On the mechanisms of oligopeptide reactions in solution and clay dispersion.

Mechanisms of the reactions of representative dipeptides (Gly2, Gly-Ala), oligopeptides (Gly3, Gly4) and the polypeptide (poly-Gly)n) in solution and clay suspensions at 85 degrees C were investigated. The reaction products and their yields were analysed and determined by means of HPLC. Interestingly, hydrolysis, where water molecules act as the reactant, was not the main reaction, even for oligopeptides. Formation of cyclic dipeptides prevailed in the reactions of dimers as well as oligopeptides. The breakdown of oligopeptide molecules proceeded via an intramolecular cyclization reaction. For example, the reaction of Gly3 led to the formation of equal amounts of cyclic dipeptide, c(Gly)2 and Gly. The presence of clay (montmorillonite) significantly increased yields in the reactions of dipeptides but it did not have much effect on the reactions of oligopeptides. However, an opposite effect of clay, protection of poly(Gly)n against decomposition, was proven.     

Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae. Roles of Arg234 and Arg242 revealed by sequence analysis and site-directed mutagenesis.

Glycogen phosphorylases (GPs) constitute a family of widely spread catabolic alpha1,4-glucosyltransferases that are active as dimers of two identical, pyridoxal 5'-phosphate-containing subunits. In GP from Corynebacterium callunae, physiological concentrations of phosphate are required to inhibit dissociation of protomers and cause a 100-fold increase in kinetic stability of the functional quarternary structure. To examine interactions involved in this large stabilization, we have cloned and sequenced the coding gene and have expressed fully active C. callunae GP in Escherichia coli. By comparing multiple sequence alignment to structure-function assignments for regulated and nonregulated GPs that are stable in the absence of phosphate, we have scrutinized the primary structure of C. callunae enzyme for sequence changes possibly related to phosphate-dependent dimer stability. Location of Arg234, Arg236, and Arg242 within the predicted subunit-to-subunit contact region made these residues primary candidates for site-directed mutagenesis. Individual Arg-->Ala mutants were purified and characterized using time-dependent denaturation assays in urea and at 45 degrees C. R234A and R242A are enzymatically active dimers and in the absence of added phosphate, they display a sixfold and fourfold greater kinetic stability of quarternary interactions than the wild-type, respectively. The stabilization by 10 mm of phosphate was, however, up to 20-fold greater in the wild-type than in the two mutants. The replacement of Arg236 by Ala was functionally silent under all conditions tested. Arg234 and Arg242 thus partially destabilize the C. callunae GP dimer structure, and phosphate binding causes a change of their tertiary or quartenary contacts, likely by an allosteric mechanism, which contributes to a reduced protomer dissociation rate.     

Modified plant plasma membrane H+-ATPase with improved transport coupling efficiency identified by mutant selection in yeast

Transport across the plasma membrane is driven by an electrochemical gradient of H⁺ ions generated by the plasma membrane proton pump (H⁺-ATPase). Random mutants of Arabidopsis H⁺-ATPase AHA1 were isolated by phenotypic selection of growth of transformed yeast cells in the absence of endogenous yeast H⁺-ATPase (PMA1). A Trp-874-Leu substitution as well as a Trp-874 to Lys-935 deletion in the hydrophilic C-terminal domain of AHA1 conferred growth of yeast cells devoid of PMA1. A Trp-874-Phe substitution in AHA1 was produced by site-directed mutagenesis. The modified enzymes hydrolyzed ATP at 200–500% of wild-type level, had a sixfold increase in affinity for ATP (from 1.2 to 0.2 mM; pH 7.0), and had the acidic pH optimum shifted towards neutral pH. AHA1 did not contribute significantly to H⁺ extrusion by transformed yeast cells. The different species of aha1, however, displayed marked differences in initial rates of net H⁺ extrusion and in their ability to sustain an electrochemical H⁺ gradient. These results provide evidence that Trp-874 plays an important role in auto-inhibition of the plant H⁺-ATPase and may be involved in controlling the degree of coupling between ATP hydrolysis and H⁺ pumping. Finally, these results demonstrate the usefulness of yeast as a generalized screening tool for isolating regulatory mutants of plants transporters.     

A comparative study of the effects of iloprost and PGE1 on pulmonary arterial pressure and edema formation in the isolated perfused rat lung model

Isolated lungs from male Wistar rats (250–350 g) were perfused at a constant flow rate (10 ml/min, non -recirculating) with Krebs-Ringerbicarbonate buffer containing 4.5 % bovine serum albumin, and were ventilated at a positive pressure (60 breaths/min). Pulmonary arterial pressure and lung weight (as a measure of edema formation) were recorded continuously. After an equilibration period of 20 minutes the various test compounds were added to the perfusion fluid and experimental recording was continued for another 60 minutes.The effects of the stable PGI₂-mimetic, iloprost, of PGE₁, and of the biologically active PGE₁-metabolite, 13,14-dihydro-PGE,, were evaluated in this model (n=6). Iloprost showed slight, but not significant vasodilation; however, lung weight remained unchanged. PGE₁ and 13,14-dihydro-PGE₁ also caused slight vasodilation, but in contrast to iloprost these compounds induced distinct pulmonary edema. The lung weight gain was discernible at concentrations of 2.8 × 10^-6 mol/1 (significant at 2.8 × 10^-5 mol/l; p 0.05) and was accompanied by increases in the wet-weight to dry-weight ratios. These findings were duplicated in a second set of experiments (n = 6) from which the same results were obtained.The results indicate that at high concentrations PGE, (and 13,14-dihydro-PGE₁), but not iloprost, can induce pulmonary edema in rats probably by increasing the permeability of the pulmonary vasculature.     

Oscillations of apoplasmic K+ and H+ activities in Desmodium motorium (Houtt.) Merril. pulvini in relation to the membrane potential of motor cells and leaflet movements

The lateral leaflets of Desmodium motorium exhibit rhythmic upward and downward movements with a period in the minute range. Apoplasmic K⁺ and H⁺ activities were monitored in situ in the abaxial part of the pulvini with ion-selective microelectrodes. An extracellular electric potential was recorded simultaneously. The apoplasmic H⁺ activity of all pulvini exhibiting a regular rhythm of the extracellular electric potential oscillated with the same period between about 10 and 20 mM. The apoplasmic K⁺ activity was high when the membrane potential of the motor cells was depolarized (about 36 mV) and the cells were shrunken. In contrast, the apoplasmic K⁺ activity was low in the swollen state of the motor cells, when the membrane potential was hyperpolarized (about -136 mV). The volatile anesthetic enflurane suppressed reversibly the movement of the leaflets. The same treatment also arrested spontaneous oscillations in the apoplasmic K⁺ activity in the pulvinus. The apoplasmic K⁺ activity oscillated roughly in phase with the K⁺ activity between pH 6.6 and 6.0. Application of white light disturbed the rhythm and increased the extracellular pH. Our results indicate that the physiological mechanism that drives the lateral leaflet movements of Desmodium motorium is closely related to the osmotic motors mediating the leaf movements of Mimosa, Samanea and Phaseolus.Abbreviations E_m membrane potential - E_ex extracellular electric potential - H_ex extracellular H⁺ activity - K_ex extracellular K⁺ activity - R_ex extracellular electrical resistance B. Antkowiak was supported by the Stiftung Volkswagenwerk.     

Evidence for organic synthesis in high temperature aqueous media — Facts and prognosis

Hydrothermal systems are common along the active tectonic areas of the earth. Potential sites being studied for organic matter alteration and possible organic synthesis are spreading ridges, off-axis systems, back-arc activity, hot spots, volcanism, and subduction. Organic matter alteration, primarily reductive and generally from immature organic detritus, occurs in these high temperature and rapid fluid flow hydrothermal regimes. Hot circulating water (temperature range — warm to >400 °C) is responsible for these molecular alterations, expuslion and migration. Compounds that are obviously synthesized are minor components because they are generally masked by the pyrolysis products formed from contemporary natural organic precursors. Heterocyclic sulfur compounds have been identified in high temperature zones and hydrothermal petroleums of the Guaymas Basin vent systems. They can be interpreted as being synthesized from formaldehyde and sulfur or HS _x ^– in the hydrothermal fluids.Other products from potential synthesis reactions have not yet been found in the natural systems but are expected based on known industrial processes and inferences from experimental simulation data. Various industrial processes have been reviewed and are of relevance to hydrothermal synthesis of organic compounds. The reactivity of organic compounds in hot water (200–350 °C) has been studied in autoclaves, and supercritical water as a medium for chemistry has also been evaluated. This high temperature aqueous organic chemistry and the strong reducing conditions of the natural systems suggest this as an important route to produce organic compounds on the primitive earth. Thus a better understanding of the potential syntheses of organic compounds in hydrothermal systems will require investigations of the chemistry of condensation, autocatalysis, catalysis and hydrolysis reactions in aqueous mineral buffered systems over a range of temperatures from warm to >400 °C.Presented in part at the International Society for the Study of the Origin of Life Meeting, Barcelona, Spain, July 1993.     

The genes of the lysosomal cysteine proteinases cathepsin B, H, L, and S map to different mouse chromosomes

The mouse genes for the lysosomal cysteine proteinases cathepsin B, H, L, and S were mapped to Chromosomes (Chrs) 14, 9, 13, and 3, respectively. Two of the DNA probes used in this study detected an additional, independently segregating locus. The cathepsin B-specific probe hybridized to a locus on Chr 2, and the cathepsin H probe to a locus on the X Chr. These loci either correspond to pseudogenes or to cathepsin B- and cathepsin H-related genes. The four cysteine proteinases mapped in this study lie within known regions of conserved synteny between mouse and human chromosomes, when compared with the corresponding positions of their human homologs. Assuming that the genes of the cysteine proteinase gene family arose from a common ancestral gene, our results suggest that these four cysteine proteinases had been dispersed over different chromosomes before separation of mouse and human in evolution. Received: 22 August 1996 / Accepted: 20 November 1996