Peptidase activities in candida albicans : comparison of yeast and filamentous forms

Author

David A. Logan

Date of Award

12-1981

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Jeffery M. Becker

Committee Members

Raymond W. Beck, W. S. Riggsby, Leaf Huang

Abstract

A study of the peptidases of the yeast and filamentous forms of Candida albicans H-317 was carried out to determine their array and specificity and to establish whether differences exist in the enzymes of the two cell forms. The results reveal the presence of at least three distinct peptidase activities in cell-free extracts of the yeast and filamentous forms. The activities were separated by gel filtration column chromatography or native polyacrylamide gel electrophoresis and measured by an enzyme-coupled colorimetric assay. The first peak of trimethionine hydrolyzing activity to elute from the gel filtration column was designated as peak I. The molecular weight of peak I peptidase(s) by gel filtration was estimated to be 550,000. The partially purified enzyme(s) has pH optima of 7.5 and 8.0 for the yeast and filamentous phase, respectively. The activity followed saturation kinetics with the K_m for both the yeast and filamentous phase equal to 1 X 10^-4 M. Peak I activity was inhibited by EDTA, o-phenanthroline, and citrate while p-chloromercuribenzoate caused a stimulation of its activity. Peak I activity from both phases was inhibited by KCl but not NaCl, however, the yeast activity was more sensitive to inhibition by KCl. Peak I was likely an aminopeptidase(s) with broad substrate specificity since it hydrolyzed (Met)₃, (Met)₅, (Met)₃ methyl ester, (Leu)₃, (Met)₂, Met-Phe, Met-Pro, and Leu-Gly whereas Acetyl-(Met)₃ was not hydrolyzed. The activity of peak I showed stereospecificity since it hydrolyzed D-methionyl-L-methionyl-L-methionine, and L-methionyl-L-methionyl-D-methionine but not L-methionyl-D-methionyl-L-methionine.

The second peak of trimethlonine hydrolyzing activity to elute from the gel filtration column was designated as peak II. The molecular weight of peak II peptidase(s) by gel filtration was estimated to be 120,000. The partially purified enzyme(s) had a pH optimum of 7.0 for the yeast phase and 7.0-8.0 for the filamentous phase. The K_m for the yeast and filamentous phase was 3.2 x 10^-4 M and 7.5 x 10^-5 M, respectively. Peak II peptidase(s) was inhibited by EDTA, o-phenanthroline and p-chloromercuribenzoate. Activity from both phases was inhibited by NaCl and KCl with the yeast activity yielding more inhibition by these salts. In addition to peak I and peak II peptidase(s), a dipeptidase activity was detected. This peptidase activity was strong toward Leu-Gly and was estimated by column chromatography to have a molecular weight of 150,000.

In summary, three peptidase activities of the yeast and filamentous phases of the dimorphic fungus Candida albicans have been detected and two of these were partially purified and characterized. No differences were noted in the partially purified enzymes from either phase with respect to molecular weight estimate, pH optimum or kinetic behavior. However, peak II peptidase(s) activity from the yeast phase showed greater sensitivity to inhibition by NaCl and KCl while peak II activity of the filamentous form yielded a greater degree of sensitivity to the metal chelators EDTA and o-phenanthroline.

Recommended Citation

Logan, David A., "Peptidase activities in candida albicans : comparison of yeast and filamentous forms. " PhD diss., University of Tennessee, 1981.
https://trace.tennessee.edu/utk_graddiss/13470