Transition metal-binding proteins from three Chesapeake Bay fish species.

Three species of Chesapeake Bay fish were collected, and endogenous levels of metal binding protein (MBP) were determined. In addition, the induction of metal-binding proteins by cadmium was studied. Livers from freshly caught fish were extracted and chromatographed on Sephadex G-75 to resolve MBP in the 5 to 20 kdalton range. All species studied exhibit measurable but varied levels of endogenous MBPs in the molecular weight range investigated, mostly as a copper protein complex. Upon induction with cadmium, the total MBP content increased in both catfish (Ictalurus punctatus) and striped bass (Morone saxatilis), with significant amounts of cadmium bound to the protein. In white perch (Morone americana), induction of MBPs with cadmium could not be demonstrated due to the large amount of constitutive Cu-BP present, although significant quantities of cadmium were bound to MBP. Electrophoresis in polyacrylamide gel was used to further identify these MBPs. Electrochemical analysis of the MBPs by polarography indicated that the wave properties of the fish MBPs resemble that of rat metallothionein. In conclusion, these studies indicate that: MBPs are present in estuarine fish from the Chesapeake Bay; concentrations of MBPs and their inducibility by exogenous cadmium vary with species, and fish MBPs may be related to mammalian metallothionein.


Introduction
Striped bass (Morone saxatilis) and white perch (Morone americana) are two species of fish coinhabiting in the Chesapeake Bay area. Considered the noblest of Bay creatures, striped bass have been a sporting as well as gourmet favorite ofmany. However, striped bass spawning population has undergone an alarming reduction in size in the last few years. The annual index of its reproductive success has reached an all time low approaching oblivion. A legislative moratorium on fishing for this species along its migratory route of the Atlantic coast and in the Chesapeake Bay has now been imposed. White perch, on the other hand, appears to thrive well. It is thus important to learn what are the causes for the decline in striped bass population. What selective differences are there between the two Morone species which account for the decline in M. saxatilis and the apparent stability of M. americana?
Several factors have been attributed to the peril of striped bass; among them acid rain, overfishing, and landleached contaminants, such as heavy metals. This study is to examine whether striped bass and other fish species in the same natural environment possess different de-*Departments of Biochemistry and Environmental Health Sciences, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205.
tAuthor to whom correspondence should be addressed.
fense mechanisms against toxicity of metals, particularly transition IB and IIB metals, copper, zinc, and cadmium. In mammals, the inducible metal-binding protein metallothionein has been suggested to have a detoxification function (1). Results to be presented in this report will show that similar, but not identical, metal-binding protein(s) exist in these and other fish species examined. ELUTION VOLUME (ml) V at -5 mV/sec. The mercury drop time was 0.5 sec and the sensitivity setting 20 ,uA/mm. The Brdicka cobalt electrolyte was used without surface-active agent.
The electrolyte was purged with high purity nitrogen for 8 min prior to addition of sample and then for an additional 2 min.

Polyacrylamide Gels of Fish Hepatic Cytosols
Fish heat-treated cytosol samples (5-10 j,L) were run on a polyacrylamide gel electrophoresis system consisting of 5% stacking gel and a 7.5% to 17% gradient gel using a Sturdier vertical slab gel unit (model SE400, Hoefer Scientific Instruments) and a Buchler 3-1500 constant power supply. The chemical polymerization of the polyacrylamide gel was performed by using ammonium persulfate and TEMED as catalyst-redox system. A small amount of glycerol was added to minimize turbulence during gradient pouring. Both stacking and gradient gel contained 0.1% SDS. Before electrophoresis all samples were diluted 1:1 with a denaturing solution containing 4% SDS and 10% 3-mercaptoethanol and heated at 80°C for 2 min to minimize negative charge differences and disrupt disulfide linkages. The running buffer for the electrophoresis also contained 0.1%o SDS. Purified rat liver metallothionein standard used as a marker protein was prepared according to Ohi et al. (4).

Results
Inspection of the Sephadex G-75 profiles for all three species of fish (Figs. [1][2][3] indicates that low molecular weight metal-binding proteins (MBP) are present in fish ,uL); (6) white perch liver cytosol ( Injections of cadmium intramuscularly results in significant quantities of cadmium binding to the MBPs. In the case of the catfish and striped bass, the total MBP content increased following cadmium induction. With the white perch it is not possible to draw a similar conclusion due to the extremely high levels of copper-BP present in the uninduced fish. Only in the case of the striped bass was DEAE-anionexchange chromatography successful (Fig. 4). Insufficient quantities of catfish MBP were available, while white perch gave low quality profiles (smeared peaks) possibly due to the presence of large quantities of copper. For the striped bass, the DEAE results indicated two major cadmium-containing peaks. The possibility of additional minor cadmium-containing peaks cannot be ruled out.
Analysis of the crude MBP preparations obtained from Sephadex G-75 chromatography by differential pulse polarography indicated that the MBPs from all three fish species exhibited characteristics similar to mammalian metallothioneins (Fig. 5). This conclusion is based on the close correspondence of the electrode voltage at the reductive wave peak between rat MT and the fish MBPs (at -1.45 V). When the polarographic response is plotted versus increasing additions of cellular proteins to the reaction vessel (Fig. 6) there is evidence for induction of BPs with cadmium injections for catfish and striped bass. Furthermore, it is apparent that either the white perch : 0 0ĩ f : : : .  Figure 7. Coomassie Brilliant Blue staining indicates that all three fish species, normal as well as cadmium induced, exhibit a band which migrates with the same characteristics as authentic rat MT-I. Significant quantities of this protein are present in normal catfish. Of particular interest is the strongly staining low molecular weight band in the induced striped bass lane. This protein may represent cadmium-induced induction of a new cellular protein. Further studies will be necessary to investigate this possibility.

Discussion
Several species of fish have been previously examined for the presence of metallothionein-related metal-binding proteins in the liver. These results are summarized in Table 1. As shown in the table, proteins with apparent molecular weight from 9 to 15.3 kdalton exists in these fish. Except in plaice, where one of the proteins has been shown to share a conserved mammalian type of metallothionein sequence, the primary structure and function of most metal-binding proteins are yet unclear.
A major finding in this study is the drastic difference between the amount of copper and metal-binding protein in two related species of the same genus of fish. White perch accumulates between 100 and 1000 times more copper in the liver than striped bass. It is thus significant to observe that the corresponding binding protein is at least 10-fold higher in the former.
Several mechanisms can explain this species variation, two of which are particularly attractive: (a) differential affinity for metals due to alteration in the intracellular binding proteins and (b) similar proteins but differential expression due to amplified gene copies. These are test-able explanations and are indeed being examined experimentally.