Document Detail


Purification of MINUS: A negative regulator of microtubule nucleation in a variety of organisms.
MedLine Citation:
PMID:  16420961     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Microtubules (MT) are important for cell behavior and maintenance, yet the factors regulating MT assembly in vivo remain obscure. In a biochemical search, we have isolated a small (4.7 kDa) acidic, phosphorylated polypeptide, which we named MINUS (microtubule nucleation suppressor) for its activity to inhibit MT nucleation [P. Fanara, B. Oback, K. Ashman, A. Podtelejnikov, R. Brandt, EMBO J. 18 (1999) 565]. Here, the purification strategy was optimized and the polypeptide purified to homogeneity from bovine brain, Drosophila, Caenorhabditis elegans and yeast. Amino acid analysis showed similar composition of MINUS from different species. In particular, MINUS was rich in glycine, threonine, isoleucine, leucine and acidic amino acids. Inductively coupled plasma mass spectrometry revealed a large peak for phosphorus confirming its identity as a phosphopeptide. For further purification, MINUS was separated as a single peak on reverse phase-HPLC (RP-HPLC). Preliminary sequence analysis suggested MINUS to be N-terminally blocked. However, conventional enzymatic digestions did not reveal differences in the peak profile compared to undigested MINUS. Hence, partial acid hydrolysis and proteinase K digestion was performed followed by RP-HPLC. The proteinase K digested peaks were subjected to Edman degradation (first peak, ser-pro-ser/gly-ser; second peak, tyr/arg-leu), mass spectrometry (no result) and MALDI analysis (no result). Collectively, the data suggest that MINUS belongs to a new class of MT assembly regulators. Sequence information and antibody development will be useful to examine its biological role in a definitive manner.
Authors:
Neelam Shahani; Srinivasa Subramaniam; Roland Brandt
Publication Detail:
Type:  Journal Article     Date:  2006-01-18
Journal Detail:
Title:  International journal of biological macromolecules     Volume:  39     ISSN:  0141-8130     ISO Abbreviation:  Int. J. Biol. Macromol.     Publication Date:  2006 Aug 
Date Detail:
Created Date:  2006-07-24     Completed Date:  2006-09-15     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  7909578     Medline TA:  Int J Biol Macromol     Country:  England    
Other Details:
Languages:  eng     Pagination:  15-22     Citation Subset:  IM    
Affiliation:
Department of Neurobiology, University of Osnabrück, Barbarastrasse 11, D-49076 Osnabrück, Germany. neelam.shahani@biologie.uni-osnabrueck.de
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MeSH Terms
Descriptor/Qualifier:
Animals
Caenorhabditis elegans
Caenorhabditis elegans Proteins / chemistry,  isolation & purification*
Cattle
Chromatography, High Pressure Liquid
Drosophila
Drosophila Proteins / chemistry
Microtubule-Associated Proteins / chemistry,  isolation & purification*
Saccharomyces cerevisiae / chemistry,  isolation & purification*
Saccharomyces cerevisiae Proteins / chemistry,  isolation & purification*
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Chemical
Reg. No./Substance:
0/Caenorhabditis elegans Proteins; 0/Drosophila Proteins; 0/Microtubule-Associated Proteins; 0/Saccharomyces cerevisiae Proteins

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