THE Jon~sh~ OF BIOLOGICAL CHEUISTRY Vol. 248, No. 22, Issue of November 25, pp. 7898-7903, 1973

P&hi in U.S.A.

Polymorphism of Rhesus Monkey Serum Prealbumin

PURIFICATIOS AND PARTIAI, STRUCTURE*

(Heceived for pltblication, April 2, 1973)

PIETEIZ VAX .JAARSVELD,$ WILLIAM T. BRANCH, AND JACOB ROBBINS~

From the Clikcal Endocknology Branch, National Institute of Arthritis, :lIetabolisnr, and Digestive Diseases, National Institutes o;f Health, Bethesda, Maryland S?OOl/,

FRANCIS J. MORGAN,T[ YOSHIKAZU KANDA, AND ROBERT E. CANFIELU

From the Department of Medicine, College of Physicians and Xwgeom, Columbia L?Gversity, -\‘ele Yolk, Sex Yodc 1003w

SUMMARY

Two genetic variants of Rhesus monkey prealbumin, Pt l-l and Pt 2-2, have been purified by ion exchange chro- matography followed by preparative polyacrylamide gel electrophoresis. Both monkey proteins have molecular weights of approximately 54,000 as determined by ultracen- trifugation, Amino acid compositions of both variants are very similar. Peptide maps of the two monkey prealbumins differ definitely from each other in only one peptide. The NH2-terminal sequences (1 to 21) suggest that each variant of Rhesus prealbumin is composed of identical subunits; an interchange of valine and isoleucine is found at residue 5 in Rhesus prealbumin. The structural features of both Rhesus prealbumins are very similar to those of human prealbumin.

There are two elcctrophoretically distinct variants of serum prealbumin in Rhesus monkeys (.lfacaca mulatia); the faster migrating type is designated Pt 1-l and the slower, Pt 2-2. The existence of tlx-o types a-as recognized by Blumberg and Robbins (I), and they were subsequently shown to result from a genetic polymorphism (2,3). Hybrids (Pt l-2) of these two types formed five electrophoretic bands, which provided the first evidence that prealbumin is composed of four subunits (2, 3). Human prealbumin has since been extensively studied (4-13). In humans only one type has been found, n-hi& corresponds elec- trophoretically to monkey Pt l-1. It is a very stable tctramer (4, 5) which binds thyroxine and also retinol-binding protein, the protein responsible for the transport of vitamin A (14-18). In this report WC describe the purification of the monkey pre-

* This lxork was supported in part by Research Grants AM 09579, AM 05968, and HL 14236 from the National Institutes of Health.

$ Permanent address, Department of Pharmacology, University of Stcllenbosch. P.O. 130x 53. Bellville. ReDublic of Hollth Africa.

$ To x%-horn requests for reirints should be addressed. (1 Present address, St. Vincent’s School of Medical IIcsearch,

Victoria Parade, Melbourne, Victoria 3065, Allstralia.

albumins and compare their structural features l-r-ith human prcalbumin. In a previous report (19) the molecular properties and binding reactions of Pt. 1-l and Pt 2-2 xere present&.

MATERIALS AXD METHODS

Small samples of blood were collected from Rhesus monkeys at the Kational Institutes of Health atld Bionct,ics Research Laboratorirs (Rockville, AId.) and the sera v,-ere scrccncd by polyacrylamide disc gel clectrophoresis to determine the pheno- type of the animal (3). Serum collected from the appropriate monkeys by repeated bleeding was pooled and kept at -20” until used. Lyophilized human prcalbumin was purchased from 13ehringwerke and further purified by preparative, poly- acrylamide gel electrophoresis (4).

Analytical polyacrylamide gel electrophorcsis Teas pel,formed as detailed previously (3). Carbohydrate was detected by the periodate-Schiff reaction (20). Elcctrophoresis in sodium dodecyl sulfate-containing media and preparative polyacryl- amide gel electrophoresis was performed as previously described

(4). The concentration of prcalbumin in individual monkey sera

was estimated from thyroxine binding capacities determined by the saturation method usin g either paper electrol~l~orcsia in 0.2 RI glycine, 0.13 nz sodium acctatc, pH 9.0 (al), or polyacryl- amide gel electrophoresis (3). 13ecause of the proximity of Pt 1-2 and Pt 2-2 to albumin, only polyacrylamide gel electro- phoresis was suitable to mcasurc the binding capacities of these prealbumin types. Prealbumin concentration was also meas- ured by single radial immunodiffusion (22) against rabbit anti- human prealbumin antiserum using Partigen plates from 13chring Diagnostics. The t,hyrosinc-binding capacity of thvrosinc- binding globulin \vas measured by reverse flow paper elcctro- phorcsis (23) using the same glycine-acetate buffer as above. The extinction coefficients of Pt 1-l and Pt 2-2 were determined from thrir absorption at 280 nm. E: F,,, values of 14.4 and 14.3 were obtained for Pt 1-l and Pt 2-2, respc‘ctively. Et& = 14.1 was used for human prcalbumin (7).

Sedimentation equilibrium expcrimcnts were performed ac- cording to the meniscus depletion method of Yphantis (4, 24).

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A value of 0.73 was calculated for v from the amino acid com- position (25).

Total amino acid analysis was performed by the method of Spackman et al. (26) after hydrolysis of the protein in 6 N IICl at 110” for 24, 48, or 72 hours. Half-cystine was determined in 24-hour IICl hydrolysates as cysteic acid after oxidation of prealbumin with performic acid (27). Tryptophan was deter- mined after hydrolysis in p-tolucnesulfonic acid (28).

Reduction and X-carboxymethylation of prealbumin was per- formed as described for human prealbumin (8).

Tryptic digestion were performed as follows: X-carboxy- methyl prealbumin in 0.1 M NH4HC03 (20 mg per ml) was heated for 10 min at 100” (10, 13) and then incubated at 37” for 8 hours with 2 o/o (w/w) tosylphenylalanyl chloromethane-treated trypsin (Worthington). A suspension of performic acid-oxidized preal- bumin in 0.1 M KHJIC03 (16 mg per ml) was incubated at 37” with aCl0 (w/w) of tosylphenylalanyl chloromethane-treated trypsin; the protein became completely soluble shortly after addition of trypsin.

Peptide mapping of trypsin hydrolysates was performed on cellulose thin layer plates (Avicel microcrystalline, 0.1.mm thickness, E. Merck). One-tenth milligram of hydrolyzed pro- tein (10 to 15 ~1) was applied to the plates and electrophoresis performed at 37.5 volts per cm in pyridine-acetic acid-water, ~1-1 6.0 (10:1:8.9, v/v). Ascending chromatography was per- formed with butanol-pyridine-acetic acid-water (15: 10 :3: 12, v/v) as the solvent (13). The air-dried plates of S-carboxy- methyl prealbumin hydrolysates were sprayed with buffered cadmium-ninhydrin, phenanthrenequinone, Pauly, and Ehrlich reagents (29). Those obtained from pcrformic acid-oxidized preparations were sprayed with phenanthrenequinone followed by ninhydrin. Autoradiography was performed by exposing the dried plate to No-Screen x-ray film.

NIB-terminal amino acid sequence determination was per- formc,d as described previously (8).

RESULTS

Thyroxine-binding Capacify and Concentration of Prealbunrin

in Serum-Freshly collected sera from nonpregnant animals previously not repeatedly bled were used and the results of thyroxine binding capacity measurements are summarized in Table I. Since no sex differences were observed, results from male and female monkeys are pooled. The concentration of prealbumin calculated from the binding capacities (Table I) agreed closely with independent measurements using single

TABLE I Thyroxine-binding capacity of thyroxine-binding globulin (TBG)

and prealbumin and concentration of prealbumin in monkeys

with prealbumin polymorphism

Values are mean f S.D.

Phenotype

Binding capacity

TBG Prealbumin

Concentration of prealbumina

pg tkyrozine/ml swum mg/lOO ml SIYZ,,~

Pt l-1 14 0.23 f .04* 3.22 i 0.41b 22.4 f 2.9 rt 1-2 13 0.22 zt .05b 2.52 f 0.43c 17.5 It 3.0 Pt 2-2 5 0.18 f .15” 1.71 f 0.49” 11.9 f 3.4

Flo. 1. Chromatography of Rhesus monkey sera containing prealbllmin types Pt l-l and Pt 2-2 on 1)1GAE-Sephadex A-50. Columns (30 X 4 cm) were equilibrated with 0.05 r~ phosphate buffer, pH 7.4. Sera (400 ml each) were dialyzed against the same buffer and applied to the columns. The linear NaCl gradients (l&liter, 0.05 to 0.4 M, pH 7.4) were started after 800 ml of the equilibration buffer had passed through the column. Fraction vohlme was 20 ml. The entire procedure took place at 4”. R.eti- nol, detected by fluorescence at 480 nm, served to indicate the position of the prealbllmin-rctinol-binding protein complex

n Calculated from the thyroxine-binding capacity (n = 1, mol wt = 54,000).

b By reverse-flow paper electrophoresis. c Bv nolvacrvlamide gel electroohoresis.

“& ” ”

radial immunodiffusion OII plates calibrated with purifictl monkey prealbumin. Preliminary studies showed that monkry pre- ablumin (I’t l-l) gave a steeper calibration (urve (area of circle versus collcentration) than human prealbumin, rompatiblc with fewer antigenic determinants on the mollke)- protein to\\-ard anti-human prcalbumin antibody. This is in agreement with recent findings with cynomolgus prealbumin (30). Double immunodiffusion showed identity between Pt 1-l and 1% 2-2.

The data in Table I showed that serum from l’t l-l niollkeys had a higher concentration of prealbumin compared to both Pt, l-2 and Pt 2-2 monkeys (p < 0.001). The concentration of Pt l-2 was also higher than Pt 2-2 (p < 0.01). The thyrosille- binding capacity of thyroxine-binding glob&l did not differ sig- nificantly in the three groups (p > 0.05).

Pur$cation of Pt 1-1 and Pt d%-13oth Rhesus prealbumins were purified by ion exchange chromatography on DE-1E- Scphadex A-50 (Fig. I), followed by preparative polyacrylamide gel electrophoresis (Fig. 3). The initial purification of Rhesus prealbumin on DUE-Sephadex is shown ill Fig. 1. The frac- tions between the arrows show the location of prealbumin, which eluted together with retinol-binding protein. Analytical gel electrophoresis of these combined fractions is shown in Fig. 2. The Pt 1-l eluate contained serum components which migrated largely with a /?- to y-globulin mobility and could easi1.v be re- moved by subsequent preparative gel electrophorcsis, as shown in Fig. 3 (upper panel). The final yield of Pt l-l n-as 40 to 507; of the serum prcalbumin.

The lower panel of Fig. 1 shows the DEAE-Sephades elution profile of Pt 2-2 serum. Pt 2-2 elutcd at a lower ionic strength than l’t l-l and consequently was contaminated by a large amount of albumin (Fig. 2). Of the [12”I]thyrosii~e-coi~taiJli~~g fractions obtained by preparative gel clectrophoresis (Fig. 3, lower panel), only those at the leading edge of t’he peak COII-

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PA FRACTION FROM Pt2-2 FRACTION FROM DEAE SEPHADEX PREP. GEL ELECTROPHORESIS

Pt 2-2

- ALB

-+ PA

t-c)

au

Before ialidase

After Slalldase

FIG. 2. Analytical polyacrylamide electrophoresis of Pt l-1 and Pt 2-2 fractions obtained from DEAE-Sephadex A-50 (Fig. 1) and Pt 2-2 fractions obtained from preparative gel electrophoresis (FIG. 3). The latter were analyzed before and after treatment for 1 hour with 0.005 units of sialidase per mg of protein. The total acrylamide concentration was 8.7% and the running gel was pH 9.2. Migration was towards the anode.

0 20 30 40 50

FRACTION NUMBER

PREP GEL ELECTROPHORESIS

Pi 2-2 A 1 -,

8

6

FIG. 3. Preparative polyacrylamide gel electrophoresis patterns of Pt 1-l and Pt 2-2 fractions obtained from DEAE-Sephadex A-50 (Fig. 1). The arrows indicate the salt front (bromphenol blue). [iZ51]Thyroxine was added in tracer amount prior to electrophore- sis. Retinol is detected by fluorescence at 480 nm. Fraction size was 2.5 ml.

Amino Acid Composition-In Table II the amino acid com- positions are given as the number of residues per subunit molec- ular weight and are compared with that for human prealbumin determined concurrently. There are few differences between the three proteins. Human prealbumin has 2 more arginine residues per subunit than both forms of Rhesus prealbumin; this difference is also seen in cynomolgus prealbumin (30). Human prealbumin also appears to have 1 additional glycine and iso- leucine, 2 additional threonines, and 1 less aspartic acid, serine, proline, and valine compared to Pt l-l.

The electrophoretic difference between Pt l-l and Pt 2-2 is probably due to a difference in net charge. However, the source of this difference is not clear from the amino acid analysis. The

tained electrophoretically homogeneous Pt 2-2. The later frac- tions, accounting for 60 to 70% of the total prealbumin, still contained large amounts of albumin and other contaminants. Fig. 2 shows a disc gel electrophoretic analysis of a pool of these fractions. The strong band which migrated just behind Pt 2-2 stained for carbohydrate; amino acid analyses of these frac- tions showed significant amounts of amino sugars. The broad band behind Pt 2-2 (Fig. 2), therefore, consisted of albumin and a carbohydrate-containing protein which most probably was the al-acid glycoprotein of serum (orosomucoid). To increase the electrophoretic mobility difference between Pt 2-2 and the contaminating glycoprotein, the pooled samples were incu- bated with Clostridium perfringens sialidase (Worthington), 0.005 unit per mg of tot,al protein, for 1 hour at 37” in 0.1 M acetate buffer, pH 5.0. Subsequent polyacrylamide gel elec- trophoresis (Fig. 2) showed a clear separation between Pt 2-2, albumin, and the desialyzed glycoprotein. Only the slowest migrating band stained for carbohydrate. Pt 2-2 was therefore purified by preparative polyacrylamide electrophoresis of the sialidase-treated fraction. The total yield of electrophoretically homogeneous Pt 2-2 was 20% of the serum prealbumin.

The electrophoretic separations shown in Fig. 3 also indicate that retinol-binding protein, dissociated from prealbumin during the procedure (14), had the same mobility in the two monkey types.

Molecular Weight-Weight average molecular weights of 54,000 and 53,400 for Pt l-l and Pt 2-2, respectively, were cal- culated from the sedimentation equilibrium data obtained at 20,000 rpm. These values are similar to that for human pre- albumin (4) and also agree with x-ray crystallographic data of human prealbumin (6).

On polyacrylamide gels containing 0.1% sodium dodecyl sulfate, the relative electrophoretic mobilities of proteins are in general a linear function of molecular weight (31, 32). This method has been employed for human prealbumin (4) and good agreement was found with the value obtained by sedimentation equilibrium. With Pt l-l a value of 53,000 was obtained by following the same procedure; however, Pt 2-2 showed anomalous behavior and migrated close to serum albumin (mol wt = 69,000). Thus sodium dodecyl sulfate does not appear to dissociate either human or the monkey prealbumins into their subunits under the conditions used. The anomalous behavior of Pt 2-2 on sodium dodecyl sulfate gels is unexplained. The relative elec- trophoretic mobilities of Pt l-l and Pt 2-2 were also determined in 6 to 10% polyacrylamide (acrylamide to bis ratio = 10: 1). Each protein gave a single sharp band at every gel concentra- tion, and the slopes of the logarithm of the relative mobility versus gel concentration (33) were parallel, indicating that Pt l-l and Pt 2-2 differ in charge but not in size.

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FZB ARGININE B TRYPTOPHAN UlIilIM HISTIDINE AND TYROSINE

TABLE II Amino acid composition of prealbumins

Amino acid - I lhesus Pt 1-l

Lysine ..................... 9.5 Histidine ................... 4.0 Arginine .................... 2.0 Aspartic acid. .............. 8.9 Threonine .................. 10.2 Serine ...................... 11.4 Glutamic acid ............... -12.3 Proline. .................... 8.0 Glycine ..................... 8.7 Alanine ..................... 11.8 Half-cystine”. .............. 1.1 Valine ...................... 14.0 Methionine ................. 1.4 Isoleucine ................... 3.8 Leucine ..................... 6.8 Tyrosine .................... 5.0 Phenylalanine ............... 4.8 Tryptophand. ............... 2.8 Totals ...................... 127

Residues/subunit”

:llesus Pt 2-2 HUIWIb -

9.6 8.5 3.7 4.0 2.3 3.7 9.2 8.1

10.4 11.8 11.2 9.9 11.6 12.0 7.9 7.0 9.2 9.7

12.2 12.0 1.0 0.8

12.8 12.6 1.1 1.0 4.7 5.2 7.0 6.9 4.8 5.2 4.9 5.1 3.2 3.3

127 128

a Values are based on a subunit molecular weight of 13,500. Figures are obtained from the average of duplicate hydrolyses at 24,48, and 72 hours. Appropriate corrections have been made for those residues whose value changes with time of hydrolysis.

b Analysed in parallel with Rhesus prealbumin. Values are in good agreement with earlier estimates from several laboratories (7, 10, 13).

c Determined as cysteic acid (27). d Determined after hydrolysis in p-toluenesulfonic acid (28). c Sum of nearest integral values.

replacement of a valine by an isoleucine is the only clearly evi- dent alteration between the monkey prealbumins.

Peptide Muppiw-Fig. 4 shows tracings of peptide maps obtained from g-hour tryptic hydrolysates of S-carboxymethyl prealbumin. The numbers to the right of the spots relate in- dividual peptides in the three prealbumin preparations. Peptide 1, seen in all three proteins, was identical with free glutamic acid. The most cathodal arginine-positive spot in the human pre- albumin map coincided with free arginine and was not present in either monkey prealbumin. The arginine-containing spot in human prealbumin with an Rp slightly greater than peptide 2 was also absent in monkey prealbumin. These differences in arginine-containing peptides are consistent with the arginine differences in the amino acid compositions (Table II). A sim- ilar variation has been found between human and cynomolgus prealbumin (30). Monkey and human prealbumin showed similarities in about half of the peptide spots.

Disc gel electrophoresis showed that 60 to 70% of the ma- terial in all three trypsin hydrolysates migrated with a mobility identical with that of the corresponding native prealbumin. Denaturation and hydrolysis procedures similar to those used here were employed by Rask et al. (10) and by Gonzalez and Offord (13) for human prealbumin. The photograph shown by Rask et al. (Fig. 8 in Ref. 10) also contains a peptide comparable to peptide 12. This peptide gives a specific positive reaction for arginine, tryptophan, histidine, and tyrosine, contains half or more of the [Wlcarboxymethyl label determined by auto- radiography, and therefore is probably unhydrolyzed preal- bumin. Nevertheless, the number of peptides found in these

l-

Pt l-l

01

pt2-2

01

HPA

T

@&I2 --_- -

O0

a

0 e 011

@312

> ELECTROPHORESIS @

FIG. 4. Tracings of peptide maps of tryptic digests (8-hour incubation) of S-carboxymethyl prealbumin preparations. The numbers to the right of each spot relate common peptides in the three hydrolysates. HPA signifies human prealbumin. The method for preparing the maps is described in the text.

incomplete hydrolysates agrees closely with the number ex- pected from the amino acid composition and it is likely that those peptide chains which are attacked by trypsin are even- tually completely hydrolyzed.

The two monkey prealbumins showed one striking difference: the Jilled spot (Fig. 4) seen in Pt 2-2 but not in Pt l-l. Since no major difference was seen in any peptide containing tryp- tophan in the hydrolysates described above, performic acid- oxidized preparations, which appear to be more fully digested by trypsin, were analyzed in an attempt to confirm this dif- ference between Pt l-l and Pt 2-2. Maps prepared from g-hour hydrolysates of performic acid-oxidized preparations did not contain ninhydrin-staining material at or near the origin. Again there was only one major peptide which differed between Pt l-l and Pt 2-2. Maps of mixed samples of Pt l-l and Pt 2-2 con- firmed the single peptide difference.

NH2-terminal Amino Acid Sequences-The partial NH2- terminal amino acid sequences of type Pt l-l and type Pt 2-2 are shown in Table III. The two NH*-terminal sequences are identical except for position 5 in the first 21 residues. In each case a unique sequence was determined, suggesting that the subunits of each prealbumin type are identical; this corresponds to the finding with human prealbumin (8). The differences seen at Steps 5 and 6 are consistent with the differences ob-

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TABLE III

NHp-lerminal amino acid sequence of Rhesus prealbumins, 1’1 1-l and Pt 2-2, and human prealbumin

Human prealbumin data are from Morgan et al. (8). Italics indicate differences between Pt l-l, Pt 2-2, and/or hllman.

1 5 10 Pt l-l Gly-Pro-Thr-GlyVaI-Asp-Glu-Ser-Lys-Cys- Pt 2-2 C;ly-Pro-Thr-(:ly-lie -Asp-Glu-Ser-Lys-Cys- Human Gly-Pro-Thr-Gly-ThvGlu -Glu-Scr-Lys-Cys-

11 15 20 Pt 1-l Pro-Lcu-Met-Val-Lys-Val-Leu-Asp-Ala-Val-Arg- Pt 2-2 Pro-Leu-i\Iet-Val-Lgs-Val-Leu-Asp-Ala-Val-Arg- Human Pro-Leu-i\let-Val-Lgs-Val-J,eu-Asp-Ala-Val-Arg-

served in the amino acid compositions and confirm the valine- isoleucine interchange in the two monkey prealbumins.

DISCUSSION

Serum prealbumin is a tetrameric protein whose properties hal-c attracted considerable interest. It combines with 1 mole- cule of thyroxine (7) and 4 molecules of retinal-binding protein (18) and provides a model for protein-protein and protein-small molecule interactions. X-ray examination of human preal- bumin crystals (6) has provided evidence for a tetrameric struc- ture, with a channel between the four subunits where the thy- roxine-binding site is presumably located. The molecular organization appears to be unusual and is reflected in the ex- treme resistance to denaturation, subunit dissociation, and enzymatic digestion (4, 10, 16).

=\lthough human prealbumin has been the more extensively studied, the genetic polymorphism in Rhesus prealbumin and the hybrid forms produced from these variants provided the initial demonstration that prealbumin was a tetramer. Evo- lutionary and genetic variants have proved useful in the study of many homologous protein systems for identifying features in the structure which have been maintained for biological roles. It was important to examine the monkey variants in this light, to assess the degree of variation between the two forms and be- tweell monkey and human prealbumin. In order to purify the monkey variants, designated Pt l-l and Pt 2-2, use was made of conventional protein separation methods and sialidase treat- mcnt to increase the electrophoretic separation of Pt 2-2, the slowmigrating variant, from orosomucoid. Prior studies with affinity chromatography utilizing fixed retinol-binding protein (34) or thyroxine (35, 36) proved unsuccessful for isolation of prealbumin.

The results in this paper, which are concerned with the pri- mary structure of these molecules, indicate that the differences which exist are of a minor nature. The molecular weight as determined by ultracentrifugation is very similar for human prealbumin and both types of monkey protein. Sodium dodecyl sulfate gel electrophoresis indicates that monkey prealbumin, like human prealbumin, is not dissociated by sodium dodecyl sulfate under our conditions. If one accepts the evidence from the ultracentrifuge that the monkey prealbumins have equal molecular weight, the anomalous behavior of Pt 2-2 on sodium dodecyl sulfate gel clectrophoresis must reflect a difference other than in size. With respect to amino acid composition, the monkey vnriauts appear to differ only in the exchange of a valine for an i,soleucine. The most striking difference between human and monkey prealbumin is the presence of 2 additional arginiue and thrconine rcsiducs in human prealbumin. The similarities

are reflected in the peptide maps. The incomplete nature of the tryptic digest of X-carboxymethyl prealbumin renders the peptide maps from this source somewhat unreliable as an ana- lytical tool. Nevertheless, the difference in arginine-containing peptides between human and monkey prealbumin is evident in these maps. When the performic acid-oxidized protein, which is hydrolyzed much more completely by trypsin, is studied, only one pcptide is clearly different between the two monkey types. Thus the peptide maps confirm the finding that these proteins have very similar primary structures. The NHz-terminal amino acid sequences are in agreement with these findings. The variations in sequence observed at position 5 in the monkey variants, and at positions 5 and 6 in human compared to monkey, are consistent with the differences noted in amino acid com- position.

The valine-isoleucine interchange in Pt 1-l and Pt 2-2 can be explained by a point mutation. However, it is not clear whether this is the only difference between these variants because it does not provide an obvious explanation for their difference in charge. Possible loci for this charge difference are in amide groups, which were not measured in this study, or in variations in molecular conformation. Our studies of the molecular properties of Pt 1-l and I’t 2-2, however, have revealed only minor differences (19). Of particular significance is the fact that both monkey prealbumin and human prealbumin have virtually identical interactions with thyroxine and with retinal-binding protein (18, 19), and thus the binding sites do not appear to be affected by the amino acid differences described in the present report.

Acknowledgments-We are indebted to Drs. G. Pucak, I. Baas, and D. Martin for collecting blood samples, and to Drs. R. S. Bernstein and R. Goebel for assistance with the purification procedures. We thank Ms. G. Sandeen and Mr. M. Brackbill for technical assistance.

1.

2.

3.

4.

5.

6.

BLUMWXG, B. S., .\SD Rons~lvs, J. (1960) Endocrinology 67, 368-378

ALPE~, C. A., ROJIIX, N. I., AND REFETOFF, S. (1969) Proc. Nat. Acad. Sci. U. S. A. 63, 775-781

BICRNSTICIN, I:. S., ROBBINS, J., BND R.ZLL, J. E. (1970) En- docriuology 86, 383-390

BR.\NCH. W. T.. ROF~JJ~NS. J.. BND KDELHOCH, H. (1971)J.UioZ. Chem.‘246, 6011-6018

BEINCH. W. T.. ROBBINS. J.. BND EDELHOCH. H. (1972) Arch. Biochbn. Uiobhys. 162, ‘144-151

I ~

BL~I~E, C. C. F., SWAN, I. I). A., RER~T, C., BERTHOU, J., Lnun~c.~~, A., .\ND REILT, B. (1971) J. lwol. Biol. 61, 217- 224

7.

8.

9.

10.

11.

12.

13.

14.

15.

16. 17.

RAZ, A., .LND GOODM.~N, Dt;W. S. (1969) J. Biol. Chem. 244,

3230-3237 MORGAN. F. J.. CINFIELD, R. E:., .\ND GOODMLN, DsW. S.

(1971) kiochik. Biophys.‘Acta 236, 798-801 GOTTO. A. &I.. LUX. S. E.. BND GOODMAN. Dr,W. S. (1972)

Biockm. Bidphys. ‘Acta 2k, 429-435 ’ R.ISIC, L., PETERSON, P. A., AND NILSSON, S. F. (1971) J. Biol.

Chem. 246, 6087-6097 NILSSON, 8. F., :LND PLTIBRSON, P. A. (1971) J. Biol. Chem. 246,

6098-6105 R.\slc, L., PETERSOX, P. A., AND BJORK, I. (1972) Biochemistry

11, 264-268 GONZALIBZ,G.,.\ND OFFOIZD, R. IX. (1971) Biochem.J. 126,309-

317 KANAI. M.. Ikz. A., .~ND GOODMSN. DEW. S. (1968) J. Clin.

Invefit. 4i, 2025-2044 Raz. A.. SHIR.\TORI. T.. AND GOODM.LN. DEW. S. (19701 J.

B;ol. bhem. 246, 180%i912 PETEJISON, P. A. (1971) J. Biol. Chem. 246, 34-43 PJSTJXZSON, P. A. (1971) J. Biol. Chem. 246, 44-49

REFERENCES

by guest on October 23, 2020

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nloaded from

7903

18. VAN JAARSVELD, P. P., EDELHOCH, H., GOODMaN, DEW. S., AND ROBBINS, J. (1973) J. Biol. Chem., 248,4698-4705

19. VAN JAARRVELL), P: P., BRANCH, W. T:, E&LHOCH, H., AND ROBBINS. J. (1973) J. Biol. Chem.. 248. 4706-4712

20. CLARKE, J.’ T. (1964j Ann. N. Y. A&d. ,ci. 121, 428-436 21. OPPENHEIMER, J. H., MARTINEZ, A., AND BERNSTEIN, G. (1966)

J. Lab. Gin. Med. 6’7, 500-509 22. MANCINI, G., CARBONARA, A. O., AAD HEREMANS, J. F. (1965)

Immunochemistry 2, 234-254 23. ROBBINS, J. (1956) Arch. Biochem. Biophys. 63, 461-469 24. YPHANTIS, D. A. (1964) Biochemistry 3, 297-317 25. COHN, E. J., AND EDSALL, J. T. (1943) Proteins, Amino Acids

and Peptides, Reinhold Publishing Corp., New York 26. SPACKMAN, D. H., STEIN, W. H., AND MOOI~E, S. (1958) Anal.

Chem. 30, 1190-1206 27. HIRS, C. H. W. (1956) J. Biol. Chem. 219, 611-621 28. LIU, T.-Y., AND CHANG, Y. H. (1971) J. Biol. Chem. 246, 2842-

2848

29. EASLEY, C. W., ZIGGERS, B. U. M., AND DEVIJLDER, M. (1969) Biochim. Biophys. Acta 176, 211-213

30. VAHLQUIST, A., AND PJ~TERSON, P. A. (1972) Biochemistry 11, 4526-4531

31. WEBIXX, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406- 4412

32. DUNKER, A. K., AND RUIXKERT, 1~. lt. (1969) J. Biol. Chem. 244, 5074-5080

33. HEDRICK, J. L., AND SMITH, A. J. (1968) Arch. Biochem. Bio- phys. 126, 155-164

34. VAHLQUIST, A., NILSSON, S. F., AND PETERSON, P. A. (1971) Eur. J. Biochem. 20, 160-168

35. PENSKY, J., AND MARSHALL, J. S. (1969) Arch. Biochem. Bio- phys. 135, 304-310

36. Pac~os, R. A., AND CBHNMANN, H. J. (1972) in Proceeclings of the Fourth International Congress of Endocrinology, Washing- ton, D.C., June l&94, p. 241

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Kanda and Robert E. CanfieldPieter van Jaarsveld, William T. Branch, Jacob Robbins, Francis J. Morgan, Yoshikazu

PARTIAL STRUCTUREPolymorphism of Rhesus Monkey Serum Prealbumin: PURIFICATION AND

1973, 248:7898-7903.J. Biol. Chem. 

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