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An apparatus for rapid kinetic analysis of isotopic efflux from membrane vesicles and of ligand dissociation from membrane proteins
ANALYTICAL
BIOCHEMISTRY
14&495-505
(1984)
An Apparatus for Rapid Kinetic Analysis of Isotopic Efflux from Membrane Vesicles and of Ligand Dissociation from Membrane Proteins BLISS FORBUSH III Department of Physiology, Yale School of Medicine, 333 Cedar Street, New Haven, Connectinrt 06510 Received December 27, 1983 A new method for the measurement of rapid isotopic release from a membrane compartment is described. Membrane vesiclesloaded with isotope, or broken membranes with bound radioactive ligand, are filtered onto the surface of a cellulose ester filter; the rate of the loss of isotope from the membrane compartment is followed continuously by collecting fluid which is passed through the filters under high pressure. A change in release rate is initiated by changing the solution or by delivering a flash of light to a photosensitive sample. The approach has been used to study rapid 22Na efflux from membrane vesiclesrich in the ouabain-sensitive Na pump, and to examine dissociation of 32P and 86Rb from membrane-bound Na,K-ATPase. Since the rate of efflux is measured, and not the total counts remaining on the filter, the technique has high sensitivity. A complete time course is obtained using only a few micrograms of membrane protein. The apparatus described is simple, inexpensive, and easily constructed; with the present device, time resolution is - 10 ms.
The study of individual reaction rates in biological processes requires the use of specialized equipment to monitor changes in the subsecond time range (1). With regard to rapid membrane transport processes, in the general case where radioactive species are used to follow the movement of solutes, the critical step is the physical separation of solute in a membrane-bounded compartment from solute in the bulk solution. Previous methods have been described, but they have limitations in sample size, sensitivity, and applicability in certain situations. A continuous-flow apparatus, in which the extracellular solution is sampled at filtration ports, has been successfully employed in transport experiments with red blood cells (2,3); however disadvantages of this method include the large amount of sample required and the possibility that membranes may become trapped on the filters. Quench-flow techniques are useful in special cases where the solute concentration can be rapidly reduced by mixing with chelators (4,5), or where transport can be quickly halted with inhibitors (6); filtration is then used to separate the solute 495
in the two compartments in the quenched mixture. As with the first method, milligram quantities of membrane protein are typically required to generate a single time course. Ionexchange resins have been used in place of filtration to remove ions from the bulk solution (7); however, without a quench reaction the time resolution of this method is probably not as good as that obtainable by filtration. This paper describes the use of pressure filtration on cellulose ester filters to continuously monitor rapid isotopic efflux from membrane vesicles, or to monitor rapid dissociation of bound ligands from membrane proteins. Efflux or ligand dissociation is followed as a function of time by continuously sampling the medium flowing past the immobilized membranes, and thus a complete time course is obtained from a single aliquot of membranes. Application of the method requires that there be a way to instantaneously initiate the rapid efflux or dissociation process-for instance, by a solution change; however it does not require that there be a way to terminate efflux or to block dissociation of ligand. Be0003-2697184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
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cause the amount liberated is measured instead of the amount bound, the signal-to-noise ratio is much better than with conventional sampling methods. Using apparatus described here, it is possible to measure these rapid efflux or ligand dissociation events with a time resolution of better than 10 ms. To use this method, a sample of membrane vesicles (or membrane protein) is first incubated in a small volume with a radioactive substance to load the vesicles (or to bind the radioactive ligand). The sample is then diluted and filtered onto the surface of a cellulose ester filter and washed briefly in a conventional vacuum-filtration setup. The filter and immobilized membranes are then quickly transferred to an apparatus in which filtration is resumed, driven by a pressure of 50- 100 psi. Fluid passing through the filter contains radioactive solute that appears as a result of efflux from the vesicles (or dissociation of ligand); this fluid squirts from the lower filtration funnel and is collected in a rapidly moving string of cuvettes below. By designing the pressure filtration apparatus with low internal volume and maintaining high flow rates, time resolution on the order of lo-20 ms is easily attained. METHODS
Two versions of the apparatus have been built to allow a perturbation to be made in the sample by either changing the composition of the filtration medium (apparatus I) or exposing the immobilized membranes to a bright flash of light (apparatus II). The use of apparatus I will be described in greatest detail followed by a description of the differences in apparatus II. Filters. Cellulose-ester filters with 0.45~pm pore size were 2.5-cm squares of Millipore (HAWP) or Gelman (GN-6) cut from sheet stock; with our present batches of filters, the latter brand gives about 30% higher flow rate. Improvements in time resolution would be obtained by using filters of larger pore size, but at the cost of poorer sample retention.
Prejltration. To remove the large amount of extravesicular (or unbound) radioisotope, the sample is diluted, filtered, and rinsed on a conventional glass-f& vacuum-filtration apparatus (Fig. 1). The hlter is held in position on an aluminum carrier ring with “doublestick” tape so that the centered sample spot will also be in the center of the funnel in the high-pressure apparatus (Fig. 4, below). The conventional glass “chimney” is replaced by an epoxy-coated brass block which restricts the sample to a 4-mm-diam area on center with regard to the filter carrier ring. Typically a 4-~1 sample containing 1 mg/ml membrane protein is diluted into 200 ~1of medium (O”C), filtered, and rinsed with 400 ~1 of medium. The vacuum is released and the filter transferred to the pressure filtration apparatus before the rinse solution has been completely filtered, so that the sample remains fully wet. Sample size (amount of membrane protein) is restricted by the tendency of protein to clog the filter. We use the time required in the prefiltration steps ( 1O-20 s) to judge flow rate, and use samples small enough that the flow rate through the sample is no less than onehalf of that through the unloaded filter. Pressure jiltration apparatus I. Schematic diagrams of the apparatus are shown in Fig. 2 and 3. This is simply a device to permit filtration of a sample, immobilized on a cellulose-ester filter under high pressure (90 psi). The sample is introduced into this device on
-Brass
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FIG. 1. Cross section of the apparatus used in preliminary filtration of the sample. Not to scale.
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FIG. 2. Schematic diagram of the rapid filtration apparatus (I), in which a solution change is used to initiate the reaction, (A and B) solutions in thermostated pressure vessels, (a and b) solenoid valves to switch solutions A and B, (c) one way valve, (d) manual valve to flush B, (e and f) upper and lower funnels of filtration chamber, (g) sample of membranes, (h) cellulose-ester filter, (i) ring of 56 cuvettes, (j) turntable, (k) Hall-effect pickup and magnet (one of two), and (1) solenoid-controlled diversion funnel.
the surface of a cellulose-ester filter after the prefiltration step described above. Fluid flows continuously from one of two pressure reservoirs (Fig. 2 (A, B)) through the sample and filter(g), (h). The filtration effluent leaving the filter funnel (e), (f) is collected in 56 cuvettes (i) spinning on a turntable (j) during one rev-
olution, and otherwise detoured by a solenoidcontrolled waste-collection arm (1). Under electronic control, keyed to the revolution of the turntable by magnetic sensors (k), the composition of the filtration medium can be changed rapidly from A to B by operation of valve (b).
FIG. 3. Diagram of rapid-filtration apparatus approximately to scale. (a-l) as in Fig. 2, (m) magnet, (n) solenoid to retract diversion funnel (I), (p) level clamp to hold lower filter funnel disc, which is not in place, (q) manually operated cam which closes the lever clamp, and (p) r stand.
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Valve
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FIG. 4. Cross section of filter funnel assembly and valve (b). Not to scale.
Solution delivery. Solutions A and B are held in thermostated pressure vessels (Amicon RC800 or modified polyethylene bottles) and compressed Nz is supplied to the two vessels at 50 psi (A) and 90 psi (B). The dual-pressure system was chosen to simplify the plumbing in the critical area of the upper Iilter funnel. With this arrangement flow could be changed from solution A to solution B with a single solenoid valve (b) with the valve seat in close proximity to the sample. Flow of solution into the A reservoir is prevented by the one-way valve (c). The advantages are simplicity and rapidity of switching; the disadvantage is that the pressure (and flow) jump that occurs when solutions are switched can produce small artifacts. Alternatively, solenoid valve (a) could be used to shut off flow in the “A” line when valve (b) is activated, thus permitting uniform pressure operation, but even then brief pressure pulses would be unavoidable. Valves and tubing were chosen so as to not present a significant restriction to flow by comparison to the filter; without the filter present, flow through the B line is 8.3 ml/s compared to 3.6 ml/s in its presence. Teflon tubing (0.8-mm i.d.) was used for lines, and No. 18 stainless-steel hypodermic tubing (0.84mm i.d.) was used for the interconnections shown in Fii 2. Valves (a) and (b) were from General VaIve (Fairfield, N. J., series 9, 200 psi, 12 V) ad the one-way valve (c) was from
Isco (Lincoln, Neb., 60-0574-082). The body of valve (b) was machined to decrease the distance between the valve seat and the filter funnel and to provide the “T” from line A (see Fig. 4). Immediately before each experimental run the “A” line was flushed thoroughly (fluid passing out through the upper filter funnel) to remove any residual B solution in the “T” area. If solution B was changed between experimental runs, the new solution was flushed through valve (b) by means of a manual flow valve ((d), Hamilton, Reno, Nev., 86781). Filter funnel assembly. The time resolution of this method is determined by characteristics of fluid flow in the region between the seat of valve (b) and the cuvette (assuming instantaneous opening of valve (b)). Time delays occur between the opening of solenoid valve (b) and the arrival of the new solution at the filter, and then again between a change in isotopic efflux at the filter and the arrival of the efflux medium at the cuvette. However, a simple delay is easily accounted for in data analysis: in the experiments shown here this is accomplished by using an internal marker for the second solution, 3H20, so that efflux events can be directly referenced to the start of the solution change. The real limitation to time resolution is dispersion along the time axis arising from longitudinal mixing within the flow path. Since tlow is turbulent rather than
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laminar in the tubing leading to and from the filter funnels (Reynolds number = -4700, see Ref. (l)), it is likely that much of the dispersion is a result of the flow pattern in the top and bottom funnels. Here some of the fluid takes the shortest path through the filter in a straight line, while some must travel radially outward and then, after it has passed through the filter, radially inward towards the outlet; clearly the time delay for these two paths must be different. To minimize dispersion it is necessary to minimize the length of the flow path and particularly to keep the filter diameter as small as possible. In addition, a smaller filter area is preferable because the mechanical force will be less, and the filter support can be less substantial, which means lower volume and less obstruction in the lower funnel. It also appears advantageous to use a centered sample area that is smaller than the active filter area as long as the filter is not clogged in that area; in this way a high-volume flow can be maintained through the apparatus and yet effluent from the sample will not enter the outer circumference of the lower funnel, where radial flow is relatively slow. The present apparatus utilizes an active filter diameter of 10 mm with a centered sample spot of 4 mm-diameter. The total fluid volume of upper and lower filter funnels is 35 ~1, including that in the tubing from the valve (b) seat to the funnel and from the funnel to the lower orifice. At 90 psi, the flow rate is 3.6 ml/s and the mean transit time following valve (b) is - 10 ms. The lower funnel body is a brass disc that is removable to allow ready insertion and removal of the filter and filter ring; it is forced tightly against the upper funnel to seal the filtration chambers, by means of a lever clamp attached to the upper funnel assembly (Fig. 3, (p) (q)). The filter support is made up of 20 stainless-steel wires (0.125-mm diam.) in tension across the face of the funnel. This was constructed by wrapping, under hand tension, a continuous steel wire over and around the brass disc, guided by slots in the brass (not shown). After protecting the wire in the active filter area by coating it with wax,
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the rest of the wire was coated with epoxy cement to secure it and to impart a smooth flat surface to the upper face of the disc. In other versions of the apparatus (e.g., apparatus II, below) the filter support was made up of a disk of stainless-steel screen (Small Parts, Miami, Fla., No. CX-60) resting on several radially directed stainless-steel wires on the bottom of the chamber. Although a direct comparison has not been made, it would seem that the tensioned-wire support is superior in regard to dead volume and minimization of flow obstruction; however it is also somewhat more difficult to construct. Since most of the pressure drop is across the filter, and not within the flow path of the funnel, the flow velocity across the filter is fairly uniform, -4.6 cm/s. On the other hand the flow velocity of the fluid leaving the tubing as it enters the upper funnel is -680 cm/s. To prevent this high-velocity solution from striking the sample directly we have routinely placed a second filter (prerinsed and prewet) on top of the sample before it is placed in the pressure filtration apparatus (shown in Fig. 4). This filter is of high porosity (Millipore, SSWP) and does not significantly decrease the overall flow rate. Sample collection. A small lightweight aluminum funnel mounted on a hinged aluminum arm is connected to vacuum ((1) in Fig. 2, 3) and intercepts the fluid leaving the lower funnel. It is retracted -5 mm by a solenoid (Ledex, Vandalia, Ohio, 5 EC) during a single revolution in which samples are collected. During that time fluid flows directly into plastic cuvettes which are continuously moving on the turntable. Approximately 95% of the fluid is collected in the cuvettes, and the small splatter due to fluid striking the edges of the cuvettes has not proven to be a problem. The turntable is a modified phonograph turntable (Garrard RC 88/4; although this is no longer available, any other turntable mechanism would be satisfactory) providing speeds of 16, 33.3, 45, and 78 rpm, and with an auxiliary drive motor we have used it at speeds up to 536 r-pm. The 56 plastic cuvettes (Sarstedt,
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Princeton, N. J.; 67.741) are placed at a radius of 11 cm, and at these speeds, fluid flows into an individual cuvette for 67, 32, 24, 14, or for as short as 2 ms, respectively. The cuvettes are taped together by strips of masking tape so that the entire set can be placed on the turntable and secured by tape in a few seconds. A different set is used for each experimental run (except when signal averaging, see below), and the samples are transferred to scintillation vials at a later time. Samples from the first and/or last cuvettes in the cycle are usually not used due to overlap in closing of the waste arm (l), lasting about 35 ms. Samples late in the cycle were sometimes combined in sets of 3-10; not only did this reduce the number of vials to be counted, but it also increased sensitivity above counter background. Timing. The operation of valves (a) and (b) and diversion funnel (1) is under electronic control and is keyed to rotation of the tumtable by two Hall effect sensors (k,), (k2) (Sprague, Concord, N. H., UGS3020T) which detect the proximity of a single magnet ((m), Fig. 3) mounted on the turntable platter. The position of (k,) on the turntable determines the start position of the cycle. Sensor (k2) is moveable and generally follows (k,) by 5-8 cuvette positions-its position determines the timing of the solution changeover (a)-(b). The sensor electronics, sequencing logic, and solenoid drivers are conventional electronic circuits and are housed in one control box (schematic diagram available upon request). A typ ical sequence of operation (at 78 rpm), with t = 0 referenced to the start of flow of the second solution, is as follows: After the sample/filter/lower filter funnel assembly is clamped into place, the electronic logic is armed by a manual push button. Following this, the first occurrence of the (k,) pulse (delayed by 500 ms) initiates opening of valve (a) (t = -353 ms), the initial effluent being collected by waste arm (1). The subsequent (k,) pulse causes retraction of the waste arm (t = -84 ms) and the (k2) pulse then results in opening of valve (b) (t = 0 ms). Finally, at the next (k,) pulse both valves are turned off
and the waste arm is dropped back into place (t = 685 ms). Signal averaging. The operation of the various solenoids and solenoid valves is fixed in relation to turntable rotation, and is reproducible to better than 2 ms. Thus it is possible to do a small amount of “signal averaging” by carrying out a number of identical filtration runs with the same set of cuvettes in position. Since all of the manipulations required for a single run are easily accomplished by two people in 2-3 min, and since very small samples are used, it is certainly feasible to average 220 runs in this way. So far we have not needed this capability for experiments with apparatus I, although we have used it extensively in conjunction with apparatus II, as ciescribed below. Pressurejltration apparatus II. In a second version of the apparatus, the sample on the surface of the filter can be illuminated by the light from a high intensity arc lamp. Since light is used to trigger the eIIlux event only a single solution is needed, and the plumbing is much simpler than for apparatus I. The filter is clamped in a vertical orientation in this apparatus, and a short bent piece of hypodermic tubing directs the fluid leaving the “lower” filter funnel downward into the cuvettes. The “upper” filter funnel has a glass cover (Coming Glassware No. WG-305) and the fluid enters radially. The geometry of the “upper” hlter funnel is relatively unimportant, as rapid solution changes are not required. Apparatus II was actually built prior to apparatus I; the volume of the “lower” filtration funnel was about 20 ~1 and the flow rate was - 1.2 ml/s; as shown below, the time resolution with this device was about 20-30 ms. Light from a continuous high-intensity arc lamp (8) was focused on the filter through a hole in a vertical skirt attached to the outside rim of the turntable. The hole was equivalent in size to one cuvette position, so that, for instance at 33.3 rpm, 32 ms of illumination occurred as the hole moved across the beam. In this way repetitive “flashes” could be delivered which were in registration with cuvette position, and signal averaging was achieved
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by collecting fluid for a number of revolutions of the turntable, usually for 15 s overall. A manually operated diversion funnel collected the fluid at the start and end of the run. Illumination of the sample can alternatively be delivered by a high-intensity flash lamp, in which case the turntable skirt is not required. Timing is then achieved by magnetic pickup as with apparatus I. We have recently employed the apparatus in this mode, although data are not presented in this article. Temperature. The temperatures of the solution reservoirs (A and B, Fig. 2) are maintained by placing the vessels in thermostated baths. Because of the high-volume flow, this results in good temperature control at the sample when flow is maintained for more than a few seconds (as with Apparatus II) as determined by a thermistor probe. In apparatus I, the solution lines and valve (b) are also thermostated to assure that solution B is at the correct temperature when flow is begun. Na, K-A TPase. Purified membrane-bound Na,K-ATPase was prepared from dog kidney outer medulla by the method of Jorgensen (9) as previously described (8). 86Rb was bound tightly to the Na,K-ATPase in an “occluded state” (10) by incubating typically 50 pg of Na,K-ATPase in 40 ~1 with 3 BtM Mg+, 0.5 mM 86Rb (4 Ci/mmol), 25 mM imidazole, 1 mM EDTA, pH 7.2, for 30 s at 20°C. The sample was then held on ice and 3- to 5-~1 aliquots were taken for each experimental run and diluted into 25 mM imidazole, 1 mM EDTA, pH 6.8 (O”C), for prefiltration. In phosphorylation experiments Na,K-ATPase was phosphorylated in the presence of 20 PM [y-32P]ATP, 3 mM MgC12, 50 tnM NaCl, 25 InM imidazole, pH 7.2, for 3 s at 0°C; diluted in 50 ~1 1 M NaCl, 20 mM imidazole, filtered, rinsed quickly with the same solution at O”C, and quickly transferred to the rapid-filtration apparatus. From 25-50% of the incorporated 32P remained on the Na,K-ATPase after the prefiltration procedure. Membrane vesicles. Right-side-out membrane vesicles rich in Na,K-ATPase were prepared from dog kidney outer medulla as pre-
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FIG. 5. Time resolution of rapid-filtration apparatus I. Sample: 4 rg Na,K-ATPase (sp act 10 rmol PJmg min) phosphorylated from [y-32P]ATP (41 Ci/mmol) as under Methods. Solution A: 100 mM Na, 25 mM imidazole, 1 mM EDTA, pH 7.2,0-YC. Solution B: same as solution A, except containing [‘H]H*O (140,000 cpm/ml); and, in the two upper panels only, with 10 mM K+ replacing an equivalent amount of Na; temperature = 37°C.
viously described (11). These were loaded with 1 mM Na, 0.1 mM KCl, 5 mM MgC12,32 mM glutathione, 5 mM caged ATP’ (8), 25 mM sucrose, 32 tnM Tris-Hepes, pH 7.2, by osmotic shock (11). The medium used for both prefiltration and filtration was 0.1 mM KCI, 125 mM sucrose, 33 mM Tris-Hepes, pH 7.2. RESULTS
The worst-case time response of apparatus I is most easily determined by examining the time required for the switchover from solution A to solution B, marked by ‘H20 in the second solution. As seen in Fig. 5A the solution change is at least 90% complete within about seven cuvette positions at 536 rpm or within about 14 ms. Note that routinely including 3H20 in the second solution also provides an internal time reference for the change, relative ’ Abbreviation used: caged ATP, 3P- l-(2-nitro)phenyIethyladenosine triphosphate, a protected analog of ATP that releases ATP on exposure to longwave ultraviolet light.
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to solute release from the sample; this relationship is unaffected by delays in the flow path or in solenoid action. At this speed only 7 ~1 is collected per cuvette and the scatter from cuvette to cuvette is greater than that which is achieved at lower speeds. Figure 5 also illustrates the rapid release of “P from the phosphorylated intermediate upon addition of K+. The dephosphorylation reaction was chosen to test the time resolution of solute release because it is known to have a rate of >300 ms-’ at 20°C (12) and should be at least severalfold faster at 37”C, much more rapid than the expected response of the apparatus. Thus the time course of 32P release seen in Fig. 5B illustrates the time dispersion of the apparatus, which is seen to be approximately 7 ms. There are probably two reasons to explain why the 32P pulse is slightly faster than the apparent switch to the second solution. First, assuming that the “leading edge” of the K-containing solution is sufficient to trigger dephosphorylation, the dispersion in the 32P data will reflect primarily mixing in the lower filter funnel, whereas the ‘H record will be affected by mixing in the upper funnel as well. Second, it should be remembered that the sample is centered on a small spot within the filter chamber and that fluid flowing through the sample thus has the shortest pathway through the chamber. On a semilog plot, not shown, a slow phase in the 32P release is clearly distinguished from the unresolved fast component. This may correspond to a previously reported slow phase of dephosphorylation ( 12). Results of a control run in which solution B had the same composition as solution A are shown in Fig. 5C. The large 32P burst of Kstimulated dephosphorylation is absent as expected; however, a very small 32P release did occur in the 5th and 6th cuvettes (a similar artifact is seen more clearly in Fig. 6, below). This is an artifact of the pressure jump that occurs when solenoid (b) is opened, presumably due to a small fraction of the membranes which are dislodged and pass through the filter. Note that this occurs earlier by about 5-10
ms than the dephosphorylation burst in Fig. 5B, consistent with the fact that the pressure pulse occurs instantaneously at the filter upon the opening of the valve, whereas solution (b) requires 5-10 ms to reach the sample. The pressure artifact, which is usually quite small, can be considerably greater if the sample is not rinsed in the prefiltration step. The size of the artifact is illustrative of the sensitivity of the method: the ‘*P in the fifth cuvette in the bottom panel of Figure 5 represents approximately 0.2% of the 32P on the filter (32PATP exhibits high background binding); although not apparent on this scale, a fivefold smaller change in the effluent rate would have been distinguishable above the background in cuvettes l-4. The rapid filtration technique has been applied to the problem of the release of tightly bound *‘Rb (or 42K) ions from the Na,KATPase. An “occluded state” has been proposed for the Na pump, in which Rb ions are held tightly within the membrane until released by the binding of ATP at a low affinity site (13). Actual measurements of occluded 86Rb on the Na,K-ATPase are consistent with this model (lo), although the time course of *‘Rb dissociation, which should be fast enough to occur within the turnover of the pump, has not been reported. Figure 6 shows the results of an experiment in which 86Rb was bound to Na,K-ATPase in the presence of Mg*+ but no ATP; after prefiltration and rinsing, the sample was exposed to 2 mM ATP in the rapid filtration apparatus (solution B). The burst of 86Rb release (Fig. 6B) is easily resolved by this method; the time constant is about 18 s-‘. This is consistent with this step as part of the pump cycle, but only if it is a rate limiting step, since the pump turnover rate is about 15 XX-’ at 2O”C.* Results of a control run, in which ATP and Mg were omitted from solution (b), are shown in Fig. 6B, and indicate only a small pressure pulse artifact. A more * The turnover of the Na,K-ATPase is about 150 s-’ at 37°C (I 6). With our enzyme we find the ratio of Na,KATPase activity at 20°C to that at 37°C to be 0.10.
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FIG. 6. Time course of 86Rb dissociation from Na,K-ATPase. Sample: 4.5 pg Na,K-ATPase, with 86Rb (4.3 Ci/mmol) bound as described under Methods. Solution A: 2 mM M&l,, 25 mM imidazole, pH 6.8, 20°C. Solution B: [‘H]H20 (33,000 cpm/ml), 75 mM NaCl, 25 mM KCI, 2 mM MgCl*, 25 mM imidazole, pH 7.2. Left panels (Fig. 6A): solution B also contained 2 mM Na-ATP. Right panels (Fig. 6B): solution B did not contain ATP.
extensive analysis of 86Rb dissociation from Na,K-ATPase will be presented elsewhere ( 14). To demonstrate the performance of the rapid filtration apparatus in a study of ion transport, 22Na efflux from membrane vesicles is shown in Fig. 7. The membrane vesicles used here are tight right-side-out membrane vesicles isolated from outer medulla of dog kidney (11). Since the catalytic site of the Na pumps are on the inner aspect of the membrane, it is necessary to supply ATP within the vesicle. This is accomplished by incorporating a photosensitive ATP precursor, caged ATP (8), within the vesicles by osmotic shock (11); ATP can then be made available by exposure to ultraviolet light. In the experiments presented here, < 100 I.~M ATP was released in each flash of light; this is an amount of ATP less than the number of catalytic sites3 (11). In Fig. 7A it is seen that following a 16ms flash of light at 37°C 22Na efflux from the vesicles occurred in a burst about 20-30 ms in length. In other experiments, not shown, after preincubation of the vesicles in the presence of 1O-3 M ouabain no burst was observed. Since the turnover rate of the pump is - 150 s-’ at 37°C (16), it is likely that the time course
in Fig. 7 reflects the resolution afforded by this version of the apparatus; this is consistent with the flow rate and geometry of the filter funnel (1.2 m&c, 3Oql filter funnel). At 15°C the burst of Na efflux is much slower, as shown in Fig. 7B, and is not limited by the apparatus. The half-time for the decay of the Na-efflux rate is about 70 ms. Further experiments regarding rapid Na efflux through the Na pump will be discussed elsewhere (15). Note that in the experiments of Figs. 7A, B, a number of cycles (16 and 8, respectively) were averaged by collecting filtrate continuously for 30 s.
3 Further evidence that only one cycle of the Na pump is fueled per flash will be presented elsewhere (15).
(a) Efflux from vesicles or ligand dissociation is measured; isotopic injlux and ligand
DISCUSSION
A new method for rapid kinetic analysis of membrane phenomena has been described. Efflux from a membrane-bound compartment or ligand dissociation from binding sites on membrane proteins can be observed with a time resolution of better than 10 ms. While there are certainly limitations to the approach, there are also striking advantages. Limitations:
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FIG. 7. Rapid 22Na efflux from membrane vesicles. Sample: 4Oqg membrane vesicles loaded with caged ATP and 22Na, as described under Methods. During a 14 or 32-ms time period (hatched) a flash was given to release ATP from caged ATP. The two panels are from different experiments. (A) 37”C, 24Na (9 Ci/ mol), 37 cycles averaged; (B) 1YC, 22Na (300 Ci/mol), 8 cycles averaged.
association rates can not be determined by this method. (b) An appropriate perturbation is required to initiate efflux or dissociation after the membranes are in place in the rapid f&ration apparatus and they have been rinsed free of solute in the bulk solution. In one example given here (Fig. 6), a change of solution to one containing ATP caused *‘Rb dissociation, and in another example (Fig. 7), release of ATP by photolysis of caged ATP within membrane vesicles initiated a cycle of the Na pump. A temperature jump when solutions are changed is an alternative which would require redesign of apparatus I with care given to the insulation of the two fluid paths. (c) There must be some condition available for which the isotopic solute will remain within the membrane vesicles (or bound to the membranes) while the free isotope is rinsed away. Although it is possible to perform the rinse in a few hundred milliseconds within the rapid filtration apparatus itself, much lower background can be obtained if the sample is rinsed in the prefiltration setup; this requires lo-25 seconds at 0°C. Since occluded 86Rb is stable for minutes at 0°C (14), as is 2ZNa in membrane vesicles (1 l), this has not been a problem in these studies. However the high rate of spontaneous breakdown of phosphorylated Na,K-ATPase limits the types of dephosphorylation experiments which can be studied by this method. (d) The sample size is limited to -40 pg of membrane protein by the tendency of the
filters to clog with the sample. On the one hand, sparing of biological material is an advantage, discussed below; on the other hand, the consequence is a requirement for relatively high density of bound ligand, and for a highspecific-activity isotope. A larger sample could be accommodated by a greater area on the filter, but this will necessarily be at some cost in time resolution. Alternatively, an increase in the number of counts can be obtained by summing a number of cycles within one set of cuvettes. (e) The time resolution of the present apparatus is < 10 ms. While with a careful redesigning of the filter support and lower funnel this could be reduced still further, it seems unlikely that, given the present filters and sample size, it will be brought much below 2 ms. Advantages: (a) The rapid-pressure filtration method delivers a continuous, or at least quasicontinuous, record of efflux, in contrast to other methods which yield on: point per experimental run. Apart from the savings in material, discussed below, scatter in the data is dramatically reduced in comparison with the case in which individual time points are obtained from separate samples. Additionally, the savings in operator time is substantial. In a typical experiment, in which 12 time courses were obtained, the time required for the experimental runs was only about 30 min (2
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persons); experiment setup and sample processing together require several hours. (b) In the method described here, it is the rate of solute efflux or release that is determined as a function of time rather than the total isotope remaining or the total isotope released; the latter quantities can be obtained as the time integral of the rate. This is a great advantage since it results in the excellent signal/noise ratio and the high sensitivity of the method. Thus even when there is a very large background of nonspecific counts on the filter, following a prerinse only a small amount of isotope will be released per cuvette because of the short sample time. For instance, in the case of ligand dissociation from Na,K-ATPase, we are generally able to distinguish a change above background when as little as 0.2% of the bound counts is released into a single cuvette. In the application of apparatus II under conditions of high non-specific binding, the sensitivity is more likely to be limited by the pressure-change artifact rather than by contribution from the background release. (c) Less than 4 pg of membrane protein is required to obtain a complete time course of 32P or 86Rb dissociation from Na,K-ATPase. This is at least 50 times less than the amount generally used to obtain a single time point with other methods (l-3) and is thus O.OOl0.01 that used to obtain a time course. 22Na efflux from membrane vesicles is readily observed with 20 pg of membrane protein, even when only 10% of the vesicles are fueled with ATP (15). (d) The rapid-pressure filtration method is well suited to signal averaging by combining, in the same set of cuvettes, the filtrate from a number of experimentrl samples (or in special cases, from a number of cycles with the same sample). While in general the sensitivity
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of the method is such that this is not necessary, we have found the procedure useful in studies with membrane vesicles in which less than 0.1% of the vesicular **Na is released per cuvette after a brief flash of light. It is also helpful when the background isotopic release is particularly “noisy”, which we have found to be the case with 22Na efflux from membrane vesicles. ACKNOWLEDGMENTS I gratefully acknowledge the expert technical assistance of Grace Jones This research was supported by NIH grants ROI-GM27920 and ROl-GM31782. REFERENCES 1. Chance, B., Eisenhardt. R. H., Gison, Q. H., and Lot&erg-Helm, K. K., eds. (1964) Rapid Mixing and Sampling Techniques in Biochemistry. Acad. Press, New York. 2. Paganelli, C. V., and Solomon, A. K. (1957) J. Gen. Physiol. 41, 259-277. 3. Brahm, J. (1977) J. Gen. Physiol. 70, 283-306. 4. Will, H., Blank, J., Smeltman, G., and Wollenberger, A. (1976) Biochim. Biophys. Ada 449, 295-303. 5. Chiesi, M., and Inesi, G. (1979) J. Biol. Chem. 254, 10370-10377. 6. Cash, D. J., and Hess, G. P. (1981) Anal. Biochem. 112,39-51. I. Tanaka, J. C., Eccleston, and Barchi, R. L. (1983) J. Biol. Chem. 258, 7519-7526. 8. Kaplan, J. H., Forbush, B., III, and Hoffman, J. F. (1978) Biochemistry 17, 1929-1935. 9. Jorgensen, P. C. ( 1974) Biochim. Biophys. Actu 356, 36-52. 10. Glynn, I. M., and Richards, D. E. (1982) J. Physiol. 330, 17-43. 11. Forbush, B., III. (1982) J. Biol. Chem. 257, 1267812684. 12. Hobbs, A. S., Albers, R. W., and Froehlich, J. P. (1980) J. Biol. Chem. 255, 3395-3402. 13. Post, R. L., Hegevary, C., and Kume, S. (1972) J. Biol. Chem. 247,6530-6540. 14. Forbush, B., III, in preparation. 15. Forbush, B., III, Proc. Nat. Acad. Sci. USA, in press. 16. Jorgensen, P. L. (1975) Quart. Rev. Biophys. 7,239274.
BIOCHEMISTRY
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(1984)
An Apparatus for Rapid Kinetic Analysis of Isotopic Efflux from Membrane Vesicles and of Ligand Dissociation from Membrane Proteins BLISS FORBUSH III Department of Physiology, Yale School of Medicine, 333 Cedar Street, New Haven, Connectinrt 06510 Received December 27, 1983 A new method for the measurement of rapid isotopic release from a membrane compartment is described. Membrane vesiclesloaded with isotope, or broken membranes with bound radioactive ligand, are filtered onto the surface of a cellulose ester filter; the rate of the loss of isotope from the membrane compartment is followed continuously by collecting fluid which is passed through the filters under high pressure. A change in release rate is initiated by changing the solution or by delivering a flash of light to a photosensitive sample. The approach has been used to study rapid 22Na efflux from membrane vesiclesrich in the ouabain-sensitive Na pump, and to examine dissociation of 32P and 86Rb from membrane-bound Na,K-ATPase. Since the rate of efflux is measured, and not the total counts remaining on the filter, the technique has high sensitivity. A complete time course is obtained using only a few micrograms of membrane protein. The apparatus described is simple, inexpensive, and easily constructed; with the present device, time resolution is - 10 ms.
The study of individual reaction rates in biological processes requires the use of specialized equipment to monitor changes in the subsecond time range (1). With regard to rapid membrane transport processes, in the general case where radioactive species are used to follow the movement of solutes, the critical step is the physical separation of solute in a membrane-bounded compartment from solute in the bulk solution. Previous methods have been described, but they have limitations in sample size, sensitivity, and applicability in certain situations. A continuous-flow apparatus, in which the extracellular solution is sampled at filtration ports, has been successfully employed in transport experiments with red blood cells (2,3); however disadvantages of this method include the large amount of sample required and the possibility that membranes may become trapped on the filters. Quench-flow techniques are useful in special cases where the solute concentration can be rapidly reduced by mixing with chelators (4,5), or where transport can be quickly halted with inhibitors (6); filtration is then used to separate the solute 495
in the two compartments in the quenched mixture. As with the first method, milligram quantities of membrane protein are typically required to generate a single time course. Ionexchange resins have been used in place of filtration to remove ions from the bulk solution (7); however, without a quench reaction the time resolution of this method is probably not as good as that obtainable by filtration. This paper describes the use of pressure filtration on cellulose ester filters to continuously monitor rapid isotopic efflux from membrane vesicles, or to monitor rapid dissociation of bound ligands from membrane proteins. Efflux or ligand dissociation is followed as a function of time by continuously sampling the medium flowing past the immobilized membranes, and thus a complete time course is obtained from a single aliquot of membranes. Application of the method requires that there be a way to instantaneously initiate the rapid efflux or dissociation process-for instance, by a solution change; however it does not require that there be a way to terminate efflux or to block dissociation of ligand. Be0003-2697184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
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cause the amount liberated is measured instead of the amount bound, the signal-to-noise ratio is much better than with conventional sampling methods. Using apparatus described here, it is possible to measure these rapid efflux or ligand dissociation events with a time resolution of better than 10 ms. To use this method, a sample of membrane vesicles (or membrane protein) is first incubated in a small volume with a radioactive substance to load the vesicles (or to bind the radioactive ligand). The sample is then diluted and filtered onto the surface of a cellulose ester filter and washed briefly in a conventional vacuum-filtration setup. The filter and immobilized membranes are then quickly transferred to an apparatus in which filtration is resumed, driven by a pressure of 50- 100 psi. Fluid passing through the filter contains radioactive solute that appears as a result of efflux from the vesicles (or dissociation of ligand); this fluid squirts from the lower filtration funnel and is collected in a rapidly moving string of cuvettes below. By designing the pressure filtration apparatus with low internal volume and maintaining high flow rates, time resolution on the order of lo-20 ms is easily attained. METHODS
Two versions of the apparatus have been built to allow a perturbation to be made in the sample by either changing the composition of the filtration medium (apparatus I) or exposing the immobilized membranes to a bright flash of light (apparatus II). The use of apparatus I will be described in greatest detail followed by a description of the differences in apparatus II. Filters. Cellulose-ester filters with 0.45~pm pore size were 2.5-cm squares of Millipore (HAWP) or Gelman (GN-6) cut from sheet stock; with our present batches of filters, the latter brand gives about 30% higher flow rate. Improvements in time resolution would be obtained by using filters of larger pore size, but at the cost of poorer sample retention.
Prejltration. To remove the large amount of extravesicular (or unbound) radioisotope, the sample is diluted, filtered, and rinsed on a conventional glass-f& vacuum-filtration apparatus (Fig. 1). The hlter is held in position on an aluminum carrier ring with “doublestick” tape so that the centered sample spot will also be in the center of the funnel in the high-pressure apparatus (Fig. 4, below). The conventional glass “chimney” is replaced by an epoxy-coated brass block which restricts the sample to a 4-mm-diam area on center with regard to the filter carrier ring. Typically a 4-~1 sample containing 1 mg/ml membrane protein is diluted into 200 ~1of medium (O”C), filtered, and rinsed with 400 ~1 of medium. The vacuum is released and the filter transferred to the pressure filtration apparatus before the rinse solution has been completely filtered, so that the sample remains fully wet. Sample size (amount of membrane protein) is restricted by the tendency of protein to clog the filter. We use the time required in the prefiltration steps ( 1O-20 s) to judge flow rate, and use samples small enough that the flow rate through the sample is no less than onehalf of that through the unloaded filter. Pressure jiltration apparatus I. Schematic diagrams of the apparatus are shown in Fig. 2 and 3. This is simply a device to permit filtration of a sample, immobilized on a cellulose-ester filter under high pressure (90 psi). The sample is introduced into this device on
-Brass
“Chimney”
Cellulose Filter
Ester Filter Carrier
Ring
Millipore Glass Frit Filter Funnel
i Vacuum
FIG. 1. Cross section of the apparatus used in preliminary filtration of the sample. Not to scale.
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FIG. 2. Schematic diagram of the rapid filtration apparatus (I), in which a solution change is used to initiate the reaction, (A and B) solutions in thermostated pressure vessels, (a and b) solenoid valves to switch solutions A and B, (c) one way valve, (d) manual valve to flush B, (e and f) upper and lower funnels of filtration chamber, (g) sample of membranes, (h) cellulose-ester filter, (i) ring of 56 cuvettes, (j) turntable, (k) Hall-effect pickup and magnet (one of two), and (1) solenoid-controlled diversion funnel.
the surface of a cellulose-ester filter after the prefiltration step described above. Fluid flows continuously from one of two pressure reservoirs (Fig. 2 (A, B)) through the sample and filter(g), (h). The filtration effluent leaving the filter funnel (e), (f) is collected in 56 cuvettes (i) spinning on a turntable (j) during one rev-
olution, and otherwise detoured by a solenoidcontrolled waste-collection arm (1). Under electronic control, keyed to the revolution of the turntable by magnetic sensors (k), the composition of the filtration medium can be changed rapidly from A to B by operation of valve (b).
FIG. 3. Diagram of rapid-filtration apparatus approximately to scale. (a-l) as in Fig. 2, (m) magnet, (n) solenoid to retract diversion funnel (I), (p) level clamp to hold lower filter funnel disc, which is not in place, (q) manually operated cam which closes the lever clamp, and (p) r stand.
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Valve
b Body
Valve
Plunger
Upper
Filter
Funnel
~v;,:ilter
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----+--
Filter Filter
Carrier
Leer
Filter
Support
Funnel
Wires
FIG. 4. Cross section of filter funnel assembly and valve (b). Not to scale.
Solution delivery. Solutions A and B are held in thermostated pressure vessels (Amicon RC800 or modified polyethylene bottles) and compressed Nz is supplied to the two vessels at 50 psi (A) and 90 psi (B). The dual-pressure system was chosen to simplify the plumbing in the critical area of the upper Iilter funnel. With this arrangement flow could be changed from solution A to solution B with a single solenoid valve (b) with the valve seat in close proximity to the sample. Flow of solution into the A reservoir is prevented by the one-way valve (c). The advantages are simplicity and rapidity of switching; the disadvantage is that the pressure (and flow) jump that occurs when solutions are switched can produce small artifacts. Alternatively, solenoid valve (a) could be used to shut off flow in the “A” line when valve (b) is activated, thus permitting uniform pressure operation, but even then brief pressure pulses would be unavoidable. Valves and tubing were chosen so as to not present a significant restriction to flow by comparison to the filter; without the filter present, flow through the B line is 8.3 ml/s compared to 3.6 ml/s in its presence. Teflon tubing (0.8-mm i.d.) was used for lines, and No. 18 stainless-steel hypodermic tubing (0.84mm i.d.) was used for the interconnections shown in Fii 2. Valves (a) and (b) were from General VaIve (Fairfield, N. J., series 9, 200 psi, 12 V) ad the one-way valve (c) was from
Isco (Lincoln, Neb., 60-0574-082). The body of valve (b) was machined to decrease the distance between the valve seat and the filter funnel and to provide the “T” from line A (see Fig. 4). Immediately before each experimental run the “A” line was flushed thoroughly (fluid passing out through the upper filter funnel) to remove any residual B solution in the “T” area. If solution B was changed between experimental runs, the new solution was flushed through valve (b) by means of a manual flow valve ((d), Hamilton, Reno, Nev., 86781). Filter funnel assembly. The time resolution of this method is determined by characteristics of fluid flow in the region between the seat of valve (b) and the cuvette (assuming instantaneous opening of valve (b)). Time delays occur between the opening of solenoid valve (b) and the arrival of the new solution at the filter, and then again between a change in isotopic efflux at the filter and the arrival of the efflux medium at the cuvette. However, a simple delay is easily accounted for in data analysis: in the experiments shown here this is accomplished by using an internal marker for the second solution, 3H20, so that efflux events can be directly referenced to the start of the solution change. The real limitation to time resolution is dispersion along the time axis arising from longitudinal mixing within the flow path. Since tlow is turbulent rather than
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laminar in the tubing leading to and from the filter funnels (Reynolds number = -4700, see Ref. (l)), it is likely that much of the dispersion is a result of the flow pattern in the top and bottom funnels. Here some of the fluid takes the shortest path through the filter in a straight line, while some must travel radially outward and then, after it has passed through the filter, radially inward towards the outlet; clearly the time delay for these two paths must be different. To minimize dispersion it is necessary to minimize the length of the flow path and particularly to keep the filter diameter as small as possible. In addition, a smaller filter area is preferable because the mechanical force will be less, and the filter support can be less substantial, which means lower volume and less obstruction in the lower funnel. It also appears advantageous to use a centered sample area that is smaller than the active filter area as long as the filter is not clogged in that area; in this way a high-volume flow can be maintained through the apparatus and yet effluent from the sample will not enter the outer circumference of the lower funnel, where radial flow is relatively slow. The present apparatus utilizes an active filter diameter of 10 mm with a centered sample spot of 4 mm-diameter. The total fluid volume of upper and lower filter funnels is 35 ~1, including that in the tubing from the valve (b) seat to the funnel and from the funnel to the lower orifice. At 90 psi, the flow rate is 3.6 ml/s and the mean transit time following valve (b) is - 10 ms. The lower funnel body is a brass disc that is removable to allow ready insertion and removal of the filter and filter ring; it is forced tightly against the upper funnel to seal the filtration chambers, by means of a lever clamp attached to the upper funnel assembly (Fig. 3, (p) (q)). The filter support is made up of 20 stainless-steel wires (0.125-mm diam.) in tension across the face of the funnel. This was constructed by wrapping, under hand tension, a continuous steel wire over and around the brass disc, guided by slots in the brass (not shown). After protecting the wire in the active filter area by coating it with wax,
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the rest of the wire was coated with epoxy cement to secure it and to impart a smooth flat surface to the upper face of the disc. In other versions of the apparatus (e.g., apparatus II, below) the filter support was made up of a disk of stainless-steel screen (Small Parts, Miami, Fla., No. CX-60) resting on several radially directed stainless-steel wires on the bottom of the chamber. Although a direct comparison has not been made, it would seem that the tensioned-wire support is superior in regard to dead volume and minimization of flow obstruction; however it is also somewhat more difficult to construct. Since most of the pressure drop is across the filter, and not within the flow path of the funnel, the flow velocity across the filter is fairly uniform, -4.6 cm/s. On the other hand the flow velocity of the fluid leaving the tubing as it enters the upper funnel is -680 cm/s. To prevent this high-velocity solution from striking the sample directly we have routinely placed a second filter (prerinsed and prewet) on top of the sample before it is placed in the pressure filtration apparatus (shown in Fig. 4). This filter is of high porosity (Millipore, SSWP) and does not significantly decrease the overall flow rate. Sample collection. A small lightweight aluminum funnel mounted on a hinged aluminum arm is connected to vacuum ((1) in Fig. 2, 3) and intercepts the fluid leaving the lower funnel. It is retracted -5 mm by a solenoid (Ledex, Vandalia, Ohio, 5 EC) during a single revolution in which samples are collected. During that time fluid flows directly into plastic cuvettes which are continuously moving on the turntable. Approximately 95% of the fluid is collected in the cuvettes, and the small splatter due to fluid striking the edges of the cuvettes has not proven to be a problem. The turntable is a modified phonograph turntable (Garrard RC 88/4; although this is no longer available, any other turntable mechanism would be satisfactory) providing speeds of 16, 33.3, 45, and 78 rpm, and with an auxiliary drive motor we have used it at speeds up to 536 r-pm. The 56 plastic cuvettes (Sarstedt,
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Princeton, N. J.; 67.741) are placed at a radius of 11 cm, and at these speeds, fluid flows into an individual cuvette for 67, 32, 24, 14, or for as short as 2 ms, respectively. The cuvettes are taped together by strips of masking tape so that the entire set can be placed on the turntable and secured by tape in a few seconds. A different set is used for each experimental run (except when signal averaging, see below), and the samples are transferred to scintillation vials at a later time. Samples from the first and/or last cuvettes in the cycle are usually not used due to overlap in closing of the waste arm (l), lasting about 35 ms. Samples late in the cycle were sometimes combined in sets of 3-10; not only did this reduce the number of vials to be counted, but it also increased sensitivity above counter background. Timing. The operation of valves (a) and (b) and diversion funnel (1) is under electronic control and is keyed to rotation of the tumtable by two Hall effect sensors (k,), (k2) (Sprague, Concord, N. H., UGS3020T) which detect the proximity of a single magnet ((m), Fig. 3) mounted on the turntable platter. The position of (k,) on the turntable determines the start position of the cycle. Sensor (k2) is moveable and generally follows (k,) by 5-8 cuvette positions-its position determines the timing of the solution changeover (a)-(b). The sensor electronics, sequencing logic, and solenoid drivers are conventional electronic circuits and are housed in one control box (schematic diagram available upon request). A typ ical sequence of operation (at 78 rpm), with t = 0 referenced to the start of flow of the second solution, is as follows: After the sample/filter/lower filter funnel assembly is clamped into place, the electronic logic is armed by a manual push button. Following this, the first occurrence of the (k,) pulse (delayed by 500 ms) initiates opening of valve (a) (t = -353 ms), the initial effluent being collected by waste arm (1). The subsequent (k,) pulse causes retraction of the waste arm (t = -84 ms) and the (k2) pulse then results in opening of valve (b) (t = 0 ms). Finally, at the next (k,) pulse both valves are turned off
and the waste arm is dropped back into place (t = 685 ms). Signal averaging. The operation of the various solenoids and solenoid valves is fixed in relation to turntable rotation, and is reproducible to better than 2 ms. Thus it is possible to do a small amount of “signal averaging” by carrying out a number of identical filtration runs with the same set of cuvettes in position. Since all of the manipulations required for a single run are easily accomplished by two people in 2-3 min, and since very small samples are used, it is certainly feasible to average 220 runs in this way. So far we have not needed this capability for experiments with apparatus I, although we have used it extensively in conjunction with apparatus II, as ciescribed below. Pressurejltration apparatus II. In a second version of the apparatus, the sample on the surface of the filter can be illuminated by the light from a high intensity arc lamp. Since light is used to trigger the eIIlux event only a single solution is needed, and the plumbing is much simpler than for apparatus I. The filter is clamped in a vertical orientation in this apparatus, and a short bent piece of hypodermic tubing directs the fluid leaving the “lower” filter funnel downward into the cuvettes. The “upper” filter funnel has a glass cover (Coming Glassware No. WG-305) and the fluid enters radially. The geometry of the “upper” hlter funnel is relatively unimportant, as rapid solution changes are not required. Apparatus II was actually built prior to apparatus I; the volume of the “lower” filtration funnel was about 20 ~1 and the flow rate was - 1.2 ml/s; as shown below, the time resolution with this device was about 20-30 ms. Light from a continuous high-intensity arc lamp (8) was focused on the filter through a hole in a vertical skirt attached to the outside rim of the turntable. The hole was equivalent in size to one cuvette position, so that, for instance at 33.3 rpm, 32 ms of illumination occurred as the hole moved across the beam. In this way repetitive “flashes” could be delivered which were in registration with cuvette position, and signal averaging was achieved
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by collecting fluid for a number of revolutions of the turntable, usually for 15 s overall. A manually operated diversion funnel collected the fluid at the start and end of the run. Illumination of the sample can alternatively be delivered by a high-intensity flash lamp, in which case the turntable skirt is not required. Timing is then achieved by magnetic pickup as with apparatus I. We have recently employed the apparatus in this mode, although data are not presented in this article. Temperature. The temperatures of the solution reservoirs (A and B, Fig. 2) are maintained by placing the vessels in thermostated baths. Because of the high-volume flow, this results in good temperature control at the sample when flow is maintained for more than a few seconds (as with Apparatus II) as determined by a thermistor probe. In apparatus I, the solution lines and valve (b) are also thermostated to assure that solution B is at the correct temperature when flow is begun. Na, K-A TPase. Purified membrane-bound Na,K-ATPase was prepared from dog kidney outer medulla by the method of Jorgensen (9) as previously described (8). 86Rb was bound tightly to the Na,K-ATPase in an “occluded state” (10) by incubating typically 50 pg of Na,K-ATPase in 40 ~1 with 3 BtM Mg+, 0.5 mM 86Rb (4 Ci/mmol), 25 mM imidazole, 1 mM EDTA, pH 7.2, for 30 s at 20°C. The sample was then held on ice and 3- to 5-~1 aliquots were taken for each experimental run and diluted into 25 mM imidazole, 1 mM EDTA, pH 6.8 (O”C), for prefiltration. In phosphorylation experiments Na,K-ATPase was phosphorylated in the presence of 20 PM [y-32P]ATP, 3 mM MgC12, 50 tnM NaCl, 25 InM imidazole, pH 7.2, for 3 s at 0°C; diluted in 50 ~1 1 M NaCl, 20 mM imidazole, filtered, rinsed quickly with the same solution at O”C, and quickly transferred to the rapid-filtration apparatus. From 25-50% of the incorporated 32P remained on the Na,K-ATPase after the prefiltration procedure. Membrane vesicles. Right-side-out membrane vesicles rich in Na,K-ATPase were prepared from dog kidney outer medulla as pre-
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FIG. 5. Time resolution of rapid-filtration apparatus I. Sample: 4 rg Na,K-ATPase (sp act 10 rmol PJmg min) phosphorylated from [y-32P]ATP (41 Ci/mmol) as under Methods. Solution A: 100 mM Na, 25 mM imidazole, 1 mM EDTA, pH 7.2,0-YC. Solution B: same as solution A, except containing [‘H]H*O (140,000 cpm/ml); and, in the two upper panels only, with 10 mM K+ replacing an equivalent amount of Na; temperature = 37°C.
viously described (11). These were loaded with 1 mM Na, 0.1 mM KCl, 5 mM MgC12,32 mM glutathione, 5 mM caged ATP’ (8), 25 mM sucrose, 32 tnM Tris-Hepes, pH 7.2, by osmotic shock (11). The medium used for both prefiltration and filtration was 0.1 mM KCI, 125 mM sucrose, 33 mM Tris-Hepes, pH 7.2. RESULTS
The worst-case time response of apparatus I is most easily determined by examining the time required for the switchover from solution A to solution B, marked by ‘H20 in the second solution. As seen in Fig. 5A the solution change is at least 90% complete within about seven cuvette positions at 536 rpm or within about 14 ms. Note that routinely including 3H20 in the second solution also provides an internal time reference for the change, relative ’ Abbreviation used: caged ATP, 3P- l-(2-nitro)phenyIethyladenosine triphosphate, a protected analog of ATP that releases ATP on exposure to longwave ultraviolet light.
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to solute release from the sample; this relationship is unaffected by delays in the flow path or in solenoid action. At this speed only 7 ~1 is collected per cuvette and the scatter from cuvette to cuvette is greater than that which is achieved at lower speeds. Figure 5 also illustrates the rapid release of “P from the phosphorylated intermediate upon addition of K+. The dephosphorylation reaction was chosen to test the time resolution of solute release because it is known to have a rate of >300 ms-’ at 20°C (12) and should be at least severalfold faster at 37”C, much more rapid than the expected response of the apparatus. Thus the time course of 32P release seen in Fig. 5B illustrates the time dispersion of the apparatus, which is seen to be approximately 7 ms. There are probably two reasons to explain why the 32P pulse is slightly faster than the apparent switch to the second solution. First, assuming that the “leading edge” of the K-containing solution is sufficient to trigger dephosphorylation, the dispersion in the 32P data will reflect primarily mixing in the lower filter funnel, whereas the ‘H record will be affected by mixing in the upper funnel as well. Second, it should be remembered that the sample is centered on a small spot within the filter chamber and that fluid flowing through the sample thus has the shortest pathway through the chamber. On a semilog plot, not shown, a slow phase in the 32P release is clearly distinguished from the unresolved fast component. This may correspond to a previously reported slow phase of dephosphorylation ( 12). Results of a control run in which solution B had the same composition as solution A are shown in Fig. 5C. The large 32P burst of Kstimulated dephosphorylation is absent as expected; however, a very small 32P release did occur in the 5th and 6th cuvettes (a similar artifact is seen more clearly in Fig. 6, below). This is an artifact of the pressure jump that occurs when solenoid (b) is opened, presumably due to a small fraction of the membranes which are dislodged and pass through the filter. Note that this occurs earlier by about 5-10
ms than the dephosphorylation burst in Fig. 5B, consistent with the fact that the pressure pulse occurs instantaneously at the filter upon the opening of the valve, whereas solution (b) requires 5-10 ms to reach the sample. The pressure artifact, which is usually quite small, can be considerably greater if the sample is not rinsed in the prefiltration step. The size of the artifact is illustrative of the sensitivity of the method: the ‘*P in the fifth cuvette in the bottom panel of Figure 5 represents approximately 0.2% of the 32P on the filter (32PATP exhibits high background binding); although not apparent on this scale, a fivefold smaller change in the effluent rate would have been distinguishable above the background in cuvettes l-4. The rapid filtration technique has been applied to the problem of the release of tightly bound *‘Rb (or 42K) ions from the Na,KATPase. An “occluded state” has been proposed for the Na pump, in which Rb ions are held tightly within the membrane until released by the binding of ATP at a low affinity site (13). Actual measurements of occluded 86Rb on the Na,K-ATPase are consistent with this model (lo), although the time course of *‘Rb dissociation, which should be fast enough to occur within the turnover of the pump, has not been reported. Figure 6 shows the results of an experiment in which 86Rb was bound to Na,K-ATPase in the presence of Mg*+ but no ATP; after prefiltration and rinsing, the sample was exposed to 2 mM ATP in the rapid filtration apparatus (solution B). The burst of 86Rb release (Fig. 6B) is easily resolved by this method; the time constant is about 18 s-‘. This is consistent with this step as part of the pump cycle, but only if it is a rate limiting step, since the pump turnover rate is about 15 XX-’ at 2O”C.* Results of a control run, in which ATP and Mg were omitted from solution (b), are shown in Fig. 6B, and indicate only a small pressure pulse artifact. A more * The turnover of the Na,K-ATPase is about 150 s-’ at 37°C (I 6). With our enzyme we find the ratio of Na,KATPase activity at 20°C to that at 37°C to be 0.10.
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FIG. 6. Time course of 86Rb dissociation from Na,K-ATPase. Sample: 4.5 pg Na,K-ATPase, with 86Rb (4.3 Ci/mmol) bound as described under Methods. Solution A: 2 mM M&l,, 25 mM imidazole, pH 6.8, 20°C. Solution B: [‘H]H20 (33,000 cpm/ml), 75 mM NaCl, 25 mM KCI, 2 mM MgCl*, 25 mM imidazole, pH 7.2. Left panels (Fig. 6A): solution B also contained 2 mM Na-ATP. Right panels (Fig. 6B): solution B did not contain ATP.
extensive analysis of 86Rb dissociation from Na,K-ATPase will be presented elsewhere ( 14). To demonstrate the performance of the rapid filtration apparatus in a study of ion transport, 22Na efflux from membrane vesicles is shown in Fig. 7. The membrane vesicles used here are tight right-side-out membrane vesicles isolated from outer medulla of dog kidney (11). Since the catalytic site of the Na pumps are on the inner aspect of the membrane, it is necessary to supply ATP within the vesicle. This is accomplished by incorporating a photosensitive ATP precursor, caged ATP (8), within the vesicles by osmotic shock (11); ATP can then be made available by exposure to ultraviolet light. In the experiments presented here, < 100 I.~M ATP was released in each flash of light; this is an amount of ATP less than the number of catalytic sites3 (11). In Fig. 7A it is seen that following a 16ms flash of light at 37°C 22Na efflux from the vesicles occurred in a burst about 20-30 ms in length. In other experiments, not shown, after preincubation of the vesicles in the presence of 1O-3 M ouabain no burst was observed. Since the turnover rate of the pump is - 150 s-’ at 37°C (16), it is likely that the time course
in Fig. 7 reflects the resolution afforded by this version of the apparatus; this is consistent with the flow rate and geometry of the filter funnel (1.2 m&c, 3Oql filter funnel). At 15°C the burst of Na efflux is much slower, as shown in Fig. 7B, and is not limited by the apparatus. The half-time for the decay of the Na-efflux rate is about 70 ms. Further experiments regarding rapid Na efflux through the Na pump will be discussed elsewhere (15). Note that in the experiments of Figs. 7A, B, a number of cycles (16 and 8, respectively) were averaged by collecting filtrate continuously for 30 s.
3 Further evidence that only one cycle of the Na pump is fueled per flash will be presented elsewhere (15).
(a) Efflux from vesicles or ligand dissociation is measured; isotopic injlux and ligand
DISCUSSION
A new method for rapid kinetic analysis of membrane phenomena has been described. Efflux from a membrane-bound compartment or ligand dissociation from binding sites on membrane proteins can be observed with a time resolution of better than 10 ms. While there are certainly limitations to the approach, there are also striking advantages. Limitations:
BLISS FORBUSH
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FIG. 7. Rapid 22Na efflux from membrane vesicles. Sample: 4Oqg membrane vesicles loaded with caged ATP and 22Na, as described under Methods. During a 14 or 32-ms time period (hatched) a flash was given to release ATP from caged ATP. The two panels are from different experiments. (A) 37”C, 24Na (9 Ci/ mol), 37 cycles averaged; (B) 1YC, 22Na (300 Ci/mol), 8 cycles averaged.
association rates can not be determined by this method. (b) An appropriate perturbation is required to initiate efflux or dissociation after the membranes are in place in the rapid f&ration apparatus and they have been rinsed free of solute in the bulk solution. In one example given here (Fig. 6), a change of solution to one containing ATP caused *‘Rb dissociation, and in another example (Fig. 7), release of ATP by photolysis of caged ATP within membrane vesicles initiated a cycle of the Na pump. A temperature jump when solutions are changed is an alternative which would require redesign of apparatus I with care given to the insulation of the two fluid paths. (c) There must be some condition available for which the isotopic solute will remain within the membrane vesicles (or bound to the membranes) while the free isotope is rinsed away. Although it is possible to perform the rinse in a few hundred milliseconds within the rapid filtration apparatus itself, much lower background can be obtained if the sample is rinsed in the prefiltration setup; this requires lo-25 seconds at 0°C. Since occluded 86Rb is stable for minutes at 0°C (14), as is 2ZNa in membrane vesicles (1 l), this has not been a problem in these studies. However the high rate of spontaneous breakdown of phosphorylated Na,K-ATPase limits the types of dephosphorylation experiments which can be studied by this method. (d) The sample size is limited to -40 pg of membrane protein by the tendency of the
filters to clog with the sample. On the one hand, sparing of biological material is an advantage, discussed below; on the other hand, the consequence is a requirement for relatively high density of bound ligand, and for a highspecific-activity isotope. A larger sample could be accommodated by a greater area on the filter, but this will necessarily be at some cost in time resolution. Alternatively, an increase in the number of counts can be obtained by summing a number of cycles within one set of cuvettes. (e) The time resolution of the present apparatus is < 10 ms. While with a careful redesigning of the filter support and lower funnel this could be reduced still further, it seems unlikely that, given the present filters and sample size, it will be brought much below 2 ms. Advantages: (a) The rapid-pressure filtration method delivers a continuous, or at least quasicontinuous, record of efflux, in contrast to other methods which yield on: point per experimental run. Apart from the savings in material, discussed below, scatter in the data is dramatically reduced in comparison with the case in which individual time points are obtained from separate samples. Additionally, the savings in operator time is substantial. In a typical experiment, in which 12 time courses were obtained, the time required for the experimental runs was only about 30 min (2
MEASUREMENT
OF RAPID ISOTOPIC RELEASE
persons); experiment setup and sample processing together require several hours. (b) In the method described here, it is the rate of solute efflux or release that is determined as a function of time rather than the total isotope remaining or the total isotope released; the latter quantities can be obtained as the time integral of the rate. This is a great advantage since it results in the excellent signal/noise ratio and the high sensitivity of the method. Thus even when there is a very large background of nonspecific counts on the filter, following a prerinse only a small amount of isotope will be released per cuvette because of the short sample time. For instance, in the case of ligand dissociation from Na,K-ATPase, we are generally able to distinguish a change above background when as little as 0.2% of the bound counts is released into a single cuvette. In the application of apparatus II under conditions of high non-specific binding, the sensitivity is more likely to be limited by the pressure-change artifact rather than by contribution from the background release. (c) Less than 4 pg of membrane protein is required to obtain a complete time course of 32P or 86Rb dissociation from Na,K-ATPase. This is at least 50 times less than the amount generally used to obtain a single time point with other methods (l-3) and is thus O.OOl0.01 that used to obtain a time course. 22Na efflux from membrane vesicles is readily observed with 20 pg of membrane protein, even when only 10% of the vesicles are fueled with ATP (15). (d) The rapid-pressure filtration method is well suited to signal averaging by combining, in the same set of cuvettes, the filtrate from a number of experimentrl samples (or in special cases, from a number of cycles with the same sample). While in general the sensitivity
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of the method is such that this is not necessary, we have found the procedure useful in studies with membrane vesicles in which less than 0.1% of the vesicular **Na is released per cuvette after a brief flash of light. It is also helpful when the background isotopic release is particularly “noisy”, which we have found to be the case with 22Na efflux from membrane vesicles. ACKNOWLEDGMENTS I gratefully acknowledge the expert technical assistance of Grace Jones This research was supported by NIH grants ROI-GM27920 and ROl-GM31782. REFERENCES 1. Chance, B., Eisenhardt. R. H., Gison, Q. H., and Lot&erg-Helm, K. K., eds. (1964) Rapid Mixing and Sampling Techniques in Biochemistry. Acad. Press, New York. 2. Paganelli, C. V., and Solomon, A. K. (1957) J. Gen. Physiol. 41, 259-277. 3. Brahm, J. (1977) J. Gen. Physiol. 70, 283-306. 4. Will, H., Blank, J., Smeltman, G., and Wollenberger, A. (1976) Biochim. Biophys. Ada 449, 295-303. 5. Chiesi, M., and Inesi, G. (1979) J. Biol. Chem. 254, 10370-10377. 6. Cash, D. J., and Hess, G. P. (1981) Anal. Biochem. 112,39-51. I. Tanaka, J. C., Eccleston, and Barchi, R. L. (1983) J. Biol. Chem. 258, 7519-7526. 8. Kaplan, J. H., Forbush, B., III, and Hoffman, J. F. (1978) Biochemistry 17, 1929-1935. 9. Jorgensen, P. C. ( 1974) Biochim. Biophys. Actu 356, 36-52. 10. Glynn, I. M., and Richards, D. E. (1982) J. Physiol. 330, 17-43. 11. Forbush, B., III. (1982) J. Biol. Chem. 257, 1267812684. 12. Hobbs, A. S., Albers, R. W., and Froehlich, J. P. (1980) J. Biol. Chem. 255, 3395-3402. 13. Post, R. L., Hegevary, C., and Kume, S. (1972) J. Biol. Chem. 247,6530-6540. 14. Forbush, B., III, in preparation. 15. Forbush, B., III, Proc. Nat. Acad. Sci. USA, in press. 16. Jorgensen, P. L. (1975) Quart. Rev. Biophys. 7,239274.