Printed in Sweden Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserocd
Experimental
CYTOCHEMICAL 3’,5’-NUCLEOTIDE
Cell Research 91 (1975) 3646
LOCALIZATION
OF ADENYLATE
PHOSPHODIESTERASE
CYCLASE
AND
IN DICTYOSTELIUM
C. J. M. FARNHAM Department
of Microbiology,
Universiiy
of Florida,
Gainesville,
Fla 32601,
USA
SUMMARY Cytochemical localizations of adenylate cyclase and 3’,5’-nucleotide phosphodiesterase were performed on aggregating Dictytostelium discoideum myxamoebae. The adenylate cyclase reaction product was localized on the inner surface of the plasma membrane. The phosphodiesterase reaction product was localized on the outer surface of the plasma membrane. Differences in enzyme activity were noted according to the state of cell (isolated or aggregated) and according to the cell position in larger aggregates. Heavy precipitation indicative of adenylate cyclase activity was not observed in isolated amoebae, but was often observed in streams and in some cells of aggregates. The precipitation indicative of phosphodiesterase activity could be found in isolated amoebae and in peripheral cells of aggregates.
Adenosine 3’,5’-monophosphate cyclic (CAMP) has been shown to figure prominently in the processesof aggregation and development in the cellular slime mold Dictyostelium discoideum [l-4]. This involvement must be attributed to the activity of at least two enzymes: adenylate cyclase, which cleaves ATP into CAMP and pyrophosphate, and a 3’,5’-nucleotide phosphodiesterase, which cleaves CAMP into 5’-AMP. Pulsesof CAMP are released into the medium serving as the signal to aggregate [4, 51. Phosphodiesterase is both secretedinto the medium, presumably to keep the CAMP concentration within the system’s range of sensitivity [6-lo] and is membrane bound, possibly involved in signal reception [9-l 11. Delineation of the regulation of the intercellular communication system which results in aggregation has been limited by the nature of biochemical investigations, i.e. information is only obtained on the average enzymatic Exptl
Cell Res 91 (1975)
activities of cells, and on the general intracellular localization of enzymes. Light microscope observations of aggregating cells [12, 131 have provided information concerning the timing of cell movements during aggregation, but cannot effectively relate that to enzyme activities of individual cells. A cytochemical investigation of this system at the electron microscope level has therefore been undertaken to provide more exact information on the intracellular localization of adenylate cyclase and phosphodiesterase, and to attempt to correlate enzymatic activity with stage of development on an individual cell basis. Adenylate cyclase can be localized by provision of a specific substrate, adenylyl imidodiphosphate (AMPPNP) [14] which can be cleaved into CAMP and imidodiphosphate (PNP) by adenylate cyclases so far investigated [15, 161, but cannot be cleaved by most adenosine triphosphatases so far investigated
Dictyostelium [17, 181. Provision of an abundance of lead nitrate permits deposition of lead imidodiphosphate near the actual site of the enzyme t151. Phosphodiesterase can be localized [19] by preincubation in 5’-nucleotidase followed by incubation with CAMP, 5’-nucleotidase, and lead nitrate. The endogenous phosphodiesterase will cleave CAMP into 5’-AMP, which will then be acted upon by the 5’-nucleotidase resulting in the release of phosphate, which can then interact with the lead to form a precipitate of lead phosphate, presumably reasonably near the site of the original phosphodiesterase activity. MATERIALS
AND
METHODS
Dictyostelium discoideum spores were streaked onto a plate of either dextrose-peptone agar or half-strength cornmeal agar in association with Escherichia co/i and allowed to grow 36 to 48 h at 23°C until medium-tolarge aggregation territories were observed, with some slugs forming. The plates were then flooded with 1 % redistilled glutaraldehyde in 0.025 M cacodylate buffer, 37 mM with sucrose, and allowed to fix at room temperature for 30 min. Cells were scraped free of the agar surface and harvested and buffer washed by centrifugation. Some plates of aggregating amoebae were overlaid with 50°C agar before fixation. so that entire aggregation territories were preserved. Cells were then immediately suspended in the reaction mixture. which for the adenvlate cvclase localization was OS’mM AMPPNP (I&), 80-mM Tris-maleate pH 7.4, 2 mM MgCl,, 4 mM Pb(NO&, and 37 mM sucrose [15]. Controls were 10 mM theophylline, 1 mM dithiothreitol (DTT). a substrateless control, and incubation buffer alone. Incubation was for I h at 30°C. For the 3’,5’-nucleotide phosphodiesterase localization [19] cells fixed, washed, and harvested by centrifuaation as ureviouslv described were susnended in a preincubatidn mixture of 80 mM Tris-maleate pH 7.4, 2 mM MgCl,, and 5 mg/ml Crotalus atrox venom (source of 5’-nucleotidase), for 30 min at room temperature, followed by 30 min incubation at 37°C in the complete reaction mixture, 80 mM Tris-maleate pH 7.4, 2 mM MgCI,, 3 mg/ml Crotulus atrox venom 2 mM Pb(NO,),, and 3 mM CAMP. There was a substrateless control, a 50 mM theophylline control, and a 1 mM DTT control. After incubation all samples were given two room temperature buffer washes, post-fixed for 1 h at room temperature in 1 % OsO,, buffer washed twice, then agar embedded (if not previously). Dehydration was through an ethanol series carried into acetone; the embedding medium was Spurr’s plastic mixture
adenylate cyclase and phosphodiesterase
31
1201.Polymerization was at 60°C for at least one day. Thin sections were cut with a DuPont diamond knife with an LKB Ultrotome III. collected on Formvarcoated grids, and viewed without poststain in a Hitachi HU 1lC electron microscope.
RESULTS In the adenylate cyclase localization lead imidodiphosphate deposition was observed on the inner surface of the plasma membrane (fig. 1) which would indicate that the enzyme is membrane-bound and that the imidodiphosphate (PNP) liberated by enzymatic cleavage of AMPPNP is released into the cytoplasm. Some nonspecific lead deposition would be expected, since there are other endogenous enzymes and substrates which can interact to release phosphate or pyrophosphate which with lead freely available would result in lead deposition. For example, ATPases as well as adenylate cyclase could interact with endogenous ATP to release pyrophosphate or phosphate, and 5’-nucleotidases could interact with endogenous 5’-nucleotides to release phosphate. The amount of deposition from any such activity would be expected to be light, however, due to the limited amount of extraneous substrates which would be available following glutaraldehyde fixation and buffer washes. Specific deposition should thus be defined as precipitation of significantly larger amounts than that obtained in the absence of specific substrate. Comparison of figs 1 and 2 shows that significantly more deposition occurred on the inner surface of the plasma membrane in the presence of AMPPNP (fig. 1) than in its absence (fig. 2) in amoebae of similar developmental stage. The amount of presumably nonspecific cytoplasmic depositionvaried from cell to cell, with no clear correlation with cell type, but could be as heavy without substrate AMPPNP as with substrate. Although theophylline has not been shown to significantly inhibit D. discoideum phosExptl
Cell Res 91 (197.5)
38
C. J. M. Farnham
Figs 1-7. D. discoideum adenylate Fig. I. Aggregated myxamoebae, Fig. 2. Aggregated myxamoebae, Fig. 3. Aggregated myxamoebae, Fig. 4. Aggregated myxamoebae,
Exptl
Cell Res 91 (1975)
cyclase localization. Arrows denote specific precipitate. standard reaction mixture. x 216 000. without AMPPNP. x 244 000. with theophylline. x 244 000. with DTT. x 244 000.
Dictyostelium
adenylate cyclase and phosphodiesterase
Fig. 5. Isolated myxamoeba, standard reaction mixture. x 216 000. Fig. 6. Aggregated myxamoebae, standard reaction mixture. x 83 000. Fig. 7. Aggregated myxamoebae, standard reaction mixture. * 83 000. Figs 8-12. D. discoideum 3’,5’-nucleotide phosphodiesterase localization. Fig. 8. Isolated myxamoeba, standard reaction mixture. x 188 000.
Arrows
39
denote specific precipitate.
Exptl
CeN Res 91 (1975)
40
Fig. Fig. Fig. Fig.
Exptl
C. J. M. Farnham
9. Aggregated 10. Aggregated II. Aggregated 12. Aggregated
myxamoebae, myxamoebae, myxamoeba, myxamoeba,
Cell Res 91 (1975)
standard reaction without CAMP. with theophylline. with DTT. x 216
mixture. x 220 000. x 216 000. x 216 000. 000.
Dictyostelium phodiesterase as it does other cyclic nucleotide phosphodiesterases [6, 151, it has been implicated in this adenylate cyclase-phosphodiesterase system [2]. A control was therefore included to detect any possible effect theophylline might have on amount or specificity of lead deposition in this localization procedure. No difference was observed, as can be seen in fig. 3. Since DTT has been shown to inhibit D. discoideum phosphodiesterase [9, lo] a control was included with DTT in addition to AMPPNP, to reduce any precipitation resulting from the action of phosphodiesterases, which could conceivably be cleaving CAMP formed by adenylate cyclase, thus providing a supply of 5’-adenosine monophosphate which could then be cleaved by 5’-nucleotidase, releasing phosphate which could then be precipitated by the available lead. The limited time of the incubation, 1 h, makes such a complex chain of reactions unlikely to lead to significant deposition, but if it were a major contributing factor inclusion of DTT might be expected to reduce its contribution. As can be seen from fig. 4, no significant reduction in deposition was obtained in the presence of DTT. Although some precipitation could be noted on the inner surface of the plasma membrane of apparently isolated amoebae, as shown in fig. 5, such precipitation was never clearly more than could be found in substrateless controls. Figs 6 and 7, which show two areas of a medium-small aggregate, illustrate the amount of variation possible in degree of precipitation according to cell position. The most consistent observation for all aggregates so far examined is that some of the peripheral amoebae are densely precipitated. Some of the central amoebae in some aggregates are densely precipitated, as are some of those cells which are in singlefile streams.
adenylate cyclase and phosphodiestevase
41
In the cyclic 3’,5’-nucleotide phosphodiesterase localization procedure, lead phosphate was deposited on the outer surface of the plasma membrane, which would indicate that the enzyme is associated with the membrane and that the phosphate liberated upon enzymatic cleavage of CAMP is released extracytoplasmically. Fig. 8 shows an isolated amoeba, standard reaction mixture, fig. 9 shows an aggregated amoeba, standard reaction mixture, whereas fig. 10 shows an aggregated amoeba, standard reaction mixture without substrate cyclic AMP. A comparison of figs 8,9, and 10 will show considerable precipitation in the cytoplasm and some precipitation on the inner surface of the plasma membrane both with (figs 8, 9) and without (fig. 10) CAMP, but only with CAMP is there heavy precipitation on the outer surface of the plasma membrane. As previously noted, theophylline has not been shown to inhibit D. discoideum phosphodiesterase, although it has been implicated in this system [2, 61. Fig. 11, an aggregated amoeba, standard reaction mixture plus theophylline, shows that theophylline indeed does not inhibit the heavy deposition of lead phosphate on the outer surface of the plasma membrane. Deposition of reaction product was considerably heavier in the presence of theophylline than in the standard reaction mixture, but there is insufficient evidence to support ascribing it a stimulatory function. One mM DTT has been shown in biochemical assays to inhibit D. discoideum phosphodiesterase [9, lo]. A cytochemical assay using standard reaction mixture plus 1 mM DTT has now shown significant inhibition of the heavy deposition on the outer surface of the plasma membrane, which would tend to support the contention that this cytochemical assay is indeed specific for phosphodiesterase. Fig. 12 shows an aggregated amoeba, standard reaction mixExptl
Cell Res 91 (1975)
42
C. J. M. Farnham
ture plus DTT; a comparison with figs 8 and 9 will show no significant reduction of cytoplasmic and inner plasma membrane precipitation, but a severe reduction in precipitation on the outer surface of the plasma membrane. Dense specific precipitation can be observed on the outer surface of the plasma membrane in both isolated (fig. 8) and aggregated (fig. 9) amoebae. In aggregates densely precipitated cells are usually found in one or more areas, from the periphery inward one to many amoebae, for part of the periphery of the aggregate. DISCUSSION Several assumptions are necessary to the acceptance of the validity of the adenylate cyclase localization procedure. The most formidable is the assumption that only adenylate cyclase can cleave PNP from AMPPNP to allow formation of lead PNP. Although this has not been shown biochemically for D. discoideum, it has been shown that AMPPNP is not hydrolysed ((2 %) by myosin or heavy meromyosin ATPases in 16 h under conditions in which ATP is 90 % hydrolysed in 10 min, although AMPPNP is the most competitive inhibitor known for Ca2+ and Mn2+ moderated heavy meromyosin ATPase [17]. Sperow et al. [21] have shown that analogs of pyrophosphate with bridge oxygen replaced by imido are not substrates for crystalline yeast inorganic pyrophosphatase. M. Rodbell has indicated in a personal communication to Yount et al. [14] that AMPPNP is not cleaved by plasma membrane ATPases or by mitochondrial ATPases. Rodbell et al. [18] have reported that compared to ATP, AMPPNP was only slowly hydrolysed during incubation with rat liver plasma membranes. Other cytochemical localization assays have also supExptl
Cell Res 91 (1975)
ported general specificity of AMPPNP cleavage by adenylate cyclase. Howell & Whitfield [15] found that the specific precipitate obtained from this procedure in rat islets of Langerhans was subject to glucagon and fluoride sensitivity, as was their adenylate cyclase. Biochemical tests confirmed that some portion (2040 %) of their adenylate cyclase activity did indeed remain after fixation. A localization by Wagner et al. [16] in isolated capillary endothelium has also supported the specificity of AMPPNP cleavage by adenylate cyclase. Our results would also tend to support specificity for adenylate cyclase, although no specific inhibitor or stimulatory substance was used to confirm this. The failure of DTT to reduce deposition on the inner surface of the plasma membrane indicates little if any contamination by phosphodiesterase activity. The role of theophylline in this system remains uncertain, since it did not affect the specific deposition. Another significant assumption which must be made is that only the adenylate cyclase which is active at the time of fixation is demonstrated by the localization procedure. It is known that the specific activity of adenylate cyclase in cell-free extracts remains the same throughout growth and fruiting, so it appears that the enzyme does not turn over at an appreciable rate [22]. Thus if adenylate cyclase is a significant factor in the regulation of this system it would have to be via modulation of its activity. Malkinson & Ashworth [IO] have shown that when the intracellular level of CAMP rises, phosphodiesterase activity soon rises, also tending to indicate that the intracellular concentration of CAMP is controlled by modulation of adenylate cyclase activity. If such modulation were not preserved by the fixation procedure, one would expect to find little difference in activity from cell to cell, whereas one finds
Dictyostelium considerable differences. Rossomando & Sussman [23] have demonstrated a possible means of modulation of adenylate cyclase activity. They have found that the adenylate cyclase from D. discoideum has an absolute requirement for 5’-AMP, and that ATP pyrophosphohydrolase, which cleaves ATP into 5’-AMP and pyrophosphate, has an absolute requirement for CAMP. ATP pyrophosphohydrolase is extremely cooperative with CAMP concentration, and could thus serve as an efficient on-off switch leading to CAMP pulses. A natural consequence of the assumption that only those enzyme molecules that are active at the time of fixation remain active is that there should be differences between cells that are responding equally according to their position relative to the signal pulse at the time of fixation. One should therefore not expect all amoebae in similar positions to exhibit identical reaction product deposition. Any meaningful interpretation of the distribution of adenylate cyclase must therefore be based on where one can observe significant precipitation, since one can find cells without significant precipitation in any position. Characterization of the stage of a cell must also be carefully determined: serial sections may reveal that cells believed to be isolated are in close contact with other cells above or below the plane of section. Apparent cytoplasmic deposition may also be actually in contact with membrane just above or below the plane of section. The sensitivity of the localization procedure is also limited by the amount of apparently nonspecific reaction product obtained in the absence of substrate: the amount of adenylate cyclase activity must be relatively high before one can accept it as real with some degree of confidence; adenylate cyclase activity actually present in a particular class of amoebae, such as isolated amoebae, may not normally
adenylate cyclase and phosphodiesterase
43
be high enough to be distinguishable from the nonspecific deposition. Extensive scanning is thus essential to even preliminary observations, and conclusions relating enzyme activity to the stage of cells in the aggregation process are necessarily less rigorous than conclusions concerning the localization of enzyme activity within the cell. One further assumption that might be made is that the enzyme activity revealed by the cytochemical localization is representative, i.e. that all active enzyme molecules are equally likely to retain their activity following fixation. This is not a completely justifiable assumption, since some portion of the enzyme molecules may be localized in a position more or less accessible to the fixative than the rest of the enzyme molecules. Nor is it a necessary assumption if one limits one’s conclusions to the observation of where enzyme activity was localized, and does not try to assert that active enzymes could not also have been localized elsewhere. The discovery of adenylate cyclase reaction product in association with the cell membrane is not surprising: such a location is consistent with its involvement in intercellular communication, and biochemical evidence has indicated it is membrane bound [lo, 221. Other adenylate cyclase localizations, in rat islets of Langerhans [15] and in rat liver [24], have clearly shown reaction product on the outer surface of the plasma membrane, although biochemical evidence has indicated that CAMP is released intracellularly [25], leading to the suggestion that CAMP and pyrophosphate might be released on different sides of the plasma membrane [24]. In D. discoideum also there is biochemical support for intracellular release of CAMP: Malkinson & Ashworth [lo] found that as cells enter stationary phase the CAMP level increases first intracellularly to almost double, and Exptl
Cell Res 91 (1975)
44
C. J. A4. Farnham
soon thereafter the extracellular concentration of CAMP rises. If CAMP is indeed released intracellularly it would seem that at least in D. discoideum the pyrophosphate is released on the same side of the membrane as CAMP. The failure to find heavy deposition in isolated amoebae, even those appearing to be responding to aggregation signals, would tend to indicate that significant adenylate cyclase activity usually begins after formation of the close intercellular contact typical of streams and aggregates, or possibly that it closely precedes the formation of such contacts. Such a change in enzyme activity upon cell association might be related to the observed changes in plasma membrane particles following cell association [26]. Light microscope observations [12] of artificially aggregating amoebae have been interpreted as indicating that signal propagation, i.e. adenylate cyclase activity, precedes formation of the close intercellular contacts of streams, but the authors did observe some minor stream formation as signal propagation began. The validity of the phosphodiesterase localization procedure rests primarily on the assumption that observed differences in deposition of reaction product are indeed due to the action of phosphodiesterase. Some factors which might participate in spurious localization have been investigated for possible contribution. Endogenous 5’-nucleotidase can contribute to deposition of reaction product. For some tissue endogenous 5’nucleotidase apparently does not survive the experimental procedures used. Florendo et al. [19] found no deposition of reaction product in rat cerebral cortex tissue preincubated without the Crotalus atrox venom, and then incubated still without the enzyme but with lead nitrate and either 5’-AMP or CAMP. Liver samples treated identically showed a typical reaction product. Higazi Exptl
CeN Res 91 (197.5)
et al. [27] found that deposition of reaction product in their light microscopic investigation of cervicovaginal epithelium of neonatal mice was only slightly reduced by omission of 5’-nucleotidase, which indicates a relatively high level of endogenous 5’-nucleotidase activity in their tissue. In any case one must assume that the relative contribution of endogenous 5’-nucleotidase observed in the control does not change significantly in the experimental conditions, for any given tissue. Penetration and selective affinity of exogenous S’nucleotidase has been considered as a possible source of localization error. When Florendo et al. [19] preincubated their rat cerebral cortex in 5’-nucleotidase, then incubated with 5’-AMP in the absence of exogenous enzyme they observed deposition throughout the sections, indicating that the exogenous 5’-nucleotidase had indeed penetrated (sine they had not found any endogenous 5’-nucleotidase activity previously) and had not bound specifically. These results also tended to exclude the possibility that selective binding of lead ions, 5’-AMP, or phosphate was contributing to the localization in their tissue. Phosphodiesterase activity could be introduced along with the 5’-nucleotidase in snake venom, since Crotalus atrox venom has been found to have 8.7 x lo3 units of phosphodiesterase activity per mg dry venom [28]. This venom, however, had 860 x lo3 units of 5’-nucleotidase activity per mg dry venom, which is a ratio of 99: 1 for 5’-nucleotidase to phosphodiesterase [28]. Florendo et al. [19] have also examined C. atrox venom, finding no detectable phosphodiesterase activity. The possibility of nonenzymatic hydrolysis of CAMP has also been investigated [29]; happily, unlike adenosine triphosphate and many other nucleoside phosphates, CAMP and 5’-AMP have not been found subject to appreciable leadcatalysed nonenzymatic hydrolysis. One or
Dictyostelium more of these factors could be responsible for the lead deposition observed here in D. discoideum in the absence of substrate CAMP, and one must assume that their relative contribution does not change significantly in the range of experimental conditions employed. If the localization procedure is valid, two conditions should be met. Phosphodiesterase activity should be biochemically demonstrable in tissue fixed, washed, and incubated as for the localization procedure. Florendo et al. [19] did indeed find about one-third of original enzyme activity was retained, and this activity was subject to about 80 % inhibition by 50 mM theophylline. Secondly, modulation of presumably specific reaction product deposition in the cytochemical localization procedure should reflect biochemically known modulation of enzyme activity. Florendo et al. [19] did indeed find little or no deposition of reaction product in the presence of 50 mM theophylline. In D. discoideum we have found 1 mM DTT, which is known to competitively inhibit phosphodiesterase activity [9] to also strongly inhibit the deposition of reaction product on the outer surface of the plasma membrane, which we have interpreted as representative of phosphodiesterase activity. Cytochemical localizations should also correspond to the distribution of activity as ascertained by biochemical procedures. Biochemical studies have indicated that some phosphodiesterase activity in D. discoideum is membrane bound they cannot distinguish [9-l 11, although between inner and outer surfaces. Although it is possible that phosphodiesterase activity may also be contributing to the deposition of reaction product on the inner surface of the plasma membrane and in the cytoplasm, it appears likely that some phosphodiesterase is localized on the outer surface of the plasma membrane. This is a rather novel location, since the previous studies, with mammalian
adenylate cyclase and phosphodiesterase
45
systems, have localized phosphodiesterase intracellularly, adjacent to the postsynaptic membrane or in close association with smooth endoplasmic reticulum and possibly microtubules in rat cerebral cortex [19], and adjacent to the plasma membrane in cervicovaginal epithelium of neonatal mice [27]. Localization on the outer surface of the plasma membrane would, however, seem to be compatible with current understanding of the mechanism of intercellular communication in D. discoideum aggregation. At least one function of phosphodiesterase is apparently reduction of CAMP concentration, presumably to keep the concentration within the sensitivity of the system. This is almost certainly true for the phosphodiesterase which is secreted, since a phosphodiesterase inhibitor is also secreted beginning early in aggregation, presumably a fine control for the system ensuring that the CAMP concentration can attain proper levels [30]. The membranebound phosphodiesterase is insensitive to this specific phosphodiesterase inhibitor [ 1 I] which might indicate another function such as signal reception, although a distinct CAMP binding protein has also been described [31]. Since the membrane-bound phosphodiesterase is associated with the outer surface of the plasma membrane it seems likely that it is indeed hydrolyzing CAMP in the medium as has been believed [25]. The observation of phosphodiesterase activity in amoebae which have not yet formed intercellular contacts is consistent with the biochemical evidence that membrane-bound phosphodiesterase increases significantly upon development of aggregation competence in amoebae shaken in buffer [l l] and may be related to other changes in the plasma membrane which precede cell aggregation, such as the acquisition of a new antigenic site on the membrane (cs A), blockage of which prevents development of end-to-end cell adhesion [32]. Exptl
Cell Res 91 (1975)
46
C. J. AL Farnham
The author is indebted to Dr Henry Aldrich and Dr Robert Cohen for their advice in the course of this investigation, and to Mr William Farnham for advice and technical assistance. Support was provided from the Brown-Hazen Fund of the Research Corporation.
REFERENCES 1. Bonner, J T, Proc natl acad sci US 65 (1970) 110. 2. Bonner, J T, Barkley, D S, Hall, E M, Konijn, T M, Mason, J W, Keefe, G 0 III & Wolfe, P B, Dev biol 20 (1969) 72. 3. Konijn, T M, Barkley, D S, Chang, Y Y & Bonner, J T, Am natur 102 (1968) 225. 4. Konijn, T M, van de Meene, J G C, Bonner, J T & Barkley, D S, Proc natl acad sci US 58 (1967) 1152. 5. Gerisch, G, Current topics in dev biol 3 (1968). 6. Chang, Y Y, Science 161 (1968) 57. Chassy, B M, Science 175 (1972) 1016. i-i: Riedel, V & Gerisch, G, Biochem biophys res commun 42 (1971) 119. 9. Pannbacker, R G & Bravard, L J, Science 175 (1972) 1014. 10. Malkinson, A M & Ashworth, J M, Biochem j 134 (1973) 311. 11. Malchow, D, Nagele, B, Schwartz, H & Gerisch, G, Em j biochem 28 (1972) 136. 12. Robertson, A, Drage, D & Cohen, M H, Science 175 (1972) 333. 13. Cohen, M H & Robertson, A, J theor biol 31 (1971) 119. 14. Yount, R G, Babcock, D, Ballantyne, W & Ojala, D, Biochemistry 10 (1971) 2484. 15. Howell, S L & Whitfield, M, J histochem cytothem 20 (1972) 873.
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16. Wagner, R C, Kreiner, P, Barrnett, R J & Bitensky, M W, Proc natl acad sci US 69 (1972) 3175. 17. Yount, R G, Ojala, D & Babcock, D, Biochemistry 10 (1971) 2490. 18. Rodbell, M, Birnbaumer, L, Pohl, S L & Krans, H M J, J biol them 246 (1971) 1877. 19. Florendo, N T, Barrnett, R J & Greengard, P, Science 173 (1971) 745. Spurr, A R, J ultrastruct res 26 (1969) 31. 2 Sperow, J W, Moe, 0 A, Ridlington, J W & Butler, L G, J biol them 248 (1973) 2062. 22. Rossomando, E F & Sussman, M, Bioched biophys res commun 47 (1972) 604. 23. Rossomando, E F & Sussman, M, Proc natl acad sci US 70 (1973) 1254. 24. Reik, L, Petzold, G L, Higgins, J A, Greengard, P & Barrnett, R J, Science 168 (1970) 382. 25. Robison, G A, Butcher, R W & Sutherland, E W, Ann NY acad sci 139 (1967) 703. 26. Aldrich, H C & Gregg, J H, Exptl cell res 81 (1973) 407. 27. Higazi, M G & Kvinnsland, S, J reprod fert 36 (1974) 135. 28. Richards, G M, Vair, G du & Laskowski, M, Biochemistry 4 (1965) 501. 29. Rosenthal, A S, Moses, H L, Beaver, D L & Schuffman, S S, J histochem cytochem 14 (1966) 698. 30. Riedel, V, Gerisch, G, Muller, E & Beug, H, J mol biol 74 (1973) 573. 31. Malkinson. A M. Kwasniak. J & Ashworth. I J M. > Biochem j 133 (1973) 601. ’ 32. Beug, H, Katz, F E & Gerisch, G, J cell biol 56 (1973) 647. Received July 15, 1974 Revised version received September 18, 1974
Experimental
CYTOCHEMICAL 3’,5’-NUCLEOTIDE
Cell Research 91 (1975) 3646
LOCALIZATION
OF ADENYLATE
PHOSPHODIESTERASE
CYCLASE
AND
IN DICTYOSTELIUM
C. J. M. FARNHAM Department
of Microbiology,
Universiiy
of Florida,
Gainesville,
Fla 32601,
USA
SUMMARY Cytochemical localizations of adenylate cyclase and 3’,5’-nucleotide phosphodiesterase were performed on aggregating Dictytostelium discoideum myxamoebae. The adenylate cyclase reaction product was localized on the inner surface of the plasma membrane. The phosphodiesterase reaction product was localized on the outer surface of the plasma membrane. Differences in enzyme activity were noted according to the state of cell (isolated or aggregated) and according to the cell position in larger aggregates. Heavy precipitation indicative of adenylate cyclase activity was not observed in isolated amoebae, but was often observed in streams and in some cells of aggregates. The precipitation indicative of phosphodiesterase activity could be found in isolated amoebae and in peripheral cells of aggregates.
Adenosine 3’,5’-monophosphate cyclic (CAMP) has been shown to figure prominently in the processesof aggregation and development in the cellular slime mold Dictyostelium discoideum [l-4]. This involvement must be attributed to the activity of at least two enzymes: adenylate cyclase, which cleaves ATP into CAMP and pyrophosphate, and a 3’,5’-nucleotide phosphodiesterase, which cleaves CAMP into 5’-AMP. Pulsesof CAMP are released into the medium serving as the signal to aggregate [4, 51. Phosphodiesterase is both secretedinto the medium, presumably to keep the CAMP concentration within the system’s range of sensitivity [6-lo] and is membrane bound, possibly involved in signal reception [9-l 11. Delineation of the regulation of the intercellular communication system which results in aggregation has been limited by the nature of biochemical investigations, i.e. information is only obtained on the average enzymatic Exptl
Cell Res 91 (1975)
activities of cells, and on the general intracellular localization of enzymes. Light microscope observations of aggregating cells [12, 131 have provided information concerning the timing of cell movements during aggregation, but cannot effectively relate that to enzyme activities of individual cells. A cytochemical investigation of this system at the electron microscope level has therefore been undertaken to provide more exact information on the intracellular localization of adenylate cyclase and phosphodiesterase, and to attempt to correlate enzymatic activity with stage of development on an individual cell basis. Adenylate cyclase can be localized by provision of a specific substrate, adenylyl imidodiphosphate (AMPPNP) [14] which can be cleaved into CAMP and imidodiphosphate (PNP) by adenylate cyclases so far investigated [15, 161, but cannot be cleaved by most adenosine triphosphatases so far investigated
Dictyostelium [17, 181. Provision of an abundance of lead nitrate permits deposition of lead imidodiphosphate near the actual site of the enzyme t151. Phosphodiesterase can be localized [19] by preincubation in 5’-nucleotidase followed by incubation with CAMP, 5’-nucleotidase, and lead nitrate. The endogenous phosphodiesterase will cleave CAMP into 5’-AMP, which will then be acted upon by the 5’-nucleotidase resulting in the release of phosphate, which can then interact with the lead to form a precipitate of lead phosphate, presumably reasonably near the site of the original phosphodiesterase activity. MATERIALS
AND
METHODS
Dictyostelium discoideum spores were streaked onto a plate of either dextrose-peptone agar or half-strength cornmeal agar in association with Escherichia co/i and allowed to grow 36 to 48 h at 23°C until medium-tolarge aggregation territories were observed, with some slugs forming. The plates were then flooded with 1 % redistilled glutaraldehyde in 0.025 M cacodylate buffer, 37 mM with sucrose, and allowed to fix at room temperature for 30 min. Cells were scraped free of the agar surface and harvested and buffer washed by centrifugation. Some plates of aggregating amoebae were overlaid with 50°C agar before fixation. so that entire aggregation territories were preserved. Cells were then immediately suspended in the reaction mixture. which for the adenvlate cvclase localization was OS’mM AMPPNP (I&), 80-mM Tris-maleate pH 7.4, 2 mM MgCl,, 4 mM Pb(NO&, and 37 mM sucrose [15]. Controls were 10 mM theophylline, 1 mM dithiothreitol (DTT). a substrateless control, and incubation buffer alone. Incubation was for I h at 30°C. For the 3’,5’-nucleotide phosphodiesterase localization [19] cells fixed, washed, and harvested by centrifuaation as ureviouslv described were susnended in a preincubatidn mixture of 80 mM Tris-maleate pH 7.4, 2 mM MgCl,, and 5 mg/ml Crotalus atrox venom (source of 5’-nucleotidase), for 30 min at room temperature, followed by 30 min incubation at 37°C in the complete reaction mixture, 80 mM Tris-maleate pH 7.4, 2 mM MgCI,, 3 mg/ml Crotulus atrox venom 2 mM Pb(NO,),, and 3 mM CAMP. There was a substrateless control, a 50 mM theophylline control, and a 1 mM DTT control. After incubation all samples were given two room temperature buffer washes, post-fixed for 1 h at room temperature in 1 % OsO,, buffer washed twice, then agar embedded (if not previously). Dehydration was through an ethanol series carried into acetone; the embedding medium was Spurr’s plastic mixture
adenylate cyclase and phosphodiesterase
31
1201.Polymerization was at 60°C for at least one day. Thin sections were cut with a DuPont diamond knife with an LKB Ultrotome III. collected on Formvarcoated grids, and viewed without poststain in a Hitachi HU 1lC electron microscope.
RESULTS In the adenylate cyclase localization lead imidodiphosphate deposition was observed on the inner surface of the plasma membrane (fig. 1) which would indicate that the enzyme is membrane-bound and that the imidodiphosphate (PNP) liberated by enzymatic cleavage of AMPPNP is released into the cytoplasm. Some nonspecific lead deposition would be expected, since there are other endogenous enzymes and substrates which can interact to release phosphate or pyrophosphate which with lead freely available would result in lead deposition. For example, ATPases as well as adenylate cyclase could interact with endogenous ATP to release pyrophosphate or phosphate, and 5’-nucleotidases could interact with endogenous 5’-nucleotides to release phosphate. The amount of deposition from any such activity would be expected to be light, however, due to the limited amount of extraneous substrates which would be available following glutaraldehyde fixation and buffer washes. Specific deposition should thus be defined as precipitation of significantly larger amounts than that obtained in the absence of specific substrate. Comparison of figs 1 and 2 shows that significantly more deposition occurred on the inner surface of the plasma membrane in the presence of AMPPNP (fig. 1) than in its absence (fig. 2) in amoebae of similar developmental stage. The amount of presumably nonspecific cytoplasmic depositionvaried from cell to cell, with no clear correlation with cell type, but could be as heavy without substrate AMPPNP as with substrate. Although theophylline has not been shown to significantly inhibit D. discoideum phosExptl
Cell Res 91 (197.5)
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Figs 1-7. D. discoideum adenylate Fig. I. Aggregated myxamoebae, Fig. 2. Aggregated myxamoebae, Fig. 3. Aggregated myxamoebae, Fig. 4. Aggregated myxamoebae,
Exptl
Cell Res 91 (1975)
cyclase localization. Arrows denote specific precipitate. standard reaction mixture. x 216 000. without AMPPNP. x 244 000. with theophylline. x 244 000. with DTT. x 244 000.
Dictyostelium
adenylate cyclase and phosphodiesterase
Fig. 5. Isolated myxamoeba, standard reaction mixture. x 216 000. Fig. 6. Aggregated myxamoebae, standard reaction mixture. x 83 000. Fig. 7. Aggregated myxamoebae, standard reaction mixture. * 83 000. Figs 8-12. D. discoideum 3’,5’-nucleotide phosphodiesterase localization. Fig. 8. Isolated myxamoeba, standard reaction mixture. x 188 000.
Arrows
39
denote specific precipitate.
Exptl
CeN Res 91 (1975)
40
Fig. Fig. Fig. Fig.
Exptl
C. J. M. Farnham
9. Aggregated 10. Aggregated II. Aggregated 12. Aggregated
myxamoebae, myxamoebae, myxamoeba, myxamoeba,
Cell Res 91 (1975)
standard reaction without CAMP. with theophylline. with DTT. x 216
mixture. x 220 000. x 216 000. x 216 000. 000.
Dictyostelium phodiesterase as it does other cyclic nucleotide phosphodiesterases [6, 151, it has been implicated in this adenylate cyclase-phosphodiesterase system [2]. A control was therefore included to detect any possible effect theophylline might have on amount or specificity of lead deposition in this localization procedure. No difference was observed, as can be seen in fig. 3. Since DTT has been shown to inhibit D. discoideum phosphodiesterase [9, lo] a control was included with DTT in addition to AMPPNP, to reduce any precipitation resulting from the action of phosphodiesterases, which could conceivably be cleaving CAMP formed by adenylate cyclase, thus providing a supply of 5’-adenosine monophosphate which could then be cleaved by 5’-nucleotidase, releasing phosphate which could then be precipitated by the available lead. The limited time of the incubation, 1 h, makes such a complex chain of reactions unlikely to lead to significant deposition, but if it were a major contributing factor inclusion of DTT might be expected to reduce its contribution. As can be seen from fig. 4, no significant reduction in deposition was obtained in the presence of DTT. Although some precipitation could be noted on the inner surface of the plasma membrane of apparently isolated amoebae, as shown in fig. 5, such precipitation was never clearly more than could be found in substrateless controls. Figs 6 and 7, which show two areas of a medium-small aggregate, illustrate the amount of variation possible in degree of precipitation according to cell position. The most consistent observation for all aggregates so far examined is that some of the peripheral amoebae are densely precipitated. Some of the central amoebae in some aggregates are densely precipitated, as are some of those cells which are in singlefile streams.
adenylate cyclase and phosphodiestevase
41
In the cyclic 3’,5’-nucleotide phosphodiesterase localization procedure, lead phosphate was deposited on the outer surface of the plasma membrane, which would indicate that the enzyme is associated with the membrane and that the phosphate liberated upon enzymatic cleavage of CAMP is released extracytoplasmically. Fig. 8 shows an isolated amoeba, standard reaction mixture, fig. 9 shows an aggregated amoeba, standard reaction mixture, whereas fig. 10 shows an aggregated amoeba, standard reaction mixture without substrate cyclic AMP. A comparison of figs 8,9, and 10 will show considerable precipitation in the cytoplasm and some precipitation on the inner surface of the plasma membrane both with (figs 8, 9) and without (fig. 10) CAMP, but only with CAMP is there heavy precipitation on the outer surface of the plasma membrane. As previously noted, theophylline has not been shown to inhibit D. discoideum phosphodiesterase, although it has been implicated in this system [2, 61. Fig. 11, an aggregated amoeba, standard reaction mixture plus theophylline, shows that theophylline indeed does not inhibit the heavy deposition of lead phosphate on the outer surface of the plasma membrane. Deposition of reaction product was considerably heavier in the presence of theophylline than in the standard reaction mixture, but there is insufficient evidence to support ascribing it a stimulatory function. One mM DTT has been shown in biochemical assays to inhibit D. discoideum phosphodiesterase [9, lo]. A cytochemical assay using standard reaction mixture plus 1 mM DTT has now shown significant inhibition of the heavy deposition on the outer surface of the plasma membrane, which would tend to support the contention that this cytochemical assay is indeed specific for phosphodiesterase. Fig. 12 shows an aggregated amoeba, standard reaction mixExptl
Cell Res 91 (1975)
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C. J. M. Farnham
ture plus DTT; a comparison with figs 8 and 9 will show no significant reduction of cytoplasmic and inner plasma membrane precipitation, but a severe reduction in precipitation on the outer surface of the plasma membrane. Dense specific precipitation can be observed on the outer surface of the plasma membrane in both isolated (fig. 8) and aggregated (fig. 9) amoebae. In aggregates densely precipitated cells are usually found in one or more areas, from the periphery inward one to many amoebae, for part of the periphery of the aggregate. DISCUSSION Several assumptions are necessary to the acceptance of the validity of the adenylate cyclase localization procedure. The most formidable is the assumption that only adenylate cyclase can cleave PNP from AMPPNP to allow formation of lead PNP. Although this has not been shown biochemically for D. discoideum, it has been shown that AMPPNP is not hydrolysed ((2 %) by myosin or heavy meromyosin ATPases in 16 h under conditions in which ATP is 90 % hydrolysed in 10 min, although AMPPNP is the most competitive inhibitor known for Ca2+ and Mn2+ moderated heavy meromyosin ATPase [17]. Sperow et al. [21] have shown that analogs of pyrophosphate with bridge oxygen replaced by imido are not substrates for crystalline yeast inorganic pyrophosphatase. M. Rodbell has indicated in a personal communication to Yount et al. [14] that AMPPNP is not cleaved by plasma membrane ATPases or by mitochondrial ATPases. Rodbell et al. [18] have reported that compared to ATP, AMPPNP was only slowly hydrolysed during incubation with rat liver plasma membranes. Other cytochemical localization assays have also supExptl
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ported general specificity of AMPPNP cleavage by adenylate cyclase. Howell & Whitfield [15] found that the specific precipitate obtained from this procedure in rat islets of Langerhans was subject to glucagon and fluoride sensitivity, as was their adenylate cyclase. Biochemical tests confirmed that some portion (2040 %) of their adenylate cyclase activity did indeed remain after fixation. A localization by Wagner et al. [16] in isolated capillary endothelium has also supported the specificity of AMPPNP cleavage by adenylate cyclase. Our results would also tend to support specificity for adenylate cyclase, although no specific inhibitor or stimulatory substance was used to confirm this. The failure of DTT to reduce deposition on the inner surface of the plasma membrane indicates little if any contamination by phosphodiesterase activity. The role of theophylline in this system remains uncertain, since it did not affect the specific deposition. Another significant assumption which must be made is that only the adenylate cyclase which is active at the time of fixation is demonstrated by the localization procedure. It is known that the specific activity of adenylate cyclase in cell-free extracts remains the same throughout growth and fruiting, so it appears that the enzyme does not turn over at an appreciable rate [22]. Thus if adenylate cyclase is a significant factor in the regulation of this system it would have to be via modulation of its activity. Malkinson & Ashworth [IO] have shown that when the intracellular level of CAMP rises, phosphodiesterase activity soon rises, also tending to indicate that the intracellular concentration of CAMP is controlled by modulation of adenylate cyclase activity. If such modulation were not preserved by the fixation procedure, one would expect to find little difference in activity from cell to cell, whereas one finds
Dictyostelium considerable differences. Rossomando & Sussman [23] have demonstrated a possible means of modulation of adenylate cyclase activity. They have found that the adenylate cyclase from D. discoideum has an absolute requirement for 5’-AMP, and that ATP pyrophosphohydrolase, which cleaves ATP into 5’-AMP and pyrophosphate, has an absolute requirement for CAMP. ATP pyrophosphohydrolase is extremely cooperative with CAMP concentration, and could thus serve as an efficient on-off switch leading to CAMP pulses. A natural consequence of the assumption that only those enzyme molecules that are active at the time of fixation remain active is that there should be differences between cells that are responding equally according to their position relative to the signal pulse at the time of fixation. One should therefore not expect all amoebae in similar positions to exhibit identical reaction product deposition. Any meaningful interpretation of the distribution of adenylate cyclase must therefore be based on where one can observe significant precipitation, since one can find cells without significant precipitation in any position. Characterization of the stage of a cell must also be carefully determined: serial sections may reveal that cells believed to be isolated are in close contact with other cells above or below the plane of section. Apparent cytoplasmic deposition may also be actually in contact with membrane just above or below the plane of section. The sensitivity of the localization procedure is also limited by the amount of apparently nonspecific reaction product obtained in the absence of substrate: the amount of adenylate cyclase activity must be relatively high before one can accept it as real with some degree of confidence; adenylate cyclase activity actually present in a particular class of amoebae, such as isolated amoebae, may not normally
adenylate cyclase and phosphodiesterase
43
be high enough to be distinguishable from the nonspecific deposition. Extensive scanning is thus essential to even preliminary observations, and conclusions relating enzyme activity to the stage of cells in the aggregation process are necessarily less rigorous than conclusions concerning the localization of enzyme activity within the cell. One further assumption that might be made is that the enzyme activity revealed by the cytochemical localization is representative, i.e. that all active enzyme molecules are equally likely to retain their activity following fixation. This is not a completely justifiable assumption, since some portion of the enzyme molecules may be localized in a position more or less accessible to the fixative than the rest of the enzyme molecules. Nor is it a necessary assumption if one limits one’s conclusions to the observation of where enzyme activity was localized, and does not try to assert that active enzymes could not also have been localized elsewhere. The discovery of adenylate cyclase reaction product in association with the cell membrane is not surprising: such a location is consistent with its involvement in intercellular communication, and biochemical evidence has indicated it is membrane bound [lo, 221. Other adenylate cyclase localizations, in rat islets of Langerhans [15] and in rat liver [24], have clearly shown reaction product on the outer surface of the plasma membrane, although biochemical evidence has indicated that CAMP is released intracellularly [25], leading to the suggestion that CAMP and pyrophosphate might be released on different sides of the plasma membrane [24]. In D. discoideum also there is biochemical support for intracellular release of CAMP: Malkinson & Ashworth [lo] found that as cells enter stationary phase the CAMP level increases first intracellularly to almost double, and Exptl
Cell Res 91 (1975)
44
C. J. A4. Farnham
soon thereafter the extracellular concentration of CAMP rises. If CAMP is indeed released intracellularly it would seem that at least in D. discoideum the pyrophosphate is released on the same side of the membrane as CAMP. The failure to find heavy deposition in isolated amoebae, even those appearing to be responding to aggregation signals, would tend to indicate that significant adenylate cyclase activity usually begins after formation of the close intercellular contact typical of streams and aggregates, or possibly that it closely precedes the formation of such contacts. Such a change in enzyme activity upon cell association might be related to the observed changes in plasma membrane particles following cell association [26]. Light microscope observations [12] of artificially aggregating amoebae have been interpreted as indicating that signal propagation, i.e. adenylate cyclase activity, precedes formation of the close intercellular contacts of streams, but the authors did observe some minor stream formation as signal propagation began. The validity of the phosphodiesterase localization procedure rests primarily on the assumption that observed differences in deposition of reaction product are indeed due to the action of phosphodiesterase. Some factors which might participate in spurious localization have been investigated for possible contribution. Endogenous 5’-nucleotidase can contribute to deposition of reaction product. For some tissue endogenous 5’nucleotidase apparently does not survive the experimental procedures used. Florendo et al. [19] found no deposition of reaction product in rat cerebral cortex tissue preincubated without the Crotalus atrox venom, and then incubated still without the enzyme but with lead nitrate and either 5’-AMP or CAMP. Liver samples treated identically showed a typical reaction product. Higazi Exptl
CeN Res 91 (197.5)
et al. [27] found that deposition of reaction product in their light microscopic investigation of cervicovaginal epithelium of neonatal mice was only slightly reduced by omission of 5’-nucleotidase, which indicates a relatively high level of endogenous 5’-nucleotidase activity in their tissue. In any case one must assume that the relative contribution of endogenous 5’-nucleotidase observed in the control does not change significantly in the experimental conditions, for any given tissue. Penetration and selective affinity of exogenous S’nucleotidase has been considered as a possible source of localization error. When Florendo et al. [19] preincubated their rat cerebral cortex in 5’-nucleotidase, then incubated with 5’-AMP in the absence of exogenous enzyme they observed deposition throughout the sections, indicating that the exogenous 5’-nucleotidase had indeed penetrated (sine they had not found any endogenous 5’-nucleotidase activity previously) and had not bound specifically. These results also tended to exclude the possibility that selective binding of lead ions, 5’-AMP, or phosphate was contributing to the localization in their tissue. Phosphodiesterase activity could be introduced along with the 5’-nucleotidase in snake venom, since Crotalus atrox venom has been found to have 8.7 x lo3 units of phosphodiesterase activity per mg dry venom [28]. This venom, however, had 860 x lo3 units of 5’-nucleotidase activity per mg dry venom, which is a ratio of 99: 1 for 5’-nucleotidase to phosphodiesterase [28]. Florendo et al. [19] have also examined C. atrox venom, finding no detectable phosphodiesterase activity. The possibility of nonenzymatic hydrolysis of CAMP has also been investigated [29]; happily, unlike adenosine triphosphate and many other nucleoside phosphates, CAMP and 5’-AMP have not been found subject to appreciable leadcatalysed nonenzymatic hydrolysis. One or
Dictyostelium more of these factors could be responsible for the lead deposition observed here in D. discoideum in the absence of substrate CAMP, and one must assume that their relative contribution does not change significantly in the range of experimental conditions employed. If the localization procedure is valid, two conditions should be met. Phosphodiesterase activity should be biochemically demonstrable in tissue fixed, washed, and incubated as for the localization procedure. Florendo et al. [19] did indeed find about one-third of original enzyme activity was retained, and this activity was subject to about 80 % inhibition by 50 mM theophylline. Secondly, modulation of presumably specific reaction product deposition in the cytochemical localization procedure should reflect biochemically known modulation of enzyme activity. Florendo et al. [19] did indeed find little or no deposition of reaction product in the presence of 50 mM theophylline. In D. discoideum we have found 1 mM DTT, which is known to competitively inhibit phosphodiesterase activity [9] to also strongly inhibit the deposition of reaction product on the outer surface of the plasma membrane, which we have interpreted as representative of phosphodiesterase activity. Cytochemical localizations should also correspond to the distribution of activity as ascertained by biochemical procedures. Biochemical studies have indicated that some phosphodiesterase activity in D. discoideum is membrane bound they cannot distinguish [9-l 11, although between inner and outer surfaces. Although it is possible that phosphodiesterase activity may also be contributing to the deposition of reaction product on the inner surface of the plasma membrane and in the cytoplasm, it appears likely that some phosphodiesterase is localized on the outer surface of the plasma membrane. This is a rather novel location, since the previous studies, with mammalian
adenylate cyclase and phosphodiesterase
45
systems, have localized phosphodiesterase intracellularly, adjacent to the postsynaptic membrane or in close association with smooth endoplasmic reticulum and possibly microtubules in rat cerebral cortex [19], and adjacent to the plasma membrane in cervicovaginal epithelium of neonatal mice [27]. Localization on the outer surface of the plasma membrane would, however, seem to be compatible with current understanding of the mechanism of intercellular communication in D. discoideum aggregation. At least one function of phosphodiesterase is apparently reduction of CAMP concentration, presumably to keep the concentration within the sensitivity of the system. This is almost certainly true for the phosphodiesterase which is secreted, since a phosphodiesterase inhibitor is also secreted beginning early in aggregation, presumably a fine control for the system ensuring that the CAMP concentration can attain proper levels [30]. The membranebound phosphodiesterase is insensitive to this specific phosphodiesterase inhibitor [ 1 I] which might indicate another function such as signal reception, although a distinct CAMP binding protein has also been described [31]. Since the membrane-bound phosphodiesterase is associated with the outer surface of the plasma membrane it seems likely that it is indeed hydrolyzing CAMP in the medium as has been believed [25]. The observation of phosphodiesterase activity in amoebae which have not yet formed intercellular contacts is consistent with the biochemical evidence that membrane-bound phosphodiesterase increases significantly upon development of aggregation competence in amoebae shaken in buffer [l l] and may be related to other changes in the plasma membrane which precede cell aggregation, such as the acquisition of a new antigenic site on the membrane (cs A), blockage of which prevents development of end-to-end cell adhesion [32]. Exptl
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The author is indebted to Dr Henry Aldrich and Dr Robert Cohen for their advice in the course of this investigation, and to Mr William Farnham for advice and technical assistance. Support was provided from the Brown-Hazen Fund of the Research Corporation.
REFERENCES 1. Bonner, J T, Proc natl acad sci US 65 (1970) 110. 2. Bonner, J T, Barkley, D S, Hall, E M, Konijn, T M, Mason, J W, Keefe, G 0 III & Wolfe, P B, Dev biol 20 (1969) 72. 3. Konijn, T M, Barkley, D S, Chang, Y Y & Bonner, J T, Am natur 102 (1968) 225. 4. Konijn, T M, van de Meene, J G C, Bonner, J T & Barkley, D S, Proc natl acad sci US 58 (1967) 1152. 5. Gerisch, G, Current topics in dev biol 3 (1968). 6. Chang, Y Y, Science 161 (1968) 57. Chassy, B M, Science 175 (1972) 1016. i-i: Riedel, V & Gerisch, G, Biochem biophys res commun 42 (1971) 119. 9. Pannbacker, R G & Bravard, L J, Science 175 (1972) 1014. 10. Malkinson, A M & Ashworth, J M, Biochem j 134 (1973) 311. 11. Malchow, D, Nagele, B, Schwartz, H & Gerisch, G, Em j biochem 28 (1972) 136. 12. Robertson, A, Drage, D & Cohen, M H, Science 175 (1972) 333. 13. Cohen, M H & Robertson, A, J theor biol 31 (1971) 119. 14. Yount, R G, Babcock, D, Ballantyne, W & Ojala, D, Biochemistry 10 (1971) 2484. 15. Howell, S L & Whitfield, M, J histochem cytothem 20 (1972) 873.
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16. Wagner, R C, Kreiner, P, Barrnett, R J & Bitensky, M W, Proc natl acad sci US 69 (1972) 3175. 17. Yount, R G, Ojala, D & Babcock, D, Biochemistry 10 (1971) 2490. 18. Rodbell, M, Birnbaumer, L, Pohl, S L & Krans, H M J, J biol them 246 (1971) 1877. 19. Florendo, N T, Barrnett, R J & Greengard, P, Science 173 (1971) 745. Spurr, A R, J ultrastruct res 26 (1969) 31. 2 Sperow, J W, Moe, 0 A, Ridlington, J W & Butler, L G, J biol them 248 (1973) 2062. 22. Rossomando, E F & Sussman, M, Bioched biophys res commun 47 (1972) 604. 23. Rossomando, E F & Sussman, M, Proc natl acad sci US 70 (1973) 1254. 24. Reik, L, Petzold, G L, Higgins, J A, Greengard, P & Barrnett, R J, Science 168 (1970) 382. 25. Robison, G A, Butcher, R W & Sutherland, E W, Ann NY acad sci 139 (1967) 703. 26. Aldrich, H C & Gregg, J H, Exptl cell res 81 (1973) 407. 27. Higazi, M G & Kvinnsland, S, J reprod fert 36 (1974) 135. 28. Richards, G M, Vair, G du & Laskowski, M, Biochemistry 4 (1965) 501. 29. Rosenthal, A S, Moses, H L, Beaver, D L & Schuffman, S S, J histochem cytochem 14 (1966) 698. 30. Riedel, V, Gerisch, G, Muller, E & Beug, H, J mol biol 74 (1973) 573. 31. Malkinson. A M. Kwasniak. J & Ashworth. I J M. > Biochem j 133 (1973) 601. ’ 32. Beug, H, Katz, F E & Gerisch, G, J cell biol 56 (1973) 647. Received July 15, 1974 Revised version received September 18, 1974
