6 Application of Radioisotopes in Biochemical Analyses : Metal Binding Proteins and Metal Transporters

Radioisotopes (RI) such as 3H, 14C, 32P, and 45Ca are excellent tools in biological research. Most RI are used as tracers in studies of primary and secondary metabolism, drug metabolism, transcription, translation, post-translational modifications such as protein phosphorylation, association of proteins with metals, and transport of metals across biomembranes. Furthermore, some experiments have used neutrons for mutagenesis of microorganisms, animals, and plants. Recent progress in the biological sciences has resulted in novel probes and labeling reagents, which has decreased the need for RI. Experiments with RI require experimental space specialized for RI, careful experimental procedures, and training. Although these are disadvantages, RI are still useful and powerful tools with high resolution compared with non-RI methods. Here, we describe the advantages of RI in biochemical assays, and detailed experimental procedures of metal-binding assays and membrane transport measurements of metal cations, especially calcium and zinc.


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methionine is added into in vitro translation mixtures that lack cold methionine. In certain experiments, 35 S is used for sulfation of peptides or proteins through tyrosine modification by an enzyme tyrosylprotein sulfotransferase (Moore, 2003;Hoffhines et al., 2006). 32 P is used for monitoring protein phosphorylation of serine and/or threonine residues.

Quantification of radioisotopes and measurement of pollution
Several methods have been developed for quantifying RI and measuring pollution with RI during experiments. Here, we introduce two instruments, a Geiger-Müller (GM) survey meter (also known as a GM counter) and a liquid scintillation counter. A GM survey meter is usually used to monitor and -rays, but not neutrons, from the surface of substances. Two electrodes are set in a cylinder filled with inert gas, such as neon, helium, or argon. A voltage is applied to the electrodes, i.e., the anode (a central wire or needle) and the cathode (the inside surface of the cylinder). GM survey meters operate under a high voltage of more than several hundred volts. When the ionizing radiation passes through the cylinder, ions and electrons are generated from some of the gas molecules. This reaction generates an electrical current pulse of constant voltage. GM survey meters are usually used for monitoring the surface pollution of RI that radiate or -rays. The meter cannot count rays efficiently and does not distinguish each isotope generating -rays.
As an efficient and practical means of quantifying -ray radiation, liquid scintillation counters are commonly used for biochemical analyses. A liquid scintillation counter measures -radiation in a solution containing a RI, fluorescent compounds (scintillators), and organic solvents such as xylene, dioxane, or toluene. As a scintillator, 2,5diphenyloxazole (DPO) and 1,4-bis(5-phenyl-2-oxazolyl)benzene (POPOP) are used. The energy of -rays ( particles) from RI excites the scintillator, and then the excited fluorescent molecules dissipate the energy by emitting fluorescence. Therefore, radiation of  particles causes a pulse of fluorescent light. Liquid scintillation counters are used for measuring ray-emitting RI including 3 H, 14 C, 32 P, 45 Ca, and 65 Zn because the counting efficiency is high even for nuclides emitting low energy -rays.

Analyses of Ca-binding proteins with 45 Ca
There are many types of Ca-binding proteins, such as calmodulin, calreticulin, and annexin (Berridg et al., 2003). Identification and quantitative characterization of Ca-binding proteins provide key information about their biochemical roles in living cells. Here, we briefly introduce biochemical methods to identify and characterize these proteins.

Identification of Ca-binding proteins
Staining of SDS-PAGE with Stains-all (commercially available from reagent companies such a s S i g m a A l d r i c h ) i s a c o n v e n t i o n a l n o n -R I m e t h o d t o i d e n t i f y C a -b i n d i n g p r o t e i n s (Campbell et al., 1983). Stains-all is a metachromatic cationic carbocyanine dye that tends to bind acidic proteins. Ca-binding proteins in particular are stained blue relatively stably (Yuasa & Maeshima, 2000). Therefore, this is a useful method to detect candidates for Cabinding proteins in crude samples prepared from organisms. Radioisotope 45 Ca is necessary to confirm that the protein(s) of interest can bind calcium. The 45 Ca overlay assay is one convenient method (Campbell et al., 1983;Yuasa & Maeshima, 2002;Ide et al., 2007;Kato et al., 2010). If a purified preparation is available, aliquots of the purified sample are spotted onto a membrane filter such as a PVDF membrane (ca. 30  40 mm). Then the membrane is incubated in a small volume (1 mL) of medium containing 45 Ca as CaCl 2 , 5 mM MgCl 2 , 60 mM KCl, and 10 mM Mes-KOH, pH 6.5, for 30 min at 25 to 30C, and then washed with 10 mL of 50% (v/v) ethanol to remove unbound 45 Ca ( Figure 1) (Nagasaki et al., 2008). The membrane is dried in air at room temperature. MgCl 2 and KCl are added into the reaction medium to mimic physiological conditions. An autoradiogram of the 45 Ca 2+ -labelled proteins on the membrane can be obtained by exposure to an X-ray film for 3 days at 80C. If the purified protein(s) is not available, proteins separated by SDS-PAGE are transferred onto a transfer membrane such as PVDF as is usually used for immunoblotting ( Figure 1). The Ca-binding protein(s) can be detected by the same method as the 45 Ca overlay assay mentioned above.
Purified proteins are spotted on a PVDF membrane (left, upper panel). In another method, a protein fraction that contains the Ca-binding protein is subjected to SDS-PAGE and then transferred to a PVDF membrane (left, lower panel). The membrane is incubated with buffer containing 45 Ca 2+ , rinsed, and then dried in air. By autoradiography, the 45 Ca 2+ -binding capacity is monitored (right, upper panel). From the membrane blotted after SDS-PAGE, the Ca-binding protein(s) is identified by autoradiography (right, lower panel).

Characterization of kinetic properties of Ca-binding proteins
The dissociation constant (K d ) for Ca 2+ and the calcium-binding number are important kinetic parameters for understanding these proteins. Several assay methods can be used to measure the Ca-binding kinetics of Ca-binding proteins. For example, there is equilibrium dialysis, flow dialysis, membrane microassay, and spectrophotometry. The former two methods are carried out using 45 Ca 2+ . Most methods require a relatively large amount of the purified Ca-binding protein.
Here, we introduce a special equilibrium dialysis using small dialysis buttons ( Figure 2A). A small well of dialysis button is filled with the Ca-binding protein(s) and is sealed with a dialysis membrane. The protein solution in the well is dialyzed against 40 mL of buffer containing 45 Ca 2+ at different concentrations. The Cabinding protein binds the 45 Ca 2+ entered into the well. After dialysis for 16 hr at 25C, the protein solution in the well of each dialysis button is collected with a needle and syringe. An aliquot of the solution is spotted on a nitrocellulose membrane (13 mm in diameter), and then the membranes are dried in air. Total radioactivity associated with the filter membrane is measured with a liquid scintillation counter. Unbound Ca 2+ i s m e a s u r e d f r o m t h e radioactivity of the external solution. The amount of Ca 2+ bound to the Ca-binding protein increases in proportion to the concentration of Ca 2+ as shown in Figure 2B.

Measurement of membrane transport of zinc and calcium
Radioactive elements such as Ca 2+ and Zn 2+ are commonly used in ion transport experiments because they provide direct evidence and quantitative information. Here, we introduce a Zn 2+ transporter and a Ca 2+ transporter, which work as metal/proton exchangers.

Determination of kinetic parameters of a Zn transporter across biomembranes
Transporters implicated in Zn transport include members of the metal tolerance protein (MTP), ZRT1/IRT1-like protein (ZIP) (also known as zinc-iron permease), and heavy-metal ATPase (HMA) (P 1B subgroup of P-type ATPase) families (Krämer et al., 2007). Here, we introduce an assay procedure for an MTP-type zinc transporter that works as a Zn 2+ /H + exchanger. Arabidopsis thaliana MTP1 is localized in the vacuolar membrane, which has two types of proton pumps, vacuolar H + -ATPase (V-ATPase) and H + -pyrophosphatase (Enrico et al., 2007). AtMTP1 actively transports excessive zinc in the cytoplasm into vacuoles (Kawachi et al., 2008;Kawachi et al., 2010). The assay procedure of AtMTP1 expressed in Saccharomyces cerevisiae cells is shown in Figure 3. In this case, an S. cerevisiae mutant that lacks endogenous zinc transporters COT1 and ZRC1 is used as a host cell for heterologous expression. When ATP is added into the vacuolar membrane vesicle suspension, a pH gradient (pH) is formed across the membrane by yeast endogenous V-ATPase. Then radioactive 65 Zn 2+ is added into the reaction mixture as ZnCl 2 . Under these conditions, AtMTP1 actively incorporates 65 Zn 2+ into membrane vesicles using a pH ( Figure 3A). The reaction medium contains 300 mM sorbitol, 5 mM MES-Tris pH 6.9, 25 mM KCl, 1 mM dithiothreitol, 5 mM MgCl 2 , 0.2 mM NaN 3 , 0.1 mM Na 3 VO 4 , and 3 mM ATP-Tris. The uptake reaction is started by adding 5 M 65 ZnCl 2 . Vacuolar membranes from plants and yeast contain metal-translocating ATPases that have the ability to transfer Zn 2+ into vacuoles. Therefore, the activities of these ATPases  (Mao et al., 2007). This method is applicable to assay the zinc transport activity of vacuolar membrane vesicles from plant tissues. Vacuolar membrane vesicles can be prepared from plant tissues such as mung bean hypocotyls by conventional differential centrifugation (Maeshima and Yoshida, 1989).
(A) Vacuolar membrane vesicles prepared from yeast cells or plant tissues are activated by adding ATP into the suspension. Vacuolar H + -ATPase (V-ATPase) acidifies membrane vesicles and generates a pH gradient across the membrane.Bafilomycin A1 is a potent inhibitor of V-ATPase and used to assess the V-ATPase-dependent (pH-dependent) activity of theZn 2+ /H + exchanger. When radioactive 65 Zn 2+ is added, membrane vesicles actively uptake 65 Zn 2+ using a pH gradient in a Zn 2+ /H + exchangerdependent manner. (B) Membrane vesicles are filtrated and washed with the buffer. (C) The radioactivity of 65 Zn 2+ membrane vesicles trapped on the membrane filter is determined by a scintillation counter.

Fig. 3. Assay of Zn 2+ transport into membrane vesicles through a Zn 2+ /H + exchanger
The catalytic domain of V-ATPase (V 1 sector) is exposed to the cytoplasm. Therefore, only right-side-out membrane vesicles, in which the V 1 sector faces to the reaction mixture, can be energized by V-ATPase. Approximately half of the membrane vesicles are right-side-out when plant tissues and yeast cells are homogenized. In the remaining half, the V 1 sector faces the vesicle lumen and cannot utilize ATP. The inside-out vesicles have no ability to uptake 65 Zn 2+ or to export zinc under this assay condition. Therefore, this experiment determines the Zn 2+ uptake activity of the right-side-out membrane vesicles. The inside-out membrane vesicles do not interfere with the Zn 2+ transport of right-side-out vesicles.
After an adequate period of uptake reaction, incorporated 65 Zn 2+ must be separated from the un-incorporated ions. Aliquots (for example, 100 L) of the membrane vesicle suspension are transferred to funnels with nitrocellulose membrane filters that are presoaked with the buffer at appropriate intervals. Filter units with a 0.45-m nitrocellulose membrane of 13 mm in diameter are easy to use for assays of multiple samples. The filter units are washed with 1.5 mL of cold wash buffer without 65 Zn 2+ . The wash buffer contains 300 mM sorbitol, 5 mM MES-Tris pH 6.9, 25 mM KCl, and 0.1 mM ZnCl 2 . The addition of cold ZnCl 2 is essential to remove 65 Zn 2+ from the surface of the membrane vesicles thoroughly. Finally, the radioactivity of 65 Zn 2+ is determined by a  scintillation counter. When measuring the zinc transport activity of plant vacuolar membranes, vacuolar H +pyrophosphatase (V-PPase) also works as a useful proton pump (Maeshima, 2001). V-PPase hydrolyzes pyrophosphate (diphosphate) instead of ATP as a substrate. Therefore, metaltranslocating ATPases do not work in the assay medium when assayed with V-PPase.
To demonstrate the active translocation of Zn 2+ by exogenous zinc transporters, the membrane sample from the yeast mutant with a vacant vector is assayed as a control. Zinc ionophore pyrithione is usually used to collapse the concentration gradient of Zn 2+ across the membrane at a concentration of 5 M (MacDiarmid et al., 2002). If Zn 2+ is actively incorporated into the membrane vesicles, the addition of pyrithione releases Zn 2+ from membrane vesicles as shown in Figure 4A. Also, uptake experiments without ATP or with 0.2 M bafilomycin A 1 is done by the same protocol. The bafilomycin A 1 -sensitive zinc uptake activities are plotted as V-ATPasedependent zinc uptake in time-course or substrate saturation analysis.
The vacuolar type Zn 2+ /H + exchanger of Arabidopsis thaliana (AtMTP1) was heterologously expressed in a yeast (Saccharomyces cerevisiae) mutant that lacks endogenous vacuolar membrane zinc transporters (zrc1 cot1 mutant). The vacuolar membrane-enriched fraction was prepared from yeast cells expressing AtMTP1 (circles) or vacant vector (closed squares) and assayed for zinc uptake activity. (A) Membrane vesicles were pre-incubated in uptake medium (1.0 mL) containing 3 mM ATP for 10 min at 25C to generate a pH gradient across the membrane as shown in Figure 3A. The same reaction media supplemented with 0.2 mM bafilomycin A1 were also prepared and assayed to measure bafilomycin A1sensitive zinc activity. The reaction was started by the addition of 5 M 65 ZnCl2 at time 0 and continued for the indicated period. Aliquots (100 L) of the reaction suspensions were filtered though a nitrocellulose membrane and washed with 1.5 mL of cold wash buffer. The radioactivity of 65 Zn 2+ in the membrane vesicles was determined. The bafilomycin A1-sensitive zinc uptake activities are plotted as V-ATPase-dependent zinc uptake. A zinc ionophore pyrithione is added into the reaction medium to make a final concentration of 5 M to confirm the active transport of zinc. (B) Zinc uptake activity is measured at the indicated concentration of 65 ZnCl2 and shown as a substrate-saturation curve.

Determination of kinetic parameters of Ca transporters across biomembranes
The Ca 2+ -ATPase (calcium pump) belongs to the P-type ATPase family that includes the Na + ,K + -ATPase (Morth et al., 2011), and actively translocates Ca 2+ across the membrane coupled with ATP hydrolysis. Ca 2+ -ATPases in eukaryotes are divided into to the ER-type and calmodulin-activated plasma-membrane-type Ca 2+ -ATPases. The Ca 2+ -ATPase is localized in the plasma membrane, ER, Golgi apparatus, and vacuole, and maintains calcium homeostasis in the cytoplasm and lumen spaces. The Ca 2+ /H + exchanger is the other type of active Ca 2+ transporter (Ueoka-Nakanishi et al., 2000;Kamiya & Maeshima, 2004). As an energy source, the exchangers use a pH gradient across the membrane that is generated by proton pumps. Plant and fungal cells have the Ptype H + -ATPase in their plasma membranes, and H + -ATPase in their vacuolar membranes as primary proton pumps. In plants, an additional proton pump, H + -pyrophosphatase (V-PPase), functions as an efficient proton pump (Martinoia et al., 2007 Background values resulting from incubations without ATP or Na 2 PPi are subtracted from the corresponding values in the presence of ATP or Na 2 PPi. Bafilomycin A 1 and carbonyl cyanide m-chlorophenylhydrazone (CCCP) dissolved in dimethyl sulfoxide (DMSO) are used to inhibit V-ATPase and collapse the pH gradient, respectively. The DMSO concentration in the assay medium should be less than 1% (by volume) to avoid artificial effects of the solvent. Calcium ionophore A23187 is also dissolved in DMSO and used to confirm the active transport of Ca 2+ through Ca 2+ -ATPase or Ca 2+ /H + exchanger. A23187 is a mobile Ca 2+ carrier produced by Streptomyces chartreusensis as an antibiotic. Figure 5 shows typical substrate-saturation curves of the Ca 2+ -ATPase and Ca 2+ /H + exchanger of vacuolar membranes prepared from mung bean hypocotyls (Ueoka-Nakanishi et al., 1999). Experiments with radioisotope 45 Ca provide quantitative information of their transport kinetics. Ca 2+ -ATPase is recognized as a high-affinity, low-capacity transporter, while the Ca 2+ /H + exchanger is low-affinity, high capacity. These two active transporters maintain calcium homeostasis in the cytoplasm through their characteristic properties.
Activity of Ca 2+ -ATPase (circles) in the vacuolar membrane was determined in the presence of 1 mM ATP, 0.1 mM bafilomycin A1, and indicated concentrations of 45 CaCl2. Ca 2+ /H + exchanger activity (squares) was determined after pre-incubation with 1 mM NaPPi for 3 min. Vmax values of Ca 2+ -ATPase and the Ca 2+ /H + exchanger were 6.9 and 21 nmol min -1 mg -1 of protein, respectively. Apparent Km values of Ca 2+ -ATPase and the Ca 2+ /H + exchanger were 2.6 and 25 M, respectively.

Conclusion
High sensitivity and accuracy in quantitative assay are the actual merits of RI. Information concerning kinetic parameters of ion transporters cannot be obtained without RI, such as 65 Zn and 45 Ca, as described for Zn 2+ /H + exchanger and Ca 2+ /H + exchanger, and Ca 2+ -ATPase. The obtained values of K m and V max are fundamental to evaluate physiological importance of individual transporters quantitatively. The data presented here are typical examples, which show advantages of RI in biochemical analyses. Although the use of RI is regulated by the laws established in each country, university and research institute, these rules keep the safety for the users and people. RI in biochemistry is one of the peaceful use of atomic energy and will be utilized as an essential tool to develop our scientific knowledge.