The release of periplasmic proteins from Pseudomonas aeruginosa and Pseudomonas putida
Date Issued
March 1, 1980
Author(s)
Sanozky, Rosemary B.
Advisor(s)
Thomas C. Montie
Additional Advisor(s)
Jeffrey M. Becker
Raymond W. Beck
Abstract
Attempts were made to isolate periplasmic glucose binding protein from Pseudomonas aeruginosa and Pseudomonas putida. Two basic shock procedures, derived from published methods, were employed. These were shocking with high magnesium or a modification of a sucrose-EDTA osmotic shock method. The activity of asparaginase, a known periplasmic enzyme in Pseudomonas, was used as a marker to evaluate the efficiencies of the different shock procedures. Release of glucose-6-phosphate dehydrogenase, a cytoplasmic marker, was used as a measure of viability and integrity of the cells that were shocked. Glucose binding activity was not detected in the shock fluid by equilibrium dialysis or by sephadex chromatography. Glucose uptake was compared in shocked vs. non-shocked cells of Pseudomonas aeruginosa PA 46 and RM 1. No significant decrease was found in the ability of the shocked cells to take up glucose. The inner and outer membranes of P. Aeruginosa RM 1 were separated in order to attempt localization of glucose dehydrogenase, an exocytoplasmic enzyme, and to test membrane fractions for binding activity. Studies with Pseudomonas putida involved using modifications of the sucrose-distilled water osmotic shock method. The degree of extractability of some known periplasmic enzymes was investigated. In P. Putida, asparaginase was found to be tightly bound; it was released to the same degree as glucose-6-phosphate dehydrogenase, the cytoplasmic marker. Attempts to preferentially release asparaginase by using high Tris concentrations and by preparing osmotically sensitive cells were not successful. Alkaline phosphatase, another periplasmic enzyme, was not released from P. Putida cells grown in a phosphate-deficient medium, although some asparaginase activity was found in the shock fluid. These data support the hypothesis that in P. Putida, and perhaps other Pseudomonas species, some "periplasmic proteins" are tightly bound and are only partially shockable, being released upon disruption of the cell.
Degree
Master of Science
Major
Microbiology
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