Date of Award

1985

Publication Type

Doctoral Thesis

Degree Name

Ph.D.

Department

Biological Sciences

Keywords

Biology, Molecular.

Rights

info:eu-repo/semantics/openAccess

Abstract

A Ca('2+)-dependent activator of bovine cyclic nucleotide phosphodiesterase (PDE) from the oomycetous water mold Achlya ambisexualis was purified and characterized; it is an electrophoretically homogeneous protein (pI 4.3), migrates slightly faster than bovine calmodulin (CaM) and shows a Ca('2+)-dependent mobility shift on SDS polyacrylamide gels. Achlya CaM competes with antibodies raised against bovine brain and rat testis CaMs. Analysis indicates 10-20 more amino acid residues than CaMs reported from other sources and the presence of a trimethyllysine residue. Achlya CaM activates PDE in a Ca('2+)-dependent manner. Its tyrosine fluorescence is unaltered by Ca('2+) or by EGTA but is enhanced by Al('3+). Vegetative hyphae contain 8-10 ng CaM/mg protein; the level doubles to 15-20 ng/mg protein during induction of asexual sporulation. About 77% of the CaM is in the soluble fraction of homogenates. Immunofluorescence study reveals that CaM is mainly cytoplasmic, with growing tips showing enhanced CaM levels. When vegetative mycelia are induced to reproduce asexually CaM becomes concentrated in the sporangial apices. After spore release bands of CaM persist around the exit pores of sporangia and cysts of Achlya and other members of the Saprolegniaceae studied. Calcofluor pulse-chase experiments indicate that sporangiogenesis involves a transition from tip growth to overall growth of walls. Anti-calmodulin drugs such as trifluoperazine (TFP) inhibit Achlya CaM-dependent activation of bovine PDE, spore germination, germ tube growth, colony formation, sporangial induction and spore release, with the latter process being the most sensitive; growing hyphae are insensitive. Rhodamine-phalloidin or anti DNAse I-stained F-actin structures observed in Achlya include peripheral plaques (near the hyphal tips), cytoplasmic filaments and intranuclear structures. Differentiating sporangia have uniformly distributed actin plaques. Intranuclear actin becomes evident only in hyphae induced to produce asexual sporangia, and persists in fresh cysts. In a germinating cyst the peripheral plaques become localized in the zone of germ tube emergence. Calcofluor pulse-chase experiments suggest that concentrations of plaques are associated with actively growing hyphal tips and new emerging branches. The actin plaques tend to adhere to the cell walls. Failure of attempts to isolate Ca('2+)-CaM-dependent PDE from Achlya suggests its absence in the water mold.Dept. of Biological Sciences. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1985 .S879. Source: Dissertation Abstracts International, Volume: 46-08, Section: B, page: 2573. Thesis (Ph.D.)--University of Windsor (Canada), 1985.

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