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Isolation of Lysyl Oxidase from Human Umbilical Cord
Author(s) -
Pestov Nikolay,
Korneenko Tatyana,
Shidlovskaya Natalia,
Petrukhin Vasily,
Modyanov Nikolay
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.721.33
Subject(s) - chemistry , lysyl oxidase , amine oxidase , biochemistry , oxidative deamination , amine oxidase (copper containing) , diamine oxidase , cadaverine , chromatography , enzyme , putrescine
Five isoforms of human lysyl oxidases (LOX, LOXL1‐4) catalyze oxidative deamination of various primary amines and ε‐amino groups of lysine residues in proteins. LOX expression is up‐regulated by hypoxia in normal and cancer tissues. Usually, LOX is isolated from bovine aorta. However, authentic human enzyme should be preferred for pharmacological studies. We have optimized the aorta LOX protocol for human umbilical cord. The procedure includes homogenization, differential washes, urea extraction, absorption on hydroxyapatite and sulfoethylcellulose, followed by chromatography on DEAE‐cellulose and gel filtration. Simultaneously, semicarbazide sensitive amine oxidase (SSAO) can be also isolated using triton extraction followed by concanavalin A and ion exchange chromatography. Amine oxidase activities were measured as hydrogen peroxide release coupled to oxidation of 10‐acetyl‐3,7‐dihydrophenoxazine by horseradish peroxidase with methylamine as a SSAO substrate and free lysine or polyallylamine for LOX. In contrast with placenta, the cord lacks monoamine oxidase and diamine oxidase. In comparison with aorta, umbilical cord is poor in SSAO, whereas the yield of active lysyl oxidase is several folds higher. Immunoblotting using polyclonal antibodies against the five lysyl oxidase isoforms indicate that the isolated preparation contains predominantly LOX. In conclusion, the human umbilical cord is an excellent source of lysyl oxidase. Supported by RFBR grant 13‐04‐01413.