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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.

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.

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.

The metabolic fate and mode of excretion of cyclohexylphenyl 4-[35S]sulphate were studied in the guinea pig. Up to 54.8% of the dose appeared in the bile, the majority as unchanged ester. Substantial amounts of hydroxylated cyclohexylphenyl 4-[35S]sulphate were also excreted in the bile together with minor amounts of the corresponding glucuronic acid conjugate. When isolated guinea-pig livers were perfused with cyclohexylphenyl 4-[35S]sulphate the biliary components were the same as those in the intact animal, although the relative concentration of the hydroxylated derivative was significantly greater. When the hydroxylated derivative was re-injected into guinea pigs it was excreted almost entirely unchanged in the bile. However, in the rat, it was excreted in the bile as a glucuronic acid conjugate. These findings are discussed in relation to studies carried out in the rat [Hearse, Powell, Olavesen & Dodgson (1969) Biochem. Pharmacol. 18, 181--195] and to differences in enzyme activities in rat and guinea-pig liver. The results are also discussed in terms of the molecular-weight threshold for the excretion of anions in guinea-pig bile.

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.

The induction of myocardial infarction in rats by ligation of the left-anterior coronary artery was confirmed by measurement of increased plasma levels of creatine kinase, aspartate aminotransferase and lactate dehydrogenase. Using this model system it has been established that intravenous administration of 125I-labelled hyaluronidase to rats resulted in a preferential uptake of the enzyme by damaged myocardium as compared to normal heart tissue.

1. The metabolism of potassium [2-14C]octan-2-sulphate and potassium octan-2-[35S]sulphate was investigated in the rat. Following oral administration, the bulk of the radioactivity was eliminated in the urine within 24 h. 2. Whole-body radioautography showed the liver to be the principal site of tissue accumulation of radiolabel following administration of 14C- or 35S-labelled DL-octan-2-sulphate. 3. Octan-2-sulphate was extensively degraded in vivo. The major urinary components are five sulphate estes, present in urine in essentially the same proportions regardless of label. The relative proportions of radioactivity associated with the urinary components showed considerable differences between male and female rats. 4. Three of the components have been identified as butanoate-3-sulphate, hexanoate-5-sulphate and octanoate-7-sulphate. The remaining metabolite was tentatively identified as an aldehyde derivative of octan-2-sulphate, a possible intermediate in the formation of octanoate-7-sulphate.

Studies on the effect of pH and ionic strength upon the activity of purified bovine testicular hyaluronidase have shown that the pH optimum for the hydrolysis of hyaluronic acid occurs at 5.2 in the presence of, and at 6.0 in the absence of NaCl. Hydrolytic activity towards various mucopolysaccharide and hyaluronate octasaccharide substrates was dependent upon the presence of strong electrolyte (LiCl, NaCl, KCl, CsCl, NaNO3 and Na2SO4), maximum activity being obtained at electrolyte strengths of 0.2. Identical weights of sulphated and unsulphated mucopolysaccharides were hydrolysed at similar rates under optimal conditions, except that double chains of chondroitin 4-sulphate were hydrolysed at twice the rate of the other polysaccharides. Hydrolytic activity towards hyaluronate hexasaccharide was favoured at pH values below 5.2 whereas transglycosylation activity was favoured at higher pH. Hyaluronate tetrasaccharide was neither a substrate for the hydrolytic or transglycosylation activity, nor was it an inhibitor of the enzymic hydrolysis of hyaluronic acid. No conformational change in hyaluronic acid was detected by CD-spectroscopy in the presence of varying concentrations of salt and the collective results suggest that the salt effect is exerted on the enzyme rather than on the substrate.

1. The biliary excretion of phenolphthalein di[35S]sulphate was studied in rats. 2. The conjugate was administered by continuous infusion at rates of 3, 4.5, 6, 9 and 12 mug/min, and kinetic analysis of the rate of biliary excretion was consistent with a two-compartment open-model system. 3. The results obtained after single injections of the ester were also consistent with the model. 4. An essential feature of the model is the presence of a compartment into which the ester may pass as an alternative to direct excretion via the bile. 5. It is suggested that such a compartment may be located within the liver.

1H n.m.r. spectra in [2H6]dimethyl sulphoxide of dodecyltrimethylammonium salts of chondroitin sulphates and hyaluronate, or sodium salts of oligomers from hyaluronate, showed unambiguous NH signals. The acetamido NH occurs in two different environments: environment I ('normal') in simple sugars, and environment II (hydrogen-bonded NH) appearing in tri- or tetrasaccharides, indicating a secondary structure in hyaluronate (and some chondroitin sulphates) involving a hydrogen-bonded acetamido NH.

1. The metabolism of potassium 10-undecenyl [35S]sulphate and potassium 10-phenyldecyl [35S]sulphate was investigated in the rat. 2. The major route for elimination of radioactivity following potassium 10-undecenyl [35S]sulphate administration was urinary. With 10-phenyldecyl [35S]sulphate the major route of elimination was biliary. 3. Whole-body radioautography and isolated liver perfusion experiments established that both esters were concentrated in the liver, the major organ of metabolism. 4. Neither surfactant was degraded to short-chain anionic derivatives but both compounds were extensively metabolized.

The metabolic fates and modes of excretion of diethylstilboestrol mono[35S]sulphate and diethylstilboestrol di[35S]sulphate were studied in the guinea pig. Comparative studies were also made with [G-3H]diethylstilboestrol and phenolphthalein di[35S]sulphate. Diethylstiboesterol di[35S]sulphate was extensively eliminated in the bile unchanged. After administration of diethylstilboestrol mono[35S]sulphate, extensive biliary elimination of radioactivity was also recorded. Radioactive components were identified as diethylstilboestrol disulphate, diethylstilboestrol monosulphate monoglucuronide and unchanged diethylstilboestrol monosulphate. When [G-3H]diethylstilboestrol was administered, 3H-labelled diethylstilboestrol monoglucuronide, diethylstilboestrol monosulphate monoglucuronide and diethylstilboestrol disulphate appeared in the bile. Phenolphthalein di[35S]sulphate was excreted unchanged in bile. These findings are discussed in relation to studies carried out in the rat [Barford, Olavesen, Curtis & Powell (1977) Biochem. J. 164, 423--430] and species differences are related to differences in enzyme activities in rat and guinea-pig liver.

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).

1. The metabolism of potassium nonan-5-[35S]sulphate, a symmetrical secondary alkylsulphate ester, was investigated in the rat. Oral administration of the radiolabelled ester was followed by the elimination of the majority of radioactivity in the urine. 2. Potassium nonan-5-[35S]sulphate is degraded in vivo to produce at least three radiolabelled sulphate esters. 3. The same metabolites were produced by isolated rat livers perfused with potassium nonan-5-[35S]sulphate. 4. The three radioactive metabolites were identified by combined g.l.c.-mass spectroscopy as the unchanged parent ester, nonan-1-ol-5-sulphate and nonanoate-5-sulphate. 5. The nature of the latter two metabolites indicates that potassium nonan-5-sulphate is metabolized by omega-oxidation only and, moreover, the alkylsulphate ester is metabolized only at one end of the molecule.

1H NMR spectra were obtained in fully deuterated dimethyl sulphoxide of sodium chondroitin 4-sulphate and of the tetrasaccharide, hexasaccharide and octasaccharide prepared from it. Two types of NH resonance were observed, at 7.6 ppm and 8.8 ppm. The latter was the only one in the polymer spectrum, whereas both were present in the oligomer spectra: N-acetylchondrosinate produced only the 7.6-ppm signal. The upfield resonance (7.6 ppm) is characteristic of "normal", monomeric acetamido sugars and disaccharides, while the downfield resonance (8.8 ppm) suggests a hydrogen-bonded NH. The elongation of disaccharide to tetrasaccharide produces a new environment, involving a hydrogen bond at the non-reducing disaccharide NH. A structure, with three hydrogen bonds per tetrasaccharide unit, is proposed which accounts for the quantitative and qualitative aspects of the NMR spectra of polymers and oligomers. A laser light-scattering study showed that sodium chondroitin 4-sulphate has the same molecular weight in water as in dimethyl sulphoxide. The observed hydrogen bonds are therefore intramolecular, as required by the proposed structure, and probably exist in water as well as in dimethyl sulphoxide.

1. A comparison was made of the metabolism of potassium D-(+)-octan 2-[35S]sulphate and potassium L-(-)-octan-2-[35S]sulphate in the rat. 2. Following administration of either enantiomer orally or i.v. the major proportion of the radioactivity was excreted in the urine within 24 h. When either enantiomer was administered i.v. to rats with bile-duct and ureter cannulae, the majority of the radioactivity was eliminated in the urine within six hours with only small amounts in bile. 3. Both enantiomers were extensively degraded in vivo. The metabolic products were identical with those previously reported (Maggs et al. 1982). 4. The major difference in the metabolite patterns was with respect to the relative amounts of hexanoate-5-sulphate: male and female urines contained approx. twice as much of this metabolite when the D-(+)-isomer was administered. In addition, isomer and sex-linked differences were observed with respect to the amounts of octanoate-7-sulphate.

The metabolic fates and modes of excretion of diethylstilboestrol mono[35S]sulphate and diethylstilboestrol di[35S]sulphate were studied in the rat. Both of the esters were desulphated to some extent in vivo. In addition, significant amounts of radioactivity appeared in the bile as diethylstilboestrol mono[35S]sulphate monoglucuronide. The percentage of the dose appearing in bile as the diconjugate was substantially greater in experiments with diethylstilboestrol mono[35S]sulphate than with diethylstilboestrol di[35S]sulphate. Whole-body radioautography and studies with isolated perfused liver confirmed the liver as the major metabolic organ for both esters. When the metabolite diethylstilboestrol mono[35S]sulphate monoglucuronide isolated from the bile was reinjected, it was excreted in the bile unchanged. Studies in vitro demonstrated that both esters were substrates for arylsulphatase C with Km values in the range 52-76 micrometer. The metabolic fates and modes of excretion of the esters are discussed in relation to the enzyme complement of rat liver.

The metabolic fates of the synthetic surfactants, sodium [1-(14)C]undecyltriethoxy sulphate and sodium [1-(14)C]dodecyltriethoxy sulphate were studied in the rat. Both compounds were extensively metabolized regardless of the route of administration, oral, intraperitoneal or intravenous. Short-chain radioactive products were eliminated in the urine: the major metabolite of the dodecyl homologue in the urine was identified as (-)O(2)C(14)CH(2)- (OC(2)H(4))(3)OSO(3) (-) by n.m.r. and g.l.c.-mass spectrometry, whereas the major metabolite of the undecyl homologue in the urine was tentatively identified as (-)O(2)CCH(2) (14)CH(2)- (OC(2)H(4))(3)OSO(3) (-). In contrast with experiments with the dodecyl derivative, when [1-(14)C]undecyltriethoxy sulphate was administered to rats, appreciable amounts of radioactivity were recovered as (14)CO(2) in expired air. Whole-body radioautography implicated the liver as the major site of metabolism of both surfactants. The nature of the metabolic products establishes that both compounds are degraded by omega,beta-oxidation. Cleavage of the ether linkage proximal to the sulphate moiety may account for the small amounts of (14)CO(2) recovered in expired air after the administration of [1-(14)C]dodecyltriethoxy sulphate. It is suggested the substantial amounts of (14)CO(2) recovered after [1-(14)C]-undecyltriethoxy sulphate administration originate from (-)O(2) (14)C(OC(2)H(4))(3) OSO(3) (-), an unstable product of omega,beta-oxidation. An n.m.r. spectrum of the metabolite identified as 2-(triethoxy sulphate)acetic acid and a mass spectrum of the trimethylsilyl derivative of the parent alcohol of that metabolite have been deposited as Supplementary Publication SUP50086 (5 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.

The growth of the mould Trichoderma viride on a defined medium containing either potassium d-glucose 6-O-sulphate or potassium d-galactose 6-O-sulphate as sole sources of both carbon and sulphur is marked by the production of an enzyme system capable of liberating inorganic SO(4) (2-) ions from either of the sulphate esters. The enzyme is not produced when the organism is grown with glucose (or galactose) and potassium sulphate or with glucose and methionine as sole sources of carbon and sulphur. Experimental conditions are described whereby inorganic SO(4) (2-) ions liberated from potassium glucose 6-O-sulphate by the growing mould appear in the culture medium after a constant lag period of 21-24hr. The enzyme has been shown to be a simple glycosulphatase that is active towards the 6-O-sulphate esters of d-glucose and d-galactose but not towards potassium glucose 3-O-sulphate. The properties of the crude glycosulphatase show the enzyme to be appreciably different from analogous molluscan enzymes that can degrade monosaccharide sulphate esters.

1. The metabolism of sodium cortisone 21-[(35)S]sulphate was investigated in rats. 2. Quantitative and qualitative experiments showed that substantial amounts of (35)SO(4) (2-) appeared in the urine of free-ranging rats receiving the ester. 3. Whole-body radioautograms indicated considerable biliary elimination of (35)S and also pointed to the liver as the site of metabolism. 4. When female rats with bile-duct cannulae received sodium cortisone 21-[(35)S]sulphate approx. 70% of the dose appeared in the bile as a doubly conjugated steroid (metabolite I). This metabolite was identified as 3alpha-(beta-d-glucopyranuronosido)- 17alpha-hydroxy-21-[(35)S]sulpho-oxy-5alpha-pregnane-11,20-dione. 5. When metabolite I was administered to a rat with a bile-duct cannula 90% of the dose appeared in the bile unchanged. After the administration (intraperitoneally or orally) of metabolite I to free-ranging rats considerable amounts of (35)SO(4) (2-) appeared in the urine. 6. The route by which (35)SO(4) (2-) might be produced from cortisone [(35)S]sulphate in free-ranging animals is discussed.

The metabolic fate of potassium dodecyl [(35)S]sulphate was studied in rats. Intraperitoneal and oral administration of the ester into free-ranging animals were followed by the excretion of the bulk of the radioactivity in the urine within 12hr., approximately 17% being eliminated as inorganic [(35)S]sulphate. Similar results were obtained in experiments in which potassium dodecyl [(35)S]sulphate was injected intravenously into anaesthetized rats with bile-duct and ureter cannulae. Analysis of urinary radioactivity revealed the presence of a new ester sulphate (metabolite A). This metabolite was isolated, purified and subsequently identified as the sulphate ester of 4-hydroxybutyric acid by paper, thin-layer and gas chromatography, by paper electrophoresis and by comparison of its properties with those of authentic butyric acid 4-sulphate. The identity of the metabolite was confirmed by isotope-dilution experiments. When either purified metabolite A or authentic potassium butyric acid 4[(35)S]-sulphate was administered to free-ranging rats the bulk of the radioactivity was eliminated unchanged in the urine within 12hr., approx. 20% of the dose appearing as inorganic [(35)S]sulphate. Whole-body radioautography and isolated-liver-perfusion experiments implicated the liver as the major site of metabolism of potassium dodecyl [(35)S]sulphate. It is suggested that butyric acid 4-sulphate probably arises by omega-oxidation of dodecyl sulphate to a fatty acid-like compound, which is then degraded by beta-oxidation.

The most recently published method for the assay of testicular hyaluronidase preparations was based on the premise that the enzyme also exhibited carboxylesterase activity towards indoxyl acetate. Studies on the relative enzyme activities of various hyaluronidase preparations towards hyaluronate and indoxyl acetate, the relative stabilities towards pH, temperature and mechanical shaking and the behaviour towards a variety of inhibitors, showed that the activities towards the two substrates reflected the presence of at least two different enzyme systems in the preparations. Gel chromatography and polyacrylamide-gel-electrophoresis experiments confirmed these conclusions and the collective findings clearly establish that methods based on the use of indoxyl acetate cannot be employed to measure testicular hyaluronidase activity.