[26] Direct methods for measuring metabolite transport and distribution in mitochondria

[26]

METHODS

[26] Direct Methods

FOR

METABOLITE

for Measuring

TRANSPORT

Metabolite

279

Transport

and

D i s t r i b u t i o n in M i t o c h o n d r i a

By

FERDINANDO

PALMIERI

and MARTIN

KLINGENBERG

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Preparations for Transport Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Loading of Mitochondria with Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Labeling of Intramitochondrial Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Incubation Conditions for Transport Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Separation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Centrifugal Sedimentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B~ Centrifugal Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Centrifugal Layer Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Sieve Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Steady-State Distribution Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Measurement of the Intramitochondrial Volume . . . . . . . . . . . . . . . . . . . . . . . . . . B. Measurement of Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Measurement of Efflux, Back Exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Kinetic Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Inhibitor Stop Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Pressure Filtration Device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Automatic Sampling Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

279 280 280 281 281 282 282 283 285 286 287 287 288 289 290 290 296 297

I. Introduction The transport o f metabolites in mitochondria is analyzed directly by measuring their distribution between the intra- and extramitochondrial space. In these transport studies the metabolites are assayed enzymatically and/or radioactively. It is possible to study (a) the e ~ u x of endogenous substrates or of substrates from previously loaded mitochondria, (b) the uptake of added metabolites, and (c) the counterexchange by following the in and out movements of the exchanging metabolites. The steadystate distribution and the kinetic of transport between the two spaces may be examined. The measurements o f metabolite distribution are made after the separation of mitochondria from the incubation m e d i u m ) It should be emphasized that the amount of metabolite within the mitochondria is relatively small, since the volume of the suspending medium is orders of magnitude larger than that o f the intramitochondrial space. In most cases, therefore, the uptake cannot be followed by measuring the decrease of the metabolite concentration in the suspending medium. Furthermore, since the separation o f the incubation medium is not complete, the mitochoni M. Klingenberg and E. Pfaff, Vol. 10, p. 680. METHODS IN ENZYMOLOGY, VOL. LVI

Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-1S1956-6

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TRANSPORT

[26]

drial extract must be corrected for the adherent extramitochondrial fluid. A serious problem is the further metabolism of the transported substrate during the incubation time and after the mitochondria have been separated. In most cases, this may be avoided by using appropriate inhibitors and by immediate quenching of the separated phases by acid. In this chapter, direct methods for studying mitochondrial transport, which are necessarily linked to the techniques for separating the mitochondria, will be described and discussed critically. Since the rates of transport reactions are higher in mitochondria than in whole cells and bacteria, special emphasis will be devoted to the techniques developed for measuring kinetics. It should be recalled, finally, that the methods described here are, in principle, applicable to any biological system, given the existence of closed spaces. In particular, they are suitable in chloroplasts. 1 II. Preparations for Transport Studies A. Loading o f Mitochondria with Metabolites

In mitochondria, most metabolite transport systems catalyze exchange reactions. 2 Even those carriers (phosphate, pyruvate, and glutamate) which catalyze net uptake, but have no counterexchange, may catalyze homologous exchange between the intra- and extramitochondrial metabolite (Pi-Pi exchange, etc.). In most cases, in order to characterize the mitochondrial transport systems, it is imperative that the externally added metabolite exchanges with a defined intramitochondrial metabolite. This requirement is achieved by loading the mitochondria with the desired metabolite and/or by inhibiting other transport reactions during the assay. It should be emphasized that, during the loading, not all endogenous substrates are substituted by the added metabolite, but the intramitochondrial concentration of the latter is increased considerably with respect to that of the other interfering endogenous substrates. For example, with malate-loaded mitochondria, it has been shown that the rates of citrate and oxoglutarate uptake are increased by the loading procedure severalfold with respect to untreated mitochondria. Procedure. The mitochondria (4-5 mg protein/ml) are incubated at 18°-22 ° for 2 min in the presence of 0.5-2 mM substrate, with which they are to be loaded, and of appropriate inhibitors of its metabolism, a-5 The pH of the reaction medium is 6.4-6.8. Under these conditions, anionic 2 M. Klingenberg, this volume [24].

a F. Paimieri, E. Quagliariello,and M. Klingenberg,Eur. J. Biochem. 29, 408 (1972). F. Palmieri, S. Passarella, I. Stipani, and E. Quagliariello,Biochim. Biophys. Acata 333, 195 (1974). 5 M. Crompton,F. Palmieri,M. Capano, and E. Quag~ariello,Biochem. J. 142, 127(1974).

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METHODS FOR METABOLITE TRANSPORT

281

substrates are accumulated severalfold in the intramitochondrial space. 6,r This procedure has been used for the following metabolites: Pi, malate, malonate, succinate, citrate, oxoglutarate, sulfate and pyruvate. After loading, the intramitochondrial content of the metabolite varies between 10 and 20/xmole/g protein. For loading with glutamate, the mitochondria are incubated in 100 mM glutamate, s After inhibition of the glutamate/OH cartier with 100 nmoles/mg protein N-ethylmaleimide, the mitochondria are washed twice. Asparate-loaded mitochondria are prepared by incubating glutamate-loaded mitochondria with oxaloacetate. 8

B. Labeling of lntramitochondrial Metabolites Mitochondria loaded with cold metabolites are used to study the uptake of labeled substrates (forward exchange), with the exception of adenine nucleotides, which rely on the endogenous pool of mitochondria. Another way to follow a counterexchange is to measure the etttux of a labeled metabolite from mitochondria incubated in the presence of unlabeled substrates (back exchange). Procedure. For this purpose, after loading, the intramitochondrial metabolite is labeled by adding to the mitochondrial stock suspension (40-50 mg protein/ml) carrier-free labeled metabolite (approximately 1 tzCi/ml of mitochondrial suspension, i.e. 0.02 lxCi/nmole and about 1 nmole/mg mitochondrial protein, except for 3zPi, which has an approximately 105 times higher specific activity). Under the same conditions, the endogenous adenine nucleotides may be labeled. Equilibration of the radioisotope between the extra- and intramitochondrial pools of the metabolite is obtained after 10-30 min incubation at 0°. The label mitochondrial stock suspension contains approximately 3 × 104 cpm/50 /xl; more than 80% of the radioactivity is located in the intramitochondrial space.

C. Incubation Conditions for Transport Studies Procedure. The commonly employed incubation mixture consists of 125 mM KC1, 1 mM EGTA, and 20 mM HEPES-Tris, but other isotonic media preserving the intactness of the inner mitochondrial membrane may also be used. The pH may be varied from 6.2 to 8.0. The appropriate temperature for kinetic measurements is usually 0-10 °, and for studies at equilibrium 25°. The amount of mitochondria to be used depends on the special technique applied and the transport problem to be investigated (back exchange, centrifugal sedimentation, sieve filtration) and varies over a broad range from 0.2 to 8 mg of protein per milliliter. For the measurement of uptake, the metabolite, usually labeled, is n F. Palmieri, E. Quagliariello, and M. Klingenberg, Eur. J. Biochem. 17, 230 (1970). r F. Palmieri, G. Genchi, and E. Quagliariello, Experientia, Suppl. 18, 505 (1971). 8 K. LaNoue, A. J. Meijer, and A. Brouwer, Arch. Biochem. Biophys. 161,544 (1974).

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TRANSPORT

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added to the medium containing 3H20 or labeled sucrose, to correct for the adherent liquid. In parallel samples, aH20 (1/zCi/ml) and 14C-sucrose (0.2/zCi/ml) are present to measure the intramitochondrial volume. For the measurement of ettlux, mitochondria loaded with the labeled metabolite are added to the medium containing externally added counteranion. In other cases ettlux of intramitochondrial metabolite is measured as a function of external pH, presence of antibiotics, uncouplers, etc. In order to prevent metabolism of the transported substrate, appropriate inhibitors are usually added to the incubation mixture: rotenone, antimycin, oligomycin, arsenite, carboxymethoxylamine, etc. In certain cases, in order to inhibit specific carriers which might interfere in the assay, the medium is supplemented by inhibitors such as N-ethylmaleimide (Pi and glutamate carriers), mersalyl (Pi and dicarboxylate carriers), benzene-l,2,3-tricarboxylate (tricarboxylate carrier) and butylmalonate (dicarboxylate and oxoglutarate carriers). III. Separation Methods All direct transport measurements require a separation of the mitochondria from the incubation medium. There are basically two types of separation procedures: centrifugation and filtration. These may be applied in a number of modifications adapted to the special transport problem. The centrifugation may be combined with a filtration through silicone oil, a procedure known as centrifugal filtration.

A. Centrifugal Sedimentation In general, a microcentrifuge or some other centrifuge adapted for handling volumes of 0.5-1.0 ml is used. The centrifuge must be cooled and must give at least 6000 g. Bench centrifuges (type Beckman, Eppendorf, etc.) may be employed, or larger cooled centrifuges (Sorval, etc.) adapted to accept the small centrifuge cups. Inexpensive one-way plastic centrifuge cups are used for incubation and centrifugation. Procedure. A typical procedure is as follows: The mitochondria are centrifuged in a bench centrifuge (Eppendorf, model 3200 or Misco microcentrifuge) operating at maximal speed for 1 min. The supernatant is removed as completely as possible and part of it immediately acidified with HCIO4 (final concentration 0.5 M). The walls of the centrifuge tubes are wiped clean and the drop of fluid on the pellet is absorbed by introducing into the tilted tubes filter papers cut out in the shapes of arrows. The pellet is suspended by the addition of 0.1 ml of HzO with the help of a Vortex stirrer, and then extracted with 50 /.d 1.6 M HCIO4. The total volume of the pellet extract is measured by weight. One hundred micro-

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M E T H O D S FOR M E T A B O L I T E TRANSPORT

283

liters of pellet extracts are taken for scintillation counting of aH and 14C. Supernatants are also assayed for radioactivity in the experiments in which the etilux is measured, whereas this is not usually necessary in uptake experiments. In order to decrease the adherent fluid in the pellet, the mitochondria may be centrifuged at 18,000 g for 5 min in a cooled centrifuge (e.g., Sorval-RC 2B) adapted for accepting 1.5 ml "Eppendorf" cups. In this case, after removal of the supernatant, the sediment may be rinsed by addition of suspension medium. Comments. A disadvantage of this method is that the intramitochondrial space is only a small fraction of the total space of the pellet. Consequently, it is applicable in uptake experiments, only when the metabolite concentration ratio between the intra- and extramitochondrial space is high, and in efllux experiments in which the metabolite leaving the mitochondria is largely diluted in the incubation medium. Generally, only measurements at equilibrium may be made, since the resolution time of this method is about 20 sec.

B. Centrifugal Filtration In the centrifugal sedimentation, the mitochondria become anaerobic in the pellet, and thus the steady-state composition of the metabolite is altered. This problem is avoided by the centrifugal filtration method, which combines sedimentation of the mitochondria with filtration through a nonaqueous layer of silicone into an acid layer. The mitochondria have been shown to stay aerobic during the passage through the silicone? The resulting immediate quenching and deproteinization prevents metabolism of the substances transported and releases the soluble constituents of the mitochondria into the extract. Another advantage is that in silicone filtration less adherent volume is carried over into the extract. Procedure. First, two procedures for relatively large volumes up to 2 ml will be described using (a) a swinging bucket rotor which keeps the layers in a position horizontal to the bottom? -1~ or (b) a bench-top fixed angle centrifuge (e.g., Eppendorf or Misco) with a sutiiciently high silicone layer to assure separation of the incubation and acid layers in view of the slanting angle. TM In the latter case (b) disposable plastic cups are conveniently used and the maximum volume is 1.0 ml as compared to 2 ml in procedure (a). In procedure (a), Pyrex glass tubes, suitable with adaptors for a swinging bucket rotor, e.g., SW-39 Spinco-Beckman, are treated with a solution •~ E. Pfaff, Ph.D. Thesis, Philipps-Universi~t, Marburg (1965). lo F. Palmieri, M. Cisternino, and E. Quagliariello, Biochim. Biophys. Acta 143, 625 (1967). 11 E. Quagliariello and F. Palmieri, Eur. J. Biochem. 4, 20 (1968). 12 K. E LaNoue, E. I. Walajtys, and J. R. Williamson, J. Biol. Chem. 248, 7171 (1973).

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of 2% silicone in CCI4. In these tubes the following solutions are layered from the bottom to the top: 0.2-0.4 ml of 1.6 M HC104, 0.4-0.6 ml of silicone AR 100 (Wacker Chemie, Munich), 0.5 to 2.0 ml incubation mixture. In procedure (b), the volumes to be layered in the Eppendorf cups are 0.1-0.2 ml of 1.6 M HC104, 0.3 ml silicone, 0.5 to 1.0 ml incubation medium. The following densities are recommended for mitochondria suspended in 0.25M sucrose (p = 1.037): acid layer containing 1.6M HCIO4, p = 1.08 g/cma, and silicone layer, p = 1.05 to 1.06 g/cm 3. It is important that the amount of mitochondria does not exceed 4 mg/ml [procedure (a)[ and 3 mg/ml [procedure (b)], and that the density of the acid layer is sufficiently higher than that of silicone oil. To increase the amount of mitochondrial protein/ml to be used, 4-6% dextran (MW ~ 60,000) must be included in the incubation mixture. The density of the silicone must be adapted to the reaction temperature and to the different incubation media by selecting the appropriate type and mixture, e.g., AR 100 (00-20 °) or AR 150 at higher temperatures. Usually the mitochondrial suspension containing the labeled substrate is layered on top of the silicone. In some cases, the reaction is initiated by the addition of mitochondria or of labeled substrate to the incubation layer. After the desired time from the start of the reaction, the sample tubes are centrifuged in the swinging-bucket rotor, or in the Eppendorf centrifuge, until a speed of 10,000 rpm or 15,000 rpm, respectively, is reached. During centrifugation, the mitochondria pass through the silicone layer and are stripped of their surrounding medium. When they reach the acid, their soluble constituents are released. The layers may be separated elegantly by means of a O-shaped metal wire which is introduced into the tubes to the bottom portion of the silicone layer. Then the tubes are quickly immersed in a mixture of petroleum ether and Dry Ice. The silicone, which has remained liquid, is carefully removed as quickly as possible so that the incubation medium does not liquify. The incubation medium is kept for enzymatic and radioactivity assays. The residual silicone in the test tube is removed keeping the acid layer frozen. The acid extract is allowed to thaw, and aliquots are taken for radioactivity and enzymatic measurements. For enzymatic assays, the perchloric acid extracts are neutralized with 10 M KOH, in the presence of 0.25 M Tris, and centrifuged in the cold to remove the. precipitated KC104. When the amount of metabolites in the pellet is too low, extracts from parallel samples may be combined. Another procedure ia using microcentrifuges permits smaller sample volumes and the processing of several parallel samples. The smaller amount of mitochondria, however, makes enzymatic assays difficult. Polyethylene tubes (0.4-0.5 ml) are used either with the Coleman model is E. J. Harris and K. Van D a m , Biochem. J. 106, 759 (1968).

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6-811 microcentrifuge or with the Beckman Microfuge centrifuge. In the plastic tubes the following solutions are layered: 30/~1 1.6M HC104 at the bottom and above this 70/zl silicone oil AR 100 or 150. On top of the silicone oil, the mitochondrial suspension (200-300 /zl), containing 4 mg/ml protein and the labeled substrate, is added. The reaction may also be initiated by addition of labeled substrate, in the volume of 10/.d, to the incubation layer. It is terminated by centrifuging the mitochondria through the silicone layer. Fifty microliters of the supernatant is directly taken for counting the radioactivity. The HC104 extract is separated by freezing the tube and cutting off the tip at the bottom part of the silicone layer nearest the acid layer. After removal of the remaining silicone, the extract is diluted in 200/xl water and centrifuged; 100-/zl aliquot are taken for counting. Comments. The shortest time obtainable for the incubation and separation of the mitochondria is about 20 to 50 sec after the start of the reaction. This includes the time for preparing the rotor and acceleration of the centrifuge until the mitochondria begin to penetrate the silicone layer (they sediment very fast through the siliconeg). For these reasons, the usefulness of this method for short-time incubation is limited. It has been employed primarily for measurements of the distribution of metabolites between the intra- and extramitochondrial space.

C. Centrifugal Layer Filtration This is a centrifugal filtration modified for shorter incubation times. 13 The upper layer is subdivided by the addition of increasing amounts of dextran into three zones: an upper "storage" zone for the mitochondrial suspension, a middle "isolating" zone, and a lower "incubation" zone in which the mitochondria are exposed to the added substances. The exposure is limited to the time required for the migration of mitochondria through the incubation zone during the centrifugation, and depends upon the length of this zone and the speed of centrifugation. It may be calibrated by measuring a metabolic function of the mitochondria which proceeds at a known rate, such as the oxidation of/3-hydroxybutyrate to acetoacetate. 14 Procedure. In general, in the glass tubes (~b 6-mm) to conditions are as follows: incubation zone 0.2-0.5 ml, isolation zone 0.25 ml, and storage zone 0.25-0.5 ml. All layers contain isotonic sucrose medium with the addition of 15 mg/ml dextran to the isolation zone and 22.5 mg/ml dextran to the incubation zone. Reagents may be added to either of the zones. The layers are prepared immediately before centrifugation in the swinging1~ M. Klingenberg, E. Pfaff, and A. Kr6ger, in "Rapid Mixing and Sampling Techniques in Biochemistry" (B. Chance ed.), p. 333. Academic Press, New York, 1964.

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bucket rotor. Usually, the incubation layer contains cold or labeled metabolite. In order to avoid the lag phase due to a temporary accumulation of the mitochondria at the phase limit medium-silicone, an additional washing layer may be placed between the incubation layer and the silicone. The temperature is kept at O°C. Incubation times as low as 10 sec may be obtained with this procedure. Comments. This method may be used for kinetic studies in the absence of a transport inhibitor suitable for the inhibitor stop method (Section V,A). It has been mostly used, however, in the determination of the stoichiometry of the exchange reactions. In the simplest case, the intraand extramitochondrial metabolites are labeled with ~H and 14C, respectively.

D. Sieve Filtration This method is characterized by the separation of the mitochondria from the incubation medium by means of filtration through microporous filters. Procedure. Mixed esters of cellulose filters from Sartorius or Millipore Corp. (0.65 txm pore size) are used in combination with any filter holder, connected to a vacuum line. The filtrate may be collected in a tube containing perchloric acid. The filters with the mitochondrial residue are dried and counted in a scintillation fluid containing 4.0 g of PPO and 0.1 g of POPOP per liter of toluene. Alternatively, the filters are immediately frozen at - 8 0 ° , ground to powder, and extracted by diluted acid. In this case both radioactivity and enzymatic measurements may be performed on aliquots of the extract. This procedure may also be used to separate the mitochondria after the transport has been terminated by an inhibitor (Section V,A). In this case, certain of the drawbacks of the sieve filtration (see below) may be overcome by wetting the filters with the medium containing the metabolite used and the inhibitor of its transport, and by washing in the presence of the inhibitor. Furthermore, controls are carried out in which the inhibitor is added to the mitochondrial suspension simultaneously with the substrate. Comments. A major disadvantage of this method in studying transport in mitochondria is that these particles undergo an appreciable loss of internal metabolites during the washing on the filter. Furthermore, the steady-state composition of intramitochondrial metabolites may be altered, since the mitochondria on the filters are separated from the external substrate and oxygen supply. If separation of the mitochondria from the incubation medium is not followed by washing, on the other hand, the adherent fluid is usually too large for accurate measurements of the

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metabolite present in the intramitochondrial space. For these reasons, the method is primarily applicable to etflux experiments, where the assays may be limited to the filtrate. The immediate deproteinization of the filtrate into the acid allows determination of the released metabolities, before further alteration by traces of enzymes present in the extramitochondrial space. A further disadvantage of this method is that filtration by suction requires about 20 sec, and its application for kinetic studies is therefore limited.

IV. Steady-State Distribution Measurements A. Measurement of the lntramitochondrial Volume For carrier-linked transport studies, the intramitochondrial space may be identified with the matrix, whereas the intermembrane space (unspecifically permeated) and the space outside the outer membrane (adherent fluid) are considered together as extramitochondrial space. The intramitochondrial volume is calculated as the difference between the total volume and the extramitochondrial volume of the pellet. The total volume is determined by measuring the distribution between the pellet and the supernatant of aH20, which is highly permeable through the outer and inner membrane. The volume of the pellet accessible to 3H20 is given by the following relationship: 1

Vp = Vex(Cpmpel/cprn~x)mg protein

(1)

where Vp is the volume of the pellet permeated by the labeled substance in/zl/mg protein; Vex is the volume of the supernatant; cpmpe~ is the total cpm in the pellet; and cpm~x is the total cpm in the supernatant. The extramitochondrial volume (also known as sucrose-permeable space) is determined by using labeled sucrose or mannitol, which are neither permeable through the inner mitochondrial membrane nor absorbed by its surface, and by applying Eq. (1). 14C-Sucrose and aH~O are often used together, in order to calculate the intramitochondrial volume [ Eq.(2)]. Vin = VH20 -- Vsue

=

cpmI - cpmlI/R A38~o

c p m l I ) gf:xtract 1 A~-'-~-~c/ 100 ~1 mg protein

(2)

where cpmI and cpmII are cpm of 100/~1 pellet extract in aH channel and 14C channel, respectively (cpm are corrected for background); A3azo and Al,c-suc are specific activities of 31-120 and 14C-sucrose in cpm//zl

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supernatant; R is the cpm in 14C channel/cpm in 3H channel of vials containing only 14C sucrose; and Vextrac t is the volume of pellet extract. The accuracy of the measurement of the intramitochondrial space depends upon the size of the adherent fluid in relation to the intramitochondrial volume, and hence, upon the technique of separating the mitochondria from the suspension medium. The adherent fluid is determined according to Eq. (1) by using a4Cdextran (MW ~ 60,000), which is impermeable to the outer mitochondrial membrane. By comparing the dextran space, the sucrose-permeable space, and the water space with that accessible to a given substance, it is possible to determine whether this permeates the mitochondrial membranes? 5 When the volume permeated by the substance results to be greater than the water space, it may be taken to have accumulated in the matrix.

B. Measurement of Uptake The amount of metabolite taken up in the intramitochondrial space is determined by subtracting the amount of metabolite present in the extramitochondrial space from that present in the whole pellet S in = (S tot -

[Sex]Vsuc)

1 mg protein

(3)

where Sin is the amount of metabolite in the intramitochondrial space/mg protein, Stot the amount in the whole pellet, Vsuc the sucrose-permeable space, and [Sex] the concentration in the supernatant. (It is assumed that the metabolite concentration in the extramitochondrial space is the same as that in the supernatant, and that the binding of the metabolite to the mitochondrial membranes is negligible). When 14C metabolite and 3H-sucrose are used, calculations are made according toEq. (4). Sin =

{ cpmlI

cpmI - cpmlI/R

\A~"~-met

AsH-sue

) Vextrac t 1 [Sex] 100/xl mg protein

(4)

where A 14c-met is the specific activity of '4C - metabolite in cpm/nmole (nmoles added are usually used to calculate A, since endogenous nmoles are comparatively negligible), A 3._sue is the specific activity of 3H-sucrose, in cpm//zl supernatant,R = cpm in 14C channel/cpm in 3H channel of vials

~5 E. Pfaff, M. Klingenberg, E. Ritt, and W. Vogell, Eur. J. Biochem. 5, 222 (1968).

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containing only 14C metabolite, and [Sex] is the concentration of the metabolite in the supernatant. For cpmI, cpmlI, and V e x t r a c t , s e e Eq. (2). The intramitochondrial metabolite concentration is given by the ratio between the amount of metabolite present in the matrix, calculated according to Eq. (3), and the intramitochondrial volume, calculated according to Eq. (2). It should be emphasized that the activity coefficients of metabolites are lower in the intra- than in the extramitochondrial phase 6 and, in most cases, they are not established.

C. Measurement of Efflux, Back Exchange The etltux of endogenous metabolites or of substrates from previously loaded mitochondria is generally initiated by the addition of appropriate counteranions to the incubation medium (back exchange). In certain cases, the efflux is caused by changes in the external pH, addition of certain antibiotics, etc? n,17 In the absence of external anion, mitochondria loaded with phosphate or sulfate lose approximately 10% of labeled intrarnitochondrial metabolite during 2 min incubation at 8° and pH 6.8 Much less or no leakage is seen with other metabolites, such as adenine nucleotides, malate, citrate, or oxoglutarate. In order to correct for this small time-dependent spontaneous leakage of radioactivity, control experiments, i.e., in the absence of external anion, are performed. The back exchange is calculated as percent exchange of the total intramitochondrial content, according to the equation Percent exchange

=

100(cpmeontrol

--

cpmassay)

(5)

Cpmeontrol

where cpm represents the radioactivity in the pellet extract. In most cases approximately 2 × 104 cprn/mitochondrial pellet is present in the control samples. Corrections may be made for the radioactivity present in the sucrose-permeable space, but these are often unnecessary due to the large dilution of the metabolite released from mitochondria in the suspension medium. The percentage exchange can also be calculated from the radioactivity released in the supernatant. This procedure is more convenient and accurate for determining small degrees of exchange, as encountered at early time of transport kinetics. In this case, the exchange is calculated with the following equation: 16 F. Palmieri and E. Quagliariello, Eur. J. Biochem. 8, 473 (1969). lr E. Quagliariello and F. Palmieri, F E B S Lett. 8, 105 (1970).

290

TRANSPORT Percent exchange = 100 Cpmassay cpmtotal

[26] --

cpmeontrol

(6)

cpmeontrol

The " a s s a y " and "control" values are obtained from supernatants, and the "total" from uncentrifuged samples. Another advantage is simplicity: only the supernatants are assayed; the pellets may be discarded and therefore the sucrose space ignored. The "back exchange" also permits the possibility of measuring the transport of metabolites which are not available in radioactively labeled form, provided that they cross the mitochondrial membrane by exchange with an intramitochondrial labeled substrate. V. Kinetic M e a s u r e m e n t s

Kinetic measurements of transport are limited by the time resolution between the start and the stop of the transport reaction. The reaction may be terminated by centrifugation, by sieve filtration, and by addition of a transport inhibitor (inhibitor stop method). The highest resolution time is achieved with the inhibitor stop method. In this case, the resolution time is limited only by the mixing time. Furthermore, the reaction time is more precisely defined than with the usual separation techniques. With the pressure filtration, a resolution time as low as 0.5 sec may be obtained with the appropriate techniques. Centrifugation is slower and therefore of limited use for kinetic studies of transport in mitochondria. Kinetics, in general, are measured as time sequence of metabolite distribution. This normally means withdrawal of aliquots (samples) from a single incubation at increasing time intervals after the start of the reaction. For faster reactions, the manual sampling must be replaced by an automatic sampling procedure. The rates are generally evaluated from the time course of metabolite transport. In certain cases, where the rate is studied as a function of certain parameters and the initial part of the time course is well established, a single time may be selected in the approximately linear phase as representative of the rate.

A. Inhibitor Stop Method This method is based on the use of a transport inhibitor to terminate the reaction. Subsequently, the mitochondria are separated by centrifugation or sieve filtration for measurement of metabolite distribution. The method is limited to the cases where an inhibitor of a transport system is available. The inhibition must be instantaneous and complete during the time necessary for the separation of the mitochondria. (Partial deviations from these requirements may be taken into account, see below). Some of

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METHODS FOR METABOLITE TRANSPORT

291

the inhibitors used (atractyloside, 1,2,3-benzenetricarboxylate) are very specific, i.e., they block only one carrier. Others, such as phenylsuccihate, are less specific. Often the inhibitors act by competing with the substrates for binding to the carders, without being transported. 1. Procedurefor Uptake, Forward Exchange. "Eppendoff" cups containing 0.95 ml medium are preequilibrated at 0°-10 ° in aluminum blocks immersed in a water bath. The mitochondria loaded with the appropriate metabolite (50/xl, approximately 2 mg) are added to the medium. After 1 rain incubation, the assay is initiated by addition of labeled metabolite (10 /zl) and rapid mixing with the mitochondrial suspension. After time t, the reaction is terminated by rapid addition of an inhibitor (10/xl). The inhibitors used to evaluate the kinetics of several metabolite carders in mitochondria are summarized in Table I. a'ls-a2 Immediately thereafter, the mitochondria with the labeled metabolite trapped insidea3 are separated from the incubation mixture by centrifugation at maximum speed for 1 min in a microcentrifuge (either Eppendorf, model 3200, or Misco). The supernatant is removed as completely as possible by suction, care being taken to absorb the aliquot remaining on the pellet by a filter paper, and to wipe the sides of the tubes. The pellet is treated as described in Section IIIA, and the radioactivity is counted on 100-~tl aliquot of the mitochondrial extract. The interval between the addition of the inhibitor and the separation of the mitochondria by centrifugation is kept as low as possible. Alternatively, the mitochondria may be separated by micropore filtration using a washing medium containing the inhibitor. Each assay (described above) is accompanied by a control sample in which the substrate and the inhibitor are added simultaneously, and the 18 E. Pfaff, H. W. Heldt, and M. Klingenberg, Eur. J. Biochem. 10, 484 (1969). 19 E. Quagliariello, F. Palmieri, G. Prezioso, and M. Klingenberg, FEBS Left. 4, 251 (1969). 2o B. H. Robinson and G. R. Williams, Biochim. Biophys. Acta 216, 63 (1970). 21 F. Palmieri, G. Prezioso, E. Quagliariello, and M. Klingenberg, Eur. J. Biochem. 22, 66 (1971). 22 M. Crompton, E Palmieri, M. Capano, and E. Quagliariello, FEBS Lett. 46, 247 (1974). 2a M. Crompton, F. Palmieri, M. Capano, and E. Quagliariello,Biochem. J. 146, 667 (1975). z4 B. H. Robinson, G. R. Williams, and M. L. Halperin, J. Biol. Chem. 246, 5280 (1971). 25 F. Palmieri, I. Stipani, E. Quagliariello, and M. Klingenberg, Eur. J. Biochem. 26, 587 (1972). 26 S. Passarella, F. Palmieri, and E. Quagliariello, Arch. Biochem. Biophys. 180, 160 (1977). 27 W. A. Coty and P. L. Pedersen, J. Biol. Chem. 249, 2593 (1974). zs H. Meisner, F. Palmieri, and E. Quagliariello, Biochemistry 11, 949 (1972). 29 G. Prezioso, F. Palmieri, and E. Quagliariello, Bioenergetics 3, 377 (1972). a0 N. M. Bradford and J. O. McGivan, Biochem. J. 134, 1023 (1973). al A. P. Halestrap, Biochem. J. 148, 85 (1975). az j. Mowbray, Biochem. J. 148, 41 (1975). aa The permeated metabolite is trapped within the mitochondria since the inhibitors block the transport without causing ettlux of internal metabolite.

292

TRANSPORT

[26] o

o

2

e~

o o

o

0

o

o

0

u~

0 o o

,'e

z 0

m

& o

~Noo "~o ~

Z m

N o

N o

N ~0 o -

c,i

m Z

0

~2 r~ ~.~

~

m <

<

0

o

~"

0

o

0

t~ '-e

z -~'~

-~

0 Z <

0

%

u

o

E

o o .~ ~ 2-~-~ ~

0

0

['~6]

METHODS

FOR METABOLITE

TRANSPORT

293

time of contact between the mitochondria and the substrate is the same as that which is obtained after the addition of the inhibitor in the assay experiment. The control samples are required to avoid possible errors introduced by any small residual transport occurring after the addition of the inhibitor. Furthermore, they account for the amount of substrate present in the sucrose-permeable space. One example of organizing an experiment with the inhibitor stop method for a series of samples, is given in the following. A stirring device which permits simultaneous addition to eight samples is used. It consists of eight stirring rods, fixed and aligned to a rigid support like a " c o m b " , with their end spoon-shaped to hold a 20 ~1 volume. To initiate the reaction, the eight stirring rods holding the substrate only in the odd-numbered positions, with the even-numbered positions empty, are rapidly mixed with the mitochondrial suspension. At an appropriate time, a second " c o m b " is used which holds the inhibitor in the odd-numbered positions and both substrate and inhibitor in the evennumbered positions. Under these conditions the shortest time of exchange measured is 3 sec. This may be decreased to 1-1.5 sec by processing each sample separately. For better time resolution, the inhibitor stop method is combined with an automatic sampling technique as described in Section V,C. Subtraction from the experimental assay samples of the amount of metabolite present in the controls (i.e., that present in the extramitochondrial space, plus that eventually permeated into the matrix in the presence of the inhibitor) gives the substrate incorporated into the matrix space during the reaction time, according to the following equation S i n ~ C p m a s s a ~, -

c p m c o n t r o I Vextrac t

Ameta~lite

1

100 tzl mg protein

(7)

where cpmassay is the cpm of 100/zl pellet extract from assay samples; cpmeontrol is the cpm of 100 /zl pellet extract from control samples; AmetaboHte is the specific activity of labeled metabolite in cpm/nmole; and Vextract is the volume of pellet extract. This is sufficiently accurate when there are no variations in the volume of adherent fluid among the various samples, i.e., in the case of separation of the mitochondria through a silicone layer or by Millipore filtration. When the mitochondria are separated by centrifugal sedimentation, aH20 or sucrose must also be present both in the assay samples and in the controls in order to correct for variations in the supernatant which remains adherent to the mitochondria. If 3H-sucrose is used in the presence of '4C- (35S-, or 3zp_) metabolite, the amount of metabolite uptake into the matrix space during the reaction time may be calculated as follows:

294

TRANSPORT

Sin = (Sto t -

[Sex]Vsuerose)

[26] 1

mg protein

This holds for both assay (A) and control (C). Therefore, one obtains Sin = (.cpmII A - cpmII C

)tl~7

- [Sex] (cpmI A - cpmII A/R) - (cpmI C - cpmII C/R)~

/

A3H Vextrac t

1

= 100 ~----~mg protein (8) where cpmI A and cpmII A = cpm in 3H channel and 14C channel, respectively, of 100/~l pellet extract from assay samples; cpmI C and cpmII C are as above, but from control samples; A3H is the specific activity of 3H-sucrose, in cpm//.d supernatant; A ,4c is the specific activity of 14C-metabolite, in cpm/nmole; R is the cpm in 14C channel/cpm in 3H channel of vials containing only 14C-metabolite; [Sex] is the concentration of the metabolite in the supernatant; and Ve×traet is the volume of pellet extract. Often 3H20, with ~4C-metabolite, has been used. It may be demonstrated that, in this case also, Eq. (8) is appropriate for the calculation of the amount of metabolite taken up in the matrix 34 (A3H = AaH2O). 2. Procedure for Back Exchange. The method for back exchange is identical to that described for forward exchange, except that mitochondria loaded with labeled metabolite are used, and the assay is initiated by the addition of unlabeled substrate. In the back exchange, the supernatants are normally used instead of the pellets for assay. The control samples with the inhibitor added before, or simultaneously with, the substrate account for unspecific leakage and for inhibitorinsensitive exchange. The amount of metabolite leaving the mitochondria during the reaction time may be calculated from the radioactivity released in the supernatant according to Eq. (9) Sex = Cpmassay -- Cpmeontrol Vsample

Asubstrate

1

Valiquot mg protein

(9)

where A substrate is the specific activity of the metabolite, determined on the mitochondrial stock suspension dividing cpm by the amount of metabolite measured enzymatically. a4 With 3H20 the total water space o f the pellet is measured, instead of the sucrosepermeable space. The latter is given by the difference between the total space and the intramitochondrial space. On deriving Eq. (8) the terms in which the internal volume appears cancel out, if it is assumed that this space is equal in both assay samples and controls.

[26]

METHODS

FOR METABOLITE

TRANSPORT

295

3. Calculations of Transport Rates. The rates of transport can be calculated from the approximately linear section of the time course: for the uptake VT = Sin/t (/zmole/min × g protein), for the effiux (back exchange) VT = Se×/t (/zmole/min × g protein) where Sin and Sex are obtained from Eqs. (8) and (9), respectively. In many cases, the time course of counterexchange in mitochondria follows approximately first-order kinetics. The uptake rate may thereby be calculated as follows: Va. = S max

÷ In \Sin~sm----S~n x )

(10)

S max is extrapolated from the uptake at equilibration after long time incubation. The efflux rate in back exchange, with the correction for "leakage" (see Section IV,C), is calculated as follows: VT = S ~ t In

=

(

t 1 - (Stx ~.,o, ( ~m In t

1

,

o

tot

Sex)/(Sin - S°x)

)

1

) Cpmcont~o0

-- ( C p m a s s a y -- C p m c o n t r o l ) / ( c p m t o t -

(ll)

or

V T -- S itot n/t x

In[lOO/(100 - E)]

E = percent exchange and S~ t is measured enzymatically. This equation is only valid with excess of added substrate, S°dd~ > > S°n. The general case reads

stot in

VT="-Tln

(.

lOO 100-E(1

) +/3)

(12)

with

s°n /3 -- SOn + SaddedO

where S ° is the concentration at t = 0. In many cases, only part of the total radioactivity incorporated in the mitochondria represents exchangeable substrate, because of enzymatic interconversions of originally added radioactively labeled substrate, and possibly because of impurities in the labeled compound. This portion must be measured with appropriate chromatographic procedures. With a equaling exchangeable and substrate attached portion of radioactivity, it becomes a - E (1 +/3)

(13)

296

TRANSPORT ¸/7¸¸¸

[26]

~,

REACTIONCHAMBER

2.

STIRRER

3,

FILTER SUPPORT

4,

BAYONETLOCK

S,

WATERJACKET

6,

MICROSHITCH

7,

WASHINGSYRINGE

8,

INLET COMPRESSED AIR

],5-50 SEC PRESET TIME INTERVAL ),5-50 SEC 'RESET PRESSURE

rIME J ~

j[~-~~WER SWT ICH 4AGNETVALVE [ 2q V

FIG. I. Pressure filtration apparatus.

The most reliable rate determinations are obtained by following the time sequence, for example with automatic sampling equipment as described in section V,C. In this case, VT is evaluated from a plot of In [100/(100 - E)] against t. A straight line is interpolated through the linear section giving the slope "k" Vr = S°n k

with k = d In [100/(100 - E)] dt

(14)

B. Pressure Filtration Device

Faster filtration may be accomplished by means of filtration under pressure. Here more sophisticated equipment must be used, in particular when the mitochondria are incubated in the same vessel as that used for filtration. 35 An example for such a device is illustrated in Fig. I. 36 a5 M. Klingenberg and B. Schmiedt, Fur. J. Biochem. 76, 553 (1977). a6 Apparatus developed by M. Klingenberg, in collaboration with H. Nakel and H. Berger, at the Institute for Physical Biochemistry, University of Munich.

[26]

METHODS FOR METABOLITE TRANSPORT

297

The apparatus assures fairly rapid addition of substances and accurate automatic timing of the reaction before the subsequent rapid filtration. At the bottom of the reaction vessel there is a Millipore filter with a support. A water jacket surrounds the reaction chamber for temperature control between 0 ° and 35°. The reaction volume may be varied between 1.5 to 6 ml. A wing-type stirrer is provided, by means of which the substrates are added to the reaction chamber. It provides efficient mixing after pushing down the stirrer with a rapid turn into the reaction solution. This movement triggers a switch which starts the timing device. After a preset time interval, the air pressure is released on top of the reaction medium for another preset time period. The filtered supernatant is collected in a reaction vessel. The filter may be removed and applied to scintillation counters or used for further analysis. The maximum air pressure applied is 6 atm. The shortest mixing time is about 300 msec. The filtration time depends on the type of preparation, the filters used, etc., and may be less than 100 msec with chloroplasts, about 200 msec with mitochondria, and about 300 msec with submitochondrial particles. The best resolution is therefore about 500 msec between addition and filtration. Rapidity of filtration is assured only by the appropriate filter selection. It has been found in particular that the use of two filters, one with larger pores in the upper and one with smaller pores in the lower layer, gives a more rapid and cleaner filtration than the use of only one filter. The capacity of the filters is limited, and the amount of particles in the reaction medium, therefore, must also be limited. With too many particles in suspension, the filters tend to become clogged too early and filtration becomes very slow.

C. Automatic Sampling Methods In order to follow more rapid transport kinetics, it may be necessary to extend the previously described methods to consecutive sampling with the highest possible time resolution. The sampling methods are adapted to the use of isotope-labeled substrates in transport studies, combined with appropriate methods for separating the particles either by sedimentation or by filtration. In a rapid, consecutive sampling method, it is necessary not only to solve the technicalities of rapid sampling but also to provide for a rapid initiation and termination of the reactions. For this purpose, rapid mixing devices must be employed in addition to the mechanics for rapid withdrawal of equal-sized samples. On the basis of the experience for measuring transport reactions in mitochondria, two different apparatus for rapid sampling have been constructed, which have so far been applied to transport and phosphorylation in mitochondria. 37 37 Apparatus developed by M. Klingenberg, in collaboration with H. Nakel, H. Berger, and W. Oellerer, at the Institute for Physical Biochemistry, University of Munich.

298

TRANSPORT

[26]

RAMQUESA and RAMPRESA. The "rapid mixing quenching sampling apparatus" (RAMQUESA) stops the reaction by withdrawing consecutive samples and rapidly mixing them with a quenching reagent, for example transport inhibitors, during sampling. In these samples the reaction is "frozen" at sampling time and can be analyzed further after centrifugation of the mitochondria. The"rapid mixing and sampling pressure filtration apparatus" (RAMPRESA) stops the reaction by rapid filtration of a discrete sample through a micropore filter. The method is applicable to chemically nonquenchable but filterable material. Both devices have some common features such as the rapid moving-mixing chamber and parts of the electronic steering. DESCRIPTION OF THE RAPID MIXING AND SAMPLING DEVICES (FIGS. 2 AND 3). Both RAMQUESA and RAMPRESA employ a circular reaction chamber which receives the bulk of the reaction fluid. This chamber, as well as the chamber containing the quenching reagent, is made of glass in order to facilitate temperature exchange with the thermostatic fluid surrounding the chamber. The moving-mixing chamber is placed on top of the reaction chamber, and may be rapidly fastened by a lever device. Thus additions to the reaction mixture may be made until 10 sec before

( 1 ) MOVINGMIXING CHA/~E ( 2 ) REACTIONCHAMBER ( 3 ) CIRCULATINGFLUID (/4 ) RAPID VALVE (5)

RAPID VALVE STEP HOTOR

(6)

QUENCHINGFLUID

(7)

VESSEL QUENCHINGFLUID VAL~

(8)

QUENCHMIXING CHAMBI

[]

( 9 ) MOTOR (10) SAMPLEVESSELflO'nEI (11) (12)

SAMPLECHANGE/4OTOR SAMPLECUPS

50 m m i

,

I

FIG. 2, Rapid mixing-quenching-sampling apparatus (RAMQUESA)

[]

[26]

METHODS FOR METABOLITE TRANSPORT

299

1) MOVING MIXING CHAMBER 2)

REACTIONCHAMBER

3 } CIRCULATINGFLUID LI) RAPIDVALVE 5)

RAPIDVALVEMOTOR

6 ) PRESSURECHAMBERCOVEI 7 ) PRESSURECHAMBER 8) MILLIPOREFILTER SUPPI 9 ) SAMPLECOLLECTINGVE$! 10) ROUNDTABLEFOR 12 GAMI 11) HEAVYSUPPORTFOR MOVING TABLE 12) GEARBELT TO HEAVYSTI MOTOR 13 ) PHOTOCELLCONTROLPOS

50tm I,,,,I

]

FIG. 3. Rapid mixing-pressure filtration-sampling apparatus (RAMPRESA).

the actual reaction is started by the mixing device, which will be described below. The mixing chamber contains a specially devised efflux tube linked to the rapid sampling valve. This valve is designed to move with high speed and no friction, closing against a small O ring. Movement is started by a rapid stepping motor, such that opening and closing is achieved in less than 2 msec. Thus almost "rectangular" opening periods of 2 msec may be obtained to assure equal sizes of the sample volumes. A pressure of about 0.3 atm in the reaction chamber drives the reaction fluid through the valves into the collecting vessels. After the sampling valves, the reaction fluid passes a small circular mixing-quenching chamber. Here the quenching fluid, originating from the thermostated quenching fluid storage vessel, is rapidly mixed with the sample. The addition of the sampling fluid is also controlled by another rapid, frictionless valve which opens and closes 2 msec within the opening and closing time of the sample valve. The volume ratio of quenching to sample fluid is about 0.5. The quenched sample mixture is then injected into the sample collecting cups (Eppendorf plastic 1.5 ml cups) which are placed into a holder accepting 12 of these cups. This holder has the shape of a cen-

300

TRANSPORT

[26]

trifuge head, slides out easily, and may be placed into an adapted Eppendoff microfuge. The sample vessel holder is advanced by a stepping motor which is also controlled by the electronic device. In RAMPRESA a similar setup is employed, but the dimensions of the reaction chamber, the moving-mixing chamber and the sampling valve are about three times larger than those in RAMQUESA. The rapid pressure filtration relies on the experience gained with the pressure filtration apparatus for single samples described above. In RAMPRESA, for consecutive sampling, the pressure filtration chamber must be separated from the reaction chamber. At preset time intervals after the reaction has been initiated with the moving-mixing chamber, each sample is withdrawn at precisely equal volumes and injected into one of twelve filtration chambers containing the micropore filters. Then the pressure chamber is tightly closed by a powerful pressure-driven lever, which presses down a tightly fitting cover. The pressure valve is subsequently opened during predetermined time for the rapid filtration. Especially rapid magnetic valves are used for the control of the filtration pressure and the closing of the pressure chamber. After pressure release and opening of the filtration chamber cover, the roundtable containing twelve filters and collecting vessels moves on. The table is temperature-controlled by means of the same circulating fluid which runs through the jacket of the reaction vessel. Movement of this table is controlled by a heavy-duty, rapid-stepping motor, translated by a gear belt, which is feedback-controlled by a photocell-counting device. The moving-mixing chamber is designed according to the same principles ~4 as developed earlier for allowing this mixer to be used in spectrophotometric cuvettes. A "Hamilton" syringe of 0.25 (RAMQUESA) or 1 ml (RAMPRESA) volume is connected to a three-way valve for filling and displacement. The tip of the syringe leads to a mixing plate with a number of ejection holes for the reagent. The mixing plate contains about twenty skewed holes through which the reaction fluid is pressed when pushing down the plate, causing strong turbulence. The head of the mixing chamber connects the two holders for the mixing plate with the plunger of the reagent syringe, so that on pushing down, the reagent is mixed in proportion to the displacement of the mixing plate through the fluid. The downward movement is agitated by air pressure and completed within less than 8 msec. The two sampling devices are controlled by elaborate electric timing devices. A steering unit controls the various time intervals, the stepping motors for the valves, and the sampling tables. An electric timing clock based on 1 MHz quartz oscillator permits one to set the opening and the waiting time for each sample. The opening time may be varied from l0 msec

[27]

MEASUREMENTS OF CATION TRANSPORT

301

to 10 sec and the waiting time from 10 msec to 1000 sec throughout the range at 10 msec intervals. The steering unit is triggered by the start of the mixing chamber. Some characteristics of R A M Q U E S A and R A M P R E S A are summarized in Table II. TABLE II SOME CHARACTERISTICS OF THE R A P I D M I X I N G AND S A M P L I N G DEVICES

RAMQUESA Separation Centrifugation Mixing time (msec) l0 Mixing ratio 20 to 50 Minimum sampling time (msec) 80 variable for each sample up to 100 sec Number of samples 12 Maximum sample volume (p.l) 600 Number of time steps/sample 5 Maximum reaction volume (ml) 6 Temperature (°C) -20 to 50

[2 7] M e a s u r e m e n t s

of Cation Transport Indicators

RAMPRESA Filtration l0 20 to 100 250 12 1000 8 12 -20 to 50

with Metallochromic

By A. SCARPA Sensitive and accurate kinetic measurements of ionized metal ions are central for the understanding of ion transport in biological systems. Among the various cations interacting with biological materials, H +, Ca 2+, and Mg 2+ have a key role in regulating enzyme activity and cellular functions. Hence, the study of H +, Ca 2+, and Mg 2+ transport acquires particular significance for the understanding o f energy transduction and cellular events, such as contraction, secretion, fusion, excitat!on, and more complex events, such as vision and hormone and neurotransmitter action. Methods for Measuring Ca "+, Mg 2+, and H + Various procedures have been described in the literature for measuring H +, Ca 2+, and Mg 2+ binding and transport in biological systems. Those most commonly used are isotope distribution, atomic absorption, specific electrodes, and photoluminescent, fluorescent, or absorbance indicators. All of these methods present advantages and disadvantages with respect

METHODS IN ENZYMOLOGY, VOL. LVI

Copyright (~ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181956-6