trans

CALCIUM 249
acidocalcisome. The acidocalcisome is of interest
because the Ca 2 reservoir is very large
compared with other intracellular pools, and
because the acidocalcisome does not appear
to have a counterpart in the mammalian host.
Acidocalcisomes have been identified in a
wide range of kinetoplastid and apicomplexan
parasites. The low pH environment of the acidocalcisome
can be identified with fluorescent
probes, such as acridine orange, that shift
their fluorescence and absorption spectra when
they accumulate in acidic compartments. Interestingly,
pyrophosphate serves as an energy
source for acidification of the compartments
in T. cruzi, T. brucei, L. donovani, P. knowelsi and
T. gondii. A plant-like vacuolar H -pyrophosphatase
has been cloned from T. cruzi and
T. gondii. This enzyme is sensitive to the
pyrophosphate analogs imidodiphosphate or
aminomethylenediphosphate (AMDP). In addition
to the pyrophosphatase, H is pumped into
the acidocalcisome with a vacuolar type H -
ATPase. This enzyme is sensitive to bafilomycin
A 1 , and bafilomycin-sensitive acidification of
acidocalcisomes has been detected in T. cruzi,
T. brucei, L. amazonensis, T. gondii and
P. berghei. A Na /H exchanger has also been
identified, and pH within the organelle can be
neutralized in the presence of elevated Na .
When present, the Na /H exchanger is sensitive
to 3,5-dibutyl-4-hydroxytoluene, but not
to the more standard inhibitor ameloride.
The H gradient is required to help retain
Ca 2 in the acidocalcisome. Inhibitors of vacuolar
ATPase (bafilomycin A 1 ) and pyrophosphate-dependent
proton pumps (AMDP) can
disrupt acidification. These agents, along with
ion exchangers that neutralize the compartment,
can cause the release of stored Ca 2 . Ca 2
release occurs as the acidocalcisome attempts
to restore the pH gradient by Ca 2 /nH
exchange. In Plasmodium, but not kinetoplastids,
Ca 2 efflux also results from exposure to
InsP 3 . By contrast, arachidonic acid releases
Ca 2 from the acidocalcisomes of procyclic
T. brucei. The transport of Ca 2 into the acidocalcisome
occurs by means of a vacuolar
PMCA-type Ca 2 -ATPase pump. The gene for
the acidocalcisome PMCA has been cloned
from T. cruzi (Tca1) and from T. gondii (TgA1).
The protein product is distinct from other
PMCA enzymes, but similar to vacuolar PMCAs
of Entamoeba and yeast in its lack of a calmodulin-binding
site. The pump is localized along
the plasma membrane as well as the acidocalcisome
in both T. cruzi and T. gondii. The
amount of Ca 2 -ATPase varies during the life
cycle of T. cruzi, with an increase detected in
amastigote forms. A corresponding increase
in nigericin-releasable Ca 2 from amastigote
forms has also been observed.
X-ray microprobe analysis of acidocalcisomes
reveals that, in addition to Ca 2 , other
metals, including Zn 2 , Mg 2 , Na and K and
phosphorus are present. Electron energy loss
studies suggest that the phosphorus is bound
to oxygen in phosphate chains. Large quantities
of long-chain phosphates within the acidocalcisomes
of T. cruzi, T. brucei, L. major and T.
gondii have been identified with 31 P-NMR. The
long-chain polyphosphates of T. cruzi accumulate
during the transition from trypomastigote
to amastigote, and are released when
cells are subjected to osmotic stress. Pyrophosphate
analogs are able to inhibit growth
of T. cruzi, P. falciparum, T. gondii and
Cryptosporidium parvum. Whether the deleterious
effects of these analogs result from disruption
of acidocalcisomes or other primary
targets remains to be determined.
Although acidocalcisomes are ubiquitously
distributed in kinetoplastids and apicomplexans,
variations in this organelle have been
observed. Within the same organism, such as
L. donovani, two distinct acidic compartments
have been identified that contain either the
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA