Elevation of the intracellular pH activates respiration and motility of sperm of the sea urchin, Strongylocentrotus purpuratus.

The internal pH (pHi) of sperm of the sea urchin, Strongylocentrotus purpuratus, was estimated by measuring the accumulation of the weak bases [14C]methylamine, [14C]diethylamine, or 9-aminoacridine under conditions where cellular respiration was activated or inhibited. When 9-aminoacridine fluorescence measurements are corrected for binding to intracellular components, the pHi estimates agree quantitatively with those obtained from [14C]amine distributions. The pHi of sperm decreased when the intracellular [K+] was elevated above the physiological value of 10 mM, or when the external pH was substantially decreased below the physiological 8.0, or when Na+ was absent from the seawater. At the decreased pHi values, sperm respiration and motility were inhibited; conversely, both respiration and motility increased when the pHi was elevated. Increased respiration occurred whether the pHi was increased by altering the external pH, [K+] or [Na+], or by the addition of NH4Cl to the medium. In all cases, the activation of respiration and motility were linked, suggesting a unitary control mechanism, some possibilities for which are presented.

Sperm are cells that face markedly different demands a t distinct portions of their lives. For example, sea urchin sperm remain quiescent in the testes for months; immediately upon spawning (and dilution) into seawater, they respire and swim at a maximal rate (30). Upon encountering a sea urchin egg, the sperm undergo the acrosome reaction (exocytosis from an apical vesicle) that allows them to bind and fuse with the homologous egg (reviewed in Ref. 1). These rapid alterations in activity that follow their dilution in the external medium require a coordination of metabolism with external demands. The mechanism of this coordination is not well understood. Sea urchin sperm provide several advantages in studies of the biochemical basis of such metabolic coordination. They can be obtained in large quantities and induced to undergo activation (increased respiration and motility) and the acrosome reaction synchronously and within seconds. Additionally, sperm are relatively simple cells, devoid of the complex machinery to replicate DNA or synthesize proteins.
The activation of respiration and motility of sperm that occurs upon dilution is sensitive to the ionic composition of seawater. For example, sperm that are diluted into seawater of low pH or elevated K' are immotile (3)(4)(5)(6)(7)(8). Na' is required * The costs of publication of this article were defrayed in part by marked "aduertisement" in accordance with 18 U.S.C. Section 1734 the payment of page charges. This article must therefore be hereby solely to indicate this fact.
§ Supported by National Institutes of Health Research Grant GM 23910 and National Science Foundation Grant PCM 7720472. for initiation of motility (2), and the acrosome reaction is associated with "Na' uptake and H+ efflux (9). At a seawater pH of 6.0 (in contrast to the normal 8.0) the respiration of sea urchin sperm is inhibited (10)(11)(12) as are the acrosome reaction (e.g. see Ref. 1) and motility. At low external pH, sperm motility can be initiated in the presence of an egg peptide (12) if Na+ is present.
Na'-dependent H+ effux could lead to an increased pH,,' which might act as an intracellular messenger to regulate the activation response of sperm. In a previous study (17), we found that the intracellular pH of sperm was acidic with respect to seawater. In the present study, we have estimated the pH, of sperm diluted into media of ionic compositions that either permit or inhibit sperm activation. These studies utilize the ability of weak acids or bases to traverse membranes in their uncharged forms and to accumulate intracellularly according to pH gradients (13)(14)(15)(16). We show in this study that increased respiration and motility are associated with an increase in pH, and suggest how both could be coordinately regulated.

MATERIALS AND METHODS
Collection of Gametes-Spermatozoa from the sea urchin, Strongylocentrotus purpuratus, were obtained by intracoelomic injection of 0.5 M KC]; they were collected as "dry" (undiluted) sperm (2-6 X IO'* sperm/&) and kept on ice. For experiments a t low Na' concentrations, unless otherwise indicated, sperm were washed by 200-500fold dilution into Na'-free medium (ChSW) and centrifugation (Sorvall, SS34, 10 min, loo0 X g); the pellet was resuspended in ChSW.
Dry sperm from the starfish, Pisaster ochraceus, were obtained by dissection of the gonads. All experiments were performed between 10 and 12 "C except where noted.
Media-ASW was of the following composition: 360 mM NaCl, 50 mM MgC12, 10 m~ CaCI2, 10 mM KCl, 30 mM 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid, pH 8.0. In some experiments the buffer was 12.5 m~ Tris and 5 m 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. For ChSW, three times recrystallized choline chloride was used instead of NaCI; fresh solutions were used. The pH was adjusted with KOH and Tris base so that K' was 10 m~. When [K+] was varied, K' was substituted for Na' in ASW, so that [K'] + [Na'] = 370 mM. Ca2+-free medium was made by omitting Ca2+ from ASW; 1 mM EGTA was added as noted in the figures and the pH was readjusted.
Sperm Respiration-Respiration rates were determined by continuous recording with a Clark oxygen electrode. A 100-fold dilution of dry sperm was made into 5-ml medium, unless indicated otherwise. The respiratory rates in ASW were constant until about 80% of the 0 2 was depleted from the medium and measurements were made only up to this point. In long term experiments, the medium was periodically reoxygenated by bubbling air through it. A 100% reading in the figures is for medium in equilibrium with air at the indicated temperature.

IntracellularpH and Respiration
Sperm Motility-Estimates of motility were obtained by microscopic observation of a thick droplet of sperm by darkfield microscopy. Only sperm in liquid suspension (i.e. those not close to the surface of the slide) were examined, since swimming artifacts occur in the vicinity of glass surfaces.' Sperm motility was scored qualitatively in five categories from complete immotility (0) to full activity (4).
Accumulation of Radioactive Amines-Dry sperm were diluted 100-fold into media containing radioactive EhNH or DM0 (17). ["C] Diethylamine (55.5 mCi/nmol from ICN) was used at a final concentration of 2-10 p~ and DM0 at 2-4 /AM. Separation of the sperm from the reaction medium was by centrifugation through silicone oil (General Electric Versilube F-50) in an Eppendorf microfuge for 30-45 s at full speed. Extracellular and total pellet Hz0 spaces were determined with tritiated water and [14C]inulin or sucrose, as previously described (45). The average value for sperm intracellular water space in 25 determinations was 0.71 +. 0.04 (S.E.) pl/105 sperm and was nearly the same for either Ca'+-free ASW or for ChSW. pH, = pH,log,, (Ar), where Ar is the ratio of internal to external amine concentration (17). EhNH was used because it reached an equilibrium level more rapidly than methylamine did. For example, when [14C]Et2NH was added 30 min after dilution of sperm into Ca*+-Na+-free sea water, a constant Ar was achieved with a t1p of 45 s; the final Ar was identical with that found when sperm were incubated in [I4C]EgNH from the time of dilution and sampled at 35 min. Thus, changes in the Ar for ['4C]EgNH that occurred during the first hour of sperm dilution at pH 8.0 (e.g. Fig. 3) reflect changes in pH, and not a slow diffusion of the amine.
Accumulation of Fluorescent Amines-For continuous monitoring of amine accumulation in a sample, we followed the quenching of fluorescence of 9-AA as it is taken up by cells (e.g. Ref. 13). Measurements were performed with a Perkin Elmer MPF 44A fluorescence spectrophotometer, with excitation of 382 nm and emission at 454 nm.
With starfish sperm, measurements were made with an Aminco-Bowman Fluorometer (380 nm excitation, 440 nm emission). In order to test whether any of the 9-AA fluorescence that remains after quenching in the suspension is from sperm which have accumulated the dye, the fluorescence was measured before and after removal of the sperm by centrifugation. Under all conditions, the fluorescence of the supernatant was within 7% of that for the sperm suspension. This demonstrates that uptake of 9-AA almost completely quenches its fluorescence. It follows that the fluorescence serves as a measure of the extracellular 9-AA remaining in solution. Over the range of concentrations used in these experiments, the fluorescence of the solution was directly proportional to the 9-AA concentration and inversely proportional to the sperm concentration.
Binding of 9-AA to intracellular components can complicate quantitative pH measurements (13). However, in sperm there is an excess of 9-AA-binding sites, and thus the ratio of bound to free intracellular 9-AA is nearly constant over the range of 9-AA concentrations used (see Appendix 3): The binding can therefore enhance the quenching due to accumulation in response to the pH gradient. Calculation of the intracellular pH with 9-AA is given by Equation 12 of Appendix 3: where Q is the fraction of fluorescence quenched in intact sperm and Q' is the fraction quenched when the ApH is collapsed by either ' M. P. Cosson and R. Christen, unpublished observations. Portions of this paper (including Appendices 1-3, part of "Results," part of "Discussion," Figs. 1-4, and Table I) are presented in miniprint at the end of this paper. Appendix 1 has equations for dealing with a two-compartment system for accumulating a weak acid and a weak base, Appendix 2 deals with the kinetics of weak base accumulation into intracellular acidic compartments, and Appendix 3 presents equations and data for obtaining quantitative estimates of intracellular pH with 9-aminoacridine even with intracellular binding of the dye. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 82M-732, cite the authors, and include a check or money order for $9.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. Triton X-100 or the ionophores nigericin or monensin. For example, for the curve in Fig. 1B at pH, = 7.9, Q' is 0.25 and Q is 0.73. These values give pH, = 7.0 (Table 11). Q' is not dependent upon the pH or the ionic compositions of seawaters used. With corrections for binding, quantitatively equivalent pHi values were obtained with 9-AA and EbNH, but the final equilibrium was reached much more slowly: with 9-AA the process took 20-30 min at pH, = 8 and about 4 h at pH, = 6.9 (see Fig. 1B). This slow uptake precluded use of 9-AA for kinetic analyses, but did permit estimates of equilibrium values of pH, to compare with data using ['4C]Et2NH.

RESULTS
Sea urchin sperm accumulate radioactive and fluorescent amines (17). The extent of accumulation is a function of the external and internal pH. The Ar may be used to calculate the pH, when the size of the internal acidic compartment is estimated, provided that the accumulation is due only to the transmembrane pH gradient and not to binding (see "Discussion"). Amines are more useful for estimates of pH, than weak acids like DMO, since DM0 concentrates within the basic mitochondrion. We may treat the sperm as a twocompartment system, with the mitochondrion as one compartment and the remainder of the cell as the second, in order to estimate the mitochondrial uptake of each probe. Using the previously obtained (17) Ar for DM0 (0.45) and EbNH (5.01, it can be calculated that 80% of the DM0 is within the mitochondrion, whereas only 5% of the EbNH is intramitochondrial (see Appendix 1). These data suggest that amines are reasonable indicators of the average cytoplasmic pH, as long as there is no significant accumulation in acidic organelles. The acrosomal granule may be such an acidic organelle (17,44), but because of its extremely small size (<0.06% of the cell volume, as calculated from electron micrographs and water volume measurements) EbNH accumulation there would be only a small fraction of the total.
Regulation of the pH, of Sperm by Seawater Ionic Composition-The pH, of sperm was affected by the ionic composition of the seawater. As shown in Tables I and 11, the pH, and Na+ concentration both influenced the pHi. In the normal 360 m~ Na+, a decrease in pH, from 8 to 7 caused a change of only a few tenths in pH, (Tables I and 11); whereas lower pH, led to further decreases in pHi. The greater stability of pH, in the presence of Na+ at pH, 7-8 was also observed by measurement of 9-AA accumulation as described in Fig. L4. Note that the quenching (uptake) of the amine was greatest at pHe = 8.3 and least at pH, = 6.9, indicating a decrease in trans-TABLE I Influence of external ionic composition on internal pH Dry sperm were diluted 100-fold into the indicated seawater at the specified pH, and incubated with [I4C]EhNH for 25 min; for Experiment 3, sperm were first washed in the same medium. Separation of the sperm from the reaction media and determination of the Ar and pH, were performed as under "Materials and Methods." Both Ar and pH, values are illustrated to indicate the size of change in the measured value (Ar) that can occur with relatively minor effect on the calculated pH,, since the Ar reflects the ApH across the membrane.

Intracellular pH and
Respiration 14883 membrane ApH as the pH, was decreased. The calculated pH, values corresponding to these data are shown in Table 11, under "ASW." In the absence of Na+ (t0.5 m~) lower values of pH, were found at each pH,, even at the normal pH of seawater, 7.9-8.0 (Tables I and 11). This is shown in Fig. lB, where approximately the same amount of uptake of 9-AA occurred at all pH, from 6.9-8.3, indicating that a relatively stable transmembrane ApH existed over this range. The calculated pHi values that correspond to each pH, are shown in Table 11, under "ChSW'. Fig. 1B shows that in the absence of Na', the kinetics of amine penetration are slower at a more acidic pH,. This is because the penetration of an amine is a passive diffusion phenomenon, so that the unidirectional influx of the amine (equal to the initial rate of influx) is proportional to the external concentration of the diffusing species, that is, the nonprotonated form of the amine (see Fig. 4 of the Miniprint, Appendix 3). Since this concentration decreases at more acidic external pH, the initial rate of uptake is lower when the external pH is more acidic. In the presence of Na+, the total amount of amine taken up by the sperm is reduced at more acidic pH, so that although the rate of penetration is slower, the plateau is reached in a short period of time. Without sodium, the same amount of amine is accumulated at equilibrium, whatever the pH, is. Since the unidirectional flux of amine entering the cells is decreased at a more acidic pH,, it takes a much longer time to achieve equilibrium (see Fig. 1 and Appendix 2 in the Miniprint). In the absence of Na', [''CC] Et2NH uptake is also slower at pH 7 than at pH 8 (not shown), but reaches an apparent equilibrium in approximately 1 h; this level is maintained for at least 3 h (<0.1 pH change) which indicates the slow accumulation of 9-AA is not due to gradual alterations of pHi. The requirement for extracellular Na' in order to maintain a relatively constant pH, has been seen in other cells (19)(20)(21)(22)(23). In sea urchin sperm at pH, = 8, the pH, increased with increasing [NaCIe in the seawater up to approximately 100 mM Na' (Fig. 2), after which a relatively constant pH, was maintained. Unlike Na+, Ca2+ had only a small influence upon pHi (Table I).
The K' concentration in seawater had a significant effect on the final value of pHi, as well as on the kinetics of the intracellular pH change upon sperm dilution (Fig. 3). Sperm that were diluted into ASW containing normal K' (10 KIM) maintained a pHi of approximately 7.5. When they were diluted into seawater with an increased K' concentration (50 m~) , a transient internal acidification was seen, followed by an adjustment toward pH 7.5. The alterations in the accumulation ratio for EbNH indicated a time-dependent alteration in pH, that occurred following sperm dilution, for they occurred on a longer time scale than that needed for Et2NH diffusion (see "Materials and Methods"). Because it binds

TABLE I1
Effect of Na' on the regulation ofpH, as determined by 9-AA accumulation Sperm were incubated as described in Fig. 1 until an apparent equilibrium was reached. The pH, was calculated after correction for binding as described in the Miniprint. No CaZ+ was present in these seawaters, which also included 1 m EGTA. within the sperm, 9-AA takes a much longer time to reach equilibrium (see Fig. 1 and Appendix 2 in the Miniprint) and cannot be used for rapid determination of internal pH. At high K' concentrations (200 and 370 m~, Fig. 3) the sperm reached an even more acidic pH,, which did not revert to an alkaline value. Regulation of Sperm Respiration and Motility by Seawater Ionic Composition-When sea urchin sperm are diluted into seawater at the normal pH (8.0), they rapidly initiate motility and respiration (2,4,6,7,10,11,26,38,46). However, when sperm were diluted into seawater at a lower pH, respiration was inhibited. The final rate of respiration decreased with lower external pH (Fig. 4A), as did sperm motility (data not shown; 26, 46). Sperm incubated at all of these pH values remained viable, as assessed by their capacity to fertilize eggs upon subsequent incubation at pH 8.0, when they began to swim and respire normally. Thus the diminished respiration at acidic pH, was not due to sperm death. Rather, the inhibition of respiration correlated with a decrease in pHi (e.g. Figs. 1 and 2; Tables I and 11).
When sperm were diluted into Na+-free seawater, their respiration was suppressed, and addition of increasing concentrations of Na' led to increased respiration (Figs. 2 and 4 B ) . The increased respiratory rate correlated with an internal alkalinization (Fig. 2). As previously reported (2), sperm are immotile in Na+-free seawater; motility was activated by concentrations of Na' similar to those that activate respiration. In our experiments both processes were activated together, usually at some pHi between 7.0 and 7.5, although the estimated pH, value for activation varied from one batch of sperm to another.
When sperm were diluted into high concentrations of K+, respiration was inhibited even if Na' was present (Fig. 4C). At intermediate concentrations of K' (25 and 50 m~) , respiration began, but only after a delay which increased with increasing K+ concentration (Fig. 4C). When the results of   activate respiration as shown in Table 111, where the effect of changing the external pH at various extracellular [Na'] was examined. Sperm respiration was triggered if the external pH was raised to a sufficient value, and the external pH required to trigger respiration was lower at higher external concentrations of Na'. For example, similar respiratory rates were observed at pH 9 and t 0 . 3 m~ Na', pH 8 and 10 m~ Na+, and pH 7 and 50 mM Na'.
Correlation between pH, of Sperm and Activation of Respiration and Motility-To test the hypothesis that alterations in pHi might lead to increased respiration and motility, we attempted to manipulate the pHi by the addition of high concentrations of amines. Since amines diffuse through membranes in their unprotonated forms, they can increase the pHi by becoming protonated intracellularly, if concentrations that overcome the buffering capacity of the cell are used. Intracellular alkalinization may be simultaneously monitored with tracer quantities of radioactive or fluorescent amines.
In choline seawater sperm did not respire, were immotile, and had an acidic pHi. They were induced to respire by the addition of NH&1 (Fig. 4B). Likewise, sperm suspended in 200 m~ KC had an acidic pHi and did not respire or swim.
Again, the addition of NH&I initiated respiration and motility (Fig. 5 ) . The maximal respiration rate in 10 m~ N&C1 in the presence of 200 mM K' was identical with that obtained in normal seawater (10 mM K+). Fig. 5 shows the dependence of activation of respiration on the concentration of NH&1 as well as the effects on the pHi. Over the range of NH4C1 that led to increased respiratory rates, there was a concomitant elevation of pH, as estimated by a decrease in the Ar for EbNH (see "Materials and Methods"). The pHi at optimal respiratory rates was identical with that obtained in normal seawater, under conditions where the sperm were likewise fully activated. Fig. 5A also shows that motility, as estimated qualitatively (see "Materials and Methods"), was stimulated over the same range of N R C l concentrations that led to increased respiration. The motility increased upon addition of 0.5 m~ NHICl, a concentration just sufficient to stimulate respiration and one that led to only a 20% decrease in Ar for Et,NH. Thus, just as shown in Fig. 3, respiration (and motility) was sensitive to very slight changes in pH,.

DISCUSSION
In this study, we found that the sperm pH, was more acidic when the extracellular K' was increased, or when no Na' was present in the dilution medium, or when the external pH was decreased. A decreased pH, has been found in another species of sea urchin sperm by 31P-NMR when the pH, is decreased or external K+ is increased (28). Changes in the internal pH of the sperm might d e c t the acrosomal reaction (17, 39), their viability (27), and also their respiration and motility (2,26,38,46). In this paper, we found a good correlation between increased pHi and activation of sperm respiration and motility, whether the pH, was altered by changing the extracellular K', Na+, or pH, or by addition of NKCl. Similar results on the correlation between motility and pH, have been found by Lee et al. (26,46) when sea urchin sperm were diluted in a sodium free medium, whereas other invertebrate sperm may control motility independently of pHi (37).
The inhibition of both sperm respiration and motility by low pH" could be caused by independent effects on the two processes, or by inhibition of one component of a tightly linked reaction. Motility of the sea urchin sperm is effected by the dynein ATPase of the flagellar axoneme (31,32). Thus, motility and respiration have the potential for being linked, since the ATP generated by respiration is used for motility,

IntracellularpH and Respiration
and the ADP so produced is required for respiration of tightly coupled mitochondria. If motility and respiration were tightly linked and axonemal movement were inhibited by low pH,, both motility and respiration would cease. In fact, both in situ and in a macromolecular aggregate, the dynein ATPase has an alkaline pH optimum, and its activity increases sharply around pH 7.5 (33), although the purified enzyme has a broader optimum (34). Axonemal motion in permeabilized sperm preparations is activated over a very narrow pH range (4), from pH 7.3 to 7.8. This is the pH range in which the respiration and motility are increased in vivo. Changes in CAMP and cGMP levels are also associated with stimulation of motility and respiration at low pH, by the peptide "speract" (la), so these nucleotides, as well as protein phosphorylation may play some role in the regulation (24, 35, 36).
The apparent pHi is expressed as a net value for the whole cell. This is a useful but unsophisticated measurement, since multiple intracellular compartments contribute to the net pHi that is obtained, and the size of the relevant compartment can not be determined with precision (15). Additional and less valid assumptions would be needed to determine the pH of any individual intracellular compartment. Still, the reported average pH, values are useful in comparing one set of experimental conditions with another, allowing estimates of changes in pH, associated with cellular activity, even if the absolute values of the cytoplasmic pH cannot be determined with certainty.
Since the pHi of the sperm is affected by both the pH, and Na' concentration, part of the regulation of pHj might involve Na+-dependent H+ movements (2, 26, 46). Such movements have been proposed to account for pH regulation in other cell types (19-23). The syngergistic effect of extracellular Na+ and pH on activation of respiration is consistent with this idea. The same respiratory rate can be obtained at lower pH, if the extracellular Na+ is elevated (Table 111). Since we have not examined the fluxes of all other ions under the conditions of these experiments, we cannot tell whether other Na+-dependent ionic fluxes, such as chloride-bicarbonate exchanges (19-21, 29) are involved in the regulation of pH,. The possibility that protons are equilibrated across the membrane according to a Donnan equilibrium seems unlikely, since depolarization of the plasma membrane with elevated K+ (17) leads to a strong acidification instead of an alkalinization. This membrane depolarization might affect the Na+-dependent H+ efflux mechanism or other ionic permeabilities needed to regulate the internal pH.
A question raised by these studies is whether the weak bases used to estimate the intracellular pH can also distribute across the membrane in their protonated form with a rate similar to the nonprotonated form. To the extent that this occurred, their utility as pH indicators would be decreased, since the mechanism of accumulation would also depend upon the membrane potential of the cell. The following observations suggest that this is not the case. Measurements of the sperm membrane potential with lipid-soluble ions (tetraphenylphos-phonium+, triphenylmethylphosphonium+, thiocyanate-) provide results which cannot explain the accumulation ratios obtained with the pH probes4 For example, depolarization of the membrane potential with increased external K' (17) would lead to a reduced accumulation of permeable cations such as Et2NH2+. The opposite effect was seen in this study; more amine accumulated in 200 m~ K+ than in 10 m~ Kf. Also, reducing the external pH of sea water from 8 to 7 has little effect on the membrane potential4 However, this decrease in pH, leads to a reduction in the initial rate of influx of the R. W. Schackmann, R. Christen, and B. Shapiro, manuscript in preparation.

12.
13. 14. amine, as expected if the nonprotonated form is the diffusing species, since its concentration is decreased 10 times by a decrease of 1 unit in the external pH. Finally, similar results are obtained with any amine used to measure the changes in the internal pH (diethylamine, methylamine, and 9-aminoacridine) . .labeled alkylamines f a r the small pHi changer that occur w t h t h e acmlone PeaCtion (17) and the Onset of r n t i l i t y and respiration. I f the binding Of 9 -M could be analyzed quantitatively, then this reagent wuld be mre useful for determination O f i n t r a c e l l u l a r pH.

44.
Altbugh the yeak acids and barer have been used to ertimpte intracellular pH (pHi)

7n
In order to explain the difference in the Uptake of 9-A1 and alkylamines in spem, R propose t h a t 9-AA traverser spem nunbraner i n i t s unprotonated form; that free intrac e l l u l a r 9-M is accmullted according to the pH gradient; and that intracellular binding sites (probably Wn) a l l w for an equilibrium betwen bund and free 9-M which increaser the accrnvlatian of mine. This study attmpts to test this hypothesis by quantitatively correcting for the binding that occurs subsequent t o uptake; with such CoWPctiOnS Ye obtain 9-AA i s b u n d r -In fact. the binding reaction serves to amplify the uptake signal. Yith Such the ram value far pHl using either 9-AA or diethylmine. even when 591. Of  Equation [3] indicates that in Ordet' to deternine the intracellular pH i n c e l l s when the w i l l be a function Of the free intracellular amine concentration. the total concentration Of acridine dye blndr. one must be able to rmasure the amunt O f dye that is bund. Binding  Such an e f f e c t was seen a t pH = 6.9 for rea urchln sperm ( Figure

Intracellular p H and Respiration
The valve (1 + C) can be detemined in the ruggest that tk unprotonated form Of the amine i s the diffusing rpecres.
The use o f 9-AA fluorescence allovs us to test one of the underlying assumptions an "hich accvmulates. In fact, because it accumulates to such a great extent; 50 ut4 9-M i s as Another asrumption ~n these studies I S that 9-AA does not alter the PHI as i t effective 1s 5 mn methylamine at inrrearlng pH-when mawred with tracer quantities Of range which IS directly proportional to the mncentratim bound, the internal concentration p H , 6.9. 7.3, 7.9, 8.3. It was suggested that nuclear bindlng did not OCCUI. based on the absence o f fluorescence in the nuclear region d l detected by flUDrelCRlCe micmscopy. Hwever. the detection of a highly quenched signal Over the nuclear region by fluorescence micmscopy l i a~ not quantitative, and since no data e r e presented on the fractional quenching that 11s obtained after colla se of the pH gradient. the issue of binding "as not directly explored In estimates O f the intracrsrmal pH o f hamster spm 9-M uptake was -2 orders of