Purification, properties, and immunological characterization of folate-binding proteins from human leukemia cells

A new matrix for affinity chromatography using pteroylglutamic acid coupled to an epoxy-activated matrix via hexanediamine resulted in negligible ligand leakage and permitted the purification of soluble and membrane-associated folate-binding proteins from human leukemia cells contained in a human spleen. Two species of membrane-associated folate-binding proteins were purified from the solubilized membrane fraction of the tissue using 2 M guanidine-HCl to elute the proteins from the affinity matrix. The higher molecular weight binding protein had an Mr of approximately 310,000 and the smaller species had an Mr of approximately 28,000 by gel filtration. By SDS-polyacrylamide gel electrophoresis the smaller species of membrane-associated protein had a molecular weight of 35,500, but the molecular weight of the larger membrane-associated species could not be determined by this method because of the high concentration of residual Triton X-100 in the sample which interfered with the silver staining of the gel. Two folate-binding proteins, which by SDS-polyacrylamide gel electrophoresis had molecular weights of 34,500 and 32,000, were purified from the 44,000 X g supernatant fraction of the tissue homogenate by acid elution from the affinity matrix. Despite the different cell components from which the soluble and membrane-associated folate-binding proteins were purified, the amino acid compositions were similar, especially with respect to the apolar amino acids. All these forms of folate-binding proteins had higher affinity for oxidized than for reduced folates, and very low affinity for 5-formyltetrahydrofolate and methotrexate. Although these proteins cross-react with one antiserum raised previously to a folate-binding protein from other human leukemia cells, they do not cross-react with the folate-binding proteins purified from two other sources of human leukemia cells, from human placenta, or from the human KB cell line.     

The isolation, characterization, and comparison of the membrane-associated and soluble folate-binding proteins from human KB cells

Human nasopharyngeal epidermoid carcinoma (KB) cells contain a membrane-associated particulate folate-binding protein which is important in the cellular accumulation of physiologic folates (Antony, A. C., Kane, M. A., Portillo, R. M., Elwood, P. C., and Kolhouse, J. F. (1985) J. Biol. Chem. 260, 14911-14917) and in the binding of methotrexate (Kane, M. A., Portillo, R. M., Elwood, P. C., Antony, A. C., and Kolhouse, J. F. (1986) J. Biol. Chem. 261, 44-49). A soluble folate-binding protein appears in media exposed to proliferating KB cells. We have purified to homogeneity both the membrane-associated and the soluble folate-binding proteins from the KB cell tissue culture system. The purified membrane-associated and soluble folate-binding proteins give single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent Mr values of 50,000 and 40,000, respectively. The membrane-associated folate-binding protein contains 45,000 g of amino acids and the soluble folate-binding protein contains 24,000 g of amino acids per mole of folate bound. Each of the purified proteins has a single folate-binding site, and the carbohydrate content is approximately 25% for each species of protein. The affinity constants for 5-methyltetrahydrofolate of the membrane-associated and soluble folate-binding proteins are 0.3 and 2.5 X 10(9) liters/mol, respectively. The affinities of various polyglutamated forms of methotrexate are similar for each protein, increase as the chain length of the polyglutamate increases (from approximately 0.004 X 10(9) liters/mol for methotrexate to 0.3 X 10(9) liters/mol for methotrexate heptaglutamate), are equal to the affinity for 5-methyltetrahydrofolate, and exceed the reported increase in affinity of methotrexate polyglutamates for dihydrofolate reductase.     

The interrelationship of the soluble and membrane-associated folate-binding proteins in human KB cells

Human KB cells produce two immunologically cross-reactive folate-binding proteins: a particulate cell-associated protein which is solubilized by Triton X-100, and a soluble protein which is released into their growth medium. This compartmentation of these two folate-binding proteins provides a convenient system for studies of their biochemical relationship. The two folate-binding proteins behave similarly to the purified particulate and soluble folate-binding proteins of human milk in analysis by radioactive folate binding, Sephacryl S-200 gel filtration profiles, polyacrylamide gel electrophoresis in either Triton X-100 or sodium dodecyl sulfate, and in Triton X-100 binding based on sucrose density gradient ultracentrifugation in H2O and D2O. The two folate-binding proteins were endogenously labeled by pulsing methionine-starved KB cells with [35S]methionine, and each protein was purified to apparent homogeneity by affinity chromatography at different times during the chase with nonradioactive methionine. The time course of the changes in specific activity (moles of [35S]methionine per mole of folate-binding protein) revealed a more rapid initial rate of synthesis and an earlier maximum in specific activity for the cell-associated folate-binding protein than for the soluble folate-binding protein released into the growth medium. Differences in the levels and specific activities of the two folate-binding proteins of cells exposed to cycloheximide compared with simultaneous controls after pulsing with [35S]methionine suggest that, whereas the cell-associated folate-binding protein is probably produced by de novo protein synthesis, the soluble folate-binding protein seems to be produced from a cellular pool of an already synthesized protein. These results combined with the immunologic cross-reactivity of the two folate-binding proteins strongly suggest a precursor-product relationship between them.     

Characterization of the folate-binding proteins associated with the plasma membrane of rat liver

Unsaturated folate-binding proteins (i.e., apo forms) have been identified with the plasma membranes of rat liver by the binding of [3H]pteroylglutamic acid. Normal rat liver contains very little of the folate-binding apoproteins, but the folate-binding capacity increases substantially when the rats are made folate-deficient. This increase appears to be due to unsaturation of the folate-binding holoproteins rather than to synthesis of additional protein, because the binding capacity of the plasma membranes from normal rat liver following dissociation of the bound folate is equivalent to the binding capacity of the preparation from folate-deficient liver. Two molecular forms of folate-binding protein were identified by gel filtration of the solubilized plasma membrane fraction, a high-molecular-weight form (Mr less than 100,000), representing 25% of the binding capacity, and a smaller protein (Mr approximately equal to 55,000), representing 75% of the binding capacity. Whereas the larger species can be solubilized only with a detergent, the smaller form appears to be hydrophilic and dissociates spontaneously from the membrane preparation. The binding of [3H]pteroylglutamic acid by the membrane preparation was specific, saturable, and pH- and temperature-dependent. Scatchard analysis of the binding could be fitted to a curvo-linear plot, indicating at least two orders of binding sites which probably correspond to the two molecular forms identified by gel filtration. Competitive inhibition by folate analogues demonstrated that the apoproteins have higher affinity for oxidized folate than for N5-methyltetrahydrofolate and virtually no affinity for N5-formyltetrahydrofolate or methotrexate.     

Isolation and characterization of a folate receptor from human placenta

While folate binding proteins have been described in serum and a variety of tissues, the function of these proteins is unknown. A particulate folate binding protein from human placenta has been isolated and characterized following solubilization with Triton X-100. The protein was purified 61,000-fold using affinity chromatography on pteroylglutamic acid-Sepharose as the major purification step. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified protein gave a single band with a Mr = 38,500. Stoichiometry of binding indicated that 1 mol of folate was bound per mol of protein. The protein was a glycoprotein that contained 12% carbohydrate. Antiserum was raised in a rabbit, and on immunodiffusion, gave a single precipitin line with the purified placental folate binding protein. Immunoprecipitation studies using this antiserum indicated that the purified placental folate binding protein shared antigenic determinants with both the large Mr and small Mr folate binding proteins from human milk. Immunofluorescent studies with this antiserum and human erythrocytes revealed the presence of an immunologically similar protein on the plasma membrane of these cells suggesting that this protein may function as a folate receptor.     

Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone

A phosphorylated glycoprotein was purified from the mixture of proteins extracted by demineralization of rat bone with 0.5 M EDTA in 4 M guanidinium chloride. A high level of purity for the preparation was indicated by a single band on sodium dodecyl sulfate (SDS)-gradient gel electrophoresis, sedimentation equilibrium ultracentrifugal data, and by automated Edman degradation results. The molecular weight of the phosphoprotein was shown to be about 44,000 by sedimentation equilibrium analyses in 4 M guanidinium chloride, even though an Mr of 75,000 was obtained by 5-15% SDS-polyacrylamide gel electrophoresis. Subsequent analysis by 15% SDS-polyacrylamide gel electrophoresis gave an Mr of 45,000. Analytical data showed that the protein contained 16.6% carbohydrate, possibly including 1 N-linked oligosaccharide and 5-6 O-linked oligosaccharides. The aspartic acid- and glutamic acid-rich protein contained about 300 amino acid residues including 1 phosphothreonine and 12 phosphoserine residues. Alkaline beta-elimination/NaBH4 reduction data showed that the phosphate obtained by complete acid hydrolysis prior to amino acid analysis was equivalent to the phosphate subject to alkaline beta-elimination. In this experiment, the losses of serine plus threonine exceeded the amount of phosphate liberated by 5-6 residues/protein. These serine and threonine residues probably represent O-linked oligosaccharides, since the protein contained about this number of N-acetyl-galactosamine residues. That the phosphoprotein is synthesized and secreted by osteoblast-like cells was shown with cultures of clonal rat osteosarcoma cells. After pulsing with 32PO4 the proteins secreted into the medium were precipitated with trichloroacetic acid and the radiolabeled proteins were immunoadsorbed. A protein migrating in the same position, on 5-15% SDS-polyacrylamide gel electrophoresis (i.e. with an Mr = 75,000) and on 15% gels (Mr = 45,000), as the phosphoprotein obtained from bone could be specifically immunoprecipitated.     

Affinity labelling of the folate-binding protein in pig intestine.

A specific transport system for folate and a high-affinity folate-binding protein have been identified in pig intestinal brush-border membranes. To determine if the binding protein plays a role in folic acid (PteGlu) uptake in to the cell, the inactivation of folate binding and transport by N-hydroxysuccinimide esters of folic acid (NHS-PteGlu) was compared. In addition, the number of brush-border proteins modified by the affinity reagent was assessed. Brush-border vesicles were incubated with various concentrations of NHS-PteGlu or NHS-methotrexate. Transport and binding of [3H]PteGlu by the vesicles were measured at 37 and 4 degrees C respectively by using the vacuum-filtration technique. NHS-methotrexate and NHS-PteGlu specifically inhibited PteGlu transport. Incubating the vesicles with 1 microM-NHS-PteGlu inactivated [3H]PteGlu transport by 60% and binding by 80%. Half-maximal inhibition of both transport and binding was observed at similar concentrations of the affinity reagent (0.05 and 0.07 microM-NHS-PteGlu respectively). Treating the vesicles with radiolabelled NHS-PteGlu followed by gel electrophoresis and autoradiography revealed a specifically labelled protein with an Mr of 56,000. These results indicate that the intestinal folate-binding and transport proteins are identical and that the function of the folate-binding protein is to transport folate into the cell.     

Homologous membrane folate binding proteins in human placenta: cloning and sequence of a cDNA

A preparation of folate binding protein purified from human placental membranes in the presence of a variety of protease inhibitors followed by deglycosylation with N-glycanase gave a sharp band at Mr approximately 28,000 following SDS-polyacrylamide gel electrophoresis. The deglycosylated protein bound [3H]folic acid as tightly as the native protein. Peptides obtained following digestion of the purified protein with staphylococcal V8 protease and HPLC purification were sequenced. Polyclonal antibodies against the protein preparation were affinity purified and used to screen a placental cDNA expression library. A full-length cDNA for a placental folate binding protein was thus obtained and the corresponding protein sequence deduced. This result, taken together with the peptide sequence data, indicates the expression of at least two homologous folate binding proteins in placenta, one of which appears to be identical with the folate binding protein from human milk and nasopharyngeal epidermoid carcinoma (KB) cells; the cDNA sequence obtained corresponds to the other protein. The deduced protein sequence is characterized by a putative 16-residue amino-terminal signal peptide that is cleaved, resulting in a 239-residue polypeptide. The mature protein exhibits two potential sites for N-linked glycosylation at Asn-99 and Asn-179, eight potential intramolecular disulfide bonds, and a stretch of hydrophobic residues at the carboxyl terminus that could form a transmembrane domain. The protein bears a 68% sequence homology with the KB cell folate binding protein and may represent a fetal folate transport protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

The isolation and properties of multiple forms of folate binding protein in cultured KB cells

The folate binding proteins (FBPs) of KB cells which were cultured in normal (N) and folate-deficient (D) medium have been characterized. The 200,000 g supernate of lysed cells contained two FBPs which could be separated by DEAE-Bio-Gel A chromatography, indicating that they differ in ionic charge although they could not be separated by gel filtration through Sephadex G-100 (apparent Mr approximately 40,000). Two species of FBP, a major form of apparent Mr approximately 160,000 and a minor form of apparent Mr approximately 40,000, were identified by gel filtration through Sephadex G-150 in the membrane component of the cells after solubilization with Triton X-100. An additional FBP was isolated and purified by affinity chromatography from the medium in which these cells were cultured. By gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent Mr of this FBP was approximately 44,000. The association constants for pteroylglutamic acid of the FBPs in the 200,000 g cell lysate supernate, culture medium, and Triton-solubilized membrane were similar and the relative affinity of folate analogs for the FBP, vis-à-vis pteroylglutamic acid, was similar for all species. An antiserum raised to the purified FBP from the culture medium precipitated the FBPs in the 200,000 g cell lysate supernate, Triton-solubilized membrane, and culture medium, indicating antigenic homology among these FBPs. There was no unsaturated FBP in the 200,000 g cell lysate supernate or medium when KB cells were cultured in N medium. However, when cells were cultured in D medium, the unsaturated FBP of the 200,000 g cell supernate and culture medium was substantial (9.2 and 14.1 pmol/mg protein, respectively). Unsaturated FBP was detected in the membrane of normal cells but this also increased when these cells were cultured in D medium (4.5 to 756 pmol/mg protein), indicating that the FBPs of these cellular compartments are normally saturated by folate. After 16 weeks of culture in D medium, the total folate binding capacity of the membrane-associated FBP was twofold greater than that of normal KB cells, indicating the induction of FBP.     

Poly(ADP-ribosyl)ation of DNA polymerase beta in vitro

DNA polymerase beta purified from bovine thymus is markedly inhibited when incubated in a reconstituted poly(ADP-ribosyl)ating reaction system. Analyses of the reaction product synthesized in this system by SDS-polyacrylamide gel electrophoresis and subsequent fluorography of the gel indicated that ADP-ribose is covalently attached to DNA polymerase beta molecule (Mr = 44,000).     

Development of a specific radioimmunoassay for the placental folate receptor and related high-affinity folate binding proteins in human tissues

High-affinity membrane-associated and soluble folate binding proteins (FBPs) from human placenta, milk, and KB cells appear to share antigenic determinants [A. C. Antony et al. (1981) J. Biol. Chem. 256, 9684-9692 and (1985) 260, 14911-14917]. Iodination of a highly purified preparation of placental folate receptor (PFR) by various techniques resulted in significant denaturation of the PFR as evidenced by additional peaks of radioactivity on Sephacryl S-200 gel filtration in 1% Triton X-100. These denatured species had similar molecular weights on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as radioiodinated and native PFR, and were also recognized, albeit with less efficiency, by specific rabbit antiserum raised against purified PFR. Since these denatured species failed to bind folate, they were specifically excluded from 125I-PFR by their inability to bind pteroylglutamate-Sepharose. This ws accomplished in a single step by iodination of PFR bound to the affinity column and elution of 125I-PFR under identical conditions that the native PFR was purified. The purified 125I-PFR comigrated with unlabeled PFR on SDS-PAGE and its elution profile on Sephacryl S-200 gel filtration was identical to radioligand bound PFR. The resulting radioimmunoassay standard curve using this affinity chromatography purified 125I-PFR, unlabeled PFR, and anti-human PFR serum had a range for measurement between 5 and 500 ng of PFR and was not affected by the concentration of folate in the sample. The practical utility of this radioimmunoassay for measuring cross-reacting material to the PFR was validated by its ability to quantitate the 40,000 and 160,000 Mr FBPs which are the two major forms of high-affinity FBPs in human tissues.     

Purification of brain D2 dopamine receptor.

D2 dopamine receptors have been extracted from bovine brain using the detergent cholate and purified approximately 20,000-fold by affinity chromatography on haloperidol-sepharose and wheat germ agglutinin-agarose columns. The purified preparation contains D2 dopamine receptors as judged by the pharmacological specificity of [3H]spiperone binding to the purified material. The sp. act. of [3H]spiperone binding in the purified preparation is 2.5 nmol/mg protein. The purified preparation shows a major diffuse band at Mr 95,000 upon SDS-polyacrylamide gel electrophoresis and there is evidence for microheterogeneity either at the protein or glycosylation level. Photoaffinity labelling of D2 dopamine receptors also shows a species of Mr 95,000. The D2 dopamine receptor therefore is a glycoprotein of Mr 95,000.     

Purification of follitropin receptor from bovine calf testes

Follitropin (FSH) receptors were solubilized from pure light membranes of bovine calf testis, using an optimum detergent to protein ratio of 0.01. The soluble FSH receptor fraction was gel filtered through Sepharose 6B to isolate an active fraction (6B-Fr-1) which behaved as a complex of FSH receptor and Gs protein. The 6B-Fr-1 was concentrated by ultrafiltration and further purified by sequential Sepharose 4B gel filtration, DEAE-cellulose chromatography (to separate the receptor from Gs protein), and wheat germ lectin affinity chromatography. The purified receptor had an FSH-binding capacity of approximately 3.47 nmol/mg of protein with a Kd of 1.9 X 10(-10) M. Yield was 526 micrograms/11.5 kg tested. Radioiodinated, as well as unlabeled purified FSH receptor, migrated on sodium dodecyl sulfate-polyacrylamide gels as a single major band of Mr approximately 240,000. This band was not affected by 8 M urea treatment prior to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but treatment with dithiothreitol induced the loss of the 240-kDa band, with appearance of an Mr approximately 60,000 band. The availability of highly purified, stable FSH receptor should allow direct studies on its structure-function relationships.     

Characterization of prolactin receptors in pig mammary gland.

Prolactin receptors present in the particulate fraction of lactating pig mammary gland were solubilized by 7.5mM-3-[(3-cholamidopropyl)dimethylammonio]-1-propane-su lph onic acid (Chaps) and purified by affinity chromatography on prolactin coupled to Affi-Gel 10. Nearly 30% of the particulate receptors were solubilized by the detergent and over a 1000-fold purification from homogenates was achieved. A water-soluble fraction rich in receptors was observed during the preparation of membranes, although this fraction has not yet been purified. Prolactin binding to the receptors was a time-dependent, reversible and saturable reaction in particulate, Chaps-solubilized and purified receptors. In all forms, receptors showed the same specificity to peptide hormones. Prolactin and human growth hormone bound to the same receptors, whereas bovine growth hormone, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone and insulin failed to bind. After solubilization, the dissociation constant (Kd) for prolactin was decreased 5-fold from 9.8 X 10(-11) M in the particulate receptors to 1.8 X 10(-11) M in solubilized and purified receptors, being due principally to an increase in the association rate constant from 1.0 X 10(9)M-1 X h-1 to (3.9-4.6) X 10(9)M-1 X h-1, respectively, with the dissociation rate constant remaining unchanged at (1.1-1.3) X 10(-2)h-1. Isoelectric focusing of the prolactin-receptor complex revealed two peaks, one at a pI of 5.5-5.6 and the other at 5.2-5.3. Microsomal receptors were covalently cross-linked to 125I-labelled ovine prolactin with ethylene glycol bis(succinimidyl succinate) and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Autoradiography of the gel revealed a major subunit of Mr 28 000-35 000 and a minor one of Mr 67 000-69 000. Anti-(prolactin receptor) antibodies raised against rabbit mammary gland prolactin receptors were equally effective in inhibiting prolactin binding to particulate, solubilized and affinity-purified receptors, suggesting that purified prolactin receptors have a structure indistinguishable immunologically from particulate receptors and rabbit mammary gland prolactin receptors. The present demonstration shows that particulate prolactin receptors from a domestic animal can be solubilized and purified without losing the original properties of high affinity and binding specificity for hormones.     

The proteins of human lung surfactant

Human pulmonary surfactant was purified from bronchoalveolar lavage of patients. The proteins present in surfactant were analyzed by SDS-polyacrylamide gel electrophoresis into serum and non-serum components. One non-serum surfactant protein (Mr = 43 000) was then identified in the 100 000 X g supernatant of a lung homogenate on the basis of phospholipid binding. This lung protein was purified and partially characterized. The presence of 3-methyl histidine and reaction in Western blot analysis with antibody against chicken muscle actin both strongly suggested that the 43 000 Da protein of human surfactant is indeed cytoplasmic actin. It is proposed that this surfactant protein is involved in the secretion and not necessarily in the function of surfactant.     

Purification of the major protein-tyrosine-phosphatases of human placenta

This report describes the purification of the major protein-tyrosine-phosphatases from human placenta. Enzyme activity was followed with a novel artificial substrate, namely reduced, carboxamidomethylated, and maleylated lysozyme, phosphorylated on tyrosine by a partially purified preparation of insulin and epidermal growth factor receptor kinases, also from human placenta. The key step in the purification of the protein-tyrosine-phosphatases was affinity chromatography on a column of thiophosphorylated, reduced, carboxamidomethylated, and maleylated lysozyme-Sepharose. Purification was carried out separately from both the soluble and particulate fractions. Whereas multiple and distinct enzyme forms were obtained from each of these, little difference could be detected between the behavior of the "soluble" enzyme subtypes and their "particulate" counterparts. The major subtypes were purified to apparent homogeneity with an approximately 23,000-fold enrichment and 10% yield from the soluble fraction and a 4,300-fold enrichment and 13% yield from the particulate fraction. Both samples migrated as bands of 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had specific activities of approximately 45,000 nmol of Pi released min-1 mg-1, at least 2-3-fold higher than that of the type 1 and 2A serine/threonine phosphatases. The level of protein-tyrosine-phosphatases in the soluble fraction of human placenta (2,000 units/g of protein) was approximately the same as protein-serine/threonine-phosphatases 1 and 2A in skeletal muscle.     

The porcine LH/hCG receptor. Characterization and purification

Porcine luteal LH/hCG receptor (LH/hCG R) was solubilized with 70-80% recovery from the crude plasma membrane fraction by Triton X-100 in the presence of 25% glycerol and protease inhibitors. The solubilized receptor maintained 90% of original activity at -60 degrees C for 90 days. Equilibrium association constant (Ka) values of 1.92, 2.22, and 2.03 X 10(10) M-1 were observed for the whole homogenate, plasma membrane fraction, and solubilized LH/hCG R preparations, respectively. The specific binding capacity for the same fractions were 49, 70, 55 fmol/mg protein, respectively. Complexes of LH/hCG R and Triton X-100 were resolved into two components with approximate Mr = 2.7 X 10(5) and 5.4 X 10(5) by gel filtration on Sepharose 6B and two glycoprotein components by chromatography on concanavalin A-Sepharose. Solubilized porcine LH/hCG R was purified by two cycles of affinity chromatography on highly purified hCG-Sepharose with an overall recovery of 30-35% of the initial activity in the Triton extract. Purified porcine LH/hCG R had a specific binding capacity of 2300 pmol/mg protein and a Ka = 1.5 X 10(10) M-1. Silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels demonstrated that the major protein in porcine LH/hCG R preparations has Mr = 68,000. A weakly staining band at Mr = 45,000 was also observed in the purified receptor preparation. Analysis of iodinated purified LH/hCG R by autoradiography has confirmed these results. Porcine LH/hCG R was purified 40,000-fold by this method.     

High performance agarose gel chromatography in sodium dodecyl sulfate of integral membrane proteins from human red cells, with special reference to the glucose transporter

Integral membrane proteins from human red cells were fractionated in sodium dodecyl sulfate solutions by high performance gel filtration on the small-bead cross-linked agarose gel Superose 6. The components were identified by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The combination of Superose chromatography with electrophoresis afforded high resolution. As expected the gel filtration elution volumes depended essentially on the molecular mass, but the elution volumes decreased stepwise as the detergent concentration was increased from 0.6 to 100 mM, with the largest decrease for the glucose transporter. The resolution increased as the flow rate was decreased from 60 to 1 ml X cm-2 X h-1. The Mr values for the anion and glucose transporters as estimated by Superose 6-chromatography at 50 mM detergent were 75-80% of the corresponding Mr values obtained by electrophoresis. At 50 mM dodecyl sulfate the proteins were resolved into four fractions (a-d) which mainly contained: (a) dimer and (b) monomer of the anion transporter, (c) the glucose transporter and (d) components of Mr below 40 000. Monoclonal antibodies that possibly are directed against the glucose transporter (Lundahl, P., Greijer, E., Cardell, S., Mascher, E. and Andersson, L. (1986) Biochim. Biophys. Acta 855, 345-356) interacted only with part of the 4.5-material in fraction c in immunoblotting (Western blotting). Superose 6-chromatography of red cell glucose transporter that had been partially purified on DEAE-cellulose and Mono Q resolved one major and two minor fractions. Electrophoretic analysis showed that components of Mr 90,000, 50,000, and 25,000 had been separated from the major Mr-55,000-4.5-material and revealed size heterogeneity within the major chromatographic fraction. Heating of the glucose transporter in the presence of dodecyl sulfate caused an unexpected retardation of monomeric transporter on Superose 6. The apparent Mr decreased from 44,000 to 29,000.     

Purification and characterization of prolactin receptors from rat ovary

Prolactin receptors were purified to homogeneity by two affinity chromatography steps using concanavalin A-Sepharose and human growth hormone (hGH)-Sepharose. The purified receptors showed specificity and high affinity for lactogenic hormones and a binding capacity of 20 nmol/mg of protein. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis revealed that purified receptors were composed of two major protein bands of Mr = 41,000 and 88,000, which were identified as radioactive bands by binding of 125I-hGH to blotted renatured receptors and by autoradiogram of free and 125I-radiolabeled purified receptors. Autoradiographic analysis of SDS-polyacrylamide gel electrophoresis of cross-linked 125I-hGH-receptor or hGH-125I-iodinated receptor complexes showed two radioactive bands of Mr = 63,000 and 106,000. Analysis of the free receptors by high performance liquid chromatography using Superose 12 revealed two peaks of binding activity for 125I-hGH eluting in the positions of Mr approximately 150,000 and 250,000. After cross-linking with 125I-hGH, SDS-polyacrylamide gel electrophoresis analysis revealed that both peak fractions contained two binding species with Mr = 63,000 and 106,000. Chromatography of 125I-hGH-receptor complexes showed two radioactive fractions with approximate Mr approximately 180,000 and 300,000. The treatment of 125I-iodinated receptors with SDS and reductant resulted in the dissociation of the higher Mr form into the lower Mr form upon gel filtration. Chromatofocusing of free receptors showed three isoforms with pI 4.0, 5.0, and 5.3. These results indicate that detergent-solubilized prolactin receptors appear to be aggregated forms of holoreceptor containing two binding species of Mr = 41,000 +/- 2,000 and 88,000 +/- 3,000.     

An ubiquinone-binding protein in mitochondrial NADH-ubiquinone reductase (Complex I)

An ubiquinone-binding protein (QP) was purified from mitochondrial NADH-ubiquinone reductase (Complex I). Complex I was separated into 3 fragments: a fraction of hydrophobic proteins, that of soluble iron-sulfur protein (IP) and soluble NADH dehydrogenase of flavoprotein by a procedure involving the resolution with DOC and cholate, followed by ethanol and ammonium acetate fractionations. About 40% of the total ubiquinone was recovered in the IP fragment which consisted of 12 polypeptides. The QP was purified from the IP fragment with a hydrophobic affinity chromatography. SDS-polyacrylamide gel electrophoresis showed that the purified QP corresponded to 14-kDa polypeptide of the IP fragment and was a different protein from the QP (12.4 kDa) in Complex III. The purified QP (14 kDa) contained one mol ubiquinone per mol. The ubiquinone-depleted IP fragment could rebind ubiquinone. These results indicate that an ubiquinone-binding site in Complex I is on the 14-kDa polypeptide of the IP fragment.