1. Comparison of l-tyrosine containing dipeptides reveals maximum ATP availability for l-prolyl-l-tyrosine in CHO cells
Natascha Verhagen, Andy Wiranata Wijaya, Attila Teleki, Muhammad Fadhlullah, Andreas Unsöld, Martin Schilling, Christoph Heinrich, Ralf Takors Eng Life Sci. 2020 Jun 8;20(9-10):384-394. doi: 10.1002/elsc.202000017. eCollection 2020 Sep.
Increasing markets for biopharmaceuticals, including monoclonal antibodies, have triggered a permanent need for bioprocess optimization. Biochemical engineering approaches often include the optimization of basal and feed media to improve productivities of Chinese hamster ovary (CHO) cell cultures. Often, l-tyrosine is added as dipeptide to deal with its poor solubility at neutral pH. Showcasing IgG1 production with CHO cells, we investigated the supplementation of three l-tyrosine (TYR, Y) containing dipeptides: glycyl-l-tyrosine (GY), l-tyrosyl-l-valine (YV), and l-prolyl-l-tyrosine (PY). While GY and YV led to almost no phenotypic and metabolic differences compared to reference samples, PY significantly amplified TYR uptake thus maximizing related catabolic activity. Consequently, ATP formation was roughly four times higher upon PY application than in reference samples.
2. A sensitive and specific assay for granulocyte elastase in inflammatory tissue fluid using L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide
H Tanaka, T Shimazu, H Sugimoto, T Yoshioka, T Sugimoto Clin Chim Acta. 1990 Feb 28;187(2):173-80. doi: 10.1016/0009-8981(90)90344-r.
Granulocyte elastase (GE, EC 3.4.21.37) is a key enzyme in tissue injury. To elucidate the role of GE in tissue injury, a new method of measuring GE activity in various inflammatory tissue fluids was developed using diazotization and the chromogenic synthetic substrate, L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide (S-2482). GE activity demonstrated first order kinetics in the range from 1.9 to 30 U/l. Other proteases, such as pancreatic elastase, trypsin, and chymotrypsin did not hydrolyze S-2484. This assay permits the determination of GE activity with a coefficient of variance less than 7.8% and 95.6 to 105.4% recovery. With this method, hydrolytic GE activity was found to be increased in bronchoalveolar lavage fluid from patients with ARDS or pneumonia, synovial fluid from patients with rheumatoid arthritis, and blister fluid from burn patients.
3. L-Pyroglutamyl-L-prolyl-L-valine-p-nitroanilide, a highly specific substrate for granulocyte elastase
J A Kramps, C van Twisk, A C van der Linden Scand J Clin Lab Invest. 1983 Sep;43(5):427-32.
L-Pyroglutamyl-L-prolyl-L-valine-p-nitroanilide was found to be a highly specific substrate for human granulocyte elastase. At pH 8.3 and 37 degrees C, its Km = 0.55 mmol/l and the value for kcat was 6 sec-1, whereas with porcine pancreatic elastase these values were approximately 2 mmol/l and less than 0.001 sec-1, respectively. It is not cleaved by trypsin or chymotrypsin. With granulocyte elastase this new substrate is 50 times more sensitive compared to succinyltrialanyl-p-nitroanilide. L-Pyroglutamyl-L-prolyl-L-valine-p-nitroanilide can also be used for the assay of granulocyte elastase inhibitors.
