Coop Eiochem. Physzol. Vol. 89C, No. 1, pp. 53-55, 1988 0306-4492/88 $3.00 + 0.00 Printed in Great Britain 0 1988 Pergamon Journals Ltd

EFFECTS OF TEMPERATURE OSCILLATIONS ON THE DISTRIBUTION OF 65Zn ON

CYTOPLASMIC PROTEINS

BARBARA WATKINS and K. SIMKISS

Department of Pure and Applied Zoology, University of Reading, PO Box 228, Reading RG6 2AJ, UK (Telephone: (0734) 875 123)

(Received 23 January 1987)

Abstract-l. Oscillating temperatures cause an increase in 65Zn uptake into the digestive gland of M. edulis when compared with the effects of constant temperatures.

2. The increased uptake is associated with a change in the distribution of @Zn onto cytoplasmic proteins. There is a fall in the percentage of metal on the >70 K dalton fraction and a rise in the 3 K dalton fraction.

3. A possible hypothesis is produced to explam these effects in terms of heat lability of metal-protein ligands.

INTRODUCTION

Metal ions play essential roles in the physiological and biochemical activities of cells. The strongly ionic and hydrophilic metal ions (H+, Na+, K+, Ca2+, Mg2+) which Ahrland et al. (1958) classified as “group a” form the bulk electrolytes in biological systems. They are often distributed unevenly by means of energy dependent pumps located in lipid membranes so as to produce considerable electro- chemical gradients across these hydrophobic cell membranes. The physiological implications of this have been extensively studied in relation to ion and osmoregulation, energy transduction and neuro sensory phenomena.

In contrast, the less strongly ionic metals which have more covalent bonding (Cu’+, Zn’+, Ni2+, Co2+) are classified as “intermediate or group b” metals and occur in relatively trace amounts in biological systems. Their distribution within cells appears to be dictated by their binding characteristics and by the use of specialised ligand proteins (Willi- ams, 1983). It has been suggested that a series of such ligands could account for the passage of these metals between compartments of the cell and into specific metalloprotein complexes (Williams, 1981).

In a recent study Watkins and Simkiss (1987) investigated how oscillating temperatures affected the uptake of zinc by the mussel Mytilus edulis. They found that variations in temperature induced a greater uptake of metal than occurred under constant conditions and interpreted this as possibly being due to the dissociation/association of a heat sensitive metal ligand. This concept suggests that a warming of the intracellular protein ZnL, causes its dissociation into Zn2+ and Li- stimulating other metal binding systems. Subsequent cooling of the animal would induce zinc ion deficiency as the complex reformed. This would stimulate the release of the metal from other cellular stores. Oscillating temperatures would

therefore favour metal accumulation by the system

Zn L*

warm I I

cold

Zn detoxification - znz++ L:- - Zn stores

This is only one of a number of possible explanations for the oscillating temperature phenomenon that was observed (Watkins and Simkiss, 1987) but it predicts that there should be a change in the distribution of zinc protein complexes in these animals. It was, therefore, decided to investigate this possibility.

MATERIALS AND METHODS

Mussels (Mytilus edulis) were collected from low water sites at Blackpool, Lancashire and Menai Bridge, North Wales. They were kept in circulating sea water and fed on the diatom Phaeodactylum tricornutum during the 7-10 day period of acclimation prior to use. During experimental treatments, animals were kept in small aquaria in constant temperature rooms set at 10°C. 15”C, 20°C and 25°C. Temperatures were oscillated over a 10°C range by placing some of the aquaria in water baths that were programmed so as to be turned on and off every 6 hr. The actual temperatures were monitored by continual recording from thermistor beads in the sea water. The sea water in the aquaria was continually filtered by an air lift pump so as to remove any gametes or faeces that were released into the water.

Measurements of metal fluxes were made by adding 0.5 pmole Zn dm-3 to sea water to which trace amounts of 65Zn (Amersham International) were added. At various time intervals mussels were removed and dissected. The digestive gland was chosen as the most suitable organ for detailed study because of its size and rate of metal metabolism (Watkins and Simkiss, 1987). The glands from four animals were pooled and homogenized in 20 mmol dmm3 Tris-HCl buffer at pH 8.6 using a motor driven pestle. The homogen- ate was centrifuged at lo5 g for 60 min at 4°C and the supematant filtered through 0.22 pm Millipore filters to

53

54 BARBARA WATKINS and K. SIMKISS

remove any particulate matter. Aliquots of 2.0cm3 were applied to a Sephadex G 75 column (2.5 x 75 cm) that had been calibrated with proteins of known molecular wt (Vit- amin B,,, insulin, ribonuclease, chymotrypsinogen A, oval- bumen and albumin). The column was deoxygenated by bubbling nitrogen through all the reagents prior to use and the void volume was identified by the use of Dextran blue. The effluent of the column was monitored with a Uvicord fixed wavelength (280 nm) spectrophotometer (LKB Instru- ments) and 3cm3 samples were collected for subsequent 65Zn analysis usmg an Ultragamma counter (LKB model 1280).

RESULTS

The results from 15 sets of analyses showed that on average 90.2% (range 82.695.0) of the 65Zn in the tissue homogenate could be accounted for in five protein fractions (mol. wts cu. 3, 6-12, 2&30, 3@40 and over 70K daltons). The results are, therefore, presented in relation to these ligands. Table 1 shows the distribution of 65Zn between these proteins when the animals are kept at lO”C, 20°C or oscillated between these temperatures for up to 3 days. In Table 2 similar data are given for lS”C, 25°C and 15-25°C treatments over the same time period.

DISCUSSION

The main zinc-binding protein of vertebrates ap- pears to be metallothionein, a heat stable protein of low molecular wt (610 K daltons) and a high (30%) cysteine content (Cheridan and Goyer, 1978). The situation in the invertebrates is more complex (Roesi- jadi, 1981) and in the molluscs at least three fractions have been isolated. These include a high molecular wt (> 30 K daltons) group that probably includes metal- loenzymes, a second group of proteins similar to the metallothioneins (l&20 K daltons) and possibly in- cluding dimers of that protein (George et al., 1979) and finally a low molecular wt fraction (3 K daltons) of poorly understood composition (Johansson et al., 1986).

The results of this study are in general agreement with those findings. The interpretation of the func- tions of these various groups of proteins is, however, controversial. It has been suggested that as the metal- lothionein protein is exposed to increasing levels of zinc there is spill-over into the high molecular wt fractions, indicating the onset of metal toxicity (Roesijadi, 1980). Alternatively Sanders and Jenkins (1984) found that the onset of metal-induced stress resulted in an increase in the metal content of the low molecular wt proteins. Central to these interpre- tations is the concept that metallothioneins regulate cytoplasmic zinc and copper ion concentrations but in molluscs zinc is not consistently observed in the metallothionein fraction (Viarengo et al., 1982) and there is as yet no clear evidence for the induction of metallothionein synthesis by zinc in molluscs (Simkiss and Mason, 1983).

The experiments on temperature oscillation re- ported in this paper may, therefore, provide a novel probe for studying the intracellular regulation of zinc. In the 1&2O”C experiments it is apparent that by day 3 the oscillating temperatures have resulted in a fall in the zinc content of the high molecular wt fraction (> 70 K daltons) and a rise in the low molecular wt fraction (3 K daltons). This suggests either that the 70 K dalton fraction is relatively heat labile or that it acts as a store for zinc that is subsequently accumu- lated in the 3 K dalton proteins (Table 1). Experi- ments at higher temperatures (15-25°C) show an essentially similar picture except that the effect occurs earlier, by day 1, with a decline in the effect by day 3 (Table 2).

In order to produce a model for subsequent experi- mentation it is suggested that the results are consis- tent with the scheme

Zn Lz (cytoplasmic metalloprotein)

heat I I

COO1

detoxified Zn +-- znz++ L:- - Zn store

(3 K d&on) (70 Kdalton)

Table 1. Distribution of sSZn (%) between the protems of mussel dlgestlve gland of ammals keut at constant 10°C or 20°C or oscdlated between these temoeratures

Apparent molecular wt

(K daltons) >70 3@40

10°C 33.5

59

Time of exposure and treatment

Day 1 Day 3

20°C 1&2O”C 10°C 20°C IO-20°C 28.9 27.1 30.3 25.6 20.3

6.1 57 3.7 7.0 6.9 2&30 8.8 4.8 6.4 11.9 8.9 1.7 612 9.1 12.9 10.1 15.6 8.4 10.2 3 32.0 34.9 33.2 30.0 34.7 50.5

Table 2. Distnbution of “Zn (%) between the proteins of the mussel dlgestlve gland of ammals keut at constant 15°C or 25°C or oscillated between these temoeratures

Apparent molecular wt

(K daltons) >70 3&40 2C-30

612 3

15°C 45.0 12.4 9.9 34

18.6

Time of exposure and treatment

Day 1 Day 3

25°C I5-25°C 15°C 25°C 15-25°C 48.2 28.3 57 7 49.8 49.1

7.7 3.8 7.9 4.7 8.0 4.5 3.8 3.3 2.3 78 3.2 4.0 38 3.9 5.0

28.8 54.2 21.6 30.3 25. I

Zinc uptake 55

This model is consistent with our present data but we would emphasize first that more extensive data are necessary to test if the designated proteins do have these functions and second that a number of other interpretations are clearly possible. In particular it is known that temperature shocks can induce heat shock proteins which interact with metal proteins, probably via protein-SH effects (Craig, 1985; Lanks, 1986). Oscillating temperatures also produce a change in the distribution of 65Zn between the cell organelles and this is clearly also relevant to these interpretations (Watkins and Simkiss, 1987). Despite the diversity of explanations that are possible, how- ever, it is clear that temperature effects have been underutilized in devising probes to study intracellular metal metabolism in ~ivo.

Acknowledgemenls-This work was supported by a research grant GR3/5723 from the Natural Environment Research Council, UK.

REFERENCES

Ahrland S., Chatt J. and Davies N. R. (1958) The relative affimties of ligand atoms for acceptor molecules and ions. Quart. Rev. Chem. Sot. 12, 256276.

Cherian M. G. and Goyer R. A. (1978) Metallothioneins and their role in metabolism and toxicity of metals. Life Sci. 23, l-10.

Viarengo A., Pertica M., Manicelli G., Palmer0 S., Zanicchi G. and Orunesu M. (1982) Evaluation of general and specific stress indices in mussels collected from popu- lations subjected to different levels of heavy metal pollu- tion. Mar. Environ. Res. 6, 235-243.

Watkins B. and Simkiss K. (1987) The effect of oscillating temperatures on the metal ion metabolism of Myrilus edulis. J. mar. biol. Ass., UK (submitted for publication).

Wilhams R. J. P. (1981) Physico-chemical aspects of inor- ganic element transfer through membranes. Phil. Trans. Royal Sot. 294B, 57-74.

Craig E. A. (1985) The heat shock response. CRC Crit. Rev. Williams R. J. P. (1983) Inorganic elements in biological Biochem. 18, 239-280. space and time. Pure App. Chem. 55, 1089-1100.

George S. G., Carpene E., Coombs T. L., Ovemell J. and Youngson A. (1979) Characterization of cadmium- binding proteins from mussels Mytilus edulis (L) exposed to cadmium. Biochem. Biophys. Acta 580, 225-233.

Johansson C., Cain D. J. and Luoma S. N. (1986) Varia- bility in the fractionation of Cu, Ag and Zn among cytosolic proteins in the bivalve Macoma balthica. Mar. Ecol. Prog. Ser. 28, 87-97.

Lanks K. W. (1986) Modulators of the eukaryotic heat shock protems. Exp. Cell. Res. 165, l-10.

Roesiiadi G. (1981) The sienificance of low molecular . , weight metallothionein-like proteins in marine inverte- brates: current status. Mar. Environ. Res. 4, 167-179.

Sanders B. M. and Jenkins K. D. (1984) Relationships between free cupric ion concentrations in sea water and copper metabolism and growth in crab larvae. Biol. Bull. mar. biol. lab. Wood’s Hole 167, 704712.

Simktss K. and Mason A. Z. (1983) Metal tons: metabolic and toxic effects. In The MoNusca Vol. 2 (Edited by Hochachka P. W.), pp. 101-164. Academic Press, New York.