An Insertion Peptide in Yeast Glycyl-tRNA Synthetase Facilitates both Productive Docking and Catalysis of Cognate tRNAs

The yeast Saccharomyces cerevisiae possesses two distinct glycyl-tRNA synthetase (GlyRS) genes: GRS1 and GRS2. GRS1 is dually functional, encoding both cytoplasmic and mitochondrial activities, while GRS2 is dysfunctional and not required for growth. The protein products of these two genes, GlyRS1 and GlyRS2, are much alike but are distinguished by an insertion peptide of GlyRS1, which is absent from GlyRS2 and other eukaryotic homologues. We show that deletion or mutation of the insertion peptide modestly impaired the enzyme''s catalytic efficiency in vitro (with a 2- to 3-fold increase in K_m and a 5- to 8-fold decrease in k_cat). Consistently, GRS2 can be conveniently converted to a functional gene via codon optimization, and the insertion peptide is dispensable for protein stability and the rescue activity of GRS1 at 30°C in vivo. A phylogenetic analysis further showed that GRS1 and GRS2 are paralogues that arose from a gene duplication event relatively recently, with GRS1 being the predecessor. These results indicate that GlyRS2 is an active enzyme essentially resembling the insertion peptide-deleted form of GlyRS1. Our study suggests that the insertion peptide represents a novel auxiliary domain, which facilitates both productive docking and catalysis of cognate tRNAs.     

Theory of filtration of mixed blood suspensions

A theory is developed for the flow of suspensions of blood cells through filters in which the properties of the cells are defined by statistical distributions. It is shown that conditions are generally transient, and computational procedures are developed to compute the pressure drop and the fraction of the pores of the filter containing cells of various types as a function of time. The computations show a large influence of very small concentrations of stiff cells which gradually collect in the filter and effectively plug the filter during the time of a typical test. It is also shown that the mean value of the resistance offered by a cell population with a limited distribution of resistances is more important than dispersion of resistances about the mean in determining the observable pressure curve. Experimental data are presented demonstrating that the drug pentoxifylline reduces the stiffness of leukocytes.     

Membrane protein phosphorylation during stomatocyte-echinocyte transformation of human erythrocytes

The normal, discoid shape of red blood cells represents an equilibrium between two opposing factors, i.e., stomatocytic and echinocytic transformations. Most stomatocytic agents were found to be inhibitors of calmodulin, a regulator of the phosphorylation of membrane proteins. We determined whether red cell shape transformations could be caused by changes in phosphorylation of membrane proteins, specifically the cAMP-dependent phosphorylation of ankyrin and band 4.1. Red blood cells were incubated with 32P and 100 microM chlorpromazine (stomatocytic transformation) or 30 mM sodium salicylate (echinocytic transformation) for various time intervals. Ghost membrane proteins were examined by polyacrylamide gel electrophoresis and autoradiography. Spectrin (beta-chain), ankyrin, band 3, band 4.1 and 4.9 were phosphorylated. No change was found in the degree and pattern of phosphorylation after stomatocytic transformation. Salicylate caused a reversible inhibition of transmembranous phosphate transport in both directions. The results indicate that the stomatocytic transformation induced by chlorpromazine and the echinocytic transformation induced by salicylate do not involve a change in phosphorylation, but that the echinocytic transformation induced by salicylate is associated with an inhibition of transmembranous transport of phosphate. Studies with salicylate suggest that the phosphorylation sites of band 3 are found mainly on the endofacial side of the membrane.     

The bulk rheology of close-packed red blood cells in shear flow

A theoretical analysis is made of the dynamical behavior and bulk rheology of close-packed red blood cell suspensions subjected to simple shear flow. The model for the polyhedral cell shapes and tank-treading membrane motion developed in the companion paper (1) is used. The flow in the thin lubricating plasma layers between cells is analyzed taking into account the mechanical properties of the membrane at the corner regions of sharp membrane curvature. This leads to predictions for the apparent viscosity as a function of hematocrit and shear rate. Good agreement with experimental results is obtained at moderate and high shear rates (above 20 s-1). At lower shear rates, a rapid rise in apparent viscosity has been found experimentally, and the mechanisms leading to this behavior are examined.     

Formation and properties of cell-size lipid bilayer vesicles

Hydration of single or mixed phospholipids or lipid protein mixtures at low ionic strength results in the formation of a population of large, solvent free, single bilayer vesicles with included volumes of up to 300 microliters/mumol lipid. Their size ranges from 0.1 to 300 microns and they can be sorted out according to size by centrifugation. When formed in distilled water their internal solution has a conductivity of 20-50 microseconds/cm-1, an osmolarity of 0.5-5 mOsM, and a density of 1.0005-1.001. The osmotic pressure produced by the internal solutes cause a surface stress of 25 dyn/cm for a 20-microns vesicle. Their elastic constant ranges from 75-150 dyn/cm. During formation they can internalize particles such as latex beads or cell nuclei. They can be impaled with microelectrodes, or patch clamped. They can also be sealed to a small Vaseline-treated hole in a thin partition between two aqueous compartments. Sealing occurs in two stages. In the first stage sealing resistance is similar to that seen with patch-clamp pipettes. In the second stage, a much tighter seal is obtained. After sealing, the smaller portion of the sealed vesicle can be selectively broken by an electric shock leaving a single membrane across the hole. The capacitance and resistance of such membranes, in the presence of 10 mM NaCl, are approximately 0.7 microF/cm2 and 10(8) omega cm2 for pure lipid vesicles. Gramicidin increases the membrane conductance and monazomycin induces voltage-dependent gating thus providing further evidence that the vesicles are bounded by a single bilayer.     

Influence of cholesterol content on red cell membrane viscoelasticity and fluidity.

The purpose of this investigation was to correlate the viscoelastic properties and lipid fluidity of the red blood cell membrane to its lipid composition. The viscoelastic properties of human red cells that had been enriched or depleted in cholesterol were determined by the micropipette technique. The lipid fluidity of the outer and inner leaflets of the erythrocyte membrane was concurrently assessed by steady state fluorescence depolarization. The elastic modulus and the viscosity moduli of the erythrocyte membrane showed no significant differences between the cholesterol-modified and the control cells. Cholesterol enrichment decreased the lipid fluidity of the outer membrane leaflet alone, and cholesterol depletion increased the fluidity mainly of the inner leaflet.