‘De novo’ centrioles originate at sites associated with annulate lamellae in sea-urchin eggs

Hypertonic stress stimulates the formation of new centrioles in sea-urchin eggs. Those centrioles which appear away from the nuclear surface originate exclusiveJy at sites associated with annulate lamellae. Although apparent when nascent centrioles become visible, the annulate lamellar association is gradually lost as nascent forms mature into centrioles.     

Mechanistic studies on aromatase and related C---C bond cleaving P-450 enzymes

Some P-450 systems, notably aromatase and 14-demethylase catalyse not only the hydroxylate reaction but also the oxidation of an alcohol into a carbonyl compound as well as a C---C bond cleavage process. All these reactions occur at the same active site. A somewhat analogous situation is noted with 17-hydroxylase-17,20-lyase that participates in hydroxylation as well as C---C bond cleavage process. The C---C bond cleavage reactions catalysed by the above enzymes conform to the general equation:

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It is argued that all three types of reaction catalyzed by these enzymes may be viewed as variations on a common theme. In P-450 dependent hydroxylation the initially formed Fe^III---O---O^. species is converted into Fe^III---O---OH and the heterolysis of the oxygen—oxygen bond of the latter then gives the oxo-derivative for which a number of canonical structures are possible; for example Fe^V = O ↔ (+^.)Fe^IV = O ↔ Fe^IV---O^.. One of these, Fe^IV---O^. behaves like an alkoxyl radical and participates in hydrogen abstraction from C---H bond to produce Fe^IV---OH and carbon radical. The latter is then quenched by the delivery of hydroxyl radical from Fe^IV---OH. The latter species may thus be regarded as a carrier of hydroxyl radical. We have proposed that the C---C bond cleavage reaction occurs through the participation of the Fe^III---O---OH species that is trapped by the electrophilic property of the carbonyl compound giving a peroxide adduct that fragments to produce an acyl—carbon cleavage. Scientific developments leading up to this conclusion are considered. In the first author's views,

“The study of mechanisms is not a scientific but a cultural activity. Mechanisms do not aim at an absolute truth but are intended to be a “running” commentary on the status of knowledge in a field. As the structural knowledge in a field advances Mechanisms evolve to take note of the new findings. Just as a constructive “running” commentary provides the stimulus for higher standards of performance, so Mechanisms call for better and firmer structural information from their practitioners”.     

Amino acid accumulation in frog muscle. II. Are cycloleucine fluxes consistent with an adsorption model for concentrative uptake of amino acid?

Cycloleucine accumulation by frog muscle was studied at o °C and 25 °C. At external concentrations less than 5 mM the distribution ratio of cycloleucine is higher at 0 °C than at 25 °C. At concentrations greater than 5 mM the converse is true due to apparent exclusion of cycloleucine from a larger portion of the cell water at 0 °C.The steady state data are consistent with an absortion model for amino acid accumulation. Flux studies provide a means to rule out this model if all the possible rate-limiting steps in the movement of amino acid into and out of the cell are considered. These steps include intra-cytoplasmic diffusion, desorption from cytoplasmic or membrane sites and passage through the cell membrane. The assumption is made that the rate-limiting step for influx and efflux is the same, allowing the use of either influx or efflux data to examine the model.Diffusion-limited flux is ruled out on the basis of“influx profile analysis” of the time course of cycloleucine entry at both 0 °C and 25 °C.At least 95% of all intracellular cycloleucine leaves frog muscle cells with a single exponential time course at both 0 °C. The rate constant of efflux does not vary with cellular concentration.These findings are shown to be incompatible with desorption-limited efflux. They are compatible with membrane-limited efflex only if (i) adsorption sites are located on membranes with direct access to the extracellular space and (ii) the rate constant for desorption is equal to the rate constant of membrane-limited efflux of free amino acid. It is considered unlikely that such a coincidence would occur at both 0 °C and 25 °C. Therefore, an absorption model for cycloleucine accumulation in frog muscle appears to be untenable.     

Tetanus toxin binds with high affinity to neuroblastoma x glioma hybrid cells NG 108-15 and impairs their stimulated acetylcholine release

Differentiated neuroblastoma x glioma hybrid cells NG 108-15 express on their surface specific binding sites for tetanus toxin. 450 sites/cell with a KD of 2 x 10(-11) M were found under "physiological" conditions of pH and salt concentrations. A Hill coefficient of 1.1 indicated noncooperative binding. Specific binding of 125I-toxin to its sites could be prevented either by preincubation of the toxin with a neutralizing monoclonal antibody or by pretreatment of the cells with neuraminidase (Vibrio cholerae). To quantify the action of tetanus toxin on the stimulated release of 14C activity from differentiated cells preincubated with [14C]choline, a new type of perfusion device was designed which could be filled with cells growing in monolayers on Cytodex-3 microbeads. Tetanus toxin inhibited the stimulated 14C release in a time- and dose-dependent manner. A greater than 50% inhibition was found after 2 h of incubation with 10(-12) M toxin. The inhibitory action of tetanus toxin could be prevented with a monoclonal antibody to the toxin or with neuraminidase treatment of the cells. These results suggest that the neuraminidase-sensitive 2 x 10(-11) KD receptors are the productive receptors for tetanus intoxication in differentiated NG 108-15 cells. The possible chemical composition of these receptors is discussed. Differentiated NG 108-15 cells provide a useful model in which picomolar tetanus concentrations produce both measurable saturable binding and inhibition of potassium-evoked, acetylcholine release under physiological conditions of pH and salt concentrations.     

Charge dependence of Fe(II)-catalyzed DNA cleavage.

The effect of charge of the Fe(II) reagent used to induce DNA strand cleavage reactions in the presence of a source of reducing equivalents is investigated using two oligonucleotide models. The first consists of the two strands dA20 and dT20, and an equimolar complex between them. The second is a short four-arm branched DNA complex composed of four 16-mer strands. In the former case, cleavage of the 1:1 complex by three reagents with different formal charge, Fe(II).EDTA2-, Fe(II).EDDA and Fe2+, is comparable in rate to that of the individual dT20 and the dA20 strands. While the three reagents show similar cleavage rates for the duplex and single stranded molecules, they give distinctive cutting patterns in the DNA tetramer, consistent with the presence of a site of excess negative charge at the branch point. Scission induced by Fe(II).EDTA2- shows lower reactivity at the branch site relative to duplex controls, whereas Fe(II)2+ shows enhanced reactivity. Formally neutral Fe(II).EDDA shows weak loss of cutting reactivity at the branch. The position of attack by Fe(II)2+ in the branched tetramer is shifted with respect to those of Fe(II).EDTA2- or Fe(II).EDDA; a slower migrating species is also detected in the scission of dA20.dT20 duplex by Fe(II) reaction. These results suggest that the Fe(II)2+ reaction proceeds by a different mechanism from the other agents. The difference in cutting profiles induced by the neutral and negatively charged chelated complexes is consistent with a local electrostatic repulsion of a negatively charged source of radicals, not a positively charged one.     

Distribution of charged residues stabilizes individual helices in myoglobins

The effect of the distribution of charged residues on stability of alpha helices in isolated peptides and in globular proteins exemplified by myoglobins from 62 different species is discussed. A highly simplified set of rules is used to account for the interaction of charged groups with the dipole of an alpha helix. Only the position and sign of a charge with respect to the center of the helix and its ability to participate in intrahelical salt bridges determine its effect. These rules lead to a linear correlation between the helicity in variant C-peptide helices from RNAse and the extent to which the charge distribution opposes the helix dipole. Of the sample of 496 helices in the myoglobins studied, 456 exhibit arrangements of charges which oppose the effective dipole moment of the helix according to this calculation. A number of variants occur which leave the backbone moment of helices A-D unchanged, or even add to it. However no such variants exist in the sequences of helices E-H. We suggest that the E, F, G and H helices in myoglobins which show the strongest reversal of the helix dipole participate in the structures of early intermediates in folding of the chain. Stable helix structures should be more likely to occur in these isolated sequences also, and introduction of charge alterations in helices E to H should affect the initial refolding rate of mutant myoglobins.