Binding of peptides with basic residues to membranes containing acidic phospholipids.

There are clusters of basic amino acids on many cytoplasmic proteins that bind transiently to membranes (e.g., protein kinase C) as well as on the cytoplasmic domain of many intrinsic membrane proteins (e.g., glycophorin). To explore the possibility that these basic residues bind electrostatically to monovalent acidic lipids, we studied the binding of the peptides Lysn and Argn (n = 1-5) to bilayer membranes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). We made electrophoretic mobility measurements using multilamellar vesicles, fluorescence and equilibrium binding measurements using large unilamellar vesicles, and surface potential measurements using monolayers. None of the peptides bound to vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) but all bound to vesicles formed from PC/PS or PC/PG mixtures. None of the peptides exhibited specificity between PS and PG. Each lysine residue that was added to Lys2 decreased by one order of magnitude the concentration of peptide required to reverse the charge on the vesicle; equivalently it increased by one order of magnitude the binding affinity of the peptides for the PS vesicles. The simplest explanation is that each added lysine binds independently to a separate PS with a microscopic association constant of 10 M-1 or a free energy of approximately 1.4 kcal/mol. Similar, but not identical, results were obtained with the Argn peptides. A simple theoretical model combines the Gouy-Chapman theory (which accounts for the nonspecific electrostatic accumulation of the peptides in the aqueous diffuse double layer adjacent to the membrane) with mass action equations (which account for the binding of the peptides to greater than 1 PS). This model can account qualitatively for the dependence of binding on both the number of basic residues in the peptides and the mole fraction of PS in the membrane.     

Endogenous read-through of a UGA termination codon in a Saccharomyces cerevisiae cell-free system: evidence for involvement of both a mitochondrial and a nuclear tRNA.

Globin mRNA, translated in a Saccharomyces cerevisiae cell-free protein synthesizing system prepared from a [psi+ rho+] strain, primarily directed the synthesis of alpha- and beta-globin. A third globin mRNA-specific polypeptide was also synthesized, representing approximately 10% of the total translation products. This polypeptide (beta') was synthesized by translational read-through of the beta- globin mRNA UGA terminator and was mediated primarily by an endogenous tRNA coded for by the mitochondria. This mitochondrial tRNA, when charged, could be preferentially bound, in high salt, to benzoylated DEAE-cellulose, a characteristic of a tRNATrp. The synthesis of beta- mediated by this mitochondrial tRNATrp was significantly reduced when the translation system was prepared from an isogenic [psi-] strain. Evidence for a nuclear-coded tRNA, also able to suppress the beta-globin mRNA UGA terminator in [psi+] but not [psi-] lysates, was also obtained. The presence of these endogenous UGA suppressor activities in the yeast cell-free system should allow successful in vitro translation of mitochondrial mRNAs.     

Fine structural analysis of membrane changes during reaggregation culture of chick optic tectum

Thin section and freeze-fracture electron microscopy have been used to characterize the changes in membrane morphology of reaggregating cultures of chick optic tectum. The cells are rounded and freely dispersed at 0 hr after dissociation. Between 2 and 6 hr the cells become closely apposed on all sides by other cells and form small aggregates. At this time punta adhaerentia junctions and focal densities are seen along the membranes of neighboring cells. Between 1 and 5 days in vitro (DIV) neurites containing growth cone regions are present. At 5 DIV the first synaptic contacts are observed. Between 7 and 14 DIV, the number of synaptic contacts increase and fewer growth cone regions are observed. As early as 7 DIV profiles are observed which strongly resemble both astrocytic and oligodendroglial cell somata and processes. Freeze-fracture analysis of aggregates at 0–4 hr reveals a sparse particle distribution on the P and E faces of apposed cells. By 1 DIV small clusters of loosely packed, large sized particles are seen on the P face of apposed cell membranes which may represent junctional contacts. Apparent coated vesicle fusion sites are common on the P face at 1–2 DIV. By 7 DIV, E face particle arrays are seen on cell bodies and neurites which correspond to specializations characteristic of excitatory synaptic junctions. By 8–10 DIV particle arrays are seen on the P face of post-synaptic membrane which may represent inhibitory synaptic contacts. Other types of particle specializations seen in freeze-fracture replicas include: specializations characteristic of gap junctions between cells and orthogonal assemblies of particles thought to be characteristic of astrocytes.     

Identification and ontogenesis of beta-adrenergic receptors in fetal and neonatal rabbit myocardium

In adult animals, beta-adrenergic receptors are involved in the regulation of myocardial contractility and heart rate. Their properties in the fetal and early neonatal period have not been adequately investigated. We have directly characterized beta-adrenergic receptors in rabbit fetal and neonatal myocardial membranes by a radioligand binding assay utilizing 125I-hydroxybenzylpindolol (I-HYP), a potent, non-specific, beta-adrenergic antagonist, I-HYP binding sites were detected as early as the 21st day of gestation (term 31 days). The binding was rapid, saturable and reversible. The dissociation constant, KD, as determined by Scatchard plots, ranged from 30 pM to 50 pM. There was a progressive increase in the density of the receptor sites with advancing gestation. Competition studies with beta-agonists and antagonists showed that the order of potency in inhibiting I-HYP binding was consistent with a beta 1-subtype of beta-adrenergic receptors. We conclude that the progressive increase in density of beta-adrenergic receptors in rabbit fetal myocardium parallels similar maturational processes occurring in other tissues with advancing gestation. It may also account for the reported developmental changes in fetal myocardial contractility.     

Bromoacetylcholine as an affinity label of the acetylcholine receptor from Torpedo californica

Bromoacetyl[methyl-³H]choline is a highly specific label for the reduced acetylcholine binding site on the acetylcholine receptor from $\text{Torpedo californica}$. Only one of two binding sites per receptor monomer is susceptible to labeling. The labeled site is on the α chain of the receptor.     

Career preferences of doctors graduating in 1974.

Questionnaires were sent to all 2348 doctors who had graduated from medical schools in England, Scotland, and Wales in 1974 asking about their career preferences. Most were in their second preregistration post, and the response rate was 86-1%. The most popular first choice of career was general practice (665 of the responders; 32-9%), followed by medicine (454; 22-5%), surgery (321; 15-9%), and paediatrics (129; 6-4%). Only 507 of the responders (25-1%), however, stated that their preference was "definite". First choices differed widely between men and women graduates and between graduates of different medical schools.     

The binding of lanthanides to non-immune rabbit immunoglobulin G and its fragments.

The binding of Gd(III) to rabbit IgG (immunoglobulin G) and the Fab (N-terminal half of heavy and light chain), (Bab')2 (N-terminal half of heavy and light chains joined by inter-chain disulphide bond), Fc (C-terminal half of heavy-chain dimer)and pFc' (C-terminal quarter of heavy-chain dimer) fragments was demonstrated by measurements of the enhancement of the solvent-water proton relaxation rates in the appropriate Gd(III) solutions. At pH 5.5 there are six specific Gd(III)-binding sites on the IgG. These six sites can be divided into two classes; two very 'tight' sites on the Fc fragment (Kd approx. 5 muM) and two weaker sites on each Fab region (Kd approx. 140 muM). Ca(II) does not apparently compete for these metal-binding sites. The metal-binding parameters for IgG can be explained as the sum of the metal binding to the isolated Fab and Fc fragments, suggesting that there is no apparent interaction between the Fab and Fc regions in the IgG molecule. The binding of Gd(III) to Fab and Fc fragments was also monitored by measuring changes in the electron-spin-resonance spectrum of Gd(III) in the presence of each fragment and also by monitoring the effects of Gd(III) on the protein fluorescence at 340 nm (excitation 295 nm). The fluorescence of Tb(III) solutions of 545 nm (excitation 295 nm) is enhanced slightly on addition of Fab or Fc.