Thermodynamics of thermal unfolding of bovine apo-alpha-lactalbumin

Thermal unfolding of bovine alpha-lactalbumin in 10 mM borate buffer at pH 8.0 in the presence of 0.01-1.0 M NaCl was studied in terms of CD ellipticity. The apoprotein changes the conformation from a native-like (N) to an unfolded (U) form, which has an appreciable amount of the secondary structure but no tertiary structure, in the two-state type. Various thermodynamic parameters of the transition were analyzed. The differences in enthalpy and heat capacity between the N and U states are similar to the corresponding differences of the holoprotein obtained with the calorimetric method by Pfeil. It is shown that one Na+ binds with a binding constant larger than 10(2)-10(3) M-1 to a specific site (probably to the Ca2+-binding site) in the molecule and the bound Na+ stabilizes the N form of the apoprotein.     

Regulation of inter- and intramolecular ligation with T4 DNA ligase in the presence of polyethylene glycol.

Polyethylene glycol (PEG) stimulates ligation with T4 DNA ligase. In 10% (w/v) PEG 6,000 solutions, only intermolecular ligation is enhanced by monovalent cations, while both inter- and intramolecular ligation occur without their presence. Similar stimulation was also caused by divalent cations or polyamines in the PEG 6,000 solutions. Such properties of the ligase could be applied to control the extent of inter- and intramolecular ligation. Ligation with cations or polyamines in 10% PEG 6,000 solutions was effective for intermolecular ligation. Ligation without cations or polyamines in 6.0% to 10% PEG 6,000 solutions was effective for intramolecular ligation.     

Depth of side-chain pocket in the S2 subsite of dipeptidyl peptidase IV

Kinetic studies of pig kidney dipeptidyl peptidase IV (dipeptidyl-peptide hydrolase, EC 3.4.14.5) were carried out using substrates possessing a side-chain of different length at the P2 position (or amino-terminal position in this case) such as Lys-, Arg-, Phe-, Met-, Ser-, His-, Glu- and Gly-Pro-pNA. The hydrolytic coefficient (Kcat/Km) has determined in the order Met- greater than Glu- greater than Ser- greater than His- greater than Phe- greater than Lys- greater than Gly- greater than Arg-, indicating a gradual increase with elongation of the side-chain from 0.03 to 0.60 nm followed by a decline when side-chain length approached 0.70 nm. Thus, the most probable depth of the side-chain pocket at the S2 subsite of the enzyme is proposed to be 0.50-0.60nm.     

Equilibrium and kinetic study of sodium- and potassium-induced conformational changes of apo-alpha-lactalbumin

Equilibrium and kinetics of Na+-and K+-induced conformational changes of apo-alpha-lactalbumin were studied by means of circular dichroism. While apo-alpha-lactalbumin was considerably unfolded in the absence of Na+ or K+ in 20 mM Tris at pH 8.0 and 25 degrees, both the monovalent cations restored the tertiary structure of the protein. Apparent binding constants of Na+ and K+ to the apoprotein were estimated from the equilibria of the Na+- and K+-induced conformational changes. Based on kinetic data of the conformational changes induced by the monovalent cations, binding mechanism of the ions to the apo-protein was examined. Bound alkali-metal ions stabilize the native-like state and an activated state in the unfolding-refolding reaction of the apoprotein.     

Carbocyclic inosine as a potent anti-leishmanial agent: the metabolism and selective cytotoxic effects of carbocyclic inosine in promastigotes of Leishmania tropica and Leishmania donovani

Carbocyclic inosine is a potent inhibitor for the growth of the promastigote form of Leishmania tropica and Leishmania donovani. In culture, the EC50 values of carbocyclic inosine are 8.3 X 10(-8) and 1.3 X 10(-7) M for the promastigotes of L. tropica and L. donovani, respectively. On the other hand, it is less toxic towards mouse mammary tumor FM3A cells: the EC50 value is 2.7 X 10(-4) M. Carbocyclic inosine is metabolized by Leishmania promastigotes to give carbocyclic adenosine-5'-triphosphate (aristeromycin-5'-triphosphate) and carbocyclic guanosine-5'-triphosphate. This metabolic conversion provides a mechanism for the parasite-selective toxicity of carbocyclic inosine.     

Metal ion inactivation and chelator stimulation of Streptococcus mitis arginine aminopeptidase

Activation of Streptococcus mitis (ATTC 9811) arginine aminopeptidase resulted in removal of the metal(s) from the enzyme molecule, and the action of the heavy metal ion in the inactivation process was shown to involve formation of a chelate complex between the enzyme molecule and metal or oxidation of functional group(s) on the enzyme surface. The enzyme also underwent activation by bovine serum albumin, amino acids, phosphate, and citric acid, which are probable physiological chelators.     

Deoxyribonucleoside-triphosphate imbalance death: deoxyadenosine-induced dNTP imbalance and DNA double strand breaks in mouse FM3A cells and the mechanism of cell death

The mechanism of deoxyadenosine (dAdo)-induced death of mouse mammary tumor FM3A cells was studied. When the cells were exposed to dAdo at 3 mM, an imbalance of intracellular dNTP pool resulted: dATP concentration was elevated 100-fold and the dGTP concentration was reduced to less than 1% of the control values. The imbalance was followed by breakage of mature DNA. DNA double strand breaks were observed in the dAdo treated cells 12 hr after the administration. We assume that the double strand breaks play an important role in the process of the dAdo-mediated cell death, and that the intracellular dNTP imbalance is the trigger of these events.     

Acetylation of tropane derivatives by Datura innoxia cell cultures

Scopine, scopoline, pseudotropine as well as tropine supplied to the culture medium were converted into the corresponding acetates by cell cultures of Datura innoxia. Tropine was esterified preferentially with endogenous acetic acid, even if other organic acids in combination with tropine were supplied exogenously. When D. innoxia cell cultures were fed with tropine and tropic acid in the presence of different kinds of auxins, no alkaloidal metabolises but acetyltropine were detected in each treatment. Apart from Datura, tissue cultures induced from 15 other species belonging to 12 families were incapable of acetylating tropine.     

Theoretical analysis on relationship between the neural activity and the EEG

Firstly, a collective oscillation mode of the neural activity is derived from the neural network system by using the multicompartment equation and the projection operator technique. This technique takes into account higher order interactions among neurons. The solution of the equation gives a chain structure with an infinite number of circuit loops in which each of them is only composed of four neurons. The obtained eigenvalues are quite similar to the spectrum of frequencies of the EEG. Secondly, the time-dependent behavior of the observed EEG is simulated by starting from the elementary process of action potential trains of neurons, which includes the effect of the collective oscillation mode mentioned above. This gives a comprehensive derivation of the EEG from the neural activity of action potentials. The simulation assumes that information of the action potential trains can be transmitted to the EEG through the intermediate states of the postsynaptic potential trains and the slow waves. The paper reports that a slightly modulated activity of a relatively small amount of neurons can cause a strong influence on the shape of the global EEG and that the calculated results reproduce the characteristic features of the EEG in a rat such as the theta rhythm, the spindle wave and the arousal wave.     

A kinetic study of the alpha-keto acid dehydrogenase complexes from pig heart mitochondria.

The kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease complexes from pig heart mitochondria were studied at pH 7.5 and 25 degrees. A three-site ping-pong mechanism for the actin of both complexes was proposed on the basis of the parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvate concentration for various levels of CoA and a level of NAD+ near its Michaelis constant value. Rate equations were derived from the proposed mechanism. Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reaction are: 2-oxoglutarate, 0.220 mM; CoA, 0.025 mM; NAD+, 0.050 mM. Those of the pyruvate dehydrogenase complex are: pyruvate, 0.015 mM; CoA, 0.021 mM; NAD+, 0.079 mM. Product inhibition studies showed that succinyl-CoA or acetyl-CoA was competitive with respect to CoA, and NADH was competitive with respect to NAD+ in both overall reactions, and that succinyl-CoA or acetyl-CoA and NADH were uncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for succinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These results are consistent with the proposed mechanisms.