Search

The autoxidation of pyrogallol was investigated in the presence of EDTA in the pH range 7.9–10.6. The rate of autoxidation increases with increasing pH. At pH 7.9 the reaction is inhibited to 99% by superoxide dismutase, indicating an almost total dependence on the participation of the superoxide anion radical, O2·−, in the reaction. Up to pH 9.1 the reaction is still inhibited to over 90% by superoxide dismutase, but at higher alkalinity, O2·− -independent mechanisms rapidly become dominant. Catalase has no effect on the autoxidation but decreases the oxygen consumption by half, showing that H2O2 is the stable product of oxygen and that H2O2 is not involved in the autoxidation mechanism. A simple and rapid method for the assay of superoxide dismutase is described, based on the ability of the enzyme to inhibit the autoxidation of pyrogallol. A plausible explanation is given for the non-competitive part of the inhibition of catechol O-methyltransferase brought about by pyrogallol.

Enzyme secretion from incubated guinea pig submandibular gland slices was induced by adrenaline, noradrenaline, dopamine, 5-hydroxytryptamine or dibutyryl cyclic AMP combined with theophylline. Effects of alpha- and beta-blocking agents on stimulated peroxidase and amylase discharge were studied. Propranolol (beta-blocker), but not phenoxybenzamine (alpha-blocker), markedly inhibited the secretion of both enzymes induced by adrenaline or noradrenaline. Dopamine and 5-hydroxytryptamine were found to be potent enzyme secretagogues. Both propranolol and phenoxybenzamine antagonized the effects of these two amines. The secretagogic effect of dibutyryl cyclic AMP in combination with theophylline was not affected by either propranolol or phenoxybenzamine. L-DOPA and 5-hydroxytryptophan were without effect on enzyme secretion. It is concluded that peroxidase and amylase are secreted in parallel from guinea pig submandibular gland and that enzyme release is mainly mediated via the beta-adrenergic receptor.

1. 1. The catalytic properties of two horseradish peroxidases, isoenzymes A2 and C2, with p I 3.9 and 8.8, have been compared. 2. 2. The rate of formation of Compound I has been determined for the hydrogen, methyl, ethyl, n -propyl, and hydroxymethyl hydroperoxides and for p -nitroperoxybenzoic acid. The elongation of the alkyl group hampered the reaction with the acidic peroxidase and stimulated the reaction with the basic peroxidase. The acidic peroxidase generally reacted more slowly. Its reaction with H 2 O 2 showed a higher energy of activation than that of the slightly basic peroxidase. 3. 3. The rate of reaction with hydrogen peroxide was slightly but definitely influenced by pH for both peroxidases. 4. 4. The rates of reaction of twelve hydrogen donors (phenols, aromatic amines, acidic substances) with the secondary peroxidase-peroxide complex of the two peroxidases could be arranged in three groups according to the chemical nature of the donors. The acidic peroxidase was consistently more active at pH 4.5 than at pH 7.0 whereas the slightly basic peroxidase showed the reverse behavior in the presence of phenols. 5. 5. It is concluded that the two peroxidases are kinetically different with distinct substrate profiles and that they may fulfill different physiological functions.

Hydrogen peroxide efficiently protects horseradish peroxidase against inactivation by hydroxymethylhydroperoxide, whereas the hydrogen donor substrate guaiacol has little protective effect. These results, and direct studies of the effects of hydroxymethylhydroperoxide on peroxidase compound II, indicate that compound II and possibly also compound I are quite resistant to hydroxymethylhydroperoxide. The inactivation of peroxidase caused by hydroxymethylhydroperoxide seems to be due to a reaction of the peroxide with free Fe 3+ peroxidase. Albumin, which protects thiol enzymes against hydroxymethylhydroperoxide, does not protect peroxidase. Neither does 2-mercaptoethanol, which also protects thiol enzymes, appear to hamper the inactivation reaction. This seems to indicate that the inactivation is not caused by a free radical released from hydroxymethylhydroperoxide. Hydroxymethylhydroperoxide inactivates peroxidase by attacking the hematin group, the ring of which is opened by way of at least three intermediates. The most stable intermediate is green (“compound IV”). H 2 C 2 attacks the methemoglobin-peroxide compound faster than hydroxymethylhydroperoxide does. These reactions are much slower than the attack of hydroxymethylhydroperoxide on peroxidase. It is suggested that the inactivation of peroxidase takes place as a side reaction in the process of forming compound I from hydroxymethylhydroperoxide and peroxidase.

1. 1.|Many reports attribute biochemical actions to bis(hydroxymethyl)peroxide. Mixtures of hydrogen peroxide and formaldehyde are known to exert a greater effect on biochemical systems than either substance alone. In both cases the effects are probably caused by hydroxymethylhydroperoxide, formed from bis(hydroxymethyl)-peroxide by hydrolysis or from hydrogen peroxide and formaldehyde by a condensation. 2. 2.|Hydroxymethylhydroperoxide is a fairly potent inhibitor of fumarate hydratase ( l -malate hydro-lyase, EC 4.2.1.2), lactate dehydrogenase ( l -lactate:NAD+ oxidoreductase, EC 1.1.1.27), and aspartate aminotransferase ( l -aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1), whereas glucose oxidase (β- d -glucose:O2 oxidoreductase, EC 1.1.3.4) and especially acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) are less affected. 3. 3.|2-Mercaptoethanol and albumin protect against the inhibitory action of hydroxymethylhydroperoxide without reducing the amount of peroxide. 4. 4.|Fumarate hydratase and lactate dehydrogenase, exposed to hydroxymethyl-hydroperoxide, are not reactivated by 2-mercaptoethanol, whereas aspartate amino-transferase slowly partially regains its activity.

Endogenous peroxidase activity was studied at the electronhistochemical level at various stages of the secretory cycle in the guinea pig submandibular gland. The histochemical findings were correlated to quantitative assays of peroxidase and amylase activity in the glands.

Tryptic digestion of catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) leads to a gradually increased peroxidatic activity towards phenolic compounds. The reactivity with ethylhydroperoxide increases 2–3 times after brief digestion but decreases thereafter. The catalatic activity and the reactivity with methylhydroperoxide disappear rapidly. The results indicate a loosening of the structure around the hematins, increasing the reactivity with peroxide and hydrogen donor substrates formerly sterically hindered. This change may occur to some extent without loss of the catalase-specific ability to react rapidly with hydroperoxides. The ability to peroxidize ethanol is apparently not loss before the loss of high reactivity with hydroperoxides. After brief exposure of catalase to pH > 12 leading to its dissociation into subunits, the enzyme recombines when the pH is lowered (Samejima, T., McCabe, W. J. and Tsi Yang, J. (1968) Arch. Biochem. Biophys. 127, 354–360). After this procedure most (∼ 70%) of the catalatic activity is recovered. The reactivity with ethylhydroperoxide is doubled and the peroxiding actifvity towards phenolic compounds is strongly increased. The results indicate an increased accessibility of the hematins of recombined catalase. However, they do not become reducible by dithionite. After longer exposure to high pH, all enzymic activities are decreased and the hematins become reducible.

Isolierte Submandibularisdrusen des Meerschweinchens wurden in physiologischer Pufferlosung mit sekretionsstimulierenden Substanzen inkubiert. Dopamin, Noradrenalin, Adrenalin und 5-HT induzieren die Sekretion von Peroxydase und Amylase, wahrend der Serotoninantagonist BOL 148 (2-Bromo-d-Lysergsaure Dietylamid) eine beinahe vollstandige Sekretionshemmung hervorruft. BOL 148 hat sich also nicht als spezifisch 5-HT-hemmend an der Meerschweinchenspeicheldruse erwiesen.

Human milk and saliva from newborn infants were analyzed for their content of lactoperoxidase and thiocyanate. The activity of lactoperoxidase in infant saliva was variable but generally higher than that found in calf saliva. In contrast, the activity in human colostrum was low (∼5%) compared with that found in cow's milk. The enzyme was resistant to gastric juice. Thiocyanate was demonstrated in infant saliva in concentrations about one-third of that in adult saliva. The amounts of lactoperoxidase and thiocyanate in infant saliva are quite sufficient to inhibit bacterial growth in in vitro systems. The importance of this system in vivo has not yet been demonstrated. The availability of this system to both newborn calves and humans (in calves provided largely by colostrum and in human babies by saliva) might be indirect evidence of its importance.

1. 1. Mouse pancreatic α-amylase (EC 3.2.1.1) was purified in a two-step procedure comprising isoelectric focusing and chromatography on DEAE-Sephadex. 2. 2. The enzyme was found to be composed of one polypeptide chain with a molecular weight of 54,000 as determined by equilibrium sedimentation and 58,000 as determined by electrophoresis in polyacrylamide gel containing sodium dodecylsulphate. 3. 3. S° 20w was found to be 4.6 · 10 −13 sec. 4. 4. The amino acid composition is presented and is found to be remarkably similar to that of rat pancreatic α-amylase. 5. 5. The isoelectric point was 7.0 as determined by isoelectric focusing in column. 6. 6. The neutral sugar content was 1.5 %. 7. 7. The specific activity was comparatively low, 211 U/mg amylase.

Since neonatal gramnegative infections seem to increase at the same time as breastfeeding diminishes, the role of breast milk in the newborns defence systems needs evaluation. We have studied the presence of antimicrobial factors in colostrum and breast milk-specific (E.coli antibodies of the classes IgA, IgM and IgG) and unepecific (lactoperoxidase, lactoferrin and complement). 1. Lactoperoxidase, which is present in high concentrations in cows milk, was demonstrated only in low quantities in breast milk. This might be compensated by the high concentrations we found in the newborn's saliva. 2. Specific E. coli O-antibodies of the IgA-claee were demonstrated in breast milk and were probably of local production since they were not present in the serum of the mother. In contrast IgG-and IgM-E.coli antibodies were present in lower concentrations in breast milk than in serum. The IgA-antibodies pass along the GI-tract and appear in faeces with retained agglutinating capacity. 3. The relation between the maternal and infantile E.coli strains is studied. Whether or not the specific E.coli antibodies in breast milk influence the selection of the E.coli strains colonizing the infant's bowel is under investigation.