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WO1996003497A1 - Nouvelle hyaluronidase - Google Patents

Nouvelle hyaluronidase Download PDF

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Publication number
WO1996003497A1
WO1996003497A1 PCT/JP1995/001452 JP9501452W WO9603497A1 WO 1996003497 A1 WO1996003497 A1 WO 1996003497A1 JP 9501452 W JP9501452 W JP 9501452W WO 9603497 A1 WO9603497 A1 WO 9603497A1
Authority
WO
WIPO (PCT)
Prior art keywords
hyaluronic acid
haase
hyaluronidase
gel
chondroitin sulfate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP1995/001452
Other languages
English (en)
Japanese (ja)
Inventor
Tatsuya Yamagata
Ryu Miura
Sadako Yamagata
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Seikagaku Corp
Original Assignee
Seikagaku Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Seikagaku Corp filed Critical Seikagaku Corp
Publication of WO1996003497A1 publication Critical patent/WO1996003497A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • the present invention relates to a novel hyaluronidase, and in particular, to a novel hyaluronidase useful for pharmaceuticals, quasi-drugs, or screening thereof. Background technology
  • Hyaluronidase (hereinafter abbreviated as “HA ase”) is a generic name given to enzymes that degrade hyaluronic acid into molecules.
  • Hyaluronic acid / chondroitin '/ 9-N-acetyl-D of chondroitin sulfate Endohexosaminidase-type enzyme (EC 3.2.1.35), which hydrolyzes hexosaminide bond
  • HAase is used as a drug in the treatment of acute phase such as cerebral edema and myocardial infarction, and is also used as an additive for subcutaneous administration or as a drug for local treatment of pleural effusion. Have been.
  • HAase is also useful in searching for a substance having an action of inhibiting the activity of HAase.
  • Hyaluronic acid retains moisture in the intercellular space of the human body, forms a matrix in the tissue to retain cells, keeps the skin lubricious and flexible, and prevents mechanical damage. It works to prevent external force and bacterial infection.
  • HAase has the activity of decomposing such hyaluronic acid. So hyal Substances that suppress the activity of HA ase, which degrades lonic acid, can be used as agents and cosmetics that keep the skin moist, prevent skin roughness, small blemishes, and dryness.
  • various uses of HAase have been proposed, but not all of its functions have been elucidated, and the emergence of a new HAase has been awaited. Disclosure of the invention
  • the present inventors have conducted a search to find a new HAase, and as a result, for the first time, have found that a novel HAase is produced in the culture supernatant of human endometrial cancer tissue, and have completed the present invention. Reached.
  • the gist of the present invention resides in a hyalinidase derived from human endometrial cancer tissue having the following physicochemical properties.
  • the hyaluronidase of the present invention was confirmed to have a molecule S of about 94 kilodalton as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using hyaluronic acid as a substrate. was done.
  • the novel HAase of the present invention can be isolated and purified from human endometrial cancer tissue according to various treatment procedures utilizing the physical and chemical properties of the target HAase. That is, treatment with a normal protein precipitant, salting, ultrafiltration, molecular sieve chromatography (gel filtration), centrifugation, electrophoresis, ion exchange chromatography, affinity chromatography, reverse Processing operations such as phase chromatography, adsorptive pore chromatography, dialysis, and a combination thereof are mentioned. Concrete Specifically, from the culture of human endometrial cancer tissue, Markusw GuntenhOner, et al., Et al. According to the method of [Matrix Vol. 12.38 88-39 (1992)], Vol. 12, No. 3, pages 388-396 (Trix) Can be separated.
  • a conventional method for preparing a polyacrylamide gel ⁇ Laemmli, UK, UK, Nayiya, Vol. 227, pp. 68-68 (1970) [Nature, 227, 680-680 (1970)]] ⁇ . That is, hyaluronic acid or modified monochondroitin sulfate is added to an aqueous solution containing acrylamide and N, N'-methylenebisacrylamide, and the mixture is uniformly mixed, for example, by placing it in a cool and dark place for 24 hours.
  • Polymerization is carried out using a polymerization initiator such as a peroxide such as persulfate or benzoyl peroxide, an azo compound such as azobisisobutyronitrile, or a redox initiator composed of an oxidizing agent and a reducing agent.
  • a polymerization initiator such as a peroxide such as persulfate or benzoyl peroxide, an azo compound such as azobisisobutyronitrile, or a redox initiator composed of an oxidizing agent and a reducing agent.
  • N, N, N ′, N′-tetramethylethylenediamine may be added as a polymerization accelerator.
  • the amount of N, N'-methylenebisacrylamide used for acrylamide is the same as that used for general polyacrylamide gels, and the amount of hyaluronic acid or modified monochondroitin sulfate used is usually 1-2. It is selected from the range of 0 ig
  • the culture supernatant of human endometrial cancer tissue is degraded by the enzyme contained in the culture supernatant. Perform electrophoresis under the conditions not used.
  • the carrier after swimming is washed with a buffer solution or the like as necessary, and then placed under ordinary oxygen reaction conditions, and the carrier after the enzyme reaction is colored with a coloring reagent such as Alshan blue or Toluidine blue. .
  • the enzyme contained in the culture medium decomposes the group K immobilized on the polyacrylamide gel, only the decomposed part is detected as white without coloration, and the activity and molecular weight of the enzyme must be identified. Can be.
  • the enzyme thus obtained is the HAase of the present invention.
  • the optimal pH of the enzyme of the present invention can be confirmed by measuring the HAase activity while varying the pH during the enzyme reaction. You.
  • FIG. 1 is a drawing showing a synthetic pathway of modified monochondroitin sulfate.
  • FIG. 2 is a drawing showing the results of (a) H A ase of the present invention detected by SDS-P AGE using hyaluronic acid as a substrate.
  • lanes 1, 2, 3, and 4 represent the electrophoresis patterns at pH 3.5, 5.0, 6.0, and 7.5, respectively.
  • lanes 1, 2, 3, and 4 represent the electrophoresis patterns at pH 3.5, 5.0, 6.0, and 7.5, respectively.
  • Chondroitin sulfate (I) derived from shark cartilage (molecular weight 40,000-48,000)
  • a 1 mm thick 8% sodium dodecyl sulfate (SDS) -polyacrylamide gel was prepared.
  • SDS sodium dodecyl sulfate
  • Electrophoresis was performed at room temperature. After electrophoresis, the gel was washed with 2.5% Triton X—100 for 1 hour at room temperature.
  • Gel fresh buffer (5 0 mM click E down monobasic N a 2 HP 0 4 and 0. 1 5 MN a C l ( p H 5. 0, 6. 0. 7. 5), or 0. 1 MHCO OH--HCO ON a and 0.15 MN a C 1 (pH 3.5))), incubate at 37 ° C for 16 hours, and then incubate at 37 ° C for 2 hours.
  • pronase solution 20 mM Tris—HCl (pH 8.0): Pronase is derived from Streptomyces griseus, and is derived from Calnochem Coop. or).
  • the incubated gel was washed with 20% ethanol and 10% acetic acid for 20 minutes, and then washed with 20% ethanol and 10% acetic acid containing 0.5% Alcian blue (A1cianblue) for 1 hour. After staining, the cells were washed with 20% ethanol—10% acetic acid.
  • Fig. 2 (a) The results are shown in Fig. 2 (a). A clear band was observed around 94 kilodaltons in the pH range of 3.5 to 7.5. It was also confirmed that the band had high hyaluronic acid-degrading activity, especially at around pH 5.0.
  • Example 2 In the same manner as in Example 1, except that the modified-chondroitin sulfate prepared in Reference Example was used instead of hyaluronic acid and added so that the final concentration in the gel was 8.5 ug / m1, The electrophoresis of the culture supernatant of human endometrial cancer tissue was performed. The results are shown in Fig. 2 (b). Within the range of pH 3.5 to 7.5, no particularly characteristic band was observed.
  • the 94-kilodalton protein has a hyaluronidase activity that acts on hyaluronic acid and has no effect on chondroitin sulfate.
  • the isoelectric point of the 94 kilodalton protein was determined by two-dimensional electrophoresis under the following conditions.
  • the HAase of the present invention is a novel enzyme and, like the conventional HAase, is useful as a drug for cerebral edema, myocardial infarction, etc., as a drug additive, and in searching for a substance that inhibits the activity of HAase. .

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Une nouvelle hyaluronidase (HAase) est isolée du surnageant d'une culture de tissu cancéreux de l'utérus humain par une électrophorèse sur un gel de dodécyle sulfate de sodium et de polyacrylamide en utilisant de l'acide hyaluronique, un sulfate de chondroïtine modifié ou similaire comme substrat. La HAase obtenue hydrolyse l'acide hyaluronique à un pH de 3,5 à 7,5, mais pas le sulfate de chondroïtine et elle est utile, par exemple, pour la mise au point de remèdes contre les ÷dèmes cérébraux, l'infarctus du myocarde et similaire, comme adjuvant pour des médicaments et comme inhibiteur de la hyaluronidase.
PCT/JP1995/001452 1994-07-22 1995-07-21 Nouvelle hyaluronidase Ceased WO1996003497A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP17120694 1994-07-22
JP6/171206 1994-07-22

Publications (1)

Publication Number Publication Date
WO1996003497A1 true WO1996003497A1 (fr) 1996-02-08

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ID=15919001

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1995/001452 Ceased WO1996003497A1 (fr) 1994-07-22 1995-07-21 Nouvelle hyaluronidase

Country Status (1)

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WO (1) WO1996003497A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6103525A (en) * 1996-10-17 2000-08-15 The Regents Of The University Of California Hybridoma cell lines producing monoclonal antibodies that bind to human plasma hyaluronidase
US6193963B1 (en) 1996-10-17 2001-02-27 The Regents Of The University Of California Method of treating tumor-bearing patients with human plasma hyaluronidase

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANALYTICAL BIOCHEMISTRY, Vol. 225, No. 2, (1995), p. 333-340. *
DEVELOPMENTAL BIOLOGY, Vol. 106, No. 2, (1984), p. 351-359. *
DEVELOPMENTAL BIOLOGY, Vol. 66, No. 2, (1978), p. 308-320. *
INFECTION AND IMMUNITY, Vol. 47, No. 2, (1985), p. 508-513. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6103525A (en) * 1996-10-17 2000-08-15 The Regents Of The University Of California Hybridoma cell lines producing monoclonal antibodies that bind to human plasma hyaluronidase
US6193963B1 (en) 1996-10-17 2001-02-27 The Regents Of The University Of California Method of treating tumor-bearing patients with human plasma hyaluronidase
US7148201B2 (en) 1996-10-17 2006-12-12 The Regents Of The University Of California Use of human plasma hyaluronidase in cancer treatment
US7781397B2 (en) 1996-10-17 2010-08-24 The Regents Of The University Of California Human plasma hyaluronidase

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