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Title:Characterization, purification, and DNA sequence analysis of a novel ATPase of the archaebacterium Methanococcus voltae
Author(s):Dharmavaram, Rita M.
Doctoral Committee Chair(s):Konisky, Jordan
Department / Program:Biology
Discipline:Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Biology, Cell
Abstract:Membrane-bound ATPase activity was detected in the methanogen Methanococcus voltae. The ATPase was inhibited by vanadate, a characteristic inhibitor of the P type ATPases. The enzyme activity was also inhibited by diethylstilbestrol. However, it was insensitive to N,N$\sp\prime$-dicyclohexylcarbodiimide, bafilomycin A$\sb1$, nitrate, ouabain and oligomycin. The enzyme displayed a high preference for ATP as substrate, was dependent on Mg$\sp{2+}$, and had a pH optimum of 7.5. The enzyme was completely solubilized with 2% Triton X-100. It was insensitive to oxygen and was stabilized by ATP.
The M. voltae ATPase was purified by a procedure that included, purification of cytoplasmic membrane by sucrose gradient centrifugation, solubilization with Triton X-100, and DEAE-Sephadex and Sephacryl S-300 chromatography. While the DEAE-Sephadex step provided a preparation consisting of two polypeptides (74 kDa and 52 kDa), the Sephacryl S-300 step yielded a product with a subunit of 74 kDa. Incubation of either membranes or purified ATPase with ($\tau-\sp{32}$P) ATP followed by acidic (pH-2.4) lithium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the vanadate-sensitive labeling a 74 kDa acyl phosphate intermediate.
Antibodies were raised against the purified ATPase and the clones containing the ATPase gene were picked from a lambda ZAP II library. The nucleotide sequence of the ATPase gene was determined. The enzyme was determined to be acidic and had a monomeric molecular weight of 59.702 kDa as determined by an analysis of the DNA sequence. A putative phosphorylation site was identified. The enzyme was determined not to be significantly related to any of the known ATPases. This is the first report of an ATPase with these properties.
Issue Date:1990
Type:Text
Language:English
URI:http://hdl.handle.net/2142/22342
Rights Information:Copyright 1990 Dharmavaram, Rita M.
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9114220
OCLC Identifier:(UMI)AAI9114220


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