Prostate alpha-protein. Isolation and characterization of the polypeptide components and cholesterol binding.

alpha-Protein, a major glycoprotein in the cytosol fraction of rat ventral prostate, has a molecular weight of about 50,000 and can be dissociated, by sodium dodecyl sulfate, into two different subunits (A and B). alpha-Protein has three different polypeptide components with apparent molecular weights of 10,000 (I), 14,000 (II), and 15,000 (III). These components were purified to homogeneity and their amino acid compositions were determined. Subunit A is composed of Components I and III, whereas subunit B is composed of Components II and III. Carbohydrate was detectable only on Component III. Component III isolated from subunit A and Component III isolated from subunit B appear to be identical. The purified alpha-protein contains 0.7-1 mol of cholesterol/mol of protein. If cholesterol was removed by acetone, about 1 mol of 5 alpha-dihydrotestosterone or pregnenolone could bind to 1 mol of alpha-protein. In the presence of 2 mM ZnCl2, alpha-protein can form dimers and tetramers. In cell-free systems, alpha-protein can inhibit binding of the androgen-receptor complex to nuclear chromatin and also can promote the release of the complex already bound to chromatin. This effect is due to polypeptide Component I.

a-Protein, a major glycoprotein in the cytosol fraction of rat ventral prostate, has a molecular weight of about 50,000 and can be dissociated, by sodium dodecyl sulfate, into two different subunits (A and B). a-Protein has three different polypeptide components with apparent molecular weights of 10,000 (I), 14,000 (11), and 15,000 (111). These components were purified to homogeneity and their amino acid compositions were determined. Subunit A is composed of Components I and 111, whereas subunit B is composed of Components I1 and 111. Carbohydrate was detectable only on Component 111. Component I11 isolated from subunit A and Component I11 isolated from subunit B appear to be identical.
The purified a-protein contains 0.7-1 mol of cholesterol/mol of protein. If cholesterol was removed by acetone, about 1 mol of 5a-dihydrotestosterone or pregnenolone could bind to 1 mol of a-protein. In the presence of 2 m~ ZnC12, a-protein can form dimers and tetramers.
In cell-free systems, a-protein can inhibit binding of the androgen-receptor complex to nuclear chromatin and also can promote the release of the complex already bound to chromatin. This effect is due to polypeptide Component I.
In 1971, we isolated a cytosol protein from the rat vental prostate which could interfere with the association of the androgen-receptor complex with isolated cell nuclei (1). Since this protein can bind androgens and other steroids, we named it "a-protein" to distinguish it from the androgen receptor protein that was called "P-protein." a-Protein may be identical with a glycoprotein studied by others in recent years. This glycoprotein is a major secretory product of the rat ventral prostate (2-5). Since the synthesis of this protein is under the electrophoresis, the protein has been considered to have two subunits (A and B), each containing two peptide chains that are covalently linked (4,(9)(10)(11).
In this article, we will describe methods for isolation of the individual polypeptide chains that form the two subunits of a-protein. We will also show that the major ligand associated with the protein is cholesterol. One of the acidic polypeptide components of a-protein is also identified as the active unit that can inhibit binding of the androgen-receptor complex to nuclear chromatin. 53-androstan-3-one; androstenedione, 4-androstene-3,17-dione; preg-The costs of publication of this article were defrayed in part by the nenolone, 3p-hydroxypregn-5-en-20-one; epitestosterone, l7u-hydroxpayment of page charges.
This article must therefore be hereby yandrost-4-en-3-one; androstanedione, androstane-3.17-dione; anmarked "advertisement" in accordance with 18 U.S.C. Section 1734 drostanediols, 3a-(or 3p) 17-p-dihydroxyandrostane; androstenediols, solely to indicate this fact. for the purification of a-k .tein and its components are shown in Fig. 1. Since a-protein accounts for about 35% of total cytosol proteins, it can be isolated easily by fractionating cytosol proteins by DEAE-Sephacel chromatography (Fig. 2, miniprint). A small amount of subunit B that was associated with a-protein could be removed by DEAE-Sephadex chromatography (Fig. 3, miniprint). The two subunits of a-protein could be separated by hydroxylapatite chromatography of the a-protein preparation. Subunits A and B were eluted from the column by 0.2 and 0.3 M sodium phosphate buffer, respectively (Fig. 4, miniprint). Trace contaminants associated with these subunits could be eliminated by Sephacryl gel and concanavalin A-Sepharose chromatography. Both subunits A and B contain carbohydrate and are retained by concanavalin A-Sepharose columns but are eluted from the column with 0.2 M a-methylmannoside.
Both subunits A and B yield two protein bands when analyzed by PAGE in the presence of SDS and j3-mercaptoethanol or dithiothreitol. One of the components, as judged by the migration pattern during electrophoresis under various conditions, appears to be common to the A and B subunits. We therefore designated the components in A as Components I and 111, and those in B as Components I1 and I11 (Fig. 5).
Isolation of Component Proteins-The component proteins could be isolated from the individual subunits by exposing the subunits to urea and dithiothreitol and fractionating the com-  ponents by DEAE-Sephacel chromatography (Fig. 1). However, we found that the following procedure could provide a better yield of the components (60-80s from a-protein) in a reproducible manner. In this alternative procedure, a-protein is chromatographed on a DEAE-Sephacel column in the presence of urea and dithiothreitol. A protein component eluted from the column by 0.05 M NaCl (Fig. 6) is identified by PAGE as Component 111. Components I and I1 are eluted together from the column by 0.15-0.25 M NaCl. These two components are separated by hydroxylapatite chromatography in the presence of SDS (Fig. 7). Minor proteins associated with these components could be removed by passing the protein preparations through a Sephadex G-100 column in the presence of SDS (results not shown).
Physical and Chemical Properties-The PAGE patterns of various purified protein fractions are shown in Fig. 5. In nondenaturing gels (gel l ) , purified a-protein gives three bands in the order of subunit A, a-protein, and subunit B from the anode (bottom end of the gel). In the presence of SDS but without p-mercaptoethanol, a-protein migrates as two bands (gel 2) having mobilities identical with that of purified subunits A and B (not shown). In the presence of SDS and pmercaptoethanol, purified a-protein exhibits three bands (gel 3) that migrate to the same position as Components I (gel 6), I1 (gel 7), and I11 (gel 8). Subunits A (gel 4) and B (gel 5), respectively, show two bands that correspond to Components I and I11 (for A) and Components I1 and I11 (for B). From SDS-PAGE, the apparent molecular weights of the component proteins are: 10,000 (I), 14,000 (II), and 15,000 (111). As shown in the accompanying paper (34), the molecular weight of Component I, as determined by the amino acid sequence, is 10,191.
The molecular weights of a-protein and subunits A and B, as determined by co-chromatography of these proteins labeled with '251 and standard proteins on a Sephadex G-150 (superfine) gel column, are 50,000, 24,000, and 26,000, respectively. In the presence of 5 m~ EDTA, the fractionation pattern was not changed. In the presence of 2 m~ ZnClz (in 20 m~ Tris-HCl buffer, pH 7.5), a-protein appears to dimerize and tetramerize to give radioactive peaks with molecular weights of about 90,000 and 180,000.
Carbohydrate analysis showed that a-protein has no detectable sialic acid. Of the three protein components, only Component I11 appears to contain carbohydrate detectable by the methods we employed (Table I, miniprint). The amino acid compositions of a-protein and its subunits and components show that they are rich in aspartic acid and glutamic acid (Table 11, miniprint), facts which are consistent with the low isoelectric points of the various components (Table I,  miniprint).
Biochemical Properties- Fig. 8 shows the ability of a-protein and its subunits and components to inhibit the retention of the radioactive androgen-receptor complex by isolated pros-  Fig. 5) was chromatographed on a hydroxylapatite coLnn (2.6 X 12 cm). Proteins were eluted by a h e a r gradient (300 ml) of 0.01-0.5 M sodium phosphate containing 0.1% SDS and 0.1% /3-mercaptoethanol. Fractions of 2.1 ml were collected. The first absorbance peak (fractions 20-50) was due to excess /3-mercaptoethanol in the protein sample. The second and third peaks were due to Components I and 11, respectively. tate cell nuclei. Subunit A was active, but subunit B was not. The inhibitory activity of subunit A appears to be due to Component I, since Component I but not Component 1 1 1 is inhibitory. In fact, on a molar basis, Component I is about 5 times more active than subunit A. a-Protein freshly isolated from the prostate could bind less than 0.2 mol of radioactive androgen per mol of protein. If the purified protein was delipidated by acetone, the steroid-binding capability of the protein was significantly increased. At steroid concentrations higher than 5 PM, about 1 mol of 5adihydrotestosterone could bind to 1 mol of protein. The association constants (K,) for delipidated a-protein toward various steroids are: androstenedione, 1.15 X lo6 M-'; pregnenolone, 1.1 X IO6 M-'; 5a-dihydrotestosterone, 0.87 X lo6 M-'; cholesterol, 0.64 X lo6 M-'; testosterone, 0.58 X lo6 M-'; and epitestosterone, 0.53 X lo6 M". As described before for the crude preparation, the purified a-protein does not bind cortisol. On a molar basis, subunit B appears to bind steroid nearly as well as a-protein, but steroid binding by subunit A is feeble.

DEAE-Sephacel chromatography -horn in
Of the three components, only Component I1 shows a signifcant steroid binding activity, but a detailed study is difficult because of the insolubility of this component in aqueous solution without SDS.
When a-protein was extracted with acetone or chloroform and the organic solvent extract was analyzed by thin layer chromatography, we found a major iodine-stainable spot that co-migrated with authentic cholesterol. On the thin layer chromatogram, we also found two minor iodine-stainable spots (less than lo%), but these minor components were not androstenedione, androstanedione, 5a-dihydrotestosterone, testosterone, androstanediols, or androstenediols. This is in line with the fact that the lipid extract was negative in the Zimmerman test (35). Analysis of the organic solvent extract of a-protein by a gas chromatograph-mass spectrometer also showed that there was only one major compound and that this major compound has a mass spectrum identical with that To assure that exclusive binding of cholesterol by a-protein was not due to the exposure of the protein to much higher concentrations of cholesterol than of other sterols during the isolation, we added tritiated cholesterol, pregnenolone, or 5adihydrotestosterone to the minced prostate before homogenization. By comparing the total cholesterol and radioactive steroids bound to isolated a-protein, we could conclude that at least 80% of cholesterol binding occurs before prostate cells are disrupted by homogenization.
In a previous article (lo), we noted that a-protein can bind radioactive spermine at pH 8.7 but not at pH 7.5. At pH 8.7, a-protein or a mixture of subunits A and B, but not subunits A or B alone, can bind spermine weakly ( K , < lo6 M").
Besides spermine, purified a-protein also binds cadaverine, spermidine, and putrescine without notable specificity. This is in contrast with the androgen-sensitive spermine-binding protein that is highly specific toward spermine and does not bind other polyamines well (33). Spermine binding by aprotein is not affected by 1 IJM 5a-dihydrotestosterone.
Immunological comparison of the various components of a-protein by double diffusion on agar plates using antibodies to subunit A revealed that Components I and I1 are antigenically similar (fused precipitin arcs). An immunological analysis of Component I11 isolated from subunits A and B revealed that these components are also antigenically related. Comparison of Components I and I1 with Component I11 revealed antigenic dissimilarity (crossed precipitin arcs).

DISCUSSION
Since the presence of a-protein in the rat ventral prostate was fist described by us (1, 22), several investigators have described major secretory proteins in this organ. These proteins were called prostatein, by Lea et al. (4), prostatic binding protein by Heyns and De Moor (2), and estramustine binding protein by Forsgren et al. ( 3 ) . A similar steroid binding protein was also described by Ichii (36). Although there are distinct differences in the estimated sizes of these proteins and their subunit components and in the steroid-binding affinities, some or all of these proteins may be identical with a-protein. A definite identification, however, cannot be made, since the protein components for these proteins have not been isolated in large amounts in pure forms for chemical analysis and for comparison with the a-protein components.
How the two subunits A and B interact with each other is not clear. During the establishment of the purification procedure, we found that Component I1 could associate with Component I through various chromatographic processes if SDS was not present; the Components I and 11, therefore, may form a core unit but are individually linked to carbohydratecontaining Component I11 by disulfide linkages (Fig. 5).
Heyns et al. (9) also isolated the two subunits of prostatic binding protein and showed, by PAGE, that they contain dissimilar polypeptide components. Our present data, based on the isolation of the individual polypeptide components and their amino acid compositions, also clearly show that Component I in subunit A and Component I1 in subunit B are clearly different. These observations are in contrast to the conclusion of Lea et al. (4) that "prostatein" contains two identical subunits (4). Judging from electrophoretic mobility, the two subunits of a-protein contain a common polypeptide (Component 111). The same conclusion was also obtained for "prostatic binding protein" (9). To provide additional support that Component I11 in subunits A and B is identical, we have isolated the component from the individual subunits. The electrophoretic mobility and the amino acid composition of the two preparations are essentially identical. They also have the same NH2-terminal sequence (Ser-Gly-Ser-Gly-) and are antigenically related.
The biological role of a-protein is not clear. It is the major protein in the prostate secretory fluid and in the cytosol fraction of the rat ventral prostate. Using antibody raised in rabbits with a-protein and subunit A, we have found that only the dorsal and lateral prostate contain significant amounts of immunologically cross-reactive protein. However, the a-protein level in these prostate lobes was less than 5% of that in the ventral lobe which we used in this study. No traces of immunologically cross-reactive proteins are detectable in the cytosol preparations of rat seminal vesicle, liver, kidney, testis, brain, and spleen, as well as human prostate. a-Protein, therefore, is a species-and organ-specific protein that may be useful as a marker protein for rat vental prostate.
Since a-protein binds steroids with an affinity several orders of magnitude lower than the androgen receptor in the same organ (1, 12), it is unlikely that a-protein can compete well with the receptor for binding active androgens. a-Protein may, however, compete well with other steroid metabolizing enzymes and retard metabolic transformation of steroids. a-Protein may also play a role in steroid accumulation in cells and extracellular spaces of the prostate. Steroids that are not recognized by the steroid receptor in the prostate may, with the help of a-protein, be retained or transported. The fact that a-protein, freshly isolated and not treated with acetone, contains no other steroid but cholesterol in nearly stoichiometric amounts is interesting, since the synthesis of cholesterol (37) and the secretory protein (38) in the rat ventral prostate are both enhanced by androgens. Whether a-protein plays a role in the synthesis, accumulation, and/or secretion of cholesterol in the prostate is an intriguing question.
Although a-protein was fiist identified in this laboratory as a steroid-binding protein that can prevent retention of the androgen-receptor complex by nuclei in vitro, the steroid-binding property is not responsible for the inhibitory effect (11). Our present study clearly shows that the inhibitory activity is due mainly to Component 1 that does not bind steroid but is 5 times more active than a-protein or subunit A.
If such an inhibitory effect does play a regulatory role in the interaction of androgen-receptor complex with nuclear chromatin in the intact cells, as we have envisioned previously (10, 11), the active unit may be Component I itself or a smaller but more potent oligopeptide derived from the component. Since the secretory component may be present in the endoplasmic reticulum adjacent to the nuclear membrane, the inhibitory unit may have to enter the nuclei through nonporous areas of the envelope or by nuclear blebbing that has been indicated to play a role in nucleocytoplasmic exchanges (39-41).