Anthony H. Olavesen

A recommended procedure for the estimation of bovine testicular hyaluronidase in the presence of human serum

A procedure is described for the assay of bovine testicular hyaluronidase in human blood following intravenous administration of the enzyme. Inhibition of hyaluronidase by the reported nonspecific serum inhibitor is minimal. However, the presence of human serum does alter the pH profile of hyaluronidase and enhances the activity of the enzyme at low pH values. Preliminary data indicates that the effects caused by serum on the pH optimum and activity of the enzyme are largely associated with the albumin fraction and are not due to the presence of endogenous serum hyaluronidase. The activation effect is not specific for any particular blood type and is independent of whether serum or citrated plasma is used. A similar effect to that of serum on hyaluronidase activity is produced by different buffer mixtures or increased NaCl concentration. It is recommended that bovine testicular hyaluronidase be measured at pH 4.0 in 0.1 m sodium citrate buffer containing 0.15 m NaCl as under these conditions the addition of human serum or citrated plasma does not alter the pH optimum of the enzyme. These recommendations necessitate certain modifications of the reducing N-acetylhexosamine assay method of Reissig et al. (J. L. Reissig, J. L. Strominger, and L. F. Leloir, 1955, J. Biol. Chem.217, 959–966).

The Fate of 2,6-Dimethoxy[U-14C]phenol in the Rat

Abstract1. The metabolic fate of 2,6-dimethoxy[U-14C]phenol, administered intravenously to rats at three dose levels (10–30 mg/kg body wt.), was investigated.2. The majority of injected radioactivity appeared in urine but significant amounts were eliminated via the bile (16.3–35.4%).3. The urinary radioactivity was associated with two metabolites identified as 2,6-dimethoxy[U-14C]phenylglucuronide and 2,6-dimethoxy[U-14C]phenylsulphate. The glucuronide conjugate predominated in the female rat while approximately equal amounts of the conjugates were produced by the male.4. The biliary metabolite was identified as 2,6-dimethoxy[U-14C]phenyl-glucuronide.

The influence of some physico-chemical factors on the biliary excretion of a series of structurally related aryl sulphate esters

Abstract The metabolic fates and modes of excretion of biphenylyl 4-35S-sulphate, cyclohexylphenyl 4-35S-sulphate and cyclohexylphenyl 2-35S-sulphate were investigated in the rat. In experiments with free-ranging animals the bulk of the administered radioactivity was recovered in the urine. The proportion of urinary radioactivity appearing as inorganic 35S-sulphate was different for each ester (biphenylyl 4-35S-sulphate, 36 per cent in the male and 19 per cent in the female; cyclohexylphenyl 4-35S-sulphate, 33 per cent in the male and 19 per cent in the female; cyclohexylphenyl 2-35S-sulphate, less than 2 per cent in each sex). In experiments in which the esters were administered to rats with bile fistulae significant biliary elimination was recorded (biphenylyl 4-35S-sulphate, up to 18 per cent; cyclohexylphenyl 4-35S-sulphate, up to 81 per cent; cyclohexylphenyl 2-35S-sulphate, up to 70 per cent). In each case a single biliary metabolite was detected and this was identified as a glucuronic acid conjugate of the administered ester. Factors which might govern the respective levels of biliary excretion are discussed.

The toxicity of dimethoxyphenol and related compounds in the cat

Abstract The metabolic fate of 2,6-dimethoxyphenol, phenol and quinol (hydroquinone) were investigated in the cat. The nature of the urinary metabolic products was dependent upon the dose of the phenol administered, although in all cases the major detoxication products were sulfate conjugates. Hydroxylation of 2,6-dimethoxyphenol and phenol to the corresponding quinols is a major pathway and at relatively high doses unconjugated quinols were found in the urine. Experiments with para-substituted phenols suggest that quinol formation is an obligatory step leading to poisoning in the cat. 2,6-Dimethoxyquinol and quinol had no effect on mitochondrial respiration in vitro whereas the corresponding quinones were potent inhibitors. Inhibition was not observed in the presence of l -cysteine.

The preparation and characterization of a series of 35S-labelled aryl sulphate esters for metabolic studies.

Abstract In order to provide a series of suitable compounds for metabolic studies, methods were developed for the preparation of crystalline potassium salts of the 35 S-labelled sulphate esters of 4-hydroxybiphenyl, 4-cyclohexylphenol, 2-cyclohexylphenol, 2-naphthol and 5,6,7,8-tetrahydro 2-naphthol in good yield. Infrared spectrophotometric analysis of the products showed strong absorption bands in the regions 1280-1220 cm −1 and 1070–1040 cm −1 characteristic of the ester sulphate grouping. Ultraviolet absorption spectra revealed a characteristic spectral shift from the absorption region associated with the parent phenols (270–310 mμ) to that associated with sulphated phenols (240–280 mμ).

The Biodegradation of some Anionic Detergents in the Rat a Common Metabolic Pathway

1. The metabolism of the anionic detergents potassium decyl [35S]sulphate and potassium octadecyl [35S]sulphate was investigated in the rat. 2. The major route for elimination of radioactivity was urinary regardless of the route of administration. 3. Both surfactants were extensively degraded in vivo to yield a common metabolite, butyric acid 4-[35S]sulphate, the major urinary radioactive component. 4. Whole-body radioautography revealed the liver as the major site of cellular accumulation of radioactivity following administration of either compound indicating the liver as the major site of metabolism. 5. It is suggested that alkyl sulphates with even-numbered carbons are degraded by a common pathway involving omega-oxidation followed by beta-oxidation.

Modification of functional arginine residues in purified bovine testicular hyaluronidase with butane-2, 3-dione.

Purified bovine testicular hyaluronidase (hyaluronate 4-glycanohydrolase, EC 3.2.1.35) was inactivated by butane-2,3-dione in either borate or Hepes buffer, pH 8.3. The presence of borate enhanced the inactivation process which followed pseudo-first-order kinetics with a calculated second-order rate constant of 13.54M-1 min-1. Using kinetic data it was estimated that the modification of 1 mol arginine per mol enzyme was sufficient for inactivation to occur, whereas amino acid analysis indicated that 4 mol arginine had been modified. The inactivation process was partially prevented by using either competitive inhibitors or substrates of the enzyme, thus indicating that the essential arginine residue is close to the active site of hyaluronidase. A full kinetic analysis of the enzyme with either hyaluronic acid or chondroitin 6-sulphate as substrate showed that the activity of hyaluronidase was uncompetitively activated by either protons or NaCl. The product obtained by reduction of the corboxyl groups of hyaluronic acid to the corresponding alcohol groups was a competitive inhibitor. The possibility that the microenvironment of hyaluronic acid was responsible for the observed kinetic effects of pH and ionic strength was dispelled. It is concluded that these data are compatible with a mechanism that involves anionic interaction between a carboxyl group on the substrate and an arginine residue on the enzyme.

The purification and some properties of pig liver hyaluronidase

Hyaluronidase (hyaluronate 4-glycanohydrolase, EC 3.2.1.35) has been isolated from pig liver and purified 1720-fold with an overall yield of 9.5%. The enzyme was purified using an acid-extraction technique followed by successive chromatography on DEAE-cellulose, two boronate affinity columns and Sephadex G-75. This final preparation, which was essentially homogeneous as determined by gel electrophoresis, was a single subunit enzyme of apparent molecular weight 70 000 with an isoelectric point of 5.0. No contaminant enzymes capable of degrading glycosaminoglycans could be detected in the final preparation. The substrate specificity of the enzyme was the same as for bovine testicular hyaluronidase; however, both the Km and V values were significantly lower for the pig liver enzyme with all of the substrates tested (hyaluronate, chondroitin 4-sulphate, chondroitin 6-sulphate). A full kinetic analysis of the enzyme using hyaluronate as a substrate showed that the activity of pig liver hyaluronidase was uncompetitively activated by either protons or NaCl.

The fate of intravenously administered highly purified bovine testicular hyaluronidase (hyalosidase) in the rat

A highly purified commercial preparation of bovine testicular hyaluronidase (GL enzyme, Hyalosidase) was labelled with 125iodine without measurable loss of enzyme activity. The labelled preparation was administered intravenously into rats and the serum half-life of hyaluronidase was determined by measurement of both radioactivity and enzyme activity. The short half-life of the enzyme in plasma could not be accounted for by excretion in the urine and bile. Tissue distribution studies showed that the major site of uptake was the liver (59.7% of the recovered dpm). This rapid uptake by the liver could be reduced significantly by the pre-administration of yeast mannan or ovalbumin (a mannose-terminated glycoprotein). This suggests that the uptake of hyaluronidase by the liver is mediated by a mannose-specific receptor. Very little radioactivity was found in the heart (0.2% of the recovered dpm).

The Biodegradation of the Surfactant Undecyl Sulphate

1. The metabolism of the odd-numbered carbon chain surfactant, potassium undecyl [35S]sulphate in the rat was investigated. 2. The major route for elimination of radioactivity was the urine, regardless of the route of administration. 3. The surfactant was extensively degraded in vivo to yield propionic acid 3-[35S]sulphate, the major radioactive component in urine. A second urinary metabolite was identified tentatively as pentanoic acid 5-[35S]sulphate. 4. Whole-body autoradiography revealed the liver as the major site of metabolism. 5. The nature of the metabolic products of undecyl sulphate suggest that it is bio-degraded by initial omega-oxidation followed by beta-oxidation.