Direct interaction between the catalytic subunit of the calmodulin-sensitive adenylate cyclase from bovine brain with 125I-labeled wheat germ agglutinin and 125I-labeled calmodulin

A calmodulin-sensitive adenylate cyclase has been purified to apparent homogeneity from bovine cerebral cortex using calmodulin-Sepharose followed by forskolin-Sepharose and wheat germ agglutinin-Sepharose. The final product appeared as one major polypeptide of approximately 135,000 daltons on sodium dodecyl sulfate-polyacrylamide gels. This polypeptide was a major component of the protein purified through calmodulin-Sepharose. The catalytic subunit was stimulated 3-4-fold by calmodulin (CaM) with a turnover number greater than 1000 min-1 and was directly inhibited by adenosine. The catalytic subunit of the enzyme interacted directly with 125I-CaM on a sodium dodecyl sulfate-polyacrylamide gel overlay system, and this interaction was Ca2+ concentration dependent. In addition, the catalytic subunit was shown to directly bind 125I-labeled wheat germ agglutinin using a sodium dodecyl sulfate-polyacrylamide gel overlay technique, and N-acetylglucosamine inhibited binding of the lectin to the catalytic subunit. Calmodulin did not inhibit binding of wheat germ agglutinin to the catalytic subunit, and the binding of calmodulin was unaffected by wheat germ agglutinin. These data illustrate that the catalytic subunit of the calmodulin-sensitive adenylate cyclase is a glycoprotein which interacts directly with calmodulin and that adenosine can inhibit the enzyme without intervening receptors or G coupling proteins. It is concluded that the catalytic subunit of adenylate cyclase is a transmembrane protein with a domain accessible from the outer surface of the cell.     

The interaction of Ca2+ with the calmodulin-sensitive adenylate cyclase from Bordetella pertussis

Bordetella pertussis, the etiologic agent of whooping cough, produces a calmodulin-sensitive adenylate cyclase which elevates intracellular cAMP in a variety of eucaryotic cells. Exogenous calmodulin added to the partially purified adenylate cyclase has been shown to inhibit invasion of animal cells by this enzyme (Shattuck, R. L., and Storm, D. R. (1985) Biochemistry 24, 6323-6328). In this study, several properties of the calmodulin-sensitive adenylate cyclase are shown to be influenced by Ca2+ in the absence of calmodulin. The presence or absence of Ca2+ during QAE-Sephadex ion exchange chromatography produced two distinct chromatographic patterns of adenylate cyclase activity. Two different forms of the enzyme (Pk1 and Pk2EGTA) were isolated by this procedure. Pk1 adenylate cyclase readily elevated intracellular cAMP levels in mouse neuroblastoma cells (N1E-115) while Pk2EGTA adenylate cyclase had no effect on cAMP levels in these cells. Gel exclusion chromatography of Pk1 adenylate cyclase gave apparent Stokes radii (RS) of 43.5 A (+/- 1.3) in the presence of 2 mM CaCl2 and 33.8 A (+/- 0.94) in the presence of 2 mM EGTA [( ethylenebis (oxyethylenenitrilo)]tetraacetic acid). These Stokes radii are consistent with molecular weights of 104,000 (+/- 6,400) and 61,000 (+/- 3,600), respectively. Pk2EGTA adenylate cyclase had an apparent RS of 33.0 (+/- 1.2) (Mr = 60,600 (+/- 2,800] in the presence of Ca2+ or excess EGTA. At 60 degrees C, Pk1 adenylate cyclase exhibited a Ca2+-dependent heat stability with a half-life for loss of enzyme activity of 10.3 min in 5 mM CaCl2 and a half-life of 2.8 min in the presence of 0.1 microM CaCl2. The stability of Pk2EGTA adenylate cyclase was not affected by changes in free Ca2+. The adenylate cyclase preparations described above were submitted to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and enzyme activity was recovered from gel slices by extraction with detergent containing buffers. The catalytic subunit isolated from SDS-polyacrylamide gels was activated 7-fold in the presence of Ca2+ with maximum activity observed at 1 microM free Ca2+. With both preparations, the apparent molecular weight of the catalytic subunit on SDS gels was 51,000 in the presence of 2 mM CaCl2 and 45,000 in the presence of 2 mM EGTA. The catalytic subunit of the enzyme was purified to apparent homogeneity by preparative SDS-polyacrylamide gel electrophoresis and resubmitted to SDS gel electrophoresis in the presence or absence of free Ca2+. The purified catalytic subunit also exhibited a Ca2+-dependent shift in its mobility on SDS gels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphorylation of lens membranes with a cyclic AMP-dependent protein kinase purified from the bovine lens

We report the phosphorylation of lens membranes with a cAMP-dependent protein kinase isolated from bovine lenses. The holoenzyme was eluted from DEAE agarose at less than 100 mM NaCl and from gel filtration columns with a relative molecular weight of 180 000. The regulatory subunit was identified with the affinity label 8-azido-[32P]cAMP. Four focusing variants with relative molecular weights of 49 000 were seen on two-dimensional gels. The catalytic subunit was purified approx. 5000-fold and migrated at 42 000 Mr on SDS gels. Based on these observations, the enzyme is classified as a Type I cAMP-dependent protein kinase. Purified lens plasma membranes were incubated with the holoenzyme or its catalytic subunit in the presence of 32P-labeled ATP. Several membrane proteins, including the major lens membrane polypeptide, MP26, were shown to be substrates for the kinase in this reaction. MP26 appears to be the major component of intercellular junctions in the lens. Studies with protease treatments on labeled membranes appeared to localize the phosphorylation sites to the cytoplasmic side of the membrane.     

Purification and characterization of a Ca2+/calmodulin-dependent protein kinase from rat brain

A soluble Ca2+/calmodulin-dependent protein kinase has been purified from rat brain to near homogeneity by using casein as substrate. The enzyme was purified by using hydroxylapatite adsorption chromatography, phosphocellulose ion-exchange chromatography, Sepharose 6B gel filtration, affinity chromatography using calmodulin-Sepharose 4B, and ammonium sulfate precipitation. On sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels, the purified enzyme consists of three protein bands: a single polypeptide of 51 000 daltons and a doublet of 60 000 daltons. Measurements of the Stokes radius by gel filtration (81.3 +/- 3.7 A) and the sedimentation coefficient by sucrose density sedimentation (13.7 +/- 0.7 S) were used to calculate a native molecular mass of 460 000 +/- 29 000 daltons. The kinase autophosphorylated both the 51 000-dalton polypeptide and the 60 000-dalton doublet, resulting in a decreased mobility in NaDodSO4 gels. Comparison of the phosphopeptides produced by partial proteolysis of autophosphorylated enzyme reveals substantial similarities between subunits. These patterns, however, suggest that the 51 000-dalton subunit is not a proteolytic fragment of the 60 000-dalton doublet. Purified Ca2+/calmodulin-dependent casein kinase activity was dependent upon Ca2+, calmodulin, and ATP X Mg2+ or ATP X Mn2+ when measured under saturating casein concentrations. Co2+, Mn2+, and La3+ could substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations. In addition to casein, the purified enzyme displayed a broad substrate specificity which suggests that it may be a "general" protein kinase with the potential for mediating numerous processes in brain and possibly other tissues.     

Immunological distinction between calmodulin-sensitive and calmodulin-insensitive adenylate cyclases

Previous studies using calmodulin-Sepharose affinity chromatography have suggested that bovine brain may contain a mixture of calmodulin-sensitive and -insensitive adenylate cyclase activities (Wescott, K. R., La Porte, D. C., and Storm, D. R. (1979) Proc. Natl. Acad. Sci. U.S.A. 82, 3086-3090). In this study, mice were immunized with a purified preparation of the calmodulin-sensitive adenylate cyclase from bovine brain, and a polyclonal antiserum was obtained which was specific to the calmodulin-sensitive form of the enzyme. The antiserum was not inhibitory and precipitated enzyme activity from a homogeneous preparation of the calmodulin-sensitive adenylate cyclase catalytic subunit. Furthermore, the antiserum did not interact with calmodulin-insensitive adenylate cyclase which was resolved from the calmodulin-sensitive form of the enzyme by calmodulin-Sepharose affinity chromatography. Since the only polypeptide specifically precipitated by the antiserum had an Mr of 135,000, which was identical to the Mr of the catalytic subunit of the enzyme, it is concluded that the antiserum interacted directly and specifically with the catalytic subunit of the calmodulin-sensitive isozyme of adenylate cyclase. Detergent-solubilized membranes from several rat tissues were examined for the presence of calmodulin-sensitive adenylate cyclase using anti-calmodulin-sensitive adenylate cyclase antiserum. Approximately 40-60% of the total adenylate cyclase activity of rat brain and kidney were immunoprecipitated by the antiserum, whereas liver and testes contained no detectable calmodulin-sensitive adenylate cyclase. Approximately 15% of the total adenylate cyclase activity in rat heart and lung was the calmodulin-sensitive form. These data indicate that the calmodulin-sensitive and insensitive adenylate cyclases from bovine brain are immunologically distinct and support the proposal that there may be two or more distinct adenylate cyclase isozymes in brain.     

Purification and studies of some physicochemical properties of glutamine synthetase of Neurospora crassa.

Glutamine synthetase (EC 6.3.1.2) of Neurospora crassa was purified to near homogeneity by chromatography on a glutamate-Sepharose affinity column. Its properties, including molecular weight, subunit structure, amino acid composition, and approximate alpha-helix content, have been examined. In the native state, this enzyme has been demonstrated by gel filtration to be an octamer of molecular weight 360,000 and as having a sedimetation coefficient of 13.2 S by sedimentation velocity measurements. Circular dichroism spectra in the far ultraviolet range suggest an approximate alpha-helix content of 23-24%. The subunit generated by treatment with urea was found to be 45,000 daltons by gel filtration methods and a molecular weight of 46,000 was calculated for the monomer obtained by sodium dodecyl sulphate (SDS) treatment and electrophoresis in SDS-polyacrylamide gels. Interprotomeric cross-linking experiments, using diimidoesters, suggest the presence of two noncovalently linked tetramers comprising the native octameric structure. Amino acid analyses revealed the presence of six tryptophans, four half cystines, and nine methionine residues per monomer of 45,000 daltons.     

Purification and properties of Bacillus subtilis aspartate transcarbamylase.

Aspartate transcarbamylase from Bacillus subtilis has been purified to apparent homogeneity. A subunit molecular weight of 33,500 +/- 1,000 was obtained from electrophoresis in polyarcylamide gels containing sodium dodecyl sulfate and from sedimentation equilibrium analysis of the protein dissolved in 6 M guanidine hydrochloride. The molecular weight of the native enzyme was determined to be 102,000 +/- 2,000 by sedimentation velocity and sedimentation equilibrium analysis. Aspartate transcarbamylase thus appears to be a trimeric protein; cross-linking with dimethyl suberimidate and electrophoretic analysis confirmed this structure. B. subtilis aspartate transcarbamylase has an amino acid composition quite similar to that of the catalytic subunit from Escherichia coli aspartate transcarbamylase; only the content of four amino acids is substantially different. The denaturated enzyme has one free sulfhydryl group. Aspartate transcarbamylase exhibited Michaelis-Menten kinetics and was neither inhibited nor activated by nucleotides. Several anions stimulated activity 2- to 5-fold. Immunochemical studies indicated very little similarity between B. subtilis and E. coli aspartate transcarbamylase or E. coli aspartate transcarbamylase catalytic subunit.     

Subunit structure and some properties of pyruvate kinase of Neurospora.

Pyruvate kinase isolated from Neurospora and purified to homogeneity has been shown to be a tetramer of molecular weight around 242 000 by gel filtration studies and 239 000 daltons by sedimentation equilibrium measurements. The monomer produced by treatment with guanidine hydrochloride is found to be 51 000-52 000 daltons by sedimentation equilibrium studies; a molecular weight of 62 000 was determined for the monomer generated by SDS treatment by electrophoresis in SDS-polyacrylamide gels. The enzyme has an isoelectric point of 6.35-6.41; Substrate saturation kinetics of PEP show a variable extent of cooperativity depending upon the buffer ions employed in the assay. ADP is the most effective phosphoryl group acceptor, GDP and IDP being poor substitutes. A divalent cation, Mg-2+, is required for activity. At low concentrations, Ca-2+ acts as an activator of pyruvate kinase but it is inhibitory at high concentrations. Fructose 1,6-diphosphate is the most potent allosteric activator, fructose 6-phosphate being next in order of effectiveness. Valine is a powerful inhibitor. Phenylalanine, tyrosine, and tryptophan are without any effect individually, but their simultaneous presence results in a considerable activation. Alanine does not affect this enzyme appreciably.     

Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum

Phospholamban, a putative regulator of the Ca2+-dependent ATPase of cardiac sarcoplasmic reticulum (SR), was purified from canine cardiac SR membranes. Cardiac SR was extracted with deoxycholate and fractionated with ammonium sulfate followed by gel permeation high performance liquid chromatography in the presence of the nonionic detergent, octa-ethylene glycol mono-n-dodecyl ether (C12E8), and KI. Further purification was achieved with CM-Sepharose CL 6B column chromatography in the presence of C12E8. The purified phospholamban showed a single band of 22,000 daltons on neutral sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412) and 27,000 daltons on alkaline SDS gels (Laemmli, U. K. (1970) Nature (Lond.) 227, 680-685). Boiling of phospholamban in 2% SDS produced total conversion into the lower molecular weight component on SDS gels (11,000 on Laemmli gel and 10,500 on Weber and Osborn gel). The apparent molecular weight of phospholamban on SDS gels was slightly increased by cAMP-dependent phosphorylation. The extent of phosphorylation catalyzed by cAMP-dependent protein kinase in the purified phospholamban preparations was about 42 nmol of phosphate/mg of protein when the protein concentration was determined by the method of Lowry et al. (Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275), or 138 nmol/mg of protein based on the protein concentration estimated by the dye absorption method. Rabbit antisera were prepared against purified phospholamban. The obtained antisera were found to bind to purified phospholamban as well as that in cardiac SR. No reaction was detected in fast skeletal muscle SR by immunofluorescent staining of Western blots. The present preparation of purified phospholamban and the antisera should facilitate further understanding of the regulatory action of phospholamban on the calcium pump ATPase.     

Purification, crystallization, and molecular properties of aspartase from Pseudomonas fluorescens

Aspartase [L-aspartate ammonia-lyase, EC 4.3.1.1] of Pseudomonas fluorescens was highly purified to homogeneity and crystallized. The purified enzyme sedimented as a monodisperse entity upon ultracentrifugation with a s0(20),w value of 8.6S. Upon polyacrylamide gel electrophoresis (PAGE), the enzyme migrated as a single band. The molecular weight of the native enzyme was 173,000 +/- 3,000, as determined by sedimentation equilibrium analysis, and that of the enzyme subunit was determined to be 50,000 +/- 1,500 by sodium dodecyl sulfate (SDS)-PAGE. Cross-linking experiments using dimethyl suberimidate followed by SDS-PAGE indicated that the native enzyme was composed of four subunits with identical molecular weight. The amino acid composition of the enzyme was determined.     

The isolation and characterization of the ATPase inhibitory protein (TN-I) from bovine cardiac muscle.

The inhibitory component of the troponin complex (TN-I) was purified from bovine cardiac muscle, using a combination of ion exchange and molecular exclusion chromatographies in the presence of urea. It has the ability to inhibit the Mg2+-activated APTase (EC 3.6.1.3) of a synthetic cardiac actomyosin preparation and this inhibition is reversed by the addition of cardiac calcium binding component of troponin (TN-C). Conventional sedimentation equilibrium experiments suggest a molecular weight for cardiac TN-I of 22 900 +/- 500. However, sodium dodecyl sulfate (SDS) gels indicate a molecular weight of 27 000 +/- 1000. The mobility of TN-I on SDS gels may be anomalous due to the high proportion of basic amino acid residues in the protein. Cardiac TN-I and TN-C interact to form a tight complex, even in the presence of 6 M urea. The results of this study invite direct comparison with results published for rabbit skeletal TN-I.     

5-Oxoprolinase from rat kidney, II. Molecular weight determinations.

The molecular weight of 5-oxoprolinase from rat kidney was estimated by gel filtration on Sephadex G-200 and G-150 to be 460 000 +/- 30 000. A value of 230 000 +/- 10 000 was obtained by zonal sedimentation in a sucrose gradient. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate yielded a molecular weightof 115 000 +/- 6 000. It is concluded that 5-oxoprolinase consists of four subunits of 115 000 daltons each. The dissociation or aggregation behavior of the enzyme seems to be influenced neither by the presence of the substrates 5-oxo-L-proline and MgATP2theta nor by the presence of the stabilizing compounds glutathione mercaptoethanol or dithioerythritol.     

Anomalous aggregation of Clostridium perfringens enterotoxin under dissociating conditions.

Polyacrylamide gel electrophoresis of highly purified Clostridium perfringens enterotoxin revealed electrophoretic microheterogeneity of the enterotoxin, apparently because of slight charge differences in the peptides. Detergent gel electrophoresis showed that purified enterotoxin formed high molecular weight aggregates in the presence of both sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide. No conditions capable of inhibiting this phenomenon were found. Although a molecular weight of 35 000 daltons has been reported in the literature, the experimentally determined molecular weight values in the presence of detergents corresponded to multiples of a theoretical subunit molecular weight of 17 500 daltons. Binding studies performed by equilibrium dialysis and ultracentrifugation methods revealed that the enterotoxin bound very small amounts of SDS per gram of protein. The evidence presented indicates possible detergent induced structural alterations of the protein.     

Purification of RNAase II by preparative polyacrylamide gel electrophoresis.

Purification of RNAase II to electrophoretic homogeneity is described. The exonuclease is activated by K+ and Mg2+ and hydrolyses poly(A) to 5'-AMP, exclusively as described by Nossal and Singer (1968, J. Biol. Chem. 243, 913--922). To separate RNAase II from ribosomes, DEAE-cellulose chromatography was used. Two additional chromatographic steps give a preparation that yields 10 bands after analytical polyacrylamide gel electrophoresis. Preparative polyacrylamide gel electrophoresis resulted in a final preparation which on analytical polyacrylamide gels gives a single band. A molecular weight of 76 000 +/- 4000 was obtained from Sephadex G-200 chromatography, with three bands from sodium dodecyl sulfate (SDS) denaturation and SDS gel electrophoresis. The subunits have a molecular weight of 40 000 +/- 2000, 33 000 +/- 2000, and 26 000 +/- 1000. The enzyme thus appears to consist of three dissimilar subunits.     

Cobalt-dependent protein phosphatases from human cord blood erythrocytes. I. Submolecular structure and regulation of activity of E3 casein phosphatase

We have identified three phosphoprotein phosphatases in the cytosol of human cord blood erythrocytes by sequential anion-exchange chromatography and gel filtration. The most abundant was E3 protein phosphatase. After rechromatography on a column of Ultrogel AcA-44 the enzyme had a molecular weight of 95,000 daltons. According to the data obtained by SDS/PAGE, the 95,000-dalton form was composed of non-identical subunits with a molecular mass of 23,000 and 16,000 daltons. Since ethanol decreased the molecular mass of the 95,000-dalton enzyme to 25,000 daltons, we suggest that the protein of 23,000-25,000 daltons represents the catalytic subunit. The decrease in the molecular weight is followed by a 2-fold increase in the Vmax value and by a change in kinetics: the negatively cooperative 95,000-dalton enzyme (h = 0.45) transforms into Michaelis-Menten kinetics (h = 1.0) in the 25,000-dalton form. Both molecular forms, 95,000 and 25,000 daltons, only dephosphorylated casein but not phosvitine and histones. Both forms were activated by CoCl2 and inhibited by organic, and most potently, by inorganic pyrophosphates to approximately the same degree. As opposed to the inorganic pyrophosphate, which affects the catalytic properties of the enzyme molecule, CoCl2 did not affect the catalytic properties of the enzymes, but it probably did affect the rate of 'E-S' complex formation. CoCl2 protected the 95,000-dalton enzyme from pyrophosphate inhibition. The data indicate that CoCl2 and pyrophosphate may take part in the regulation of the activity of both forms of E3 phosphatase.     

Influence of salts on the activity and the subunit structure of ornithine decarboxylase from rat liver

The influence of salts on the subunit structure and the kinetics of purified rat ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) was examined. Salts were found to cause subunit dissociation of the enzyme, producing the monomeric form of molecular weight 55 000 in the presence of 0.25 M NaCl/10 mM sodium phosphate buffer (pH 7.0): the molecular weight was estimated to be 150 000 in 10 mM and 250 000 in 1 mM sodium phosphate buffer. Inclusion of NaCl in kinetic assays of rat ornithine decarboxylase had little effect on maximal velocity. However, the Km value for L-ornithine was dramatically increased with increasing sodium chloride concentration: the presence of 0.25 M NaCl resulted in a 10-fold increase of the Km. Thus, the presence of salts caused dramatic changes both in the subunit structure and in the catalytic property of the enzyme, although a direct correlation between both the changes was not evidenced.     

Rat hair follicle and epidermal transglutaminases. Biochemical and immunochemical isoenzymes

Epidermal and hair follicle transglutaminases (1,4-alpha-D-glucan: orthophosphate alpha-D-glucosyltransferase EC 2.4.1.1) were differentially isolated and subsequently purified from newborn or 4-5-day-old rats. Both enzymes migrated identically on ion-exchange chromatography but were widely separated by block electrophoresis, with the epidermal enzyme migrating further toward the anode. Each enzyme was finally purified by gel filtration. Epidermal transglutaminase had an apparent molecular weight of 56 000-58 000 in this medium and in gels containing sodium dodecyl sulfate (SDS), while hair follicle transglutaminase had a molecular weight of 52 000-54 000 and was reduced to two apparently identical subunits of a molecular weight of 27 000 by denaturing media. Antiserum specific to each transglutaminase was produced in chickens; when conjugated to fluorescein these antisera localized the enzymes to the granular layer of epidermis and the inner root sheath of follicles, respectively.     

Quaternary structure of NAD-kinase from rabbit liver

A procedure for isolation of NAD-kinase from rabbit liver resulting in 4000-fold purification and the activity yield of 50-60% is described. The molecular weight of the NAD-kinase subunit determined by SDS electrophoresis is 30 000. The purified enzyme is a dimer. Partially purified preparations of NAD kinase contain multiple forms with mol. Weights ranging from 650 000 to 180 000 and have complex kinetic behaviour. A thermostable activator of NAD-kinase which, when added to the homogeneous enzyme preparation, destroys the linear dependence of the enzyme specific activity on concentration, was detected. The nature of multiple forms of NAD-kinase and the possible role of the activator in their formation is discussed.     

Affinity purification and some properties of nucleoside diphosphatase from rat liver cytosol.

Nucleosidediphosphatase (nucleosidediphosphate phosphohydrolase, EC 3.6.1.6) of rat liver cytosol was purified up to 336--fold by the procedure including affinity chromatographies of concanavalin A- and alanine-Sepharose. The final purified enzyme showed a single protein band upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its native form was found to be a tetramer with molecular weight of 120 000 which consists of subunit with molecular weight of 30 000. The enzyme was found to be a glycoprotein on the basis of its chromatographic behaviour with concanavalin A-Sepharose and positive staining with periodate-Schiff reaction in polyacrylamide gels. Furthermore, the two molecular forms with isoelectric points of 4.7 and 5.0 were demonstrated by electrofocusing, though they did not show any significant difference with respect to their catalytic properties.     

The apparent molecular weight of androgen-binding protein (ABP) in the blood of immature rats differs from that of ABP in the epididymis

When androgen-binding protein (ABP) in unfractionated immature (20-day old) male rat serum was covalently labeled with the site-specific photoaffinity ligand [3H]17 beta-hydroxy-4,6-androstadien-3-one and analyzed on 5.6% polyacrylamide tube gels containing SDS (SDS-PAGE), a protein of Mr 33,700 +/- 1200 was shown to be specifically labeled. Rat epididymal ABP from unfractionated cytosol analyzed under identical conditions exhibited two androgen-specific peaks of radioactivity, Mr 49,900 +/- 600 and Mr 44,100 +/- 800, which correspond to the previously described subunits of ABP. The apparent molecular weight differences between serum and epididymal ABP were further assessed on preparations of serum ABP that had been partially purified by chromatography on Affi-Gel blue (to remove albumin) and on Sephadex G-150 (to remove other proteins). When these preparations of ABP were photolabeled and analyzed by SDS-PAGE as above, two subunits of Mr 61,700 +/- 1300 and Mr 47,100 +/- 700 were resolved. Serum and epididymal ABP were further purified by androgen affinity chromatography. When these preparations were subjected to SDS-PAGE on slab gels containing 10% polyacrylamide and identified by fluorography of photolabeled ABP or by immunochemical localization following electrophoretic transfer to nitrocellulose, differences in the apparent molecular weight of ABP from the two sources persisted. Immunochemical localization studies on ABPs that had been desialylated with neuraminidase indicated that there was an increased mobility of the subunits, as one would anticipate from removal of carbohydrate. Differences in apparent molecular weight of ABPs from the two sources are likely due to differences in glycosylation.