Alkaline phosphatase inhibition by copper: Implications to phosphorus nutrition and use as a biochemical marker of toxicity1

Transcription

1 Notes 743 Limnol. Oceanogr., 28(4), 1983, 1983, hy the merican Society of Limnology Ind Oceanography, Inc. lkaline phosphatase inhibition by copper: Implications to phosphorus nutrition and use as a biochemical marker of toxicity1 bstruct-lkaline phosphatase activity is inhibited by copper in phytoplankton cultures, cell-free enzyme preparations, and natural populations of phytoplankton. Inhibition in all these systems is rapid and a function of cupric ion activity, supporting the hypothesis that the cellular enzyme is directly exposed to the aqueous medium. These characteristics of the enzyme allow two applications: purified bacterial enzyme can be used to assay the cupric ion activity in filtered water samples, and the inhibition of the activity in natural populations is a biochemical marker for overall toxicity. lkaline phosphatase catalyzes the hydrolysis of dissolved organic phosphate (DOP) esters by phytoplankton (Kuenzler and Perras 1965). The enzyme is required to hydrolyze DOP introduced from the sediments in littoral regions (Taft et al. 1975), from the breakdown of particulate organic matter, and from upwelled subthermocline water (Perry 1972). These sources of DOP can be important in phosphorus nutrition. During phosphate uptake phosphorus is rapidly cycled through several compartments, including a DOP fraction, before it ends up in the phytoplankton (Lean and Nalewajko 1976). The amount of the enzyme in algae is controlled by their nutritional status: phosphorus-replete cells do not synthesize this enzyme; phosphorus starvation results in increased alkaline phosphatase activity. Because of this physiological response, the alkaline phosphatase activity of natural populations has been inversely correlated to their phosphorus nutritional status (Perry 1972). lkaline phosphatases from many organisms are inhibited by trace metals Supported by Portland State University Research and Publications Committee and Electric Power Research Institute contract RP (Dixon and Webb 1964). These phosphatases are usually zinc metalloenzymes that undergo a displacement of the required zinc by other trace metals (Cu, for example) resulting in loss of activity (Vallee 1959; Foy et al. 1978). lkaline phosphatase is associated with the surface of algae (Kuenzler and Perras 1965), specifically the plasmalemma (Rivkin and Swift 1980). This means that the enzyme is directly exposed to the free metal ion activity of the medium and cannot be protected from metal toxicity by binding to the many sites within the cells. Copper toxicity could thus affect the net phosphorus nutrition of phytoplankton by blocking sources and cycles of phosphate mediated by alkaline phosphatase. I have examined this interaction between trace metal chemistry and macronutrition in terms of a simple working hypothesis: alkaline phosphatase is on the surface of phytoplankton and will be inhibited immediately by increased cupric ion activity in the media. I studied the inhibition of the enzyme activity in three increasingly complex environments in order to sort out chemical and biological effects: in vitro with purified enzyme; in cultures with phosphate-starved diatoms, to examine short term effects; and in vivo with natural assemblages of phytoplankton over a period of weeks. I thank D. Robinson for help with the enzyme analysis, J. Sweet for the in situ enclosure study, and R. Petersen for advice and comments on the manuscript. lkaline phosphatase activity was measured by the method of Kuenzler and Perras (1965), in which the increase in absorbance at 410 nm is monitored after adding n-nitrophenylphosphate (pnpp) (Sigma Chemical). The marine diatom Thalassiosira pseudonanu (3H), obtained from R. R. L. Guil-

2 744 Note& Table 1. Computed cupric ion activity for given copper concentrations. For the quil media here (with 10d3 M Tris + 5 x 10e6 M EDT), the negative log of the cupric ion activity (pcu*) was computed using MINEQL (Morel et al. 1979; Westall et al. 1976). pch* Total Cu CM) 1.6 x 1O-5 1 x x 10-G 4 x G none lard, was grown in quil, a chemically defined medium (Morel et al. 1979) containing 4 x 10e5 M Si(OH),, 3 X lop4 M N03-, 2 x 1O-6 M P043-, 5 x lo- j M EDT, and different copper concentrations to yield different cupric ion activities as calculated by the program MINEQL (Westall et al. 1976) (Table 1). The background zinc concentration for zinc in quil has been estimated to be lo-l0 M (Rueter and Morel 1981). Except for phosphate uptake and release experiments in which the medium was filter-sterilized (0.4~pm Nuclepore filter) to minimize trace metal contamination, complete sterile technique was not used. ll manipulations of cultures were done in a laminar-flow hood. Soluble reactive phosphate and enzyme-hydrolyzable DOP concentrations were measured in reduced volumes by the methods of Strickland and Parsons ( 1972). lthough the bacterial enzyme used to determine the enzyme-hydrolyzable DOP is inhibited by copper, increased temperatures (30 C) and longer reaction times (2 h) allowed for complete hydrolysis. Two sources were used for cell-free enzyme studies: commercially purified enzyme from E. coli (Sigma), and crude extracts from T. pseudonuna prepared by centrifuging 1 liter of culture, sonicating the pellets in synthetic ocean water (Morel et al. 1979) containing 5 x lo- EDT for 90 s at 40 W, and recentrifuging the suspension. The purified bacterial en- 0 I IO 8 PCU Fig. 1. lkaline phosphatase activity (as percent of control) in two cell-free preparations vs. computed cupric ion activity (as pcu*). ctivity of commercially purified bacterial enzyme (0) showed more sensitivity than did cell-free extract from The luasiosiru pseudonam (). zyme was added to solutions of known cupric ion activity or to freshly filtered water samples. fter the enzyme had equilibrated with the copper solution (about 30 min), 26 PM of the substrate pnpp (just high enough to give maximum velocity in the control) were added, and the enzyme activity was monitored as above. Natural phytoplankton populations exposed to a range of copper concentrations were obtained as part of an in situ bag study in a reservoir (Sweet et al. in prep.). Polyethylene bags were filled with water, spiked with a range of copper concentrations, and monitored for alkaline phosphatase activity for 3 weeks. Filtered and unfiltered water samples were collected. The filtered samples were spiked with purified bacterial enzymes, equilibrated, and monitored for pnpp hydrolysis. The cells in the unfiltered samples were con-

3 Notes Orthophosphate g --- Enzyme-Hydrolyzable 12 IO 8 PCU Fig. 2.. lkaline _ phosphatase activity for whole cultures of the marine diatom vs. cupric ion activity. Enzyme velocity is computed for cultures with 4.13 ( * 1.5) x lo3 cells. ml- to which copper was added. centrated 20x by centrifugation, spiked with pnpp, and monitored for natural enzyme activity. These experiments were done immediately after copper introduction and then on samples collected over 3 weeks. The cupric ion activity was measured in fresh samples from these bags with an Orion cupric ion selective electrode calibrated for each set of measurements with a range of Cu-oxalate buffers. Cell concentrations and species composition, nutrients, major ions, and primary productivity were determined. The activity of both the purified bacterial enzyme and the crude cell-free extract from the diatom were related to the / I I I I 5 IO Minutes Fig. 3. Orthophosphate concentration vs. time after a culture of T. pseudonunu was spiked with 2 PM PO, -. There is a rapid increase in any enzymehydrolyzable compound that disappears with phosphate depletion. Mean and range of duplicate cultures is shown. cupric ion activity of the reaction mixture, showing that the bacterial and diatom enzymes are both sensitive to cupric ion (Fig. 1). In phosphate-starved cultures of T. pseudonana, alkaline phosphatase activity decreased with higher total copper concentrations (and thus cupric ion activities: Fig. 2). This decrease in enzyme activity can explain the change in patterns of phosphorus utilization. Control cultures, when spiked with phosphate, exhibited a rapid uptake of phosphate along with the excretion and eventual reuse of enzyme-hydrolyzable DOP (Fig. 3). Copper-inhibited cultures showed a similar pattern of phosphate uptake with the important exception that the DOP residual after 45 min was 35% greater, dem- onstrating that this enzyme-catalyzed step was significantly inhibited.

4 Notes /,I none Log Total Cu Fig. 4. lkaline phosphatase activity in natural samples vs. total Cu added to in situ bags. No copper was added to control bags. Enzyme rate is in terms of /*M of pnpp utilized per day. I- c E 2 zo.3 w + >,.- 0 Cu > 0.2 iii >, N I 1- [I I a I/I I I I // none Log Total Cu lkaline phosphatase activities of the concentrated natural populations (Fig. 4) and the activities of bacterial enzyme in filtered samples (Fig. 5a) were functions of the copper added to the bags. Enzyme activity can also be related to the measured cupric ion activity (Fig. Sb). Both of these sets of samples were taken immediately after copper was added. This demonstrates that the impact of copper on the natural populations and the purified enzyme is similar. Furthermore, the population decreased with time in these enclosures so that after 47 days the algal density was also a function of the total copper added (Sweet et al. in prep.). -; 0.3 \ 2 a I ), " c, t -g 0.21 z > ai E ), N c t w.. IC b I4 I2 IO 8 6 Measured pcu Fig. 5. Enzyme activity in filtered samples from the same bags as Fig. 4. Purified bacterial enzyme was added to 10 ml of sample and allowed to equil- ibrate for 30 min. Samples were then spiked with 15 PM pnpp and monitored at 410 PM for 5-10 min. Enzyme rate shows same relationship to total added copper (a) as did the whole samples (but with much higher activity) and (b) is correlated to cupric ion activity measured on site in the bags with an r2 value of 0.94.

5 Notes 747 The inhibition of alkaline phosphatase activity by copper has the same characteristics in three different systems: purified enzyme (or crude algal enzyme preparation) in both salt and freshwater media; phosphate-starved cultures of a marine diatom; and natural freshwater populations captured in large plastic bags. Because of these similarities, the immediate inhibition of the enzyme can be extrapolated to the eventual inhibition of the cells and thus serve as a biochemical marker for toxicity that would be useful in monitoring and managing natural populations. Trace metals may also affect phosphorus nutrition of the algae at concentrations that may not be acutely toxic to the whole organism. In these studies, alkaline phosphatase was sensitive to copper within the range of cupric ion activities estimated for seawater, g.6 M (Rueter et al. 1979; Sunda and Guillard 1976; nderson and Morel 1978). This toxicity can either block the direct utilization of sources of DOP (sediments, organic matter, etc.) or interfere with the use of DOP involved in cycles of phosphate uptake, release, and reutilization (Lean and Nalewajko 1976). In either case, copper toxicity results in lower alkaline phosphatase activities and lower availability of phosphorus to the cells. The reduction of alkaline phosphatase activity (Perry 1972) and its variability (Rivkin and Swift 1980) in natural marine systems could be caused by repression by orthophosphate as well as trace metal toxicity. These two mechanisms have different implications for the nutrient status of cells; phosphate repression means the cells have sufficient orthophosphate and do not require any other sources, whereas Cu toxicity would reduce the available phosphorus, possibly resulting in growthlimiting conditions. Mixing of subthermocline water into the euphotic zone would be expected to add copper as well as phosphate (Boyle et al. 1977), so that both processes may be operating simultaneously. Toxic and nutritional responses may well act on different time scales which would be of interest in con- siderations of the patchiness of phytoplankton in upwelled water. The relationship between zinc ion activity and cupric ion activity is a compli- cating Factor not dealt with here. This ratio is important because the general mechanism proposed for Cu toxicity is by displacement of the active zinc (Vallee 1959), and under equilibrium conditions the amount of displacement is a function of both Cu and Zn, i.e. the activity of the enzyme is protected by high zinc ion activities in the medium. The activity of the purified bacterial enzyme responds to the Cu2+:Zn2+ ratio; however the enzyme activity in living cells (over the same period of exposure) is only inhibited by Cu2+, and Zn*+ shows no beneficial effect. This may result from the buffering effect of the zinc content of the cell and is under further investigation with zinc-limited cultures. The purified alkaline phosphatase system behaves in its response to Cu2+:Zn2+ like the silicic acid uptake system in T. pseudonana, suggesting that Si uptake may also be mediated by a zincdependent system (Rueter and Morel 1981). The work reported here suggests a simple method for determining cupric ion activity based on the correlation between cupric ion activity measured with an ion selective electrode and the inhibition of activity of added enzyme (Fig. 5b). If the background zinc concentration can be fixed, or assumed to be constant, then the enzyme inhibition can be related to changes in cupric ion activity. This method is easily calibrated with cupric ion buffer systems (Tris and oxalate, for example) and has two advantages for field studies of toxicity: analytical simplicity (only needing timed spectrophotometric measurements) and theoretical consis- tency, that is, a physiologically function is being measured. Department of Biology Portland State University P.O. Box 751 Portland, Oregon important John G. Rueter, Jr.

6 748 Notes References NDERSON,D. M., ND F.M. MOREL Copper sensitivity of Gonyaulux tamarensis. Limnol. Oceanogr. 23: BOYLE,E..,F.R. SCLTER,ND J.M. EDMOND The distribution of dissolved copper in the Pacific. Earth Planet. Sci. Lett. 37: DIXON,M.,ND E.C.WEBB Enzymes.cademic. Fou,C.D., R. L.~HNEY,ND M.C. WHITE The physiology of metal toxicity in plants. nnu. Rev. Plant Physiol. 29: KUENZLER, E. J., ND J. P. PERRS Phosphatases of marine algae. Biol. Bull. 128: LEN, 1). R., ND C. NLEWJKO Phosphate exchange and organic phosphorus excretion 1,~ freshwater algae. J. Fish. Res. Bd. Can. 33: MOREL, F. M., J. G. RUETER, D. M. NDERSON, ND R. R. GUILLRD quil: chemically defined phytoplankton culture medium for trace metal studies. J. Phycol. 15: PERRY, M. J lkaline phosphatase activity in subtropical central North Pacific waters using a sensitive fluorometric method. Mar. Biol. 15: RIVKIN, R. B., ND E. SWIFT Characterization of alkaline phosphatase and organic phosphorus utilization in the oceanic dinoflagellate P!yrocystis noctilucu. Mar. Biol. 61: l-8. RUETER,J.G.,J.J.MCCRTHY,ND E.J. CRPEN- TER The toxic effect of copper on Oscillatoria (Trichodesmium) theihautii. Limnol. Oceanogr. 24: , ND F. M. MOREL The interaction between zinc deficiency and copper toxicity as it affects the silicic acid uptake mechanisms in Thulassiosiru pseudonunu. Limnol. Oceanogr. 26: STKICKLND,J. D.,ND T. R. PRSONS practical handbook of seawater analysis, 2nd ed. Bull. Fish. Res. Bd. Can SUND, W. G., ND R. R. GUILLRD Relationship between cupric ion activity and the toxicity of copper to phytoplankton. J. Mar. Res. 34: TFT, J. L.,W.R. TYLOR,NDJ.J.MCCRTHY Uptake and release of phosphorus by phytoplankton in the Chesapeake Bay estuary, U.S.. Mar. Biol. 33: VLLEE, B. L Metal and enzyme interactions: Correlation of composition, function a!ld structure. Physiol. Rev. 39: 443. WESTLL, J.C.,J.L. ZCHRY,ND F.M. MOREL MINEQL: computer program for the calculation of chemical equilibrium composition of aqueous systems. Mass. Inst. Technol., R. M. Parsons Lab. Tech. Note 18. Submitted: 7 pril 1982 ccepted: 14 October 1982