Effectiveness of acylation of protein amino groups with fatty acid chlorides in a system of reversed micelles of aerosol OT in octane

The characteristics of water-soluble enzyme (alpha-chymotrypsin) modification with [3H] palmitoyl chloride in the reversed Aerosol OT micelles in octane were determined. The degree of enzyme modification depends on the molar ratio [palmitoyl chloride]/[protein]. The modification reaction is characterized by the wide pH-optimum range and proceeds with high speed.     

A new strategy for the study of oligomeric enzymes: gamma-glutamyltransferase in reversed micelles of surfactants in organic solvents

A heterodimeric enzyme (gamma-glutamyltransferase) was studied in the reversed micellar medium of Aerosol OT (AOT) in octane. As was shown earlier, the size (radius) of inner cavity of the AOT-reversed micelles is determined by their hydration degree, i.e., [H2O]/[AOT] molar ratio, in the system. Owing to this, the dependence of hydrolytic, transpeptidation and autotranspeptidation activities of the enzyme on the hydration degree was investigated using L- and D-isomers of gamma-glutamyl(3-carboxy-4-nitro)anilide and glycylglycine as substrates. For all of the reaction types, the observed dependences are curves with three optima. The optima are found at the hydration degrees, [H2O]/[AOT] = 11, 17 and 26 when the inner cavity radii of reversed micelles are equal to the size of light (Mr 21,000) and heavy (Mr 54,000) subunits of gamma-glutamyltransferase, and to their dimer (Mr 75,000), respectively. Ultracentrifugation experiments showed that a change of the hydration degree resulted in a reversible dissociation of the enzyme to light and heavy subunits. The separation of light and heavy subunits of gamma-glutamyltransferase formed in reversed micelles was carried out and their catalytic properties were studied. The two subunits catalyze hydrolysis and transpeptidation reactions; autotranspeptidation reaction is detected only in the case of the heavy subunit. These findings imply that the reversed micelles of surfactants in organic solvents function as the matrices with adjustable size permitting to regulate the supramolecular structure and the catalytic activity of oligomeric enzymes.     

A new way in homogeneous immunoassay: reversed micellar systems as a medium for analysis

Possibilities of a new principle for the homogeneous enzyme immunoassay utilizing the systems of surfactant reversed micelles in organic solvents have been demonstrated taking thyroxine determination as an example. The catalytic activity of an enzyme, solubilized in such systems, is determined by the ratio of geometric dimensions of the micellar matrice and the enzyme molecule. The addition of antibodies against thyroxine to the peroxidase-thyroxine conjugate, solubilized in the system of reversed micelles of aerosol OT in octane, leads to the formation of the immune complex whose size differs substantially from that of the initial enzyme-antigen conjugate. This induces changes in the peroxidase catalytic activity. The addition of free thyroxine to the system stimulates the conjugate release from the immune complex and, consequently, the reduction of the peroxidase activity to the initial level. Sensitivity of the analysis in reversed micellar systems can be regulated by changing the surfactant hydration degree. Substances of different nature (both hydrophobic and hydrophylic) can be solubilized in reverse micellar systems under standard conditions, which allows determination of water insoluble antigens.     

Water Balance in Cucumis Plants, Measured by Nuclear Magnetic Resonance, I

In this paper we demonstrate the study of plant water balanceby the non-invasive measurement of tissue water content andwater flow using proton nuclear magnetic resonance (NMR). Sapvelocity and flux were measured independently in the presenceof an excess of stationary tissue water. The instrumentationdescribed allows automated and unattended measurement of flow-and water content-variables in a well-defined region of theplant over periods of several days, with a time resolution betweensuccessive measurements of c. 5 s. Using this apparatus theeffect of changes in light intensity (day/night rhythm) andrelative humidity on stem tissue water content as well as onthe velocity and flux of xylem sap in the stem were investigatedin a cucumber plant. The results are in agreement with predictionsfrom a simple model for plant water balance, which is basedon water potential, flow rate and resistance to flow. As longas only transpiration is varied, flow rate and water content(or potential) are affected in opposite ways as demonstratedin this paper. In contrast, the model predicts that changesin uptake (resulting from changes in, for example, root resistance)will induce changes in water content and flow in the same direction.An experimental verification of this prediction is given ina subsequent paper, where, in addition, the NMR results arecompared to those obtained with a dendrometer. Key words: Water balance model, Cucumis sativus L., flow, water content, NMR, water balance measurement     

Artificial oligomerization of enzymes--a new way of regulating their catalytic activity in reversed micellar systems]

Comparative studies were carried out in the catalytic activity regulation of native alpha-chymotrypsin and its artificially produced hexameric form as an example of non-dissociating oligomeric enzyme (covalently cross-linked by means of succinimidyl-3-(2-pyridylthiopropionate] in the Aerosol OT reversed micelles in octane. Native (monomeric) alpha-chymotrypsin exhibits maximal catalytic activity in the reversed micelles at the hydration degree w0 = 10, when the radius of the micelle inner cavity is equal to the radius of the alpha-chymotrypsin globule. For the alpha-chymotrypsin hexamer, optimum is observed at w0 = 45, with the inner micellar cavity radius (r = 68 A) being approximately equal to the radius of the sphere surrounding the octahedral combination of the six monomeric alpha-chymotrypsin molecules (r = 61 A). Thus, construction of the corresponding oligomeric structures is made easy, with the optimal catalytic activity in a preset range of the hydration degrees.     

TASTE GROUP THRESHOLDS AND SENSORY EVALUATION OF SPANISH WINE VINEGARS

Wine vinegar is a product obtained from wine acidification which contains at least 5% by wt. of acetic acid, in general without any additives or colorings.
Aspects studied in this work include: the determination of the taste group thresholds (geometric mean of the individual best-estimate thresholds "BET") of two different acids (citric and acetic acids) in aqueous solution and spanish vinegars produced from table and sherry wines. The results obtained suggest that wine vinegar can be considered something more than just an acidulant agent.
In order to evaluate differences among wine vinegars, discriminant tests for twenty-five spanish vinegars (sherry, table and flavored vinegars) were applied. Six of the twelve attributes freely chosen by assessors allowed grouping of the spanish wine vinegars according to their sensory aspects.     

Arginine deprivation in KB cells: I. Effect on cell cycle progress

When exponentially growing KB cells were deprived of arginine, cell multiplication ceased after 12 h but viability was maintained throughout the experimental period (42-48 h). Although tritiated thymidine ([(3)H]TdR) incorporation into acid-insoluble material declined to 5 percent of the initial rate, the fraction of cells engaged in DNA synthesis, determined by autoradiography, remained constant throughout the starvation period and approximately equal to the synthesizing fraction in exponentially growing controls (40 percent). Continous [(3)H]TdR-labeling indicated that 80 percent of the arginine-starved cells incorporated (3)H at some time during a 48-h deprivation period. Thus, some cells ceased DNA synthesis, whereas some initially nonsynthesizing cells initiated DNA synthesis during starvation. Flow microfluorometric profiles of distribution of cellular DNA contents at the end of the starvation period indicated that essentially no cells had a 4c or G2 complement. If arginine was restored after 30 h of starvation, cultures resumed active, largely asynchronous division after a 16-h lag. Autoradiographs of metaphase figures from cultures continuously labeled with [(3)H]TdR after restoration indicated that all cells in the culture underwent DNA synthesis before dividing. It was concluded that the majority of cells in arginine-starved cultures are arrested in neither a normal G1 nor G2. It is proposed that for an exponential culture, i.e. from most positions in the cell cycle, inhibition of cell growth after arginine with withdrawal centers on the ability of cells to complete replication of their DNA.     

Subunit separation in reversed micelle system reveals the existence of active centers both on light and heavy gamma-glutamyltransferase subunits.

Regulation of supra-macromolecular composition and catalytic activity of a heterodimeric enzyme, gamma-glutamyltransferase, in the system of Aerosol OT (sodium bis(2-ethylhexyl) sulfosuccinate) reversed micelles in octane were studied. Variation of the surfactant hydration degree (parameter, determining dimensions of the polar inner cavity of the micelle) causes a reversible dissociation of the enzyme to light and heavy subunits. Both enzyme subunits possess catalytic activity. The light and heavy subunits of the enzyme were separated on a preparative scale in a reversed micelle system using ultracentrifugation. The active centers of gamma-glutamyltransferase were studied using its irreversible inhibitor--AT-125 (L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid). Separation of the gamma-glutamyltransferase subunits results in the 'opening' of a new active center located at the heavy subunit. In the dimer form of the enzyme this center is masked and it is not accessible to both substrate and inhibitor molecules.     

Modulation of membrane activity of an enzyme in reversed micelle system with a change of media pH (using alkaline phosphatase as an example)]

Regulation of the membrane active properties of alkaline phosphatase from calf intestinal mucosa in reversed micelles of Aerosol OT (AOT) in octane was studied. The dependence of the catalytic activity on the surfactant concentration at the constant hydration degree, which characterises the membrane activity of enzymes, is modulated through pH variation. The variation may cause conformational changes of the protein molecule, resulting in exposition of anchor groups which provide the interaction of the enzyme with the micellar matrix.     

DNA interpolyelectrolyte complexes as a tool for efficient cell transformation.

A tool was developed for enhancement of plasmid penetration into an intact cell, based on increasing DNA hydrophobicity via inclusion into a soluble interpolyelectrolyte complex (IPC) with polycations. The characteristics of formation of DNA IPC with synthetic polycations [poly(N-ethyl-4-vinylpyridinium)bromide (PVP) and PVP modified with 3% of N-cetyl-4-vinylpyridinium units (PVP-C)] were studied using ultracentrifugation and polyacrylamide gel electrophoresis methods. The conditions were established under which the mixing of DNA and polycation aqueous solutions results in the self-assembly of soluble IPC species. Incorporation of DNA into IPC results in the enhancement of DNA binding with isolated Bacillus subtilis membranes. A considerable increase in the efficiency of transformation of B. subtilis cells with pBC16 plasmid resulted from incorporation of the plasmid into the IPC with PVP and CVP.