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Dilution of digestive fish proteases with protein-free blocking reagents prevents loss of catalytic activity during microquantification

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  • Chemistry and Biochemistry
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Abstract

The activity of proteases may be lost during microquantification because of their nonspecific binding to tubes and pipette tips. In this study, the effects of blocking reagents (blockers) in dilutions of digestive proteases of yellowtail, Seriola quinqueradiata, were compared. EzBlock Chemi (EBC; protein free), Western BLoT Blocking Buffer (WBB; protein free), casein-based blocker, and bovine serum albumin were evaluated by the measurement of trypsin, chymotrypsin, and aminopeptidase activities using fluorogenic substrates. Protease activities were linear in extracts of pyloric caeca at dilution rates of up to at least 1/6400 with the four blockers. However, the activities of pancreatic enzymes differed, and the highest levels were observed with EBC and WBB. Extracts diluted with EBC and WBB were incubated for up to 24 h at 25 °C. Pancreatic enzyme activities increased with time in extracts diluted with EBC, but for those diluted with WBB there was no clear trend. The activities of chymotrypsin and aminopeptidase in whole-body extracts of individual yellowtail larvae extracted with EBC were significantly higher than in those extracted with 150 mM NaCl, suggesting that EBC inhibits the loss of enzyme activity that may otherwise occur during extraction. Thus, EBC is considered to be the most effective diluent for the microquantification of proteases amongst the four blockers tested.

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References

  • Andoh T (2007) Amino acids are more important insulinotropins than glucose in a teleost fish, barfin flounder (Verasper moseri). Gen Comp Endocrinol 151:308–317

    Article  CAS  Google Scholar 

  • Chang BS, Mahoney RR (1995) Enzyme thermostabilization by bovine serum albumin and other proteins: evidence for hydrophobic interactions. Biotechnol Appl Biochem 22:203–214

    CAS  PubMed  Google Scholar 

  • Conceição L, Aragão C, Rønnestad I (2011) Proteins. In: Joan H, John G (eds) Larval nutrition. Wiley, Hoboken, pp 83–116

    Chapter  Google Scholar 

  • Gamboa-Delgado J, Le Vay L, Fernández-Díaz C, Cañavate P, Ponce M, Zerolo R, Manchado M (2011) Effect of different diets on proteolytic enzyme activity, trypsinogen gene expression and dietary carbon assimilation in Senegalese sole (Solea senegalensis) larvae. Comp Biochem Physiol B Biochem Mol Biol 158:251–258

    Article  Google Scholar 

  • Harada T (1965) Studies on propagation of yellowtail (Seriola quinqueradiata T. & S.): with special reference to relationship between feeding and growth of fish reared in floating net craw. Mem Fac Agric Kinki Univ 1965:1–275

    Google Scholar 

  • Hirji KN, Courtney WAM (1982) Leucine aminopeptidase activity in the digestive tract of perch, Perca fluviatilis L. J Fish Biol 21:615–622

    Article  CAS  Google Scholar 

  • Kawabata S, Miura T, Morita T, Kato H, Fujikawa K, Iwanaga S, Takada K, Kimura T, Sakakibara S (1988) Highly sensitive peptide-4-methylcoumaryl-7-amide substrates for blood-clotting proteases and trypsin. Eur J Biochem 172:17–25

    Article  CAS  Google Scholar 

  • Kolena J, Jezová M, Vranová J, Scsuková S (1999) Structure-stabilizing effect of albumin on rat ovarian LH/hCG receptors. Biochim Biophys Acta 1416:208–216

    Article  CAS  Google Scholar 

  • Kurokawa T, Suzuki T (1995) Structure of the exocrine pancreas of flounder (Paralichthys olivaceus): immunological localization of zymogen granules in the digestive tract using anti-trypsinogen antibody. J Fish Biol 46:292–301

    Article  CAS  Google Scholar 

  • Kurokawa T, Suzuki T (1998) Development of intestinal brush border aminopeptidase in the larval Japanese flounder Paralichthys olivaceus. Aquaculture 162:113–124

    Article  CAS  Google Scholar 

  • Molinari I, Souare K, Lamireau T, Fayon M, Lemieux C, Cassaigne A, Montaudon D (2004) Fecal chymotrypsin and elastase-1 determination on one single stool collected at random: diagnostic value for exocrine pancreatic status. Clin Biochem 37:758–763

    Article  CAS  Google Scholar 

  • Murashita K, Matsunari H, Kumon K, Tanaka Y, Shiozawa S, Furuita H, Oku H, Yamamoto T (2014) Characterization and ontogenetic development of digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae. Fish Physiol Biochem 40:1741–1755

    Article  CAS  Google Scholar 

  • Navarro-Guillén C, Rønnestad I, Jordal AO, Moyano FJ, Yúfera M (2017) Involvement of cholecystokinin (CCK) in the daily pattern of gastrointestinal regulation of Senegalese sole (Solea senegalensis) larvae reared under different feeding regimes. Comp Biochem Physiol A 203:126–132

    Article  Google Scholar 

  • Olsen J, Kokholm K, Norén O, Sjöström H (1997) Structure and expression of aminopeptidase N. In: Ansorge S, Langner J (eds) Cellular peptidases in immune functions and diseases. Springer, Berlin, pp 47–57

    Chapter  Google Scholar 

  • Ribeiroa L, Zambonino-Infante JL, Cahub C, Dinisa MT (1999) Development of digestive enzymes in larvae of Solea senegalensis, Kaup 1858. Aquaculture 179:465–473

    Article  Google Scholar 

  • Rønnestad I, Yúfera M, Ueberschär B, Ribeiro L, Sæle Ø, Boglione C (2013) Feeding behaviour and digestive physiology in larval fish: current knowledge, and gaps and bottlenecks in research. Rev Aquacult 5:S59–S98

    Article  Google Scholar 

  • Rungruangsak-Torrissen K, Moss R, Andresen LH, Berg A, Waagbø R (2006) Different expressions of trypsin and chymotrypsin in relation to growth in Atlantic salmon (Salmo salar L.). Fish Physiol Biochem 32:7–23

    Article  CAS  Google Scholar 

  • Sawada H, Yokosawa H, Hoshi M, Ishii S (1983) Ascidian sperm chymotrypsin-like enzyme; participation in fertilization. Experientia 39:377–378

    Article  CAS  Google Scholar 

  • Srivastava AS, Kurokawa T, Suzuki T (2002) mRNA expression of pancreatic enzyme precursors and estimation of protein digestibility in first feeding larvae of the Japanese flounder, Paralichthys olivaceus. Comp Biochem Physiol 132A:629–635

    Article  CAS  Google Scholar 

  • Yasuike M, Iwasaki Y, Nishiki I, Nakamura Y, Matsuura A, Yoshida K, Noda T, Andoh T, Fujiwara A (2018) The yellowtail (Seriola quinqueradiata) genome and transcriptome atlas of the digestive tract. DNA Res 25:547–560

    Article  CAS  Google Scholar 

  • Zambonino-Infante JL, Cahu CL (2001) Ontogeny of the gastrointestinal tract of marine fish larvae. Comp Biochem Physiol C 130:477–487

    CAS  Google Scholar 

  • Zeytin S, Schulz C, Bernd Ueberschär B (2016) Diurnal patterns of tryptic enzyme activity under different feeding regimes in gilthead sea bream (Sparus aurata) larvae. Aquaculture 457:85–90

    Article  CAS  Google Scholar 

  • Zimmerman M, Yurewicz E, Patel G (1976) A new fluorogenic substrate for chymotrypsin. Anal Biochem 70:258–262

    Article  CAS  Google Scholar 

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Acknowledgements

Yellowtails were kindly provided by T. Hotta, K. Yoshida, and Y. Fujinami from the Seikai National Fisheries Research Institute, Japan Fisheries Research and Education Agency, Japan. Ms. A. Ikimi and Y. Masuda helped with the sampling and measurements. This work was supported in part by funds from the Japan Science and Technology Agency.

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  1. Seikai National Fisheries Research Institute, Japan Fisheries Research and Education Agency, 1551-8 Taira-machi, Nagasaki, 851-2213, Japan

    Tadashi Andoh

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Andoh, T. Dilution of digestive fish proteases with protein-free blocking reagents prevents loss of catalytic activity during microquantification. Fish Sci 86, 543–550 (2020). https://doi.org/10.1007/s12562-020-01422-4

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