title: Synergistic Effect of Neutral Protease and Clostripain on Rat Pancreatic Islet Isolation.
abstract: BACKGROUND
  Islet isolation currently requires collagenase, neutral protease and other components. Thermolysin (TL) from Bacillus thermoproteolyticus is the gold standard neutral protease. However, we speculated that neutral protease derived from Clostridium histolyticum (Ch; ChNP) would be biologically superior for islet isolation. Tryptic-like activity has also been reported to be important. Therefore, we focused on clostripain (CP), since it is one of the main proteases in Clostridium histolyticum which possesses tryptic-like activity. We then examined the synergistic effects of highly purified ChNP and CP on rat islet isolation.
  
  
  METHODS
  The same amount of collagenase was used in all four groups (TL, ChNP, TL+CP and ChNP+CP; n = 12/group). The efficiency was evaluated by the islet yield and function. An immunohistochemical analysis, in vitro digestion assay for each enzyme component and evaluation of the activation of endogenous exocrine proteases during islet isolation were also performed.
  
  
  RESULTS
  The islet yield of the TL group was significantly higher than that of the ChNP group (P < 0.01). The islet yield was dose dependently increased in the ChNP+CP group, but was decreased in the TL + CP group. The islet yield in the ChNP + CP group was significantly higher than that in the TL group, but their islet function was similar. Different specificities for laminin, especially laminin-511, were observed in the TL, ChNP, and CP groups.
  
  
  CONCLUSIONS
  Clostripain had a strong synergistic effect with ChNP, but not with TL. Therefore, ChNP and CP, in combination with collagenase derived from the same bacteria, may effectively increase the isolation efficiency without affecting the quality of islets.
authors: 1Division of Advanced Surgical Science and Technology, Tohoku University School of Medicine, Sendai, Japan. 2New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan. 3Graduate school of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo, Japan. 4Graduate school of Biomedical Engineering, Tohoku University, Sendai, Japan. 5Department of Integrative Genomics, Tohoku Medical Megabank Organization (ToMMO), Tohoku University School of Medicine, Sendai, Japan. 6Department of Biochemistry, Center for Regulatory Epigenome and Diseases, Tohoku University Graduated School of Medicine, Sendai, Japan.
authors: Mami
authors: M
authors: Dendo
authors: Hiroshi
authors: H
authors: Maeda
authors: Youhei
authors: Y
authors: Yamagata
authors: Kazutaka
authors: K
authors: Murayama
authors: Kimiko
authors: K
authors: Watanabe
authors: Takehiro
authors: T
authors: Imura
authors: Akiko
authors: A
authors: Inagaki
authors: Yasuhiro
authors: Y
authors: Igarashi
authors: Yasutake
authors: Y
authors: Katoh
authors: Masayuki
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authors: Ebina
authors: Keisei
authors: K
authors: Fujimori
authors: Kazuhiko
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authors: Igarashi
authors: Noriaki
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authors: Ohuchi
authors: Susumu
authors: S
authors: Satomi
authors: Masafumi
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authors: Goto
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D047009:Hathewaya histolytica / Q000201:enzymology* / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D007515:Islets of Langerhans / Q000201:enzymology*; D003012:Microbial Collagenase / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D011917:Rats, Inbred Lew; D011994:Recombinant Proteins / Q000378:metabolism; D013820:Thermolysin / Q000378:metabolism; D013997:Time Factors; D020858:Tissue and Organ Harvesting / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C019913:clostripain; D013820:Thermolysin; D003012:Microbial Collagenase