Cell Swelling Activates Phospholipase A2 in Ehrlich Ascites Tumor Cells

Ehrlich ascites tumor cells, loaded with ³H-labeled arachidonic acid and ¹⁴C-labeled stearic acid for two hours, were washed and transferred to either isotonic or hypotonic media containing BSA to scavenge the labeled fatty acids released from the cells. During the first two minutes of hypo-osmotic exposure the rate of ³H-labeled arachidonic acid release is 3.3 times higher than that observed at normal osmolality. Cell swelling also causes an increase in the production of ¹⁴C-stearic acid-labeled lysophosphatidylcholine. This indicates that a phospholipase A₂ is activated by cell swelling in the Ehrlich cells. Within the same time frame there is no swelling-induced increase in ¹⁴C-labeled stearic acid release nor in the synthesis of phosphatidyl ¹⁴C-butanol in the presence of ¹⁴C-butanol. Furthermore, U7312, an inhibitor of phospholipase C, does not affect the swelling induced release of ¹⁴C-labeled arachidonic acid. Taken together these results exclude involvement of phospholipase A₁, C and D in the swelling-induced liberation of arachidonic acid. The swelling-induced release of ³H-labeled arachidonic acid from Ehrlich cells as well as the volume regulatory response are inhibited after preincubation with GDP_βS or with AACOCF₃, an inhibitor of the 85 kDa, cytosolic phospholipase A₂. Based on these results we propose that cell swelling activates a phospholipase A₂—perhaps the cytosolic 85 kDa type—by a partly G-protein coupled process, and that this activation is essential for the subsequent volume regulatory response. Received: 23 July 1996/Revised: 17 June 1997     

Proton Inhibition of Sodium Channels: Mechanism of Gating Shifts and Reduced Conductance

Extracellular acidosis affects both permeation and gating of the expressed rat skeletal muscle Na⁺ channel (μ1). Reduction of the extracellular pH produced a progressive decrease in the maximal whole-cell conductance and a depolarizing shift in the whole-cell current-voltage relationship. A smaller depolarizing shift in the steady-state inactivation curve was observed. The pK of the reduction of maximal conductance was 6.1 over the pH range studied. An upper limit estimate of the pK of the shift of the half-activation voltage was 6.1. The relative reduction in the maximal whole-cell conductance did not change with higher [Na⁺] _o . The conductance of single fenvalerate-modified Na⁺ channels was reduced by extracellular protons. Although the single-channel conductance increased with higher [Na⁺] _o , the maximal conductances at pH 7.6, 7.0 and 6.0 did not converge at [Na⁺] _o up to 280 mm, inconsistent with a simple electrostatic effect. A model incorporating both Na⁺ and H⁺ binding in the pore and cation binding to a Gouy-Chapman surface charge provided a robust fit to the single-channel conductance data with an estimated surface charge density of 1e⁻/439?². Neither surface charge nor proton block alone suffices to explain the effects of extracellular acidosis on Na⁺ channel permeation; both effects play major roles in mediating the response to extracellular pH. Received: 14 May 1996/Revised: 19 September 1996     

Calpain: A Cytosolic Proteinase Active at the Membranes

Conclusion  Membrane association is essential for GRK function and because of this the GRKs have evolved complex regulatory mechanisms for associating with the membrane. Although the GRKs are highly homologous, each kinase utilizes a distinct mechanism for associating with the membrane, which makes it unique within the family. Initially, the carboxyl terminus of the GRKs was identified as the “membrane association domain” but recent evidence suggests that the amino terminus may also play a critical role in localizing the kinases to the membrane (Murga et al., 1996; Pitcher et al, 1996). It is within these two domains that the GRKs are most variable at the amino acid level. The GRKS exhibit an absolute requirement for phospholipids not only for association with the membrane but also for activity. There are differences in preference and binding sites for the phospholipids within the GRK family, which may reflect differential targeting of the GRKs to G protein-coupled receptors situated in different lipid environments. There are hundreds of G protein-coupled receptors and only six known GRKs. All the GRKs appear to phosphorylate the same receptor substrates in vitro (Sterne-Marr & Benovic, 1995; Premont et al., 1995). Receptor specificity, in a cellular     

Gating and Conduction Properties of a Sodium-activated Cation Channel from Lobster Olfactory Receptor Neurons

The gating and conduction properties of a channel activated by intracellular Na⁺ were studied by recording unitary currents in inside-out patches excised from lobster olfactory receptor neurons. Channel openings to a single conductance level of 104 pS occurred in bursts. The open probability of the channel increased with increasing concentrations of Na⁺. At 210 mm Na⁺, membrane depolarization increased the open probability e-fold per 36.6 mV. The distribution of channel open times could be fit by a single exponential with a time constant of 4.09 msec at −60 mV and 90 mm Na⁺. The open time constant was not affected by the concentration of Na⁺, but was increased by membrane depolarization. At 180 mm Na⁺ and −60 mV, the distribution of channel closed times could be fit by the sum of four exponentials with time constants of 0.20, 1.46, 8.92 and 69.9 msec, respectively. The three longer time constants decreased, while the shortest time constant did not vary with the concentration of Na⁺. Membrane depolarization decreased all four closed time constants. Burst duration was unaffected by the concentration of Na⁺, but was increased by membrane depolarization. Permeability for monovalent cations relative to that of Na⁺ (P _X /P _Na ), calculated from the reversal potential, was: Li⁺ (1.11) > Na⁺ (1.0) > K⁺ (0.54) > Rb⁺ (0.36) > Cs⁺ (0.20). Extracellular divalent cations (10 mm) blocked the inward Na⁺ current at −60 mV according to the following sequence: Mn²⁺ > Ca²⁺ > Sr²⁺ > Mg²⁺ > Ba²⁺. Relative permeabilities for divalent cations (P _Y /P _Na ) were Ca²⁺ (39.0) > Mg²⁺ (34.1) > Mn²⁺ (15.5) > Ba²⁺ (13.8) > Na⁺ (1.0). Both the reversal potential and the conductance determined in divalent cation-free mixtures of Na⁺ and Cs⁺ or Li⁺ were monotonic functions of the mole fraction, suggesting that the channel is a single-ion pore that behaves as a multi-ion pore when the current is carried exclusively by divalent cations. The properties of the channel are consistent with the channel playing a role in odor activation of these primary receptor neurons. Received: 17 September 1996/Revised: 15 November 1996     

Deuterostomic Actin Genes and the Definition of the Chordates: cDNA Cloning and Gene Organization for Cephalochordates and Hemichordates

The evolutionary relationship of muscle and nonmuscle actin isoforms in deuterostomia was studied by the isolation and characterization of two actin genes from the cephalochordate Branchiostoma lanceolatum and two from the hemichordate Saccoglossus kowalevskii The Branchiostoma genes specify a muscle and a nonmuscle actin type, respectively. Together with earlier results on muscle actins from vertebrates and urochordates, a N-terminal sequence signature is defined for chordate muscle actins. These diagnostic amino acid residues separate the chordates from the echinoderms and other metazoa. Although the two Saccoglossus actins characterized so far lack the diagnostic residues, in line with the presumptive phylogenetic position of hemichordates outside the chordates, a definitive conclusion can only be expected once the full complement of actin genes of Saccoglossus is established. Comparison of the intron patterns of the various deuterostomic actin genes shows that intron 330-3, which is present in all vertebrate genes, is conspicuously absent from nonvertebrate genes. The possible origin of this intron is discussed. Received: 4 July 1997 / Accepted: 29 August 1997     

Structural Comparisons of Muscle and Nonmuscle Actins Give Insights Into the Evolution of Their Functional Differences

Actin is a highly conserved protein although many isoforms exist. In vertebrates and insects the different actin isoforms can be grouped by their amino acid sequence and tissue-specific gene expression into muscle and nonmuscle actins, suggesting that the different actins may have a functional significance. We ask here whether atomic models for G- and F-actins may help to explain this functional diversity. Using a molecular graphics program we have mapped the few amino acids that differ between isoactins. A small number of residues specific for muscle actins are buried in internal positions and some present a remarkable organization. Within the molecule, the replacements observed between muscle and nonmuscle actins are often accompanied by compensatory changes. The others are dispersed on the protein surface, except for a cluster located at the N-terminus which protrudes outward. Only a few of these residues specific for muscle actins are present in known ligand binding sites except the N-terminus, which has a sequence specific for each isoactin and is directly implicated in the binding to myosin. When we simulated the replacements of side chains of residues specific for muscle actins to those specific for nonmuscle actins, the N-terminus appears to be less compact and more flexible in nonmuscle actins. This would represent the first conformational grounds for proposing that muscle and nonmuscle actins may be functionally distinguishable. The rest of the molecule is very similar or identical in all the actins, except for a possible higher internal flexibility in muscle actins. We propose that muscle actin genes have evolved from genes of nonmuscle actins by substitutions leading to some conformational changes in the protruding N-terminus and the internal dynamics of the main body of the protein. Received: 15 March 1996 / Accepted: 14 July 1996     

Phylogenetic Position of the Mitochondrion-Lacking Protozoan Trichomonas tenax,Based on Amino Acid Sequences of Elongation Factors 1α and 2

Major parts of amino-acid-coding regions of elongation factor (EF)-1α and EF-2 in Trichomonas tenax were amplified by PCR from total genomic DNA and the products were cloned into a plasmid vector, pGEM-T. The three clones from each of the products of the EF-1α and EF-2 were isolated and sequenced. The insert DNAs of the clones containing EF-1α coding regions were each 1,185 bp long with the same nucleotide sequence and contained 53.1% of G + C nucleotides. Those of the clones containing EF-2 coding regions had two different sequences; one was 2,283 bp long and the other was 2,286 bp long, and their G + C contents were 52.5 and 52.9%, respectively. The copy numbers of the EF-1α and EF-2 gene per chromosome were estimated as four and two, respectively. The deduced amino acid sequences obtained by the conceptual translation were 395 residues from EF-1α and 761 and 762 residues from the EF-2s. The sequences were aligned with the other eukaryotic and archaebacterial EF-1αs and EF-2s, respectively. The phylogenetic position of T. tenax was inferred by the maximum likelihood (ML) method using the EF-1α and EF-2 data sets. The EF-1α analysis suggested that three mitochondrion-lacking protozoa, Glugea plecoglossi, Giardia lamblia, and T. tenax, respectively, diverge in this order in the very early phase of eukaryotic evolution. The EF-2 analysis also supported the divergence of T. tenax to be immediately next to G. lamblia. Received: 15 February 1996 / Accepted: 28 June 1996     

Silica, Alumina, and Clay-Catalyzed Alanine Peptide Bond Formation

The peptide bond formation of alanine (ala), ala + glycine (gly), ala + diglycine (gly₂), and ala + gly cyclic anhydride (cyc-gly₂) in drying/wetting cycles at 80°C was studied. Silica, alumina, and representative smectites—montmorillonite and hectorite—were used as catalysts, and the dependence of reaction yields on the available amount of water in the reaction systems was evaluated. Silica and alumina catalyze the formation of oligopeptide mainly in temperature fluctuation experiments, whereas higher amounts of water in the reaction system support clay-catalyzed reactions. Silica and alumina are much more efficient for amino acid dimerization than clays. Whereas only 0.1% of ala oligomerized on hectorite and no reaction proceeded on montmorillonite, about 0.9 and 3.8% alanine converted into its dimer and cyclic anhydride on silica and alumina, respectively. Clay minerals, on the other hand, seem to more efficiently catalyze peptide chain elongation than amino acid dimerization. The reaction yields of ala-gly-gly and gly-gly-ala from ala + gly₂ and ala + cyc-gly₂ reached about 0.3% on montmorillonite and 1.0% on hectorite. The possible mechanisms of these reactions and the relevance of the results for prebiotic chemistry are discussed. Received: 15 December 1996 / Accepted: 1 May 1997     

Molecular Evolutionary Analysis of the Thiamine-Diphosphate-Dependent Enzyme,Transketolase

Members of the transketolase group of thiamine-diphosphate-dependent enzymes from 17 different organisms including mammals, yeast, bacteria, and plants have been used for phylogenetic reconstruction. Alignment of the amino acid and DNA sequences for 21 transketolase enzymes and one putative transketolase reveals a number of highly conserved regions and invariant residues that are of predicted importance for enzyme activity, based on the crystal structure of yeast transketolase. One particular sequence of 36 residues has some similarities to the nucleotide-binding motif and we designate it as the transketolase motif. We report further evidence that the recP protein from Streptococcus pneumoniae might be a transketolase and we list a number of invariant residues which might be involved in substrate binding. Phylogenies derived from the nucleotide and the amino acid sequences by various methods show a conventional clustering for mammalian, plant, and gram-negative bacterial transketolases. The branching order of the gram-positive bacteria could not be inferred reliably. The formaldehyde transketolase (sometimes known as dihydroxyacetone synthase) of the yeast Hansenula polymorpha appears to be orthologous to the mammalian enzymes but paralogous to the other yeast transketolases. The occurrence of more than one transketolase gene in some organisms is consistent with several gene duplications. The high degree of similarity in functionally important residues and the fact that the same kinetic mechanism is applicable to all characterized transketolase enzymes is consistent with the proposition that they are all derived from one common ancestral gene. Transketolase appears to be an ancient enzyme that has evolved slowly and might serve as a model for a molecular clock, at least within the mammalian clade. Received: 13 September 1995 / Accepted: 14 November 1996