Effect of Ionic Strength and pH on the Properties of Purified Bovine Testicular Hyaluronidase

Gorham, S. D.; Olavesen, Anthony H.; Dodgson, K. S.

doi:10.3109/03008207509152337

Cited by 37 publications

(21 citation statements)

References 22 publications

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“…In contrast to previous investigations (94) the purified enzyme appeared to degrade the various substrates at similar rates, though the salt dependence may vary with the substrate used (101). However, the K M for hyaluronate was only about i of that for chondroitin-4-sulfate (106).…”

contrasting

confidence: 87%

“…Depending on the assay conditions, testicular hyaluronoglucosaminidase is optimally active between pH 4.4 and 6 (100)(101)(102)(103). It was shown that the pH optimum for the hydrolysis of hyaluronic acid occurs at pH 5.2 in the presence and at pH 6.0 in the absence of NaCl (101).…”

Section: Mammalian Enzymes Degrading Glycosaminoglycansmentioning

confidence: 99%

“…It was shown that the pH optimum for the hydrolysis of hyaluronic acid occurs at pH 5.2 in the presence and at pH 6.0 in the absence of NaCl (101). It is not yet clear whether NaCl or other strong electrolytes (101) activate hyaluronoglucosaminidase by disruption of intermolecular hyaluronic acid interactions or by the ability to enb.…”

Section: Mammalian Enzymes Degrading Glycosaminoglycansmentioning

confidence: 99%

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Glycosaminoglycan Degradation

Kresse¹,

GlüSSL²

1987

Advances in Enzymology - And Related Areas of Molecular Biology

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contrasting

confidence: 87%

Section: Mammalian Enzymes Degrading Glycosaminoglycansmentioning

confidence: 99%

Section: Mammalian Enzymes Degrading Glycosaminoglycansmentioning

confidence: 99%

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Glycosaminoglycan Degradation

Kresse¹,

GlüSSL²

1987

Advances in Enzymology - And Related Areas of Molecular Biology

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“…The optimal conditions for the hydrolysis of HA by commercial BTH are pH 4.0 and the presence of NaCl, whereas for transglycosylation they are pH 7.0 and the absence of NaCl [9,11].…”

Section: Introductionmentioning

confidence: 99%

Mechanism for the hydrolysis of hyaluronan oligosaccharides by bovine testicular hyaluronidase

Kakizaki¹,

Ibori²,

Kojima

et al. 2010

The FEBS Journal

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Synthetic hyaluronan oligosaccharides with defined structures and their pyridylaminated derivatives were used to investigate the mechanism of hydrolysis of hyaluronan by bovine testicular hyaluronidase. The products of the hydrolysis were analyzed by HPLC and ion‐spray mass spectroscopy (MS). It was confirmed that the minimum substrate for bovine testicular hyaluronidase is the hyaluronan hexasaccharide, even though it is a poor substrate that is barely cleaved, even on prolonged incubation. When hyaluronan octasaccharide was the substrate, increasing amounts of tetrasaccharide and hexasaccharide were produced with increasing time of incubation. Whereas disaccharide was not detectable in the reaction mixture by HPLC, MS analysis revealed trace amounts. The data suggest that the enzyme generates a disaccharide intermediate from hyaluronan oligosaccharide, the majority of which is transferred to the nonreducing ends of other oligosaccharides, only traces being released as free disaccharide. When hyaluronan octasaccharide, with an unsaturated glucuronic acid at the nonreducing end, was used as a substrate, only a tetrasaccharide was detected by HPLC. However, MS showed that the product was a mixture of equal amounts of two tetrasaccharides, one with and the other without the unsaturated glucuronic acid. This suggests that, in the case of substrates with a double bond at the nonreducing end, a tetrasaccharide is cleaved off instead of a disaccharide. The results of the experiments with pyridylaminated oligosaccharides were entirely consistent with these conclusions, and in addition showed the importance of the reducing end of the substrate for the enzyme to recognize the length of the saccharide.

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“…Hyaluronan (10 mg) was degraded into oligosaccharides by testicular hyaluronidase (1 mg/ml) in 10 ml of 0.1 M sodium acetate buffer pH 5.2, 0.15 M NaCl for 16 h at 37 ° C, yielding the hyaluronan tetrasaccharide as the main digestion product [23] . The enzyme was inactivated at 100 ° C for 3 min and precipitated by centrifugation for 3 min at 14,000 rpm.…”

Section: Preparation Of Fluorescent Hyaluronan and Measurement Of Itsmentioning

confidence: 99%

Cystic Fibrosis Transmembrane Conductance Regulator Can Export Hyaluronan

et al. 2010

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Objectives: Hyaluronan, a major water binding component of the extracellular matrix, is synthesised within the cytosol and exported across the plasma membrane by the ABC-transporter MRP5 in fibroblasts. Although its synthesis is vital for embryogenesis, MRP5-deficient mice are without phenotype, suggesting that another transporter had substituted for the MRP5 protein. Thus, we searched for a compensatory exporter in fibroblasts from MRP5 deficient mice and found that cystic fibrosis transmembrane conductance regulator (CFTR) mRNA was upregulated. Methods: Hyaluronan export was measured in cell culture. The CFTR transporter was knocked out using si-RNA. Blockers of the ABC-transporter family were used to ascertain the hyaluronan transport capabilities functionally. Results: CFTR specific siRNA inhibited hyaluronan export. The tetrasaccharide was exported in undegraded form only from normal human epithelial cells and not from human epithelial cells carrying ΔF508 CFTR. The CFTR inhibitors GlyH-101 and CFTR₁₇₂ reduced hyaluronan export from CFTR-expressing mouse fibroblasts and from human breast cancer cell lines. Bronchial secretions from patients with cystic fibrosis that consist mainly of necrotic epithelia contained at least 40-fold higher concentration of hyaluronan than secretions from patients with acute bronchitis. Conclusions: CFTR transports hyaluronan across the plasma membrane of epithelial cells and this transport mechanism is defective in cystic fibrosis patients.

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Effect of Ionic Strength and pH on the Properties of Purified Bovine Testicular Hyaluronidase

Cited by 37 publications

References 22 publications

Glycosaminoglycan Degradation

Glycosaminoglycan Degradation

Mechanism for the hydrolysis of hyaluronan oligosaccharides by bovine testicular hyaluronidase

Cystic Fibrosis Transmembrane Conductance Regulator Can Export Hyaluronan

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