Purification of Calmodulin and Quantification of Its Levels in Mammary Tissue and Isolated Cells During Pregnancy and Lactation (Acini, Secretory, Myoepithelial)

Calmodulin has been purified to apparent homogeneity from lactating bovine mammary tissue. Calmodulin was purified by heat treatment and sequential anion exchange and gel exclusion chromatographies. The molecular weight of the purified protein was estimated by gel electrophoresis to be 18,000 daltons. The purified protein produced a 10-fold stimulation of the activity of bovine brain cyclic nucleotide phosphodiesterase (PDE) in a calcium dependent manner. This report details a method for the assay of biologically active calmodulin from lactating mammary tissue based on stimulation of PDE. The present study also reports the calmodulin content of rat mammary tissue and isolated cells obtained by the PDE assay and a radioimmunoassay (RIA) procedures. The calmodulin content of rat mammary tissue increased 2.5-fold near the time of parturition, remained at the elevated level during lactation, then, after the onset of involution, decreased to values similar to those measured from tissue of pregnant rats. When tissue from 15 animals in different stages of pregnancy, lactation and involution were compared, the RIA gave 2.6-fold higher results than the PDE assay. Secretory and myoepithelial cells were enzymatically dissociated from rat mammary tissue during pregnancy, lactation and involution. Protein, DNA, lactose, glucose-6-phosphate dehydrogenase and alkaline phosphatase were assayed to characterize the cell fractions. By using RIA, the calmodulin content per mg of protein in isolated secretory cells was high near parturition, then decreased and remained relatively constant during lactation. The amount of calmodulin per mg of DNA in secretory cells did not show a marked change near parturition, suggesting a constant amount of calmodulin per cell. The calmodulin content of myoepithelial cells measured by using RIA was 6-fold lower than in secretory cells. The changing levels of calmodulin in rat mammary tissue during lactogenesis are suggested to be related to proliferation and destruction of secretory cells, events that occur near parturition and involution respectively.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Riss, Terry Lee

• Biology, General

Language dc:language

eng

Identifier

(UMI)AAI8422140

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/77639