Colicin A and colicin E1 lysis proteins differ in their dependence on secA and secY gene products.

The export of colicin A and of colicin E1 is not equally affected in both secA and secY mutants of Escherichia coli: release of colicin A occurs slowly while that of colicin E1 is blocked. Processing and functioning of Cal, the colicin A lysis protein, seem to be slightly or not at all modified in these mutants, whereas synthesis and assembly of CelA, the colicin E1 lysis protein, are highly inhibited. These variations observed in the dependence of the two lysis proteins on secA and secY gene products are interpreted as being either the cause or the consequence of the differences observed in their rate of biogenesis.

Pectin methylesterase, metal ions and plant cell-wall extension. Hydrolysis of pectin by plant cell-wall pectin methylesterase.

The hydrolysis of p-nitrophenyl acetate catalysed by pectin methylesterase is competitively inhibited by pectin and does not require metal ions to occur. The results suggest that the activastion by metal ions may be explained by assuming that they interact with the substrate rather than with the enzyme. With pectin used as substrate, metal ions are required in order to allow the hydrolysis to occur in the presence of pectin methylesterase.

Pectin methylesterase, metal ions and plant cell-wall extension. The role of metal ions in plant cell-wall extension.

The study of pectin methylesterase and wall-loosening enzyme activities in situ, as well as the estimation of the electrostatic potential of the cell wall, suggest a coherent picture of the role played by metal ions and pH in cell-wall extension. Cell-wall growth brings about a decrease of local proton concentration because the electrostatic potential difference (delta psi) of the wall decreases. This in turn activates pectin methylesterase, which restores the initial delta psi value.

Construction, expression and release of hybrid colicins.

Colicins A and E1 are two pore-forming colicins sharing homology in their C-terminal domains but not in their N-terminal or central domains. Using site-directed mutagenesis, restriction sites were inserted at the proper locations to allow recombination of these domains. Six different constructs were obtained.

Synthesis and functioning of the colicin E1 lysis protein: comparison with the colicin A lysis protein.

The colicin E1 lysis protein, CelA, was identified as a 3-kDa protein in induced cells of Escherichia coli K-12 carrying pColE1 by pulse-chase labeling with either [35S]cysteine or [3H]lysine. This 3-kDa protein was acylated, as shown by [2-3H]glycerol labeling, and seemed to correspond to the mature CelA protein. The rate of modification and processing of CelA was different from that observed for Cal, the colicin A lysis protein.

Thermodynamics of information transfer between subunits in oligomeric enzymes and kinetic cooperativity. 2. Thermodynamics of kinetic cooperativity.

The principles of structural kinetics allow one to define the thermodynamic conditions that are sufficient to generate a certain type of kinetic behavior. If subunits are loosely coupled, that is if no quaternary constraint exists between them, the kinetic behavior of the polymeric enzyme is qualitatively defined by the behavior of an ideal dimer. The nature and the extent of the kinetic cooperativity are defined by the energy of interaction, delta G rho, between two subunits.

Thermodynamics of information transfer between subunits in oligomeric enzymes and kinetic cooperativity. 1. Thermodynamics of subunit interactions, partition functions and enzyme reaction rate.

The strength of quaternary constraints between two subunits of a polymeric enzyme depends upon the number of neighboring subunits and upon whether these subunits are liganded or not. These quaternary constraints between two subunits of a complex polymeric enzyme may be expressed, however, in terms of quaternary constraints that exist within ideal dimers. The influence of quaternary constraints on the reaction rate of a complex polymeric enzyme may thus be expressed in terms of the intersubunit strain that exists within dimers.

Thermodynamics of information transfer between subunits in oligomeric enzymes and kinetic cooperativity. 3. Information transfer between the subunits of chloroplast fructose bisphosphatase.

The theory and the methods that have been described in the two preceding papers in this journal have been used to analyze the kinetic properties of chloroplast fructose bisphosphatase. The enzyme is a tetramer made up of apparently identical subunits and displays a sigmoidal kinetics with respect to its substrate, fructose bisphosphate. The free ionic species, magnesium and fructose bisphosphate bind to the enzyme and the chelate fructose-bisphosphate-magnesium does not affect the sigmoidicity of the rate curves.

Competitive inhibition of lipolytic enzymes. I. A kinetic model applicable to water-insoluble competitive inhibitors.

It is now becoming clear from the abundant lipolytic enzyme literature that any meaningful interpretation of inhibition data has to take into account the kinetics of enzyme action at the lipid/water interface. We attempt in the present paper to provide a kinetic model applicable to water-insoluble competitive inhibitors, in order to quantitatively compare the results obtained at several laboratories. We derived kinetic equations applicable to the pre-steady state as well as steady state.

Purification and reconstitution into liposomes of an integral membrane protein conferring immunity to colicin A.

The immunity protein to colicin A protects producing cells from the action of this pore-forming toxin. It is located into the cytoplasmic membrane. This protein has been 'tagged' with an epitope from the colicin A protein for which a monoclonal antibody is available.

High-level expression of the colicin A lysis protein.

Two plasmids that overproduce the colicin A lysis proteins, Cal, are described. Plasmid AT1 was constructed by a deletion in the colicin A operon, which placed the cal gene near a truncated caa gene in such a way that both gene products wer synthesized at high levels following induction. Plasmid CK4 was constructed by insertion of the cal gene downstream from the tac promoter of an expression vector.

Topology and function of the integral membrane protein conferring immunity to colicin A.

The topology of the integral membrane protein Cai (colicin A immunity protein), which is required to protect producing cells from the pore-forming colicin A, was analysed using fusions to alkaline phosphatase. The properties of these fusion proteins support the model for Cai topology previously proposed on theoretical grounds. The protein was found to contain four transmembrane sequences and its N- and C-terminal regions were found to be directed towards the cytoplasm.

Human milk bile-salt stimulated lipase: further investigations on the amino-acids residues involved in the catalytic site.

The bile-salt-stimulated lipase purified from human skim milk was modified with diisopropyl phosphofluoridate (DFP), N-ethyl-5-phenylisoxazolium-3'-sulfonate and ethoxyformic anhydride. These chemical modifications lead to the following results: (1) the inhibition of the enzyme by DFP is due to the phosphorylation of a single residue, probably a serine residue, which may represent the acylable group of the enzyme; (2) carbethoxylation of histidine residues leads to inhibition of the enzyme activity. Among the nine modified histidine residues, only one is essential for enzyme activity; (3) a free carboxyl group with a pKa of 5.

Renewal of goblet cell mucus granules during the cell migration along the crypt-villus axis in rabbit jejunum: an immunolabeling study.

A monoclonal antibody against intestinal mucins (5H7) was obtained and used immunolabel thin frozen sections and Epon-embedded sections of rabbit jejunum. It recognized a mucin oligosaccharide, the synthesis of which increased during goblet cell migration along the crypt-villus axis. During the earliest steps in their differentiation, the goblet cells located at the bottom of crypts synthesized mucins devoid of the 5H7 epitope, thus generating unlabeled granules.

Functional domains of colicin A.

A large number of mutations which introduce deletions in colicin A have been constructed. The partially deleted colicin A proteins were purified and their activity in vivo (on sensitive cells) and in vitro (in planar lipid bilayers) was assayed. The receptor-binding properties of each protein were also analysed.

The membrane channel-forming colicin A: synthesis, secretion, structure, action and immunity.

The study of colicin release from producing cells has revealed a novel mechanism of secretion. Instead of a built-in 'tag', such as a signal peptide containing information for secretion, the mechanism employs coordinate expression of a small protein which causes an increase in the envelope permeability, resulting in the release of the colicin as well as other proteins. On the other hand, the mechanism of entry of colicins into sensitive cells involves the same three stages of protein translocation that have been demonstrated for various cellular organelles.

Purification of pancreatic carboxylic-ester hydrolase by immunoaffinity and its application to the human bile-salt-stimulated lipase.

A column of immobilized antibodies directed against pure human pancreatic carboxylic (cholesterol) ester hydrolase was used to purify in a single step the enzyme from human pancreatic juice as well as carboxylic-ester hydrolases from other species (rat, dog). This immunoaffinity method was also used for the purification of the related bile-salt-stimulated lipase from the human skim milk. The enzymes were homogeneous on SDS-PAGE.

Precise localization of an overproduced periplasmic protein in Escherichia coli: use of double immuno-gold labelling.

The subcellular localization of beta-lactamase produced by a secretion-cloning vector pIN-III was studied by immunolabelling of frozen thin sections of Escherichia coli. Using double immuno-gold detections and internal reference proteins, it is shown here that beta-lactamase encoded by this vector can be exported and that its overproduction leads to aggregation within the periplasm. This aggregation induces the appearance of electron-dense areas immunolabelled by the antiserum directed against the beta-lactamase at the external side of the cytoplasmic membrane.