Hyaluronidase activity

Boiler et al. [11] demonstrated the presence of hyaluronidase activity in various mammalian tissues. They showed foot this type of hyaluronidase differed from... 

These techniques allow one to detect hyaluronidase activities in crude biological samples or extracts from bacteria], animal, or human sources. They make possible... 

T. Hirsyama, T. Hasegawa, and M. Hiioi. The measurement of hyaluronidase activity in human spermatozoa by substrate slide assay and its clinical application. Fen Stcr. 51 330(1989). 

B. Steiner and D. Grace. A lymographic assay for detection of hyaluronidase activity on polyacrylamide gels and its application to enzymatic activity found in bacteria. ajua mwewm. uu huj (im/. 

Cultures of isolated keratinocytes have facilitated the study of epithelial HA metabolism. Basal keratinocytes synthesize copious quantities of HA. When Ca++ of the culture medium is increased, from 0.05 to 1.20 mM, these cells begin to differentiate, HA synthesis levels drop,148 and there is an onset of hyaluronidase activity.149 This increase in calcium that appears to simulate in culture the natural in situ differentiation of basal keratinocytes parallels the increasing calcium gradient observed in the epidermis. There may be intracellular stores of calcium that are released as keratinocytes mature. 

Alternatively, the calcium stores may be concentrated by lamellar bodies from the intercellular fluids released during terminal differentiation. The lamellar bodies are thought to be modified lysosomes containing hydrolytic enzymes, and a potential source of the hyaluronidase activity. The lamellar bodies fuse with the plasma membranes of the terminally differentiating keratinocytes, increasing the plasma membrane surface area. Lamellar bodies are also associated with proton pumps that enhance acidity. The lamellar bodies also acidify, and their polar lipids become partially converted to neutral lipids, thereby participating in skin barrier function. 

Cultured cells secrete hyaluronidases into the culture media, away from the cells. Such a phenomenon does not occur within tissues. The production of unopposed hyaluronidase activity would cause great havoc in tissues. Simultaneous deposition of hyaluronidases and their inhibitors is a reasonable scenario, one that parallels control of the matrix metalloproteinases by their TIMPs (tissue inhibitors of MMPs). 

Lokeshwar, V.B., Soloway, M.S., and Block, N.L., Secretion of bladder tumor-derived hyaluronidase activity by invasive bladder tumor cells, Cancer Lett., 131,21, 1998. 

Frost, G.I. and Stern, R., A microtiter-based assay for hyaluronidase activity not requiring specialized reagents, Anal. Biochem., 251, 263, 1997. 

Wilkinson, C.R., Bower, L.M., and Warren, C., The relationship between hyaluronidase activity and hyaluronic acid concentration in sera from normal controls and from patients with disseminated neoplasm, Clin. Chim. Act., 256, 165, 1996. 

Triggs-Raine, B. et al., Mutations in HYAL1, a member of a tandemly distributed multigene family encoding disparate hyaluronidase activities, cause a newly described lysosomal disorder,mucopolysaccharidosis IX, Proc. Natl Acad. Sci. USA, 96, 6296, 1999. 

Li, M.W. et al., Inhibition of monkey sperm hyaluronidase activity and heterologous cumulus penetration by flavonoids, Biol. Reprod., 56, 1383, 1997. 

Kakegawa, H. et al., Inhibitory effects of tannins on hyaluronidase activation and on the degranulation from rat mesentery mast cells, Chem. Pharm. Bull., 33, 5079, 1985. 

Wolf, R.A. et al., Heparin inhibits bovine testicular hyaluronidase activity in myocardium of dogs with coronary artery occlusion, Am. J. Card., 53, 941, 1984. 

Natowicz, M.R. and Wang, Y., Plasma hyaluronidase activity in mucolipidoses II and III marked differences from other lysosomal enzymes, Am. J. Med. Genet., 65, 209, 1996. 

The first vertebrate hyaluronidase activity to be identified was derived from a testicular extract [188], active at neutral pH. A similar enzyme, but active at acidic pH, was later documented in human serum [189]. Another thirty years passed before sufficient purification was achieved for sequence analysis [172]. 

Hyaluronidase activity is present in the venoms of a surprisingly wide variety of organisms. These include bees, wasps, hornets, spiders, scorpions, as well as certain species of fish, snakes, and lizards [203,204]. Some of these venoms, particularly those from snakes, have stretches of sequence with 36% identity with testicular hyaluronidase, PH-20. Interestingly, these hyaluronidases have absolute specificity for HA [205]. Hyaluronidase in venoms may act to facilitate penetration of other venom active ingredients. However, other evolutionary selective advantages of venom hyaluronidases may exist that have not yet been identified. 

In a study of human sperm, Echinacea was found to inhibit the motility of the sperm only at high concentrations and after 24 hours. One potential effect of Echinacea is thought to be the inhibition of hyaluronidase activity. Hyaluronidase is localized on the sperm head and helps the sperm to penetrate the oocyte. This potential inhibition could prevent sperm from fertilizing oocytes, but further studies are needed to confirm this potential interaction (38). 

Lin, Y Mahan, K., Lathrop, W.F., Myles, D.G., and Primakoff, P. (1994). A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg. J. Cell. Biol. 725 1157-1163. 

Bollet et al. [11] demonstrated the presence of hyaluronidase activity in various mammalian tissues. They showed that this type of hyaluronidase differed from the testicular type concerning pH optimum and pH range of activity. Subsequent studies revealed that the enzyme was present in the lysosomal fraction of the tissues [12]. The liver is an especially rich source [13]. Degradation of hyaluronan leads to the same end products as testicular hyaluronidase [11]. Lysosomal hyaluronidase from rat liver degrades chondroitin-4- and -6-sulfate, but not dermatan sulfate, desulfated dermatan sulfate, heparan sulfate, keratan sulfate, or heparin [14], Lysosomal hyaluronidase has an acid pH optimum and a narrow pH range of activity [14]. This difference in pH profile of activity has commonly been used to differentiate between testicular and lysosomal hyaluronidase. A similar acid-active hyaluronidase is present in human serum [15]. 

Animal venoms usually possess hyaluronidase activity [17]. The enzymatic properties, including hyaluronidase, of snake venoms have been extensively studied by Tan et al. [18]. Snake hyaluronidase acts on hyaluronan, chondroitin, and chondroitin-4- and -6-sulfate, producing various oligosaccharides, mainly tetrasaccharides [1]. 

Hyaluronidase from bee venom has about the same substrate specificity as snake hyaluronidase [19]. Venom of social wasps was found to contain high levels of hyaluronidase activity, whereas venom from ants contains low levels of activity [20]. Lizard venom contains a hyaluronidase that acts almost specific on hyaluronan, i.e., it has no activity toward chondroitin-6-sulfate, dermatan sulfate, or heparin and only weak activity toward chondroitin-4-sulfate [21]. 

Table 1 presents a survey of hyaluronidase activity in the venom of several animal species. Transglycosylation properties have been observed with hyaluronidase from snake venom (Crotalus terrificus) [8], but not with hyaluronidase from bee venom [19]. 

Table 1 Hyaluronidase Activities of Some Animal Venoms...

Hyaluronidase activity has been demonstrated in streptococcal phages [34] and in infective hookworm (Ancylostoma) larvae [35], The latter has been shown to possess specific activity toward hyaluronan. 

This assay is used to investigate hyaluronidase activities from a variety of sources or to estimate the inhibitory capacities of compounds on the enzyme. The assay is based on the determination of the liberated /V-acetyl-D-glucosamine end groups from hyaluronan after digestion by hyaluronidase [60]. A comparison has been made between the Moigan-Elson and neocuproine assays and of the influence of inhibitors [138]. 

For this assay plates or petri dishes consisting of hyaluronan dispersed in agar and buffered to the appropriate pH are used. Cylindrical holes are punched in the gel and the samples (unknowns or standards) are added. The plates are incubated at 37°C for several hours. After incubation, a solution of a quaternary ammonium compound is poured over the gel, and after reaction the areas of digested hyaluronan become visible as clear rings. This technique is a simple assay for hyaluronidase activity in biological samples and has been used to estimate hyaluronidase activity in hymenoptera venoms [143], in semen, and in seminal plasma [146],... 

This technique is a histochemical procedure to evaluate the hyaluronidase activity of spermatozoa. Using this technique, Waibel et al. evaluated the inhibition of mouse testicular hyaluronidase by sodium aurothiomalate [147]. Hirayama et al. 

Stem et al. [149] made use of a hyaluronan-binding protein obtained from the tryptic digestion of the proteoglycan core protein of bovine nasal cartilage to detect hyaluronidase activities in urine samples from Wilms tumor patients. The pathophysiology of this tumor is associated with major alterations in the metabolism of hyaluronan, and it is thought that urinary hyaluronidase can be used as an additional marker. 

Most authors apply native polyacrylamide gels without sodium dodecyl sulfate, because this detergent would reduce enzymatic activity. Cevallos et al. [66] used SDS-PAGE to analyze hyaluronidase samples. This technique allows the demonstration of hyaluronidase activity and the determination of the molecular weight of the proteins present in a sample. Steiner and Cruce [151] developed a zymographic assay to evaluate hyaluronidase activities in biological samples. 

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