Phosphorylation of Escherichia coli enolase

In vivo labeling of Escherichia coli JA200 pLC 11-8 resulted in 32P incorporation into enolase as demonstrated by immunoaffinity chromatography and electrophoresis followed by autoradiography. Complete acid hydrolysis, followed by thin layer chromatography was employed for determination of the phosphoamino acid residue. Comparison with phosphoamino acid standards resulted in the identification of a labeled residue corresponding to phosphoserine. In vitro labeling of cell extracts from glucose and acetate grown cells resulted in differential labeling of enolase. When specific radioactivities of in vivo labeled enolase were compared, 7 times more label was incorporated at late log phase in glucose grown cells than in late log acetate grown cells. At stationary phase, only 2.5 times more label was incorporated into glucose compared to acetate. When 32P-labeled enolase from glucose grown cells was subjected to treatment with potato acid phosphatase, dephosphorylation of the enzyme could be observed. Monitoring enzyme activity during the acid phosphatase treatment revealed a 70% decrease for the forward enzyme reaction, and a 3-fold increase, followed by a gradual decrease to almost zero, for the reverse enzyme reaction. Complete reversal of the changes in activity was possible by adding an aliquot of partially purified enolase kinase plus ATP.     

Elastolytic activity of Pseudomonas aeruginosa elastase

Elastolysis of insoluble elastin by Pseudomonas aeruginosa elastase was found to be less specific (higher apparent Km value) but more active (higher activity) than with pancreatic elastase. Furthermore, pancreatic and P. aeruginosa elastases act synergistically during the initial stages of elastolysis. After extensive hydrolysis, the size distribution of digestion products was lower with P. aeruginosa than with pancreatic elastase. The higher extent of hydrolysis may be explained by the fact that, if pancreatic elastase needs at least six sub-sites for activity, P. aeruginosa elastase may hydrolyse tetrapeptides such as tetraalanine, or synthetic substrates such as furylacryloyltripeptides FA-X-Leu-Y, X and Y being Gly and/or Ala.     

Accelerated transbilayer movement of phosphatidylcholine in sickled erythrocytes. A reversible process

The transbilayer mobility of phosphatidylcholine (PC) molecules in the membrane of homozygous reversible sickle cells (RSCs) was studied using a PC-specific exchange protein from beef liver. In deoxygenated RSCs, all of the PC present in the membrane of the intact cell is rapidly available for exchange, mediated by this protein. Since a substantial amount of the PC is present in the inner membrane leaflet of these cells, this observation implies that the PC molecules in their membranes do experience rapid transbilayer movements. To determine the actual rate of transbilayer movement of the PC, radioactive PC was introduced into the outer monolayer of oxygenated RSCs using the PC-specific exchange protein. Subsequently, the cells were incubated at 37 degrees C under oxy- and deoxygenating conditions to enable the PC to equilibrate within the bilayer. At various time intervals, samples were taken and treated with phospholipase A2, which selectively degrades the PC in the outer monolayer. Analysis of the specific radioactivities of the lyso-PC thus produced, as well as of the residual PC, enabled us to follow the fate of the radioactive PC previously introduced into the outer membrane layer. The half-time value for transbilayer equilibration of the PC in deoxygenated RSCs was determined to be 3.5 h, which is about four times lower than that for oxygenated RSCs. This increased transbilayer mobility of PC, observed in deoxygenated RSCs, is immediately restored to the normal low rate upon reoxygenation of the cells, indicating a complete reversibility of this phenomenon.     

A calorimetric study of a polymer–monomer complex formed by polyuridylic acid 3′,5′-cyclic AMP

The existence of a soluble complex formed by polyuridylic acid (poly (U)) and 3′,5′-cyclic AMP (cAMP) is demonstrated by u.v. extinction vs. temperature curves, optical rotation, equilibrium dialysis, and reaction calorimetry. The complex hasthe stoichiometry of 2 poly (U)-cAMP and its formation is accompanied by an enthalpy change of ?13.0 kcal/mole of base triplet. The introuction of an empirical factor α in the equations given by Damle² and Crothers² leads to the evolution of a ΔH value of ?13.4 keal/mole. The parameter α is considered as a correction factor for the concentration dependence of the binding process. There is no relation between α and the reduction of monomer activity due to self-association of monomers. The study of the binding process at several temperatures showed that the cooperativity parameter, σ, is independent of temperature and its value of 6.5 × 10^?3 is in good agreement with σ = 5 × 10^?3 for the poly (U)·poly(A) system.³     

The cationic composition of incubated cerebral cortex slices

1. A new type of cutting table is described. It makes use of the elastic properties of a nylon thread, 0.08 mm thick, in which longitudinal vibrations greatly increase its ability to cut through soft tissue. Two slices of cerebral cortex may thus be obtained within 3-4 min after the death of an animal. 2. The extent of the swelling of a brain slice, as well as the ionic shift, is directly related to the amount of oxygen in the incubating fluid. Under the best conditions of oxygen supply, the swelling of a first slice was close to 12.2 +/- 4.3 per cent after 5 hr of incubation. The corresponding values for the Na+ and K+ contents were respectively 119.3 +/- 6.9 and 70.9 +/- 5.0 microequiv./g of final fresh weight (P2). 3. Incubation in complete anoxia leads to a considerable shift in the cation content of the slice, which acquires a composition close to that of the incubating fluid. This suggests that a large part of the cell population is still metabolically active when oxygen is present. 4. The inulin space represents 47.1 +/- 5.8 per cent of the initial fresh weight. It is independent of the amount of fluid taken up by the slice as well as of anoxia. 5. The cation content of the non-inulin space, calculated by assuming that the inulin space has the same composition as the incubation medium, was 77.7 microequiv./ml and 160.3 microequiv./ml for Na+ and K+ respectively. 6. The meaning of the inulin space, as well as the physico-chemical state of the cations in the slice, are discussed.     

Mode of action of an oleophilic nutrient in enhancing 14C-hexadecane mineralization by a marine bacterial community

Hexadecane mineralization by a bacterial consortium isolated from a non heavily contaminated coastal seawater was largely increased by an enrichment with Inipol EAP 22 (10% w/w). In the conditions of the experiment, the efficiency of this oleophilic nutrient is linked to the organic N and P sources (urea and lauryl-phosphate) and, to a lesser extent, to the presence of a readily available carbon source (oleic acid). Otherwise, the micro emulsion nature of the formulation and its physical properties seem to play a major role in accelerating hexadecane mineralization.     

Partial Purification and Immunocytocharacterization of the Plasma Membrane ATPase of Jerusalem artichoke (Helianthus tuberosus L.) Tubers in Relation to Dormancy

Plasmalemma ATPase from Jerusalem artichoke tubers was studiedin relation to the dormancy of tubers. After partial purification,one peptide of 110 kDa appeared on SDS PAGE electrophoresisfrom dormant and non-dormant materials. ATPase specific activitywas twice higher on dormant material in the crude and solubilizedfractions, but was the same in both materials after partialpurification. Immunolabeling of this enzyme was made using aspecific antibody raised against the C terminal portion of theH⁺-ATPase from Arabidopsis thaliana. Immunolabeling was morepronounced in dormant material, in vitro and in situ. Severalworks had shown that the C terminal part of the enzyme couldbe involved in its regulation. The results presented are discussedin relation to the hypothesis according to which an internaleffector could modulated the plasmalemma ATPase activity, duringdormancy breaking. (Received October 25, 1993; Accepted September 6, 1994)     

The making of the minibody: An engineered β-protein for the display of conformationally constrained peptides

Conformationally constraining selectable peptides onto a suitable scaffold that enables their conformation to be predicted or readily determined by experimental techniques would considerably boost drug discovery process by reducing the gap between the discovery of a peptide lead and the design of a peptidomimetic with a more desirable pharmacological profile. With this in mind, we designed the minibody, a 61-residue β-protein aimed at retaining some desirable features of immunogloblin variable domains, such as tolerance to sequence variability in selected regions of the protein and predictability of main chain conformation of the same regions, based on the ‘canonical structures’ model. To test the ability of the minibody scaffold to support functional sites we also designed a metal binding version of the protein by suitably choosing the sequences of its loops. The minibody was produced both by chemical syntyhesis and expression in E. coli and charactgerized by size exclusion chromatography, UV CD (circular dichroism) spectroscopy and metal binding activity. All our data supported the model, but a more detailed structural characterization of the molecule was impaired by its low soubility. We were able to overcome this problem both by further; mutagenesis of the framework and by addition of a solublizing motif. The minibody is being used to select constrained human IL-6 peptidic ligands from a library displayed on the surface of the f1 bacteriophage.