Saddleback Journal of Biology - Saddleback College

Fall 2009 Biology 3B Paper
Velasquez, M.T., Menitove, J.E., Skelton, M.M.,
Cowley, A.W. 1987. Hormonal responses and blood
pressure maintenance in normal and hypertensive
subjects during acute blood loss. Hypertension 9:
423-428.
Knockdown and Effect of Lactational Hormones on CTR-1 and Ceruloplasmin
Michael Wan, Christine Kassisa, Maria Linder
Department of Chemistry and Biochemistry
California State University Fullerton
A series of experiments were performed to understand what mediates the transport of
copper into and across the mammary gland during lactation. This was achieved by
attempting to determine 1) the effects of lactational hormones (prolactin, insulin, and
dexamethazone) on the expression of the copper transporter CTR1 and on the production
and secretion of ceruloplasmin by mammary epithelial cells 2) the effects of knocking down
CTR1 expression on uptake of copper by mammary epithelial cells. Copper uptake studies
were performed with radioactive 64 Cu introduced into PMC42 cells. SDS PAGE and
Western Blotting allowed us to characterize CTR1 and ceruloplasmin expression while
Real Time PCR allowed us to quantify the expression of mRNA. The results suggest: 1)
CTR1 may not be involved in copper uptake; 2) lactational hormones appear to increase
the expression of CTR1 at the protein level. The effects of lactational hormones on
ceruloplasmin at the protein level and CTR1 at the mRNA level remain unclear and
require further investigation.
Introduction
Copper is an essential nutrient for that is
required in the growth and development of all living
organisms. In mammals, copper is absorbed primarily
in the small intestine via CTR1 (Linder and Azam,
1996). CTR1 is a transmembrane protein/channel that
is responsible for cellular copper uptake and transport
of copper across the plasma membrane (Nose, 2006
and Sinani et al., 2007). It exists as a trimer (105 kDa)
and is highly specific for Copper (Guo et al., 2004).
Like all essential metals, copper is potentially toxic at
increased concentrations. Thus, homeostatic
mechanisms have evolved to avoid copper toxicity
while providing sufficient copper for metabolic
requirements. As copper enters the blood, it is
immediately bound to albumin and transcuprein - blood
plasma proteins (Linder and Azam, 1996). Copper
bound to the plasma proteins is then transported and
deposited to organs, namely the liver. In the liver,
copper is incorporated into ceruloplasmin and secreted
back into the blood bound to ceruloplasmin where it is
then ready for distribution to target organs.
Ceruloplasmin is one of the major copper
transporting proteins and holds the majority of copper
(90-95%) in human blood plasma, conducting most of
the delivery to tissues in the body (Takahashi et al.,
1984). It is an enzyme synthesized in the liver
containing 6 atoms of copper bound tightly at defined
sites in its structure. It has a molecular weight of
approximately 132 kDa, is composed of 1046 amino
acids. Ceruloplasmin binds copper in the liver, and
traffics it to necessary tissues and organs in the body.
During lactation, about 50% of copper ions entering the
blood are transported to the mammary glands (in rats)
where it crosses the mammary epithelial cells and
enters the milk (Linder et al., 1998). Although it is still
unclear how copper is donated to CTR1, ceruloplasmin
is suspected to be the copper donating protein
(Zatulovsky et al., 2007). Decreased serum
ceruloplasmin levels are characteristic in the genetic
disorder Wilson disease, also known as
64
Saddleback Journal of Biology
Spring 2010