomal A site.

70 S ribosomes were programmed with initiator tRNA and messenger oligonucleotides AUG(U)n and AUG(C)n, where n = 1, 2 or 3. The binding of the ternary complexes [Phe-tRNA X EF-Tu X GTP] and [Pro-tRNA X EF-Tu X GTP] to the programmed ribosomes was studied. If codon-anticodon interaction is restricted to only one basepair, the ternary complex leaves the ribosome before GTP hydrolysis. Two basepairs allow hydrolysis of GTP, but the aminoacyl-tRNA dissociates and is recycled, resulting in wastage of GTP. Three basepairs result in apparently stable binding of aminoacyl-tRNA to the ribosome. The antibiotic sparsomycin weakens the binding by an amount roughly equivalent to one messenger base, while viomycin has the reverse effect.

Antibiotics, Antineoplastic↗
PubMed1983

Structural and functional studies on protein S20 from the 30-S subunit of the Escherichia coli ribosome.

Fragments resistant to proteolysis have been obtained from the ribosomal protein S20. They provide evidence for a structural domain stretching from the middle of the protein to its C terminus. With the exception of a large fragment of this protein lacking only 14 residues at the N terminus, all fragments had lost their ability to bind to 16-S rRNA. The protein in the S20 . 16-S-RNA complex was highly protected against enzymic digestion, indicating that the entire protein is involved in interaction with the nucleic acid. Circular dichroism showed a high alpha helix content (36%) for the intact protein and a low alpha helix content (2%) for the large fragment. Intrinsic fluorescence studies demonstrated that the single tyrosine residue in protein S20 is exposed to the solvent in the intact protein and is not exposed in the S20 . 16-S-RNA complex. Irreversible thermal denaturation of the protein was followed by fluorescence of the tyrosine and was found between 50 degrees C and 70 degrees C.

Chemical Phenomena↗
PubMed1983

Theory of measurement of Förster-type energy transfer in macromolecules.

We describe the theoretical basis of an unconventional method for the determination of the amount of energy transferred between two fluorophores by the Förster mechanism. The method involves an internal comparison made by separation of the fluorophores in situ (i.e., in the optical cell), for example, by means of enzymic digestion; it eliminates several important sources of error and it simplifies calculation while making maximal use of the information contained in the fluorescence spectra. The validity of the method is demonstrated by determination of the known distance between two modifiable sites on the transfer RNA molecule, and its usefulness is exemplified by its application to triangulation of the ribosome of Escherichia coli.

Journal Article↗
PubMed1982

Structure of ribosomal protein L6 from Escherichia coli. A fluorescence study.

Protein L6 from the 50-S ribosomal subunit has been investigated using fluorimetric techniques. The intrinsic fluorophore Trp-61 and fluorescent labels (acetylaminoethyl-dansyl and acetylaminofluorescein) attached to the residue Cys-124 were used. It proved possible to incorporate fluorescence-labelled L6 into the 50-S ribosome. Trp-61 is exposed to solvent, as shown by its emission wavelength and by quenching experiments; the latter also show that it lies in a pocket with a high positive charge due to the basic residues in the N-terminal fragment. Cys-124 lies in a less strongly positive region. Upon incorporation into the 50-S subunit, the label on Cys-124 becomes less accessible for quenching but its positive potential rises, showing the absence of direct contact with 23-S RNA. Analysis of anisotropy data indicates a considerable degree of asphericity of free L6. Energy transfer between Trp-61 and the dansyl label on Cys-124, measured by donor quenching and acceptor enhancement, reveals a separation of 3.5 +/- 0.4 nm (35 +/- 4 A) between fluorophores.

Bacterial Proteins↗
PubMed1982

Distance measurement by energy transfer: the 3' end of 16-S RNA and proteins S4 and S17 of the ribosome of Escherichia coli.

Escherichia coli ribosomal proteins S4 and S17 were specifically labelled at their thiol groups with the acetylaminoethyl-dansyl and/or bimane fluorophores. Each formed a complex with 16-S RNA and, when the other 30-S ribosomal proteins were added, a complete 30-S subunit with at least partial activity. If the 3' end of the RNA was also labelled (with fluorescein) then the distance between the two fluorophores could be measured by Förster-type energy transfer. The result for S4 was 6.0 nm (60 A) in the ribonucleoprotein complex and 5.6 nm (56 A) in the 30-S subunit, and for S17 6.3 nm (63 A) in the complex and 6.2 nm (62 A) in the subunit. There is no evidence for a major change in the relative disposition of the 3' and 5' ends of the 16-S RNA during formation of the 30-S subunit. Sources of error are discussed, including the question of multiple labelling. In order to measure more accurately the extent of energy transfer a procedure based upon enzymic digestion was developed and is detailed in this paper.

Bacterial Proteins↗
PubMed1981

The binding of dansylated initiation factor 3 to the 30-S subunit of Escherichia coli: a fluorimetric and biochemical study.

Initiation factor 3 (IF-3) has been labelled using dansyl (1-dimethylaminonaphthalene-5-sulphonyl) chloride under conditions designed to preserve the biological activity of the factor. The sites of modification of the IF-3 have been determined by peptide mapping and sequencing: about six lysines (73, 80, 91, 96, 99, 112) react in various proportions. However, if an IF-3 molecule bears more than one dansyl group on average then its activity is lost. The extent of incorporation is proportional to the amount of dansyl chloride used in the reaction. Spectrofluorimetric analysis of the dansyl-IF-3 leads to the following conclusions. (a) The motion of the dansyl label does not change greatly upon binding to the 30-S subunit. (b) The label is not close enough to any tryptophan group of the ribosome in the 30-S-subunit . IF-3 complex to allow energy transfer. (c) The IF-3 chain is folded so as to bring the tyrosine groups close to the dansyl-binding sites. (d) The stoichiometry of the binding of IF-3 to 30-S ribosomal subunits is close to 1:1 and the binding constant is 2 x 10(7) M-1. IF-3 also binds non-covalently the fluorescent indicator 8-anilinonaphthalene 1-sulphonate (ANS) with an apparent binding constant of approximately 8000 M-1. An interaction between ANS and poly(A-U-G), both bound to IF-3, was demonstrated. Combining these results with those for dansyl-IF-3 leads to a model for the interaction between IF-3 and the 30-S subunit involving a combination of 'hydrophobic' and electrostatic attraction between the factor and ribosomal RNA.

Amino Acid Sequence↗
PubMed1979

The binding of a neutral aromatic molecule to a negatively-charged lipid membrane. I. Thermodynamics and mode of binding.

This paper presents a detailed study of the binding of the fluorescent dye N-phenyl naphthylamine (NPN) to bilayers composed of the negatively-charged phospholipid methylphosphatidic acid. Binding to the liquid-crystalline membrane is enthalpy-driven. It is shown by determination of the binding constant and confirmed by n.m.r. that most of the dye ("guest") molecules reside between the lipid hydrocarbon chains at a fixed distance from the head-group, and are not distributed uniformly throughout the hydrocarbon phase. Each guest molecule is surrounded by about four lipid molecules. Transition of the membrane from the liquid-crystalline to the crystalline state results in almost total expulsion of the bound NPN into the water phase. Electrostatic theory is developed to find the effect of electrostatics upon the binding of a neutral molecule to charged membranes. Although the charge product is zero, electrostatic interactions play a part in determining the strength of binding, if each guest molecule incorporated increases the area of the membrane. For NPN this increase was found to be ca. 41 A(2).

Journal Article↗
PubMed1979

The binding of a neutral aromatic molecule to a negatively-charged lipid membrane. II. Kinetics and mechanism.

The kinetics of the binding of the fluorescence indicator N-phenyl naphthylamine to bilayer vesicles of C12-methyl-phosphatidic acid have been investigated by means of the temperature-jump relaxation technique utilizing fluorescence light detection. Single-exponential relaxation curves were observed, with time constants in the range 0.2-3 ms. The concentration dependence of the relaxation time yielded an apparent association rate constant (expressed in terms of monomeric phospholipid) of k(on) = 5 x 10(6) M(-1) s(-1) in aqueous solution at 25 degrees . The activation energy and viscosity dependence associated with the binding rate show that this process is actually diffusion-controlled. The theory of diffusion-controlled reactions then allows a determination of the average size of the bilayer vesicles and of the true rate constant for the association of the indicator molecules with the vesicles. Assuming spherical geometry for the vesicles, the values are: r(ves) = 190 A, which corresponds to 20000 lipid molecules per vesicle and k'(on) = 1 x 10(11) M(-1) s(-1) (25 degrees). The correctness of this size-determination was confirmed semi-quantitatively by electron microscopy. Since in fact a distribution of vesicle sizes must be present, a discussion is included of the relaxation function which the system is expected to take in the general case. Biological implications of diffusion control for the transport of non-polar substances and for lipid mixing are indicated.

Journal Article↗
PubMed1979

Electrostatic interactions at charged lipid membranes. Calcium binding.

A quantitative study of calcium-ion binding by the negatively-charged phospholipid methylphosphatidic acid is presented. Experimental results are compared with the predictions of the Gouy-Chapman theory, taking into account both the ions bound at the membrane surface and the ions held in the diffuse layer. This theory suffices to explain the titration of the calcium/lipid system, but fails to explain completely the behaviour of the ordered-fluid transition temperature, which shows a splitting that according to electrostatic theory alone should not occur. The dependence of the calcium-lipid binding constant. upon 1: 1 electrolyte concentration is correctly predicted by the theory; the latter however gives equations which can only be solved numerically. A simple, approximate equation is therefore given (in the text, eq. 34) for the prediction of the degree of calcium binding to a negatively-charged lipid membrane.

Journal Article↗
PubMed1978

Phase I studies on chlorozotocin.

A phase I investigation of chlorozotocin, a new-water soluble chloroethylnitrosourea, was undertaken to define its pharmacologic effects in man. Forty-three patients received single intravenous doses ranging from 5 to 175 mg/m2 every 6 wk. No signs of toxicity were observed at doses of under 120 mg/m2, but thrombocytopenia occurred at higher doses. The thrombocytopenic nadir appeared to be dose-dependent and occurred 4 wk after treatment. Platelet transfusions were required in 2 patients who had previously received intensive chemotherapy. No significant leukopenia occurred. A mild reversible and delayed elevation of hepatic transaminases was found in 25% of courses of 120 mg/m2 or more. No renal toxicity was observed and gastrointestinal toxicity was mild. Investigation of clinical pharmacology revealed a rapid triphasic plasma clearance with initial t1/2S of 3, 15, and 30 min. The concentration of N-nitroso intact drug at 1 hr was 10% of the initial peak level. Renal excretion accounted for half of the dose. No significant concentration of N-nitroso intact or radiolabeled drug was detected in the cerebrospinal fluid of 2 patients in whom it was examined. There were objective signs of therapeutic activity in 5 patients, 3 of whom had melanoma. Based on these studies, the recommended dose for phase II investigation of chlorozotocin is 120 mg/m2 every 6 wk.

Adolescent↗
PubMed1976

Electrostatic interactions at charged lipid membranes. I. Effects of pH and univalent cations on membrane structure.

Electrostatic interactions at charged lipid membranes make a significant contribution to the free energy of the system, and can be varied within a wide range by alteration either of the membrane's surface charge density or of the concentration of electrolytes in the surrounding medium. Changes in the charged membrane's structure, such as the ordered in equilibrium fluid phase transition, can thus be induced at constant temperature by variations in pH and salt concentration. An adequate quantitative description of these phenomena is obtained from the Gouy--Chapman theory. The good agreement between theory and experiment confirms that the expression derived for the electrostatic free energy especially in respect of its positive sign is correct. The classical expression derived for the electrostatic free energy, especially in respect of its positive sign, is correct. The classical expression for the "free energy of the double layer" derived by Verwey and Overbeek, which has a negative sign, is not applicable to lipid membranes with ionizable polar groups.

Cations, Monovalent↗
PubMed1975

Models for metal ion function in carbonic anhydrase.

A model catalyst is described which has properties in common with carbonic anhydrase. The model demonstrates the availability of a mechanism, previously only hypothetical, for the action of the enzyme. It also shows, however, that this mechanism alone is not adequate to produce the high activity of the enzyme.

Acetaldehyde↗