An enzymatic method for radiolabeling vertebrate vitellogenin

ANALYTICAL

BIOCHEMISTRY

14&372-379

(1984)

An Enzymatic Method for Radiolabeling LEE OPRESKO’

AND H. STEVEN

Vertebrate

Vitellogenin

WILEY

Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84132 Received January 19, 1984 Phosphoprotein kinases from Xenopus and chicken liver have been purified and these enzymes have been used to label Xenopus vitellogenin, a phosphoprotein, to high specific activity with [y-32P]ATP. The enzymes were isolated by (NH&SO4 fractionation followed by chromatography on DE-52 cellulose and phosphocellulose. This procedure resulted in greater than 20,000-fold enrichment for the enzymes. Roth enzyme preparations were used to selectively label vitellogenin in the serum of estrogen-treated animals. Thus, isolation of the vitellogenin prior to radiolabeling was not necessary. The [32P]vitellogenin labeled in situ was incorporated by oocytes at a rate similar to [‘*P]vitellogenin labeled in vivo, was translocated to the yolk platelets, and was correctly processed into the yolk proteins. KEY WORDS: protein kinase; enzyme purification.

The oocytes of nonmammalian vertebrates are excellent model systems in which to study ligand binding and internalization. In the latter stages of their development these cells specifically incorporate enormous quantities of the yolk precursor protein, vitellogenin [up to 500 ng * oocyte- ’ - h- ’ in the case of Xenopus laevis oocytes (l)]. Once internalized, vitellogenin ( VTG)2 undergoes a proteolytic cleavage which yields the yolk proteins (2). These proteins are stored in these cells until utilized during embryogenesis. Because the intemalized hgand is not degraded in oocytes, it is possible to study its intracellular compartmentation (3) as well as its binding kinetics. A deterrent to these types of studies has been the lack of a suitable procedure for labeling VTG to relatively high specific activity. Typical in vitro procedures employed for other proteins, such as reductive methylation or iodination, have proven impractical for VTG (4). In the former case, ‘H-labeled VTG is unable to bind to the VTG receptor, while ’ To whom correspondence should be addressed. * Abbreviations used: VTG, vitellogenin; SDS, sodium dodecyl sulfate. 0003-2697184

$3.00

Copyright Q 1984 by Academic Fwss, Inc. All rights of reproduction in any form reserved.

372

12%labeled VTG appears to be deiodinated after internalization. Although it is possible to label VTG in the animal using injections of radiolabeled precursors, it is difficult to obtain high-specific-activity preparations. The use of radioisotopes in hepatic tissue culture has been tried on occasion, but this complex procedure yields only limited amounts of protein. Since VTG is a normally highly phosphorylated protein (5), we decided to develop a procedure to label the molecule with [T-~~P]ATP. Phosphate transfer was accomplished initially by a phosphoprotein kinase isolated from X. laevis liver and later by the use of a similar protein from chicken liver. Use of either enzyme in conjunction with highspecific-activity [T-~~P]ATP resulted in VTG preparations having specific activities of 60280 rCi/mg, depending upon the amount of protein and radiolabeled ATP used. Importantly, we found that enzymatically labeled [32P]VTG was incorporated by cells at a rate similar to that of [32P]VTG labeled in vivo and, subsequent to internalization, was translocated to the yolk platelets and correctly processed into the yolk proteins.

VITELLOGENIN

MATERIALS

AND METHODS

The care and maintenance of X. luevis was as previously described (6). Livers were obtained from both estrogen-treated (l-4 mg /3estradiol) and normal females and males that were exsanguinated prior to liver removal. The excised organs were rinsed in saline solution O-R2 (7) containing 0.07 M sodium citrate prior to storage at -20°C. Chicken livers were obtained from Pel-Freeze, while beef liver was provided by a local slaughterhouse; both were stored at -20°C. Isotopes were obtained from either Amersham (Na’251) or New England Nuclear ([T-~~P]ATP and [32P]orthophosphoric acid). Most chemicals were from Sigma Chemical Company with the exception of ultrapure (NH4)2S04 from Schwarz/Mann. Vitellogenin was either isolated from the plasma of estrogen-treated animals (8) or labeled directly, using serum from estrogenized animals (9) without prior isolation. Phosvitin was isolated from amphibian ovaries through the (NHJ2S04 step (10). Protein was determined by the method of Bradford (11) with bovine y-globulin as a standard. Assay of phosphoprotein kinase activity. Samples were assayed in microtiter plates (with covers) in a total reaction volume of 100 ~1. The reaction mixture was 50 mM Tris/HCl (pH 8), 5 mM MgC12, 50 mM KCl, 5-10 PM ATP, and contained 2.5-5 mg/ml VTG. The reaction was initiated by the addition of 10 ~1 of sample and then incubated at 20°C. Under these conditions the reaction was linear for 2 h and the rate was directly proportional to the enzyme concentration. For rate measurements 20-~1 aliquots of the reaction mixture were spotted at intervals on Whatman 3MM filter paper disks which were then placed in 10% (w/v) trichloroacetic acid containing 5 mM sodium pyrophosphate at 4°C. After processing ( 12) the disks were placed in 10 ml 0.6% 2,5diphenyloxazole in toluene and counted in a scintillation counter. Protein kinase isolation. Frozen livers (50100 g) were broken into small pieces with a mallet and homogenized in a Waring blender

RADIOLABELING

373

(prechilled to -20°C) with buffer (250-500 ml) containing 100 mM sucrose, 50 mM KCl, 2 mM EDTA, 100 mM Tris/HCl (pH 8). After blending for 3 min the homogenate was centrifuged for 25 min at 10,OOOg. The resulting supematant (minus the lipid cap) was slowly brought to 1.2 M (chicken) or 1.6 M (X. laevis and bovine) (NHJ2S04 at 0°C with stirring. After a 1-h equilibration the solution was centrifuged at 12,000g for 30 min and the pellet discarded. The supematant was raised to an (NH4)2S04 concentration of 2.8 M and, after equilibrating for 1 h, centrifuged at 12,000g for 30 min. The resulting pellet was dissolved in 100 ml of 50 mM Tris/HCl (pH 7.5) and dialyzed against two changes (3 liters each) of 50 mM Tris/HCl (pH 7.5). After dialysis the sample was clarified by centrifugation and then pumped, at a rate of 300 ml * h-‘, onto a 5 X 45-cm column of DE-52 or DEAEcellulose equilibrated with 50 mM Tris/HCl (pH 7.5). The protein kinase was eluted with a 1500-ml, O-O.6 M KC1 gradient in 50 mM Tris/HCl. The active fractions were then pooled a ad adjusted to a concentration of 0.2 M KC1 and pumped at a rate of 25-35 ml. h-’ onto a 1.2 X IO-cm column of phosphocellulose equilibrated with 0.2 M KCl, 50 mM Tris/HCl (pH 7.5). After loading, the protein kinase was eluted with a 150-ml, O-O.5 M potassium phosphate gradient in 0.2 M KCl, 50 mM Tris/HCl (pH 7.5). The active fractions were then pooled and concentrated to a 2-ml volume using an Amicon ultrafiltration cell with a PM- 10 filter. After dialysis against 50 mM Tris/HCl, pH 7.5, the material was stored at -20”. Labeling procedures. Vitellogenin was labeled in vivo by injection of 1 mCi [32P]orthophosphoric acid into a male Xenopus 1 week after administration of 1.5 mg &estradiol. The animal was bled by heart puncture 24 h later and the serum dialyzed against solution O-R2 and stored at 0°C. The concentration of VTG in the serum was determined by the method of Wallace et al. (9). To label VTG in vitro, an aliquot of serum containing 3-10 mg VTG was placed in a

374

OPRESKO

AND WILEY

reaction mixture containing 25-75 ~1 concentrated protein kinase, OS-l.0 mCi [y32P]ATP, 50 mM Tris/HCl (pH 8.0), 50 IYIM KCl, and 5 tYIM MgC12. The reaction was terminated after 90-120 min at 20°C either by precipitation of VTG (8) or by dialyzing the entire mixture against 2000 vol of O-R2. ‘251Labeled VTG was prepared using VTG-containing male serum, 1.0 mCi Na12’I and iodogen (13). The 12Uabeled serum was dialyzed against solution O-R2. Oocyte culture and gel electrophoresis. Oocytes (0.90-0.98 mm in diameter) were manually dissected under sterile conditions from their surrounding follicles using watchmaker forceps. They were incubated in solution O-R2 containing 2.5 mg/ml VTG labeled in vivo or in vitro for 24 h at 20°C and then washed in solution O-R2 prior to transfer to solution O-R2 containing 2.5 mg/ml unlabeled VTG. After 20 h they were removed and placed in ice-cold acetone for 18 h, rinsed in one change of acetone, and then dried. The oocytes were boiled, in groups of 12, in 2% SDS, 10 mM dithiothreitol, and then alkylated with iodoacetimide ( 14). Samples containing 50- 100 pg of protein were placed on 5- 10% gradient polyacrylamide slab gels as described (3). After electrophoresis the gels were fixed,

stained, and dried and then exposed for autoradiography using a Dupont Cronex Lightning-Plus intensification screen and Kodak XAR-5 X-ray film at -70°C. RESULTS

We initially attempted to phosphorylate VTG in vitro using commercially available CAMP-dependent protein kinase. When these enzymes yielded low-specific-activity preparations we sought to isolate a more suitable protein kinase from vertebrate liver. For such an enzyme to be useful it had to be relatively easy to purify, have no proteolytic activity, have a low K, for ATP, be active at 20°C and have a good specificity for phosphoproteins. The enzymes isolated from both X. laevis and chicken liver met all of these criteria. The purification procedure consists of (NH,)2S04 fractionation of liver postmitochrondrial supematants followed by chromatography on DE-52 or DEAE-cellulose and phosphocellulose. This protocol was first applied to Xenopus liver and subsequently found to be applicable to tissue from other vertebrates. A summary of the isolation characteristics of AT.laevis and chicken phosphoprotein kinase is shown in Table 1. Both prep

TABLE CHARACTERISTICS

OF F%OSPHOPROTEIN

KINASE

I

PREPARATIONS

FROM

X. laevis AND CHICKEN

X. Iaevis

LIVER

Chicken

Active (NH&SO, fraction Ease of handling (NH&SO4 precipitates

1.6-2.8 M 1.6 M Pellet partiatty floats; care must be taken in removal of supematant.

1.2-2.8 M All pellets are at bottom of tube.

Stability of (NH&SO,-active fraction

Relatively stable at 4°C for 48 h.

Relatively unstable; place on DE-52 or DEAEcelhtlose within 24 h.

DE-52 elution position

0.16-0.19 M KC1 and 0.34-0.38 M KC1

0.26-0.34 M KCI

Phosphocellulose elution

0.14-0.18 M K phosphate

0.09-O. 13 M K phosphate

K,,, for ATP

2.2 PM

3.0 PM

Specificity for VTG (in serum)

Excellent

Very good, a few minor, non-VTG proteins arc also labeled.

VITELLOGENIN

375

RADIOLABELING

arations are quite similar, with a few minor exceptions. The initial (NH&SO4 pellet derived from Xenopus liver is difficult to handle since it floats, and care must be taken to remove the liquid (subnatant) free of lipid contamination. The enzymatic activity in the 1.22.8 M (NH&SO4 pellet from chicken liver is not stable and the sample should only be dialyzed overnight. The X. luevis enzyme is more stable and displays a gradual decline in phosphoprotein kinase activity over a period of days. Finally, two distinct peaks of activity are found during DE-52 or DEAE-cellulose chromatography of X. luevis preparations (compared to only one for chicken liver). The peak eluting at the lower KC1 concentration increased when livers from estrogen-treated animals were used. However, it appears that the majority of this material does not adhere to the phosphocellulose column used in subsequent purification steps. The final protein kinase preparations do not appear to be homogeneous (as judged by gel electrophoresis) but do represent a 20,000fold purification. A summary of the purification of the X. laevis protein kinase is presented in Table 2. Although not homogeneous the enzyme preparations do lack protease activity. As shown in Fig. 1, when VTG-containing serum was incubated in various protein kinase preparations and [Y-~~P]ATP for 90 min at 22°C the labeled molecule appeared as undegraded as [32P]VTG labeled in vivo. Since the protein kinase preparations dem-

onstrated a specificity for phosphoproteins it seemed likely that they should utilize vitellogenins from other vertebrates as substrates. This supposition was tested by comparing the ability of the Xenopus protein kinase to label equal concentrations of Xenopus and chicken VTG. Chicken VTG was isolated from normal chicken plasma by DEAE-cellulose chromatography (8). The VTG was concentrated by vacuum dialysis (15) and the protein concentration determined by uv absorbance at 280 nm using a specific absorbance of 0.75 (5). One milligram of chicken and Xenopus VTG was labeled using 10 ~1 of protein kinase and 50 &i of [T-~~P]ATP for 90 min at 22°C. The specific activities of the Xenopus and chicken VTG were 3.48 X 10’ and 3.14 X 10’ cpm/mg, respectively. Thus the protein kinase was able to effect labeling of chicken VTG to almost the same extent as it did for Xenopus VTG, demonstrating the general usefulness of the enzyme for labeling VTG from different vertebrate sources. Both the Xenopus and the chicken liver enzyme(s) displayed low K,,,‘s for ATP, 2.2 and 3.0 pm, respectively, while the bovine enzyme had a K,,, of - 100 pm. A low K,,, for ATP is a necessity for a suitable protein kinase since commercial preparations of ATP with high specific activities yield exceeding low concentrations when added to the final reaction mixture. The high Km for ATP displayed by the bovine enzyme and poor specificity for phosphoproteins together with a relatively low en-

TABLE 2 PURIFICATION

Fraction Initial supematant 1.6-2.8

Volume (ml) 425

OF PHOSPHOPROTEIN

Protein (mg) 1318

Total activity (units)

KINASE

FROM X.

laevis LIVER

Specific activity (units mg protein-‘)

1700

1.27

Purification factor

Percentage recovery

-

loo

M (NH&SO,

precipitate after dialysis DEAE-cellulose eluate Phosphocellulose eluate

288 988 32

720 32.6 0.103

1300 7650 2790

1.82 235 27,100

1.43 185 21,300

76 450 164

376

OPRESKO I.

2.

X. loevis ----

X.laevis

3. --In viva

4.

5.

Chicken

1251

-

VTG

-

ALBUMIN

FIG. 1. A comparison of different techniques for labeling vitellogenin in serum. Samples of VTG-containing serum were radiolabeled with [‘*P]orthophosphoric acid in vivo (lane 3) or labeled in vifro with iodine (lane 5) or through the action of protein kinase preparations from Xenopus (lanes 1 and 2) or chicken liver (lane 4). The Xenopus protein kinases were obtained from either estrogen-treated (lane 1) or normal animals (lane 2). Aliquots of the labeled seracontaining equal amounts of radioactivity were applied to 5-10% gradient polyacrylamide gels containing SDS. Following electrophoresis, the dried gel was exposed for alltoradiography. The markers on the right indicate the tion position of vitellogenin, 190,000 Da, and Xenad albumin, -70,000 Da.

zymatic activity at 20°C made it unsuitable for VTG labeling. In contrast the Xenopus enzyme preparation was quite specific for polyphosphorylated proteins, as shown in Table 3. Both the Xenopus and chicken enzyme(s) were able to selectively label VTG in whole serum, as seen in Fig. 1. While neither enzyme was as specific as in vivo labeling using [32P]orthophosphate injections, both were far more selective than iodination (lane 5). The most specific enzyme preparation was derived from normal Xenopus liver (lane 2) followed by that from estrogen-stimulated Xenopus liver (lane 1). The chicken liver preparation (lane 4) labeled several low-molecular-weight proteins that are apparently present in the enzyme

AND WILEY

preparation itself since they are also labeled in the absence of any added substrate. To ascertain whether VTG labeled in vitro had properties similar to in viva-labeled VTG, we compared their ability to be specifically incorporated by oocytes and to be correctly processed into the yolk proteins. Oocytes were incubated for 24 h with a near-saturating concentration (2.5 mg/ml) of [32P]VTG labeled via protein kinases, 1251-labeled VTG, or [32P]VTG labeled in vivo. The I’,.,,, of incorporation of all of the VTG preparations was found to be identical (140 ng . oocyte * h f 5%). To ensure that the K,,, for VTG uptake was unaltered by the enzymatic phosphorylation of VTG in vitro we compared the amount of ligand incorporated by oocytes incubated for 6 h in serial dilutions (3.0-o. 1875 mg/ml) of [32P]VTG labeled in vitro or in vivo. The K,,, for VTG uptake was determined by constructing a Woolf-Augustinssen-Hofstee plot of the data (16,17). The plots were linear for each VTG preparation and yielded a Km of 8.72 X lo-” M for VTG labeled via Xenopus protein kinase and 1.06 X 10e9 M for VTG labeled in vivo. The small difference (~20%) between these values is probably not significant. However, these values do indicate that TABLE 3 SPECIFICITY OF X. PHOSPHOPROTEIN

Substrate Vitellogenin Albumin (BSA) Aldolase a-Casein Ovalbumin Phosphorylase b Phosvitin Ribonuclease A

laevis LIVER

cm 9,478 35 15 9,016 0 28 13,517 0

KINASE

Percentage of vitellogenin 100
Note. The indicated substrates were incubated under standard conditions at a concentration of 1 mg . ml-’ with equal amounts of enzyme for 20 min at 2O’C. Indicated are the trichloroacetic acid-precipitable counts corrected for background.

VITELLOGENIN

the introduction of additional phosphates onto the VTG molecule does not decrease the affinity of the receptor for the ligand. Having determined that the different VTG preparations were incorporated with similar kinetics we next examined the ability of the oocyte to localize the internalized VTG in the yolk platelets. This was done by sucrose gradient fractionation of oocytes exposed to [32P]VTG labeled via Xenopus protein kinase. Oocytes were incubated for 1 h or for 24 h followed by 24 h in unlabeled medium and then homogenized and applied to 19-50% sucrose gradients (3). The results of this experiment are shown in Fig. 2. After a l-h incubation the majority of the labeled material was found in a preyolk platelet compartment that sediments between the mitochondria and the yolk platelets; this compartment has been called the transitional yolk body (3). After a 24-h incubation in [32P]VTG followed by

377

RADIOLABELING

24 h in unlabeled medium almost all of the labeled material was localized in the yolk platelets. The small amount of radioactivity found near the top of each gradient appears to represent labeled proteins released from organelles broken during homogenization. These proteins are not membrane bound (3) and comigrate with VTG or with the yolk proteins on SDS-polyacrylamide gels. Results similar to these have been reported for [32P]VTG labeled in liver organ culture (18). Finally, to ensure that the different labeled VTG preparations were correctly processed into the yolk proteins subsequent to internalization, we performed the following experiment. Oocytes were incubated in 2.5 mg/ ml of [32P]VTG labeled via protein kinases, ‘251-labeled VTG, or [32P]VTG labeled in vivo for 24 h followed by an additional 20-h incubation in unlabeled medium. The cells were dissolved and aliquots containing equal amounts of radioactivity were analyzed by polyacrylamide gel electrophoresis as shown in Fig. 3. It is apparent that very little of the radioactivity originally present in the ‘251-labeled VTG is retained in the yolk proteins (lane 4). This is consistent with our previous observations that iodinated proteins are deiodinated after internalization by oocytes (4). In contrast, the enzymatically phosphorylated VTG was processed into the same yolk proteins as was the in vivelabeled VTG. However, the enzymatic labeling procedure resulted in a more uniform labeling of the yolk proteins than resulted from in vivo labeling. In VTG, most of the protein phosphorus is associated with the phosvitin/phosvette (19) region of the molecule, and this is reflected in the in viva-labeling pattern. DISCUSSION

FIG. 2. [“P]VTG labeled in vitro via Xenopus protein kinase is internalized by oocytes and transferred to the yolk platelets. Oocytes were incubated in [3ZP]VTG for 1 h (0) or for 24 h followed by 24 h in unlabeled medium (0) prior to homogenization and subsequent sucrose gradient fractionation. The solid line indicates the uv absorbance profile of the fractionated gradient. Each gradient contained 35 oocytes.

Unlike the majority of physiological ligands utilized in binding and internalization studies, vitellogenin can readily be labeled in vivo by simply injecting radiolabeled precursors into hormonally stimulated animals. VTG labeled in this manner is theoretically the best ligand

378 I. X. laevis --

OPRESKO 2.

3.

4.

lnvivo --

Chicken

1251

-

VTG

-

LV,

-

PV

3

LVZ

-

PVTI

3 PVTn

FIG. 3. Intracellular processing of radiolabeled vitellogenin. Oocytcs were incubated in various VTG preparations for 24 hand then maintained for 20 h in unlabeled media. The cells were then solubilized in SDS and aliquots applied to a 5-10% gradient polyacrylamide gel. The autoradiogram shows the labeled proteins resulting from the intracellular cleavage of the parent, VTG molecule. Oocyteswere exposed to VTG labeled using Xenopus protein kinase (lane I), in viva-labeled VTG (lane 2), VTG labeled using chicken protein kinase (lane 3), or iz51-labeled VTG (lane 4). Equal amounts of total radioactivity were placed in each lane. The markers on the right indicate the migration positions of the different yolk proteins. LV, = lipovitellin I, PV = phosvitin, LVI = Iipovitellin 2, PVT, = phosvette I and PVT,, = phosvette II.

to use for investigations of the mechanism of vitellogenin binding, internalization, and processing by oocytes. While it is extremely difficult and expensive to achieve the specific activities required for either receptor-binding or short-term internalization studies, the in viva-labeled VTG offers an excellent standard by which to judge suitable methods for labeling the protein to high specific activities. Using this approach we have found that VTG labeled

AND WILEY

in vitro by enzymatic phosphorylation is virtually indistinguishable from in vivelabeled vitellogenin in its ability to be bound, internalized, and processed into the yolk proteins. Although VTG labeled by radioiodination was bound and internalized normally by oocytes, virtually all of the radioactive “tag” was removed subsequent to internalization. Thus, while 1251-labeled VTG might be suitable for receptor-binding studies, it cannot be utilized for investigations on the postendocytotic compartment&ion and processing of the molecule. There are a number of different features of the enzymatic phosphorylation method that make it particularly suitable for labeling VTG. The method for isolating the protein kinase is simple and requires a minimum of steps and equipment. The method can label large quantities of VTG (5- 12 mg) to a reasonably high specific activity. Since phosphate transfer is very efficient even at very low ATP concentrations, the limiting factors in the specific activities achieved are the amounts of VTG and radiolabeled ATP utilized in the reaction. In addition, the vitellogenin molecule is very uniformly labeled in the reaction. This is particularly important for studies of the processing of the VTG molecule into the yolk proteins, since the VTG molecule is large (MI - 200,000) and contains very distinct and restricted domains of protein phosphorus and amino acid composition. Thus, enzymatically phosphorylated VTG is actually superior to in viva-labeled VTG for these types of studies. One aspect of the enzymatic phosphorylation procedure that may seem somewhat unusual is the necessity to label VTG while it is present in serum as opposed to isolated VTG. This is necessary because isolated VTG is insoluble in the presence of Mg2+, which is required for the activity of the protein kinases. It has been proposed that there are cofactor(s) in serum that serve to keep divalent cation salts of VTG soluble (20). Although labeling VTG in situ may seem a serious drawback to the technique, we have actually found it to be a distinct advantage. Since the protein kinases

VITELLGGENIN

described here are very specific for VTG, very little labeling is seen in other serum proteins. In addition, these other proteins are apparently not bound or internalized by oocytes since, even after prolonged incubations in labeled serum (44 h), none are seen in association with the oocytes. Since the isolation of the VTG is not required prior to labeling the molecule, there is very little chance of damage or denaturation of the molecule, which might take place during isolation procedures. Thus, after labeling, the serum containing the VTG can be used without further manipulation, except for dialysis to remove the unreacted [32P]ATP. This kind of preparation is desirable since it has been shown that the presence of serum has a beneficial effect on VTG uptake by oocytes (9). However, if pure VTG is desired it can be isolated from the reaction mixture using the precipitation method of Wiley et al. (8) with an efficiency of 6575% (depending upon the amount of starting material). The most selective protein kinase preparation was obtained from Xenopus liver since that from chicken liver undergoes a limited amount of self-phosphorylation. At this time, however, it appears that these minor contaminants are not internalized (to any appreciable extent) by oocytes. Certainly they are not ap parent after either short (15 min) or prolonged (24 h) incubations. While Xenopus liver would be the best source of protein kinase, it is generally not available except to laboratories maintaining large Xenopus colonies. Chicken liver is an excellent second choice that is readily available commercially. Indeed, the isolation of a phosvitin kinase from rooster liver was reported over a decade ago (2 1). Owing to the similarities between that enzyme and the one described here it appears that the two are identical, although obtained using different isolation procedures. Since the Xenopus and the chicken protein kinases are specific

379

RADIOLABELING

for polyphosphorylated proteins and since VTG is the only phosphoprotein found in vertebrate serum, this procedure should be useful for labeling VTG from any vertebrate source. ACKNOWLEDGMENTS We thank Dr. Robert Bast for his generous gilt of chicken plasma. This work was supported in part by a grant from the R. J. Reynolds Foundation.

REFERENCES Wallace, R. A., and Misulovin, Z. (1978) Proc. Natl. Acad. Sci. USA 75,5534-5538. Bergink, E. W., and Wallace, R. A. (1974) J. Biol. Chem. 249,2897-2903. Opresko, L., Wiley, H. S., and Wallace, R. A. (1980) Cell 22, 47-57. 4. Opresko, L., Wiley, H. S., and Wallace, R. A. (1980) Proc. Natl. Acad. Sci. USA 77, 1556-1560. 5. Wallace, R. A. (1970) Biochim. Biophys. Acta 215, 176-183. 6. Wallace, R. A., and Jared, D. W. (1968) Canad. J. Biochem. 46,953-959. 7. Wallace, R. A., Jared, D. W., Dumont, J. N., and Sega, M. W. (1973) J. Exp. Zool. 184, 321-334. 8. Wiley, H. S., Opresko, L., and Wallace, R. A. (1979) Anal. B&hem. 97, 145-152. 9. Wallace, R. A., Misulovin, Z., and Wiley, H. S. (1980) Reprod. Nutr. Dt!vlop. 20,699-708. IO. Wallace, R. A., Jared, D. W., and Eiscn, A. Z. (1966) Canad. J. Biochem. 44, 1647-1655. 11. Bradford, M. M. (1976) Anal. B&hem. 12,248-252. 12. Mans, R. J., and Novelli, G. D. (196 1) Arch. B&hem. Biophys. 94,48-53. 13. Salacinski, P. R. P., McLean, C., Sykes, J. E. C., Clement-Jones, V. V., and Lowry, P. J. (1981) Anal. Biochem. 117, 136-146. 14. Lane, L. C. (1978) Anal. Biochem. 86, 655-664. 15. Wallace, R. A. (1965) Anal. Biochem. 11, 297-31 I. 16. Hofstce, F. H. J. (1959) Nature (London) 184, 12961298. 17. Dowd, J. E., and Riggs, D. S. (1965) J. Biol. Chem. 240, 863-869. 18. Jared, D. W., Dumont, J. N., and Wallace, R. A. (1973) Dev. Biol. 35, 19-28. 19. Wiley, H. S., and Wallace, R. A. (1981) J. Biol. Chem. 256, 8626-8634. 20. Wiley, H. S. (1979) Ph.D. Thesis, Univ. ofTennessee. 21. Goldstein, J. L., and Hasty, M. A. (1973) J. Biol. Chem. 248,6300-6307.