Canine submandibular-gland hyaluronidase. Purification and properties

1. Methods for the purification of dog submandibular-gland hyaluronidase from sedimentable and non-sedimentable portions of a homogenate and from the whole homogenate are presented. The method consists of three main steps: removal of mucin by acid precipitation or gel filtration on Sephadex G-200, ammonium sulphate precipitation and CM-cellulose chromatography. By this method specific activities of up to 1.28 and 0.78mumoles of N-acetylglucosamine/min./mg. of protein were obtained for the purified freeze-dried non-sedimentable hyaluronidase and for the sedimentable hyaluronidase respectively. 2. A comparison of some of the properties of the non-sedimentable and the sedimentable hyaluronidase preparation indicated that there was little difference between the two and that they both resembled lysosomal hyaluronidase from rat liver.     

Canine submandibular-gland hyaluronidase. Identification and subcellular distribution

1. Submandibular glands from four species of mammal have been shown to contain a hyaluronidase active at acid pH; glands from dog and cat had a much higher content of this enzyme than has been found in other sources. 2. Product formation from hyaluronate after 24hr. incubation was almost the same as with testicular hyaluronidase, indicating that the enzyme is an endo-poly-β-hexosaminidase. 3. When submandibular-gland homogenates were fractionated by the scheme developed for liver by de Duve, Pressman, Gianetto, Wattiaux & Appelmans (1955), all the enzymes assayed, except cytochrome c oxidase, were found to occur partly in the soluble fraction and partly in the particulate fractions. Among the particular fractions, the highest specific activity was found in the heavy-mitochondrial fraction for cytochrome c oxidase, in the microsomal fraction for alkaline phosphatase and in the light-mitochondrial fraction for acid phosphatase, β-N-acetylhexosaminidase and acid-active hyaluronidase. 4. Release of the enzyme activity from the sedimentable fractions occurred in 0·1% Triton X-100 or after high-speed homogenization. 5. Stimulation of dogs by pilocarpine was found to decrease the hyaluronidase content of the submandibular gland by 5% and to cause the occurrence of a corresponding amount of acid-active hyaluronidase in the submandibular saliva. 6. The results are discussed in relation to the subcellular localization of hyaluronidase.     

Myocardial depression in streptococcal cellulitis.

A previously healthy woman developed streptococcal cellulitis of the leg after falling and lacerating her knee. On admission her blood pressure was unrecordable and her respiratory rate greater than 60 beats/minute. Mechanical ventilation was necessary, and plasma volume expansion was started, with little effect. Infusion of dopamine restored the blood pressure to 150/90 mm Hg, but interrupting the infusion for as little as 30 seconds resulted in profound hypotension. Insertion of a second central venous catheter allowed the dopamine to be given continuously, and the infusion was stopped successfully four days after her admission. She eventually made a complete recovery. This case highlights the need for intensive supportive treatment in many streptococcal infections and, in particular, the need for inotropic support.     

A new potential spreading factor: Streptomyces koganeiensis hyaluronidase. A comparative study with bovine testes hyaluronidase and recombinant human hyaluronidase of the HA degradation in ECM

BackgroundRecombinant human hyaluronidase has been used in the interstitial matrix to promote the dispersion of therapeutics. The production and isolation of an extracellular hyaluronidase from Streptomyces koganeiensis (rHyal_Sk) has recently been described.MethodsThe specificity of rHyal_Sk has been assessed against heparan sulfate, chondroitin sulfates and sulfated HAs. The oligomers generated by HA degradation have been investigated by MALDI-TOF MS analysis. rHyal_Sk has been compared with BTH and PH20 in vitro, against cross-linked HA (ACP) and HA–aggrecan complex, and in vivo, by means of a diffusion assay in nude mice.ResultsDepolymerization of HA by rHyal_Sk gave tetra-, hexa- and octasaccharides in high yields. The reaction mechanism and the high HA specificity were demonstrated. The in vivo diffusion assay, supported by the in vitro tests, evidenced an initially enhanced enzymatic activity of rHyal_Sk compared to BTH and PH20.ConclusionsrHyal_Sk, compared to BTH and PH20, showed higher substrate specificity and no inhibition from GAGs sulfate, together with a superior performance for HA depolymerization in ECM. As better predictive tests for the in vivo activity of hyaluronidase we developed two assays based on the degradation of ACP or of the HA–aggrecan complex.General significancerHyal_Sk is a new potential spreading factor for intradermal drug administration. Hyaluronidases of distinct classes, that show equivalent activities in a common turbidimetric assay, could have different potencies and dose-efficacies in vivo which influences the therapeutic effect. The new proposed in vitro tests are designed to obtain a predictive characterization of the enzyme activity in vivo.