Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.36 (hyaluronidase)
 4,606 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A beta-N-acetylhexosaminidase [EC 3.2.1.30] was isolated from internal organs of the sea-squirt, Styela plicata. The enzyme was purified 1,560-fold in 5% yield. The preparation was fairly homogeneous as examined by disc and SDS polyacrylamide gel electrophoresis and Sephadex G-200 chromatography. The molecular weight of the enzyme was estimated to be 132,000 by gel chromatography and 66,000 by SDS polyacrylamide gel electrophoresis. Therefore, this beta-N-acetylhexosaminidase was considered to be a dimer. The optimum pH for activity was 4.0 but the enzyme was stable in the pH range from 5 to 6. The isoelectric point was 4.99. This enzyme was inhibited by Fe2+, Hg2+, Ag+, and PCMB but not by acetate. The isolated enzyme hydrolyzed both p-nitrophenyl-N-acetyl-beta-D-glucosaminide and p-nitrophenyl-N-acetyl-beta-D-galactosaminide. The hydrolysis rate of p-nitrophenyl-N-acetyl-beta-D-galactosaminide was 43% of that of p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The enzyme liberated N-acetylhexosamine from asialodegalactosyl ovomucoid glycopeptide, asialodegalactosyl fetuin glycopeptide and the fragment of hyaluronic acid prepared by hyaluronidase treatment.
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PMID:Purification and characterization of a beta-N-acetylhexosaminidase of sea-squirt. 711 68

In this study several activities of the venom of Ornithorhynchus anatinus have been investigated. Whole venom induced local oedema after subplantar injection and produced relaxation of the rat uterus in vitro. The relaxant activity was partially purified by gel permeation HPLC and subsequent analyses by SDS-PAGE revealed that this activity was associated with a 4200 mol. wt peptide. The N-terminal partial sequence of this peptide exhibited substantial identity with human and porcine C-type natriuretic peptide (CNP). Three other major proteins isolated from the venom had mol. wts of 140,000, 55,000 and 16,000. None was found to have any sequence homology with proteins listed in the SwissProt database. The 140,000 mol. wt protein exhibited hyaluronidase activity but the nature of the 55,000 and 16,000 mol. wt proteins remains to be determined. Platypus venom also exhibits protease activity, although the concentration of proteolytic enzymes was too low to be visualised by SDS-PAGE using Coomassie staining.
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PMID:A pharmacological and biochemical investigation of the venom from the platypus (Ornithorhynchus anatinus). 759 19

The venom of the wandering spider Cupiennius salei was analysed biochemically by gel filtration, cation exchange chromatography, RP-HPLC, IEF, SDS-PAGE and TLC-electrophoresis. The native venom contains high levels of Na+, K+, Ca2+, histamine and taurine. It shows considerable activity of hyaluronidase, but not proteolytic activity. Thirteen peptides (CSTX-1 to CSTX-13) with an apparent mol. wt between 2.6 and 12.5 kDa causing differently strong toxic, effects were purified. Toxicity data of the crude venom (insects and mouse) are given and compared with the toxicity of CSTX-1, which causes most of the crude venom's toxicity. CSTX-1 has a mol. wt of 8352.6 and its amino acid sequence of 74 amino acids is given.
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PMID:Purification of toxic peptides and the amino acid sequence of CSTX-1 from the multicomponent venom of Cupiennius salei (Araneae:Ctenidae). 801 51

In vitro transdifferentiation of retinal pigmented epithelial cells of the chick embryo into lens cells can be markedly enhanced by culture in the presence of testicular hyaluronidase and phenylthiourea. Since the commercial preparations of hyaluronidase that had previously been used were very crude, a search for the actual effective molecule(s) enhancing lens transdifferentiation was conducted. First, we purified the enzyme and tested the effect of the purified hyaluronidase. Highly purified hyaluronidase itself did not enhance lens transdifferentiation. The crude hyaluronidase was then separated according to affinity with heparin, considering the possibility that the fibroblast growth factor (FGF) is contained in the crude hyaluronidase. Transdifferentiation-enhancing activity was detected in the fraction which was bound to heparin and eluted with 2 M NaCl, where no hyaluronate-degrading activity existed. Analysis of the fraction by SDS-PAGE revealed the existence of an 18 kDa protein whose NH2-terminal sequence was identical to that of basic FGF. The basic FGF derived from bovine brain also enhanced lens transdifferentiation of pigmented epithelial cells. These findings suggest that basic FGF must play a major role in enhancing transdifferentiation of pigmented epithelial cells to lens cells.
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PMID:Basic fibroblast growth factor as one of the essential factors regulating lens transdifferentiation of pigmented epithelial cells. 839 79

In the preovulatory follicle, the oocyte is surrounded by approximately 1000 closely associated cumulus cells forming the compact form of the cumulus cell-oocyte complex (COC). In response to the gonadotropin surge, the COC in a follicle destined for ovulation undergoes expansion when the cumulus cells synthesize and organize an extensive extracellular matrix enriched in hyaluronan. Successful expansion of the COC appears to be essential for ovulation and ultimately for fertilization. We studied this process in vitro by isolating compact COCs from preovulatory mouse follicles and incubating them under conditions which promote COC expansion by retention of newly synthesized hyaluronan (HA in the extracellular matrix around the cells. [3H]-Leucine and [35S]sulfate were used as precursors to label macromolecules synthesized by the cells that may be necessary for organizing the HA in this matrix. After labeling, expanded COCs were washed to remove medium and any labeled molecules that were not associated with the matrix. Macromolecules selectively associated with the matrix were then solubilized by digesting the expanded COCs briefly with Streptomyces hyaluronidase, an enzyme that specifically cleaves HA. Cells were removed by centrifugation, and the digest supernate was analyzed by molecular sieve chromatography and SDS-PAGE. A dermatan sulfate proteoglycan of large hydrodynamic size ( > 1 million Da) and a approximately 46-kDa protein were the predominant labeled species identified. The proteoglycan has properties similar to proteoglycans such as aggrecan and versican which interact specifically with HA. The approximately 46-kDa protein has the same molecular size as the link protein which interacts with HA and HA-binding proteoglycans to form stable ternary complexes in a variety of extracellular matrices. We propose that the dermatan sulfate proteoglycan and the approximately 46-kDa protein synthesized by the cumulus cells form similar ternary complexes that are necessary for retaining HA in the COC matrix and hence are required for successful COC expansion.
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PMID:Proteoglycans and proteins in the extracellular matrix of mouse cumulus cell-oocyte complexes. 856 97

In these experiments, we have characterized the bifunctional sperm protein PH-20 in macaque sperm and studied its hyaluronidase activity. Intact sperm were evaluated before the acrosome reaction (AR), and a soluble form of PH-20 released during acrosomal exocytosis was also investigated. Western blots of SDS-PAGE of acrosome-intact sperm extracts revealed a 64-kDa form of PH-20 was recognized by a polyclonal antibody (R-10) raised in rabbits against purified, recombinant cynomolgus macaque sperm PH-20. The soluble components released during the AR which were recognized by the R-10 antibody included both the 64-kDa form and a 53-kDa form of PH-20. An ELISA-like procedure for determining PH-20 hyaluronidase activity indicated that acrosome-intact sperm exhibited two peaks of hyaluronidase activity near pH 4 and > or = pH 7. The majority of enzyme activity in acrosome-intact sperm extracts occurred at neutral pH, while the soluble hyaluronidase activity released at the AR was predominantly acid-active. Hyaluronidase activity of PH-20 at different pH optima was investigated using hyaluronic acid substrate gel electrophoresis, and results indicated that the 64-kDa polypeptide had a broad range, with the majority of activity at neutral pH (pH 7). The 53-kDa polypeptide in sperm extracts only exhibited activity at acid pH (pH 4). The hyaluronidase activities of both enzymes could be inhibited by apigenin. The soluble PH-20 hyaluronidase activity released during the AR was primarily of the acid-active 53-kDa form. Fine structural localization of PH-20 using Fab fragments of R-10 IgG demonstrated that PH-20 was associated not only with sperm membranes, but also with the dispersing acrosomal contents. These data suggest that the more neutral-active form of PH-20 (64 kDa) is present on the plasma and inner acrosomal membranes and gives rise to the soluble acid-active form at the time of the AR. The generation of the soluble form of PH-20 may result from the action of acrosomal enzymes, which could include proteases, glycosidases, and phospholipases.
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PMID:The PH-20 protein in cynomolgus macaque spermatozoa: identification of two different forms exhibiting hyaluronidase activity. 860 61

A glycosaminoglycan (GAG) depolymerase that acts on chondroitin sulphate A (CS-A), chondroitin sulphate C (CS-C) and hyaluronic acid (HA) was purified to apparent homogeneity from a culture of Streptococcus intermedius, strain UNS 35, grown in minimal medium supplemented with CS-A as the sole carbon source. The enzyme was purified by ammonium sulphate precipitation followed by serial chromatography on DEAE Trisacryl M, CM Trisacryl M and heparin-agarose. SDS-PAGE analysis of the purified enzyme yielded a single band with a mol.wt of c. 83000. The purified GAG depolymerase was unusual in its substrate specificity. The enzyme was initially regarded as a CS depolymerase because of its induction by CS-A. However, the GAG depolymerase exhibited greatest activity against HA, whereas the degradation rates of CS-A and CS-C were c. 8% and 2%, respectively, of the rate with HA. On this basis the enzyme could be classified as a hyaluronidase rather than a CS depolymerase. The pH optimum was around neutrality and the enzyme was unusual in having a high pI of approximately 9.3.
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PMID:Purification and properties of a novel glycosaminoglycan depolymerase from Streptococcus intermedius strain UNS 35. 863 53

The rat liver sinusoidal endothelial cell (LEC) hyaluronan (HA) receptor was previously identified using a photoaffinity HA derivative (J. Biol. Chem., 267, 20451-20456, 1992). Two polypeptides with M(r) = 175,000 and 166,000, were consistently crosslinked, suggesting that the LEC HA receptor is an oligomer. Whether one or both subunits participate in HA binding, was not determined. Here we investigate the HA-subunit interactions and the potential oligomeric nature of the LEC HA receptor. When Sephacryl-400 gel filtration chromatography was used to enrich the HA receptor, the 175 kDa polypeptide was the major band seen by SDS-PAGE analysis. Little staining was seen at 166 kDa, suggesting that the 175 kDa protein could be separated from the 166 kDa protein and still retain HA-binding activity. A ligand blot assay was used to determine if each individual subunit could bind HA. LEC proteins were separated by nonreducing SDS-PAGE, and then immobilized onto nitrocellulose. 125I-HA bound to a 175 kDa polypeptide but not to the 166 kDa protein. A high molecular weight band of approximately 300,000 also bound 125I-HA. 125I-HA binding to the 175 and 300 kDa proteins showed the same specificity of competition with a panel of carbohydrates as the bona fide LEC HA receptor. The 175 kDa HA-binding subunit may be nonglobular (asymmetric), since its apparent size by SDS-PAGE is dependent on the polyacrylamide gel pore size; M(r) increases as porosity decreases. LECs were crosslinked to an 125I-labeled photoaffinity HA derivative and the HA saccharides were then released with hyaluronidase. After SDS-PAGE without reduction, radiolabeled bands were seen at 175 and 166 kDa (3:1 ratio), and a high MW (approximately 300,000) species was also detected. These data support an oligomeric model of the LEC HA receptor, and show that the 175 kDa protein possesses HA-binding activity independent from the 166 kDa polypeptide.
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PMID:Identification of a 175 kDa protein as the ligand-binding subunit of the rat liver sinusoidal endothelial cell hyaluronan receptor. 906 60

PH-20 is a sperm plasma-membrane protein that has been shown to have hyaluronidase activity in several mammalian species including nonhuman primates. In this investigation, the PH-20 protein was characterized in noncapacitated human sperm and in capacitated human sperm. Two forms of PH-20 were observed in immunoblots of sodium dodecylsulfate polyacrylamide-gel electrophoresis (SDS PAGE) using a polyclonal antibody to recombinant PH-20: a major band of 64 kDa appeared in noncapacitated and capacitated sperm extracts and a 53-kDa band that appeared only in the acrosome-reaction supernatant of acrosome-reacted sperm. Using hyaluronic acid substrate gel analysis, we demonstrated that noncapacitated sperm extracts, capacitated sperm extracts, and the acrosome-reaction supernatant had hyaluronidase activity at neutral pH (pH 7) and acid pH (pH 4). The 64-kDa form in all samples had hyaluronidase activity at both neutral and acid pH, but the 53-kDa form was only active at acid pH. Total hyaluronidase activity, as measured by a microplate assay, was higher at pH 7 than at pH 4. Very low hyaluronidase activity was detected in the acrosome-reaction supernatant. Transmission electron microscopy and immunogold labeling showed that PH-20 of acrosome-intact human sperm was located on the plasma membrane over the entire head but not on the sperm midpiece and tail. After the acrosome reaction, PH-20 was also located on the inner acrosomal membrane. The biochemical characteristics and the ultrastructural localization of PH-20 in human sperm suggest that this protein is the human sperm hyaluronidase and, therefore, has an important function during fertilization.
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PMID:The PH-20 protein in human spermatozoa. 915 9

We previously reported the first cloning of a functional glycosaminoglycan synthase, the hyaluronan synthase (HAS) from Group A Streptococcus pyogenes (spHAS) (DeAngelis, P. L., Papaconstantinou, J., and Weigel, P. H. (1993) J. Biol. Chem. 268, 19181-19184). Group A spHAS was unrelated to a putative Group C HA synthase reported by others (Lansing, M., Lellig, S., Mausolf, A., Martini, I. , Crescenzi, F., Oregon, M., and Prehm, P. (1993) Biochem. J. 289, 179-184). Here we report the isolation of a bona fide HA synthase gene from a highly encapsulated strain of Group C Streptococcus equisimilis. The encoded protein, designated seHAS, is 417 amino acids long (calculated molecular weight, 47,778; calculated pI, 9.1) and is the smallest member of the HAS family identified thus far. The enzyme migrates anomalously fast in SDS-polyacrylamide gel electrophoresis (approximately 42,000 Da). The seHAS protein shows no similarity (<2% identity) to the previously reported Group C gene, which is not an HA synthase. The seHAS and spHAS protein and coding sequences are 72 and 70% identical, respectively. seHAS is also similar to eukaryotic HAS1 (approximately 31% identical), HAS2 (approximately 28% identical), and HAS3 (28% identical). The deduced protein sequence of seHAS was confirmed by reactivity with a synthetic peptide antibody. Recombinant seHAS expressed in Escherichia coli was recovered in membranes as a major protein (approximately 10% of the total protein) and synthesized very large HA (Mr >7 x 10(6)) in the presence of UDP-GlcNAc and UDP-GlcA. The product contained equimolar amounts of both sugars and was degraded by the specific Streptomyces hyaluronidase. Comparison of the two recombinant streptococcal enzymes in isolated membranes showed that seHAS and spHAS are essentially identical in the steady-state size distribution of HA chains they synthesize, but seHAS has an intrinsic 2-fold faster rate of chain elongation (Vmax) than spHAS. seHAS is the most active HA synthase identified thus far; it polymerizes HA at an average rate of 160 monosaccharides/s. The two bacterial HA synthase genes may have arisen from a common ancient gene shared with the early evolving vertebrates.
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PMID:Molecular cloning, expression, and characterization of the authentic hyaluronan synthase from group C Streptococcus equisimilis. 940 67


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