Biomedical subjects
Publications and source records attributed to Y Nosoh.
Studies with small, monomeric proteins indicate that, to some extent, the effects of amino acid substitutions can be predicted. However, conformational and other changes may complicate the prediction. Site-directed mutagenesis is leading both to a better understanding of protein stability and to the production of more stable proteins.
Acetamidination of pig heart lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) with ethyl acetimidate resulted in an increase of thermostability, and covalent bridge formation between pairs of lysine residues is observed. Guanidination with O-methylisourea of the enzyme also increases the thermostability, but such a bridge seems not to be formed. Increased thermostability of guanidinated enzyme is considered to be due to the shift of the pK values of the lysine residues from 10.5 to 12.5 after guanidination. Modification experiments with carbodiimide reveals that the enzyme contains 4.6 pairs of neighboring lysine and carboxyl residues per subunit, and amide bonding between 3.2 pairs results in an increase of thermostability. Guanidination of 4.6 Lys/subunit of the enzyme yields an enzyme derivative with considerably increased thermostability. Salt bridge formation between the 4.6 pairs of neighboring carboxyl and guanidinated lysine residues per subunit might make a major contribution to the increased thermostability of the guanidinated enzyme.
The activity of glutamine synthetase [EC 6.3.2.1] from Bacillus stearothermophilus decreased slightly on modification with ethyl acetimidate. Acetamidination of 25--26 of the 2 epsilon-amino groups/subunit of the enzyme affected the maximum velocity, but not the Michaelis constant. The thermostability of the enzyme was considerably increased on acetamidination. Acetamidination of the enzyme did not affect the circular dichroism, the tryptophan fluorescence or the quenching effects of KI and acrylamide on the tryptophan emission. The fluorescence spectrum of p-toluidinylnaphthalene sulfonate bound to the enzyme changed on acetamidination.
Glutamine synthetase [EC 6.3.2.1] from Bacillus stearothermophilus was modified with diethyl malonimidate (DEM), dimethyl adipimidate (DMA), and dimethyl suberimidate (DMS). DMA modified most epsilon-amino groups. On modification with DMA, formation of 3 to 4 cross-links/subunit resulted in a large increase in thermostability. The activity, allosteric properties and fluorescence spectrum of the enzyme were not changed on cross-linking. The SDS-polyacrylamide gel electrophoretic profiles of DEM-, DMA-, and DMS-modified enzymes suggested that the interaction berween six subunits in each of the two hexagonal rings of the protein are heterologous and are different from those between the piled subunits on different rings.
Thiobacillus thiooxidans was acidostable even in the absence of its respiratory substrate, elementary sulfur. This suggests that the acidostability of the bacterium was enery-independent. The organism was subjected to osmotic shock with 0.75 M sucrose at 0 degrees C and then treated with snail intestinal juice in the presence of 0.3 M sucrose. The decrease in the optical density of the sample thus prepared on dilution with deionized water and electron microscopic observation of the sample showed that spheroplasts were formed from the bacterium by this procedure. Spheroplasts were able to respire sulfur and their respiratory activity was acidostable. Spheroplasts, when treated with Nagase, proteolytic enzyme, lost their acidostability, and some protein components disappeared from the membrane fraction. This suggests that the acidostability of the bacterium may be related to protein conponents of the membrane.
Bacillus stearothermophilus ATCC 12016 was unable to grow at temperatures below 40 degrees C. On incubating the bacteria at the temperatures, ATP in cells disappeared, ADP was accumulated and ATPase (EC 3.6.1.3) was inactivated. When the purified ATPase was incubated at the temperatures for 1 h with 0.17 mM ADP in the presence of MgCl2, the enzyme was completely inactivated. The inactivated enzyme was reactivated on dilution or dialysis or on warming at 65 degrees C. During the incubation of the enzyme sample, the absorbance spectrum of the enzyme changed. On further incubating the sample over 1.5 h, the second step of spectral change occurred together with the change of the circular dichrosim and the dissociation into a lower molecular weight species of the protein. When the enzyme was treated with ADP-MgCl2 at 65 degrees C, the inactivation and conformational change of the enzyme was not observed.
A sulfate-reducing bacterium, a strain of Desulfovibrio vulgaris, when lactate or pyruvate was supplied as an electron and energy source, reduced acetylene only in the presence of sulfate. Acetylene reduction started after a lag of 1 h. H2 which acts as an electron and energy source for sulfate reduction was unable to reduce acetylene even in the presence of sulfate. It was suggested, from the measurements of the amounts of adenine nucleotides of the bacterium incubated under various conditions, that the bacterium is able to reduce acetylene at a high ATP level or a high ATP/ADP ratio.
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The amino acid incorporation and alpha-amino-isobutyric acid (AIB) uptake of an alkalophilic Bacillus grown at pH 8.2 (the pH 8-bacteria) were much less pH dependent (less alkalophilic) than those of the organisms grown at pH 10.0 (the pH 10-bacteria), respectively. The rate of AIB uptake of the pH 10-bacteria was almost the same as that of the pH 8-bacteria, while the rate of amino acid incorporation of the pH 10-bacteria was higher than that of the pH 8-bacteria in alkaline environments. The colloidal titration with clupein showed that the amount of negative charge on the pH 10-bacteria was greater than that of the pH 8-bacteria in alkaline environments. Considerable difference in protein composition was observed between the membranes of the pH 8- and 10-bacteria while no difference was observed in phospholipid composition.
One of the obligate thermophilic bacteria, Bacillus stearothermophilus, was unable to grow at temperatures below 35 degrees C. About 80% of the population in the bacterial culture died at the temperatures, and the same extent of loss in either of the activities of oxygen consumption or synthesis of protein or nucleic acid of the organisms was observed. With the progress of death of the organisms, reduced nicotinamide-adenine dinucleotide came to be oxidized by the organisms, enzymes such as fructose-1,6-diphosphate aldolase, when the organisms were washed with phosphate buffer, were leaked out of the organisms, and an increasing amount of ribonucleoprotein was released into the culture medium. The change of the membrane state was then suggested to be one of the possible causes for the death of the organisms at the temperatures.
Threonine deaminase [EC 4.2.1.16] was highly purified from Bacillus stearothermophilus. The enzyme exhibited maximum activity at 65 degrees and at pH 9.2--9.6. It was inactivated on dilution and on storage at 4 degrees, but was protected by egg albumin. The enzyme was labile at 65 degrees, but became stable in the presence of egg albumin and isoleucine at pH 7.0. The substrate saturation curve for the enzyme reaction at 40 or 65 degrees was hyperbolic, but in the presence of isoleucine, the curve became sigmoidal (n = 2). The enzyme was more sensitive to isoleucine at 40 degrees than at 65 degrees, while valine slightly inhibited the enzyme at both 40 and 65 degrees. Inhibition of the enzyme by isoleucine was antagonized by valine at 40 and 65 degrees. These properties were essentially similar to those of the enzymes from mesophilic and thermophilic bacteria. The enzyme existed in two forms with different molecular sizes, 1.5-5 X 10(6) and 2 X 10(5) daltons, at pH 7.0 and at temperatures below 40 degrees. The larger component disaggregated into the small one at pH 8.5 or above, at temperatures above 50 degrees or in the presence of isoleucine and valine.
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The SH groups of glutamine synthetase [EC 6.3.1.2] from Bacillus stearothermophilus were modified with 5, 5'-dithiobis(2-nitrobenzoic acid) in order to determine the number of SH groups in the molecule as well as the effect of the modification on the enzyme activity. Three SH groups per subunit were detected after complete denaturation of the enzyme with 6 M urea, one of which was essential for the enzyme activity in view of its reactivity with 5, 5'-dithiobis(2-nitrobenzoic acid) on addition of MgCl2 with loss of the activity. The CD spectra of the modified enzyme in the near ultraviolet region changed from that of the native enzyme, indicating that aromatic amino acid residues were affected by modification of the SH group. The fluorescence derived from tryptophanyl residue(s) was quenched depending on the extent of modification of the SH group, suggesting that the tryptophanyl residue(s) was located in the proximity of the SH group. The thermostability of the enzyme was remarkably decreased by modification of the SH group.