Jun'ichi Hase

Effect of acyl residues of hydroxamic acids on urease inhibition

Abstract 1. 1. As regards the relationship between carbon number of acyl- or p- alkoxybenzohydroxamic acids and their inhibitory powers on urease (urea amidohydrolase, EC 3.5.1.5) activity, heptylo- and caprylohydroxamic acids in the series of the former and p- methoxybenzohydroxamic acid in the series of the latter showed the maximum inhibitory power. An increase in the number of carbon atoms of the acyl- or alkoxymoieties led to a marked decrease in inhibitory power, which might be attributed to the decrease of their hydrophilic properties. 2. 2. Substitution with various groups at the meta - or para -position of benzohydroxamic acid did not affect the inhibitory power. Ortho -substituted derivatives, however, were markedly less inhibitory. These observations cannot be explained as being due to the effect of electronic polarization, but can be accounted for as being brought about by the “ortho effect”, in the sense that a steric hinderance was caused by ortho -substitution in the benzohydroxamic acid at the active site of urease. 3. 3. o- Aminobenzohydroxamic acid, a unique example among ortho -substituted derivatives, was found to be one of the most powerful inhibitors. Therefore a certain electronegatively charged group might possibly be located close to the active site of urease. However, methylation of the o- amino group of the compound reduced markedly its inhibitory power, this observation probably being attributable to the increase of steric size in the ortho -position. 4. 4. Among hydroxamic acids derived from pyridine carboxylic acid, the position of the hydroxamic acid moiety influenced significantly the inhibitory power on the urease activity. The α-amino group of hydroxamic acid derived from some α-amino acids did not affect the inhibitory power. 5. 5. Compared with various related compounds of hydroxamic acid and urea on their effect on urease activity, it is very probable that -CONHOH- is the group which is absolutely necessary in the chemical structure for the inhibition of urease activity. Both the properties of hydroxamic acids to form a coloured complex with Fe 3+ and their ionization constants had no correlation with their inhibitory powers on urease activity.

The quaternary structure of carp muscle alkaline protease

Abstract Carp muscle alkaline protease consists of four kinds of subunits, and its composition was assumed to be ( αβγ 2 δ 2 ) 4 . It dissociated in the presence of 2-mercaptoethanol into an enzyme and α-subunits which upon removal of 2-mercaptoethanol rapidly aggregated to form a precipitate. The composition of the 2-mercaptoethanol-treated enzyme was ( βμ 2 δ 2 ) 4 . The pH of a 2-mercaptoethanol-treated enzyme solution was lowered to 4.5 by the addition of acetic acid in the presence of 0.4 M LiCl and centrifuged to separate the precipitate formed; this exhibited little activity and was mainly composed of β-subunits. The supernatant fluid recovered 53% of activity and contained an enzyme, whose composition was ( γ 4 δ 4 ) 4 . The temperature-activity curve of the native enzyme was the same as that of the 2-mercaptoethanol-treated enzyme and both were unable to hydrolyze casein at all below 55°C. However, the temperature dependence for activity of the LiCl-treated enzyme was ordinary: it hydrolyzed casein at physiological temperatures. When the 2-mercaptoethanol-treated enzyme was incubated with 4.5 M urea at 45°C for 20 min and this was followed by column chromatography, a little activity was recovered and the amount of recovery was parallel with the amount of δ-subunit in the fractions. These findings suggest; (1) the α-subunit does not take any part in activity but is a protein necessary for binding between subunits or between the enzyme and some functional proteins in the cells, (2) the β-subunit is used as inhibitor in the quaternary structure of the enzyme, (3) the δ-subunit is the catalytic one, and (4) binding with the γ-subunit is necessary for the δ-subunit to retain its active comformation.

Ring formation of perfringolysin O as revealed by negative stain electron microscopy.

Abstract Perfringolysin O revealed ring- and arc-shaped structures in the absence of cholesterol by negative staining electron microscopy, while before activation with cysteine it showed indistinct arcs and irregularly curved sticks but no rings. These structures were observed only at high concentrations (more than 17 000 hemolytic units per ml) and seemed to be particle associates with 20–28 particles (about 4 nm per particle) linked in a circle. The toxin produced an inactive and high molecular weight complex in the presence of phosphotungstic acid, which was isolated by Sephadex gel filtration. These findings suggest that the rings are the toxin-phosphotungstic acid complexes produced during specimen preparation on a grid in vacuo. The toxin lost the properties necessary for ring formation though moderate modification with glutaraldehyde, showing spindle- and egg-shaped particles of about 4 nm in minor and 5 nm in major axis by negative staining. These facts suggest that the aldehyde modifies the binding sites for phosphotungstic acid, which probably are the basic groups of the toxin molecules. In the presence of cholesterol, even at a low concentration, the toxin revealed rings and arcs by negative staining and also by carbon shadowing electron microscopy, although the toxin itself did not show any characteristic structure without phosphotungstic acid. These observations suggest that the rings are the toxin-cholesterol complexes themselves. The toxin-phosphotungstic acid complexes seemed to have a structure of a single layer of particle associates, while that of the toxin-cholesterol complexes may consist of double or triple layers of the associates because its border was thicker and more distinct.

The limited proteolysis of rabbit muscle aldolase by cathepsin B1.

Abstract Rabbit muscle aldolase is inactivated by cathepsin B1 to approximately 10 percent of the original activity for fructose-1, 6-bisphosphate cleavage without change in the fructose-1-phosphate cleavage activity. Activity loss is related to release of one mole of the dipeptide, alanyl-tyrosine, per mole of the enzyme. The additional three moles of the peptide are released without further loss of the residual activity.

Effects of cholesterol evulsion on susceptibility to perfringolysin o of human erythrocytes

Human erythrocytes preincubated with a phosphatidylcholine suspension (preincubated cells) showed decreased susceptibility to perfringolysin O, the decrease being strongly affected by preincubation time and temperature, and the phosphatidyl choline concentration. The binding of the toxin to the preincubated cells also decreased with the preincubation time and reached minimum at 37 degrees C for 6 h. Through this preincubation, about 30% of cholesterol was removed from cells without lysis. The susceptibility of preincubated cells to the toxin seemed to be affected by the amount of cholesterol removed from cells, but not by the cholesterol content of cell membranes. This indicates that most of the cholesterol interactive with the toxin is removable from cell membranes by preincubation with phosphatidylcholine suspension, and that the residual cholesterol is firmly constituted in the membrane structure and cannot interact with the toxin. After cholesterol evulsion by the preincubated plasma method (Murphy, J.R. (1962) J. Lab. Clin. Med. 60, 86-109 and 60, 571-578), cells also exhibited lower susceptibility to the toxin and to saponins, but higher susceptibility to lysophosphatidylcholine.

Effect of fructose 1,6-bisphosphate on the activity of liver pyruvate kinase aftter limited proteolysis with cathepsin B

Treatment of rat liver-type pyruvate kinase with rabbit liver cathepsin B at pH 7.0 caused loss of activity in the standard assay with 0.6 mM of phosphoenolpyruvate. The modified enzyme exhibited about 10% of the original activity when assayed with 2.0 mM of the substrate. No detectable change in the subunit molecular weight of the enzyme occurred during inactivation. On addition of 4 microM fructose 1,6-bisphosphate the activity of the treated enzyme was restored to that of the original enzyme. Limited proteolysis of the enzyme by cathepsin B appears to enhance the requirement for the positive effector, fructose 1,6-bisphosphate.