It has been shown that the oligomeric state of the Ca-ATPase has a direct impact upon its enzymatic function (Shi et al., 1996; Squire et al., 1988a; Voss et al.,1991; Karon & Thomas 1993). In other words, there are a large number of pumps in each SR, and if they aggregate in the bi-layer, their activity is decreased. Previous studies have shown that this aggregation and ensuing inhibition occur in the presence of various local anesthetics; hexanol, halothane, and diethyl ether (Kutchai et al., 1997). Activity assays performed by Merck Research Laboratories (Coll el al., 1999) suggest that ellagic acid (EA), a nonvolatile nucleotide mimic, may have a similar inhibitory effect. The Thomas Lab, at the University of Minnesota, decided to perform further research to find EA掇 effect on the Ca-ATPase oligomerization. The purpose of this study is twofold. First, to discover whether ellagic acid can be used to oligomerize the Ca-ATPase. If EA has this effect, EA will be labeled as a nonvolatile chemical that can be used to regulate the Ca-ATPase and control muscle movement. Second, to study the relationship between oligomeric state and enzymatic function of the Ca-ATPase.
In order to measure ellagic acid掇 impact on the Ca-ATPase, oligomeric state and enzymatic activity of the protein were examined. Oligomeric state of the Ca-ATPase is directly associated with its amount of mobility within the membrane. Ca-ATPase mobility was monitored by labeling with a phosphorescent dye, erythrosin 5-isothiocynate (ERITC), exciting this dye with a laser, and comparing the intensity of vertical and horizontally polarized light emissions over time. The oligomeric state of the Ca-ATPase was then determined by comparing the movements of Ca-ATPase samples under varying concentrations of ellagic acid. The measurement and comparison of the intensity of emitted light is called anisotropy. Enzymatic activity was evaluated by indirectly measuring the rate of ATP utilization.
Based on Merck掇 report (Coll el al., 1999) there was reason to believe that ellagic acid would cause aggregation of Ca-ATPase pumps and therefore inhibit their enzymatic function. Furthermore, increasing concentration of EA should cause greater inhibition.
Ca-ATPase samples were labeled with phosphorescent dye, erythrosin 5-isothiocynate (ERITC) which was obtained from Molecular Probes Inc. and was stored at -20。. All stock solutions and buffers were prepared at the Thomas Lab. 10 mM ellagic acid stock was prepared in di-methyl sulfoxide (DMSO).
Preparation of Ca-ATPase
Light SR (LSR) with embedded Ca-ATPase was provided by the Thomas Lab and was obtained from the fast twitch skeletal muscle of a white rabbit. LSR is composed of approximately 80% Ca-ATPase. LSR was prepared by the methods previously described in (Karon et al., 1994) and was stored at -80。.
Assays
Ca-ATPase activity was measured at 25。 by the method described in the Thomas Lab protocol for ATPase Activity Assays. This procedure uses a coupled enzyme method. The rate of the Ca-ATPase cleaving ATP is indirectly measured. Pyruvate kinase (PK), an enzyme that changes ADP back to ATP, reacts with ADP immediately after it is produced from the Ca-ATPase. To initiate this reaction, PK cleaves a phosoenolpyruvate (PEP) molecule, turning the PEP into pyruvate. A second enzyme, lactate dehydrogenase (LDH) takes the newly produced pyruvate and turns it into lactate, by oxidizing NADH. The oxidation of NADH results in a color change measurable at 340nm in a spectrophotometer (see Figure 2). The reaction of PEP -> pyruvate -> lactate is faster than the cleaving of ATP; therefore, the rate of these reactions is dependent upon the Ca-ATPase activity. This means that the rate of ATPase activity can be determined by measuring the rate of NADH loss. ATPase activity was measured with ellagic acid concentrations ranging from 0然-300然 (p[EA]= -log [Ellagic acid] ranging from 7 to 3.5). The amount of activity was determined by comparing the percent of activity under different ellagic acid concentrations. This experiment was repeated three times and the results for all three trials were averaged to produce Figure 4.
Labeling Procedure
Labeling of Ca-ATPase was achieved by incubating 1 mg of LSR with an excess amount of ERITC at 25。 for an hour. The labeled LSR solution was then cleansed of excess dye by adding bovine serum antigen (BSA) which binds to the dye allowing it to be separated by centrifugation. The labeled SR was then assayed to determine whether there was a 1:1 ratio between dye and protein.
Time-resolved Phosphorescence Anisotropy
Using a phosphorescence spectrometer (a laser that emits light of a specific polarization and intensity over a very short time period) the labeled samples were exposed to pulses of both horizontally and vertically polarized light (See Figure 3a).
Oligomeric state of the Ca-ATPase was determined by measuring anisotropy. Time-dependent anisotropy, r(t), can be defined by the equation:
where Ivv is the intensity of light emitted from the label in the vertical direction and Ivh is the intensity of light emitted in the horizontal direction.
The intensity of the light emitted from the label was then measured over a time of 1000 microseconds. The two intensities emitted in two different polarizations, Ivv and Ivh, were measured and compared by using the equation above. Ca-ATPase movement was then determined: If the Ca-ATPase is unrestricted, Ivh will change dramatically over time, while growing closer to Ivv. In this case, the r(t) graph, the ratio of Ivv to Ivh, will approach zero over time and r(
), the tail end of the graph, will approach zero. If the Ca-ATPase has restricted motion (like it does when positioned in an unaggregated bi-layer) Ivh will change little over time, growing only slightly closer to Ivv. The r(t) graph, of the somewhat restricted Ca-ATPase, will approach some value between zero and the horizontal line y=r0 (See figure 3b) . If Ca-ATPase motion is completely restricted, like it is when completely aggregated, the Ivh intensity will be negligible compared to the Ivv intensity, because if it is aggregated, the Ca-ATPase will not move and Ivh will not change. The r(t) graph will then be a horizontal line, y=r0.
As the Ca-ATPase becomes more aggregated, its motion becomes more restricted. Therefore, by comparing the difference of r( ) values for each ellagic acid concentration graph to the control graph (no ellagic acid) we are able to determine a numerical value for the difference in oligomeric state:
Anisotropy of ERITC labeled Ca-ATPase was measured with EA concentrations of 0然, 5然, 10然 and 100然. 15 loops of 1000 laser pulses were fired at the labeled sample and anisotropy was measured every microsecond for 1000 microseconds. The average phosphorescence anisotropy decays, r(t), for all 15 loops was automatically calculated and graphed using Microsoft Origin, a data plotting program.
Effect of ellagic acid on the enzymatic activity of the Ca-ATPase
Figure 4, the activity assay results recovered in the Thomas Lab, shows that EA produces 10% inhibition of Ca-ATPase at 1然 (p[Ellagic Acid] 7.0) and 95% inhibition at 320然 (pEA 3.5). Notice that this graph shows a linear relationship between EA concentration and % of normal activity. Also, notice that greater ellagic acid concentration causes greater inhibition.
Effect of ellagic acid on the oligomeric state of the Ca-ATPase
In figure 5, the TPA results, the r( ) value increases as the concentration of EA increases. The comparison of r( ) values of each ellagic acid samples to the 0然 control (Figure 6) clearly shows that greater concentrations of EA produces greater aggregation of the Ca-ATPase. 100然 (pEA 4.0) EA causes 85% aggregation of the ATPase, almost complete aggregation. 5然 (pEA 5.3) EA causes 40% aggregation.
The effect of ellagic acid on both oligomeric state and enzymatic function
pEA 4.0 (100然) causes both 85% aggregation and 77% inhibition. pEA 7.0 (1然) causes 17% aggregation and 5% inhibition. This roughly 10% difference between aggregation and inhibition was expected because not all pumps that were aggregated were also inhibited. Pumps that do not clump together do not necessarily shut off. The results show that 10% of aggregated SR Ca-ATPase pumps remain activated.
Future direction
The ultimate goal of testing the effect of ellagic acid on the Ca-ATPase is to discover whether EA can be used as an anaesthesia. Although this study does give a preliminary report on the effect of EA on the Ca-ATPase pump, a few more experiments must be preformed before EA can be marketed as such. Most importantly, the same methods must be performed on cardiac SR Ca-ATPase (CSR). Surprisingly, Merck reported (Coll el al., 1999) that EA activates CSR at low concentrations and then inhibits it at lower concentration. Because of Merck掇 exciting report, I plan to study ellagic acid掇 effect on CSR next summer.
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