The mechanism of regulation of the Ca²⁺ pump ATPase of the human
erythrocyte membrane by calmodulin, cyclic AMP and the anion channel was
studied using membrane fragments, resealed "ghosts", inside-out vesicles
and a Triton X-100 solubilized enzyme preparation.
The (Ca²⁺ + Mg²⁺ )-ATPase activity in erythrocyte membranes or a
Triton X-100 solubilized enzyme preparation showed biphasic (high and low
affinity) Ca²⁺ activation kinetics. The anionic calcium binding protein, calmodulin, increased both the calcium sensitivity (Kca²⁺) and the maximum velocity (Vmax ) of the enzyme. Certain polyanionic agents (poly-L-aspartic acid, poly-L-glutamic acid), alicyclic sulfonic acids (HEPES,N-2-hydroxyethylpiperazine-N¹-2-ethanesulfonic acid, MES,2-N-
(morpholinoethanesulfonic acid)), and aromatic carboxylic acids (benzoic
and salicylic acids) increased the Kca²⁺ but not the Vmax of (Ca²⁺ +
Mg²⁺ )-ATPase in erythrocyte membranes and Triton X-100 solubilized enzyme
preparations. Trifluoperazine (30 μM) antagonized activation of the
enzyme by calmodulin and poly-L-aspartic acid, but not by sodium-HEPES
or sodium-MES. Limited trypsin proteolysis of (Ca²⁺ + Mg²⁺ )-ATPase in the erythrocyte membrane abolished activation by calmodulin, poly-L-aspartic acid and sodium-HEPES. These results suggest that the modulation of the Ca²⁺ sensitivity of (Ca²⁺ + Mg²⁺ )-ATPase by calmodulin may
be associated with the anionic properties of this protein, and that this
property can be mimicked by some other anions, probably by interacting
at an anion-regulatory site on the enzyme.
Cyclic AMP (5 μM) was found to inhibit the (Ca²⁺ + Mg²⁺)-ATPase
activity (approx. 20%) in erythrocyte membranes, probably via endogenous
cyclic AMP protein kinase, since this effect could be blocked by cyclic
AMP protein kinase inhibitor (PKI) from the rabbit skeletal muscle,
By contrast, bovine heart PKI stimulated (Ca²⁺ + Mg²⁺ )-ATPase activity
(approx. 100%) by increasing the Kca²⁺ but not the Vmax of the enzyme
in membrane or Triton X-100 solubilized preparations. At a low calcium
concentration the stimulation by bovine heart PKI and saturating levels
of calmodulin was additive, suggesting that the two effectors acted by
distinct mechanisms. The stimulation of (Ca²⁺ + Mg²⁺ )-ATPase activity
by bovine heart PKI was not solely due to its antagonism of the protein
kinase because a) modification of arginine residues of bovine heart PKI
abolished its inhibition of cyclic AMP protein kinase, but had no effect
on the stimulation of (Ca²⁺ + Mg²⁺ )-ATPase; b) trifluoperazine (20 μM)
antagonized the stimulation of (Ca²⁺ + Mg²⁺ )-ATPase by PKI, similarly to
its antagonism of calmodulin stimulation, but it did not affect the
inhibition of protein kinase by PKI. It is suggested that different
mechanisms are involved in the inhibition of cyclic AMP protein kinase
and the stimulation of (Ca²⁺ + Mg²⁺ )-ATPase by bovine heart cyclic AMP
PKI.
Next, the role of anion channel blockers on the (Ca²⁺ + Mg²⁺ )-
ATPase was studied. The photolabeling reagent N-(4-azido-2-nitrophenyl)-
2 aminoethylsulfonate (NAP-taurine) was found to inhibit the (Ca²⁺+
Mg²⁺ )-ATPase of fragmented red cell membranes. Half maximal inhibition
occurred between 25 μM and 50 μM. At these concentrations Mg²⁺ -ATPase
and (Na⁺ + K⁺)-ATPase activities in the membranes were not affected.
The reversible inhibition of (Ca²⁺ + Mg²⁺ )-ATPase produced by NAP-taurine in the dark became irreversible after photolysis in the presence
of this reagent. Incubation of the membranes with Ca²⁺ , Mg²⁺ , ATP or
calmodulin, prior to photolysis in the presence of NAP-taurine, did not
protect the enzyme from Inhibition. Limited trypsin proteolysis of
(Ca²⁺ + Mg²⁺ )-ATPase in fragmented membranes, which abolished activation
by calmodulin, did not affect the inhibition by NAP-taurine.
NAP-taurine was found to Inhibit the (Ca²⁺ + Mg²⁺ )-ATPase activity from the cytoplasmic side of the membrane, as determined from the following
experiments. Addition of NAP-taurine (50 μM) to resealed erythrocyte
ghosts inhibited less than 5% of the (Ca²⁺ + Mg²⁺ )-ATPase activity,
compared to 50-60% Inhibition in ghosts resealed in the presence of 50 μM
NAP-taurine. Furthermore, NAP-taurine inhibited ATP-dependent Ca²⁺ -
transport into inside-out vesicles at a similar concentration (50 μM).
The inhibition of the (Ca²⁺ + Mg²⁺ )-ATPase activity of membranes by NAP-taurine appeared to be a direct action on the enzyme, rather than through
inhibition of the anion channel, as (Ca²⁺ + Mg²⁺ )-ATPase activity was not
inhibited in membranes made from red blood cells reacted irreversibly
with 50 μM NAP-taurine or the anion channel blocker 4,4'-diisothiocyano-
2,2' stilbene disulfonate (DIDS) (5 μM) or in membranes assayed in the
presence of another anion channel blocker, probenecid (125 μM). This is
the first reported selective antagonist of the Ca²⁺ pump, and it is suggested that NAP-taurine could be a useful tool for studying the Ca²⁺-
transport ATPase in a variety of cells. / Pharmaceutical Sciences, Faculty of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/23912 |
| Date | January 1982 |
| Creators | Minocherhomjee, A. M. |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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