Svetlana E. Soboleva1, Evgeniya E. Burkova1,2, Pavel S. Dmitrenok3, Dmitrii V. Bulgakov4, Natalia I. Menzorova3, Valentina N. Buneva1,2, Georgy A. Nevinsky1,2
- SB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia
- Novosibirsk State University, Novosibirsk, Russia
- G. B. Elyakov Pacific Institute of Bioorganic Chemistry FEB RAS, Vladivostok, Russia
- Far Eastern Branch of Russian Academy of Sciences, Federal Scientific Center of the East Asia Terrestrial Biodiversity, Vladivostok, Russia
It was proposed that most biological processes are performed by different protein complexes. In contrast to individual proteins and enzymes, their complexes usually have other biological functions, and their formation may be important system process for the expansion of diversity and biological functions of different molecules. Identification and characterization of embryonic components including proteins and their multiprotein complexes seem to be very important for an understanding of embryo function. We have isolated and analyzed for the first time a very stable multiprotein complex (SPC; approximately 1100 kDa) from the soluble fraction of extracts of the sea urchin embryos. By fast protein liquid chromatography (FPLC) gel filtration the SPC was well separated from other extract proteins. Stable multiprotein complex is stable in different drastic conditions but dissociates moderately in the presence of 8M urea + 1.0M NaCl. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis data, this complex contains many major, moderate and minor proteins with molecular masses from 10 to 95 kDa. The SPC was destroyed by 8M urea or SDS, and its components were separated using thin layer chromatography, ion-exchange chromatography, gel filtration, and reverse phase chromatography. Using matrix-assisted laser desorption/ionization mass spectrometry of partially dissociated SPC, it was shown that the complex contains not only proteins (10-95 kDa) but also few dozens of peptides with molecular masses from 2 to 9.5 kDa. Short peptides form very strong complexes, which at the treatment of SPC with urea or SDS can be partially break down into smaller complexes having different peptide compositions. Reverse phase chromatography of these complexes after all type of abovementioned chromatographies led to detection from 6 to 11 distinct peaks corresponding to new complexes containing up to a few dozens of peptides. The SPCs possess alkaline phosphatase activity. Progress in the study of embryos protein complexes can help to understand their biological functions.
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Stable multiprotein complexes obtained from sea urchins were boiled for 5 minutes in 20mM Tris-HCL with a pH of 7.5 and containing 0.15M NaCL, 1% SDS and 50nM DTT (buffer K). The samples were diluted and filtered three times until three fractions were obtained that contained proteins and peptides 30 to 100, 10 to 30, and <10 kDa. SDS-PAGE, reverse-phase chromatography (RPC), and MALDI mass spectrometry were used to analyze these fractions. A thin layer chromatography assay was also performed with the separated products being extracted from the TLC plates with water and methanol. The combined solution was centrifuged in order to remove traces of silica gel particles and then the supernatant was used for compound analysis using MALDI mass spectrometry. RPC was used to separate compounds corresponding to multiple spots after TLC and then fractions of different peaks were collected, the water was evaporated and the fractions were analyzed by MALDI mass spectrometry. 5 separate peaks were obtained by subjecting the stable multiprotein complex to gel filtration with 3 of these peaks possessing relatively low MMs. Stable multiprotein complex proteins that had MMs greater than 10 kDa were analyzed in the positive ion mode of a MALDI TOF mass spectrometer modified with a CovalX HM1 detection system. Based on the results of MALDI mass spectrometry, it was determined that three of the fractions (2,3 and 7) that were analyzed contained mostly peptides with MMs ranging between 2 and 9 kDa. The fourth contained two major proteins and the 5, 6, and 8 also contained many other proteins with the largest being found in the sixth.