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Metal ion/buffer interactions. Stability of binary and ternary complexes containing 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris) and adenosine 5'-triphosphate (ATP).

The interaction of 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris) with the metal ions (M2+) Mg2+, Ca2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ was studied by potentiometry and spectrophotometry in aqueous solution (I = 0.1 or 1.0 M, KNO3, 25 degrees C). Stability constants of the M(Tris)2+ complexes were determined; those constants which were measured by both methods agreed well. Ternary complexes containing ATP4- as a second ligand were also investigated and it is shown that in the presence of Tris, mixed-ligand complexes of the type M(ATP)(Tris)2- are formed. The values for delta log KM, where delta log KM = log KM(ATP)M(ATP)Tris--log KMM(Tris), are all negative, thus indicating that the interaction of Tris with M(ATP)2- is somewhat less pronounced than with M2+. However, it should be noted that even in mixed-ligand systems complex formation with Tris may still be considerable, hence great reservations should be exercised in employing Tris as a buffer in systems which also contain metal ions. Distributions of the complex species in dependence on pH are shown for several systems, and the structures of the binary M(Tris)2- and the ternary M(ATP)(Tris)2- complexes are discussed. The participation of a Tris-hydroxo group in complex formation is, at least for the M(Tris)2- species, quite evident.

Adenosine Triphosphate

[The complexing ability of gangliosides for Ca2, I. Influence of mono- and divalent cations and of acetylcholin (author's transl)].

The binding of Ca2 to single ganglioside species (GGtet1NeuAc, GGtet2aNeuAc, GGtet 3aNeuAc), to their free, reducing sialyl-oligosaccharides and to ganglioside mixtures from chicken brain was investigated by means of ion-sensitive electrodes (potentiometry). Unlike the sialyl-oligosaccharides and free N-acetylneuraminic acid, gangliosides were found to possess two different modes of binding for Ca2, depending on the total concentration of Ca2. This was mainly indicated by a release of up to 75% of previously bound Ca2 after raising the total Ca2-concentration above a critical level of about 5--9 X 10(-5)M. Addition of acetylcholin (9mM), Li, K, and Na, respectively, caused a release of Ca2 from ganglioside-Ca2-complexes (0.1mM Ca2, 0.1mM ganglioside-NeuAc) in the sequence: acetylcholin : Li : K : Na = 4 : 3 : 1 : 1 composed on molar basis (9mM).

Acetylcholine

Secondary structure in very low density and intermediate density lipoproteins of human serum.

We have studies the secondary structures of the protein moieties of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL) of human serum by circular dichroism (CD). Two potential complications in the application of this technique to lipoproteins have been evaluated. First, using chronographic potentiometry in CD measurements of VLDL fractions of different mean particle diameters, we have analyzed statistically the CD signals in order to define the limits imposed by light scattering with respect to both particle diameter and wavelength. We found that CD measurements can be made to as low as 210 nm on particles of 520 A or smaller, and to 194 nm on particles of 450 A and below. Second, we have evaluated the CD contribution of lipid chromophores. Despite the high ratio of lipid to protein, the relative CD effect of the lipids is smaller than for low density lipoproteins (LDL). due to the extremely small ellipticity of natural VLDL triglycerides. Thus, CD measurements can be obtained with confidence on the preponderant bulk of normal VLDL. For the first time we report the CD spectra of human VLDL and IDL. In contrast with human LDL and the lipoproteins of the hypercholesterolemic rabbit, the entire CD SPECTRUM OF HUMAN VLDL shows increased ellipticity with decreasing temperature, which is completely reversible. We have found that the protein moieties of human VLDL and IDL contain substantially more helix (approximately 50%) than does that of human LDL.

Animals

[Enzymatic hydrolysis of fat in the presence of Lamblia duodenalis (in vitro)].

The influence of Lamblia on the hydrolysis of fat by lipase was studied in vitro. The hydrolysis rate of fat in the presence of live Lamblia and without them was determined colorimetrically by the quantity of the formed glycerine. In addition, the kinetics of this reaction was studied by the method of compensating potentiometry by neutralization of fat acids with alkali. The intact organisms were found to cause an inhibition of fermentative hydrolysis of fat. The importance of this fact from the point of view of interaction in the host-parasite system is discussed.

Giardia

[Comparative study of the rate of glucose and oxygen consumption by bacteria normally and under the action of low temperatures].

The effect of freezing on the rate of respiration and glucose assimilation in Escherichia coli M-17 incubated in 2 mM phosphate buffer, physiological solution, and media containing 1% and 10% glycerol was studied by potentiometry and delayed fluorescence of bacterial cells. When the cells were frozen to --70 degrees C and the rate of freezing-thawing was 10--15 degrees C per 1 min, the rate of respiration and glucose assimilation was below the stationary level, and the restoration took 20--60 min depending on the incubation medium. The low activity of the cells is attributed to the disturbed permeability of bacterial membranes, in particularly, for H+ ions.

Cell Membrane Permeability

[Role of water in the thermal instability of DNA].

We studied structural changes of DNA heated for long times at temperatures between 20 to 100 degrees C at different relative humidities (range 0--100%). DNA structure was analyzed by the following criteria: solubility changes, optical melting, ultraviolet spectroscopy, potentiometry and equilibrium dialysis. It has been found that irreversible changes in DNA secondary structure take place at around 37 degrees C, temperature optimal for the function in many living systems. These irreversible changes may be interpreted as a consequence of formation of interchain contacts (non-covalent "cross-links"). We demonstrate that the whole range of relative humidities may be divided onto three regions. For each of these regions DNA has somewhat different molecular structure and peculiarities are observed in the stabilizing and destabilizing action of water on DNA three-dimensional structure. Irreversible changes of DNA secondary structure as a function of bound water are represented by a bell-shaped curve. Similarity between this curve and a curve for the survival microorganisms is discussed.

DNA