Effects of the intensity of the suckling stimulus and ovarian steroids on pituitary gonadotropin-releasing hormone receptors during lactation

These studies examined whether the decrease in pituitary responsiveness to gonadotropin-releasing hormone (GnRH) observed during lactation in the rat results from a change in pituitary GnRH receptors. GnRH binding capacity was determined by saturation analysis using D-Ala6 as both ligand and tracer. During the estrous cycle, the number of GnRH binding sites increased from 199 +/- 38 fmol/mg protein on estrus to 527 +/- 31 fmol/mg protein on the morning of proestrus, whereas there was no change in receptor affinity (Ka, 6-10 X 10(9) M-1), During lactation, females nursing 8 pups on Days 5 or 10 postpartum had 50% fewer GnRH receptors (109-120 fmol/mg protein) than observed during estrus or diestrus 1 (199-242 fmol/mg protein) although receptor affinity was similar among all the groups. No deficits in pituitary GnRH receptors were observed in females nursing 2 pups on Day 10 postpartum. Removal of the 8-pup suckling stimulus for 24 or 48 h resulted in a dramatic increase in GnRH receptor capacity by 24 h from 120 +/- 16 to 355 +/- 39 fmol/mg protein. The rise in GnRH receptors after pup removal was accompanied by an increase in serum luteinizing hormone (LH) and estradiol concentrations. To assess the role of ovarian steroids in determining GnRH receptor capacity during lactation, females were ovariectomized (OVX) on Day 2 postpartum. Suckling of a large litter (8 pups) completely blocked the postcastration rise in serum LH and in pituitary GnRH receptors on Day 10 postpartum (OVX+ 8, 77 +/- 12 fmol/mg protein; OVX+ 0, 442 +/- 38 fmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization and partial purification of the high-affinity gonadoliberin receptor from the bovine pituitary gland

The high-affinity gonadoliberin (GnRH) receptor contained in a membrane preparation from frozen bovine anterior pituitary glands has been solubilized in Triton X-100 and the binding properties of the solubilized product have been examined. The radioreceptor-binding assay, using the GnRH agonist [D-Ser(t-Bu)⁶] des-Gly¹⁰GnRH N-ethylamide (GnRH-A) as radioligand, demonstrated that the kinetics of association and dissociation, the binding constants, as well as the specificity of receptor were not altered in the solubilized receptor preparations. Affinity chromatography on a concanavalin A-Sepharose column, with elution of adsorbed material using a solution of α-methyl-d-mannoside, allowed a 33-fold purification of the receptor. The K_a of the receptor thus purified was of the same order as that of the starting material, although slightly higher values were found. Only about one-half of the total receptor activity applied to the column was retained in spite of several recyclings. The other half was found in the nonadsorbed fraction. It is postulated that the detergent-solubilized fraction contains two forms of the GnRH receptor. The nonadsorbed fraction probably contains a partially or totally deglycosylated form. It is possible that the detergent-solubilization process somewhat alters the physicochemical properties of a part of the GnRH receptor molecules. Electrophoretic analysis of the purified receptor preparations, with a subsequent GnRH-binding assay, suggests that the apparent molecular mass of the high-affinity GnRH receptor, or of its monomeric form, is approximately 60,000 Da.     

Identification of a gonadotropin-releasing hormone-like factor in the rabbit fetal placenta

The biological and immunological gonadotropin-releasing hormone (GnRH)-like activities in rabbit fetal placentas collected at Day 18 of gestation were investigated. Both crude and partially purified acid extracts of placental tissue were tested. A similarly prepared liver extract served as a control. Immunological GnRH-like activity, determined through a GnRH radioimmunoassay was 1.3-2.0 pg/mg protein for the crude placental extract, 7.1-9.2 pg/mg protein for the partially purified placental extract and was nondetectable for liver extract. Both the crude and partially purified placental extracts increased (P less than 0.01) luteinizing hormone (LH) release by dispersed rabbit pituitary cells, whereas the liver extract had no effect. The (Ac-D-p-Cl-Phe1,2, D-Trp3, D-Arg6, D-Ala10)-GnRH antagonist was used to determine whether the biological GnRH-like activity in the placental extract was mediated through GnRH receptors. All three doses of antagonist (10, 100 and 1000 ng) suppressed the biological GnRH-like activity in the placental extracts. Molecular sieve chromatography of the partially purified placental extract showed that the immunoreactive GnRH-like factor eluted in the same fractions as the GnRH standard. These data indicate that the rabbit fetal placenta has both immunological and biological GnRH-like activity.     

Purification of A1 adenosine receptor from rat brain membranes

The A1 adenosine receptor from rat brain membranes has been purified about 50,000-fold to apparent homogeneity by sequential use of affinity chromatography on immobilized xanthine amine congener-agarose, hydroxylapatite chromatography, and reaffinity chromatography. The overall yield starting from the membranes was approximately 4%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified preparation gave a broad single band of an apparent molecular weight of 34,000 either by silver staining or autoradiogram after radioiodination. The purified receptor bound approximately 24 nmol of 8-cyclopentyl-1,3-[3H]dipropylxanthine/mg of protein with a dissociation constant of 1.4 nM. This maximum specific binding value is consistent with the expected theoretical specific activity (29.4 nmol/mg) for a protein with a molecular mass of 34,000 daltons if it is assumed that there is one ligand-binding site/receptor molecule. Affinity-labeling experiments using [3H]p-phenylenediisothiocyanate-xanthine amine congener showed that the Mr = 34,000 protein band contained the ligand-binding sites. The purified receptor gave a typical A1 adenosine receptor pharmacological specificity similar to that of unpurified receptor preparations.     

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.     

Solubilization and purification of rat pituitary gonadotropin-releasing hormone receptor

Gonadotropin-releasing hormone (GnRH) receptors were solubilized from rat pituitary membrane preparations in an active form by using the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid). The solubilized receptor exhibits high affinity, saturability, and specificity. The soluble supernatant retained 100% of the original binding activity when stored at 4 or -20 degrees C in the presence of 10% glycerol. The receptors were resolved into two components on the basis of chromatography on wheat germ agglutinin-agarose. Homogeneous receptor preparation was obtained by two cycles of affinity chromatography on immobilized avidin column coupled to [biotinyl-D-Lys6]GnRH. The overall recovery of the purified receptor was 4-10% of the initial activity in the CHAPS extract, and the calculated purification -fold was approximately 10,000 to 15,000. Analysis of iodinated purified GnRH receptors by autoradiography indicated the presence of two bands, Mr = 59,000 and 57,000. This was confirmed by photoaffinity labeling of the partially purified receptors and suggests that both components can specifically bind the hormone.     

Purification of low density lipoprotein receptor from liver and its quantification by anti-receptor monoclonal antibodies.

The low density lipoprotein (LDL) receptor has been purified to homogeneity from rabbit liver by a combination of DEAE-Sephacel chromatography, LDL-Sepharose 4B chromatography and preparative SDS/polyacrylamide-gel electrophoresis. The receptor protein had a pI of 4.45 and an Mr of 120 x 10(3)-125 x 10(3) in SDS gels under non-reducing conditions. Incubation of the LDL receptor with neuraminidase decreased its Mr to 105 x 10(3)-110 x 10(3) and increased its pI from 4.45 to 5.25. The purified receptor exhibited all the properties of the membrane-bound receptor including Ca2+-dependent binding of rabbit and human LDL but not of methylated LDL or high density lipoprotein. The amount of LDL receptor present in rabbit liver was measured by a quantitative blotting procedure employing a newly developed rat anti-receptor monoclonal antibody. The affinity and specificity of this monoclonal antibody allowed the quantification of the LDL receptor in detergent extracts of liver homogenate, thus eliminating the loss of receptor associated with the preparation of membrane fractions prior to receptor assay. Livers from adult female New Zealand White rabbits contained 149 +/- 13 ng of LDL receptor/mg of liver protein. Administration of pharmacological doses of 17 alpha-ethinyloestradiol raised the concentration of LDL receptor in liver to 312 +/- 25 ng/mg of liver protein.     

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.     

Affinity Chromatography of Thymidine Kinase from a Rat Colon Adenocarcinoma

Thymidine kinase from a transplantable colon adenocarcinoma, induced by 1, 2-dimethylhydrazine and maintained in CDF rats, was purified by affinity chromatography using thymidine-3′(4-amino-phenylphosphate) coupled to carboxyhexyl-Sepharose. Most of the contaminating protein passed through the column; non-specifically adsorbed protein was washed from the column by 0.1 M KC1 in 0.01 M Tris-HCl, pH 7.5. Thymidine kinase was eluted with 0.1 mM thymidine, 0.1 M KC1 in 0.01 M Tris-HCl, pH 7.5. The purified enzyme accounted for about 267. of the applied activity, the specific activity of the purified material (peak fraction) was 3, 500 nmoles TMP formed per mg protein per 10 min., a 1, 800-fold purification of the applied extract. The preparation is free of nucleoside phosphotransferase, but contains other protein impurities. Purification was completed in less than 1 hour, making this a useful procedure for isolation of this unstable enzyme.     

Characterization and subcellular localization of GnRH analog binding in rat brain

The binding of a degradation-resistant analog of gonadotropin-releasing hormone, [D-Phe6]GnRH, to rat brain crude particulate preparation was studied. The binding of this analog at 0 degrees C was saturable and Scatchard analysis revealed the presence of 2 binding sites: one with KD = 1.39 x 10(-7) M and Bmax = 265 pmole/mg protein, and another of lower affinity but higher capacity with KD = 5.58 X 10(-6) M and Bmax = 1734 pmoles/mg protein. The binding at 0 degrees C was substantially higher than that obtained at 37 degrees C, due to binding site-inactivation processes occurring at 37 degrees C. The binding sites exhibited a considerable degree of specificity for GnRH as unrelated peptides (with the exception of ACTH) display a much weaker affinity than GnRH and GnRH analogs. Subcellular fractionation demonstrated that most of the binding was associated with the mitochondrial fraction.     

Purification and protein sequence analysis of rat liver prolactin receptor

Prolactin receptors were purified from rat liver membranes by single-step immunoaffinity chromatography using a specific monoclonal antibody to the rat liver prolactin receptor. Scatchard analysis of 125I-human growth hormone binding to the purified receptor revealed two classes of specific binding sites with Ka = 18.5 x 10(9) and 1.2 x 10(9) M-1. Considering that both classes of binding sites are responsible for high affinity prolactin binding, the partially purified receptor preparation had a binding activity of 1.69 nmol/mg protein, representing 1000-fold purification over microsomal receptors with a recovery of 52%. From three separate purifications, 6 mg of partially purified prolactin receptor were obtained with a purity of approximately 4 to 6.5%. Thus, the use of monoclonal antibody for affinity chromatography resulted in a large improvement of prolactin receptor purification compared to previous hormone affinity chromatography (300-fold purification, 15% recovery). The purified receptor was run on preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a homogeneous preparation of prolactin receptor was obtained by electroelution from gel slices corresponding to Mr 38,000-43,000. Immunoblot analysis using a radiolabeled monoclonal antibody revealed two separate but closely located bands of Mr 42,000 and 40,000 in microsomal, partially purified, and electroeluted preparations. The homogeneous receptor protein was extensively digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone trypsin, and 10 internal amino acid sequences of the rat liver prolactin receptor were determined by gas-phase sequence analysis. Oligonucleotide probes were prepared against two of these internal sequences, and a prolactin receptor cDNA was isolated from a rat liver library using one of these probes (Boutin, J. M., Jolicoeur, C., Okamura, H., Gagnon, J., Edery, M., Shirota, M., Banville, D., Dusanter-Fourt, I., Djiane, J., and Kelly, P. A. (1988) Cell 53, 69-77). The amino acid sequence deduced from the cDNA reveals three potential sites of N-linked glycosylation, two of which were confirmed during protein sequencing. The prolactin receptor was characterized by affinity labeling with 125I-human growth hormone. Cross-linking of microsomes revealed a single band for the hormone-receptor complex with Mr 62,000. On the other hand, cross-linking of Triton X-100-solubilized or partially purified receptor with labeled hormone resulted in the appearance of two bands with Mr 62,000 and 102,000, suggesting the existence of a subunit structure of the prolactin receptor, or alternatively, the existence of two types of prolactin receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oxytocin binding sites in lactating rabbit mammary gland.

Material which specifically binds oxytocin was prepared from a crude preparation of lactating rabbit mammary gland by purification on a sucrose density gradient. On examination of activities of enzyme markers and the molar ratio of cholesterol to phospholipid, this material was considered to be a highly purified plasma membrane fraction. For the determination of specificity and time course of oxytocin binding, a Scatchard plot analysis was carried out for the crude and purified fractions. Dissociation constant (Kd) and binding capacity values were found to be as follows: crude, Kd equals 1.83 X 10(-9) M, capacity equals 670 fmol/mg protein; purified, Kd equals 2.8 X 10(-9) M, capacity equals 1700 fmol/mg protein. Treatment of the purified material with different detergents resulted in loss of all [3H]oxytocin binding capacity. However, preincubation of this material with [3H]oxytocin prior to detergent treatment resulted in solubilization of a receptor-hormone complex. This complex remained in the supernatant even after centrifugation at 210 000 X g for 30 min. Using oxytocin analogs, we have shown this solubilized complex to be oxytocin specific.     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

Atrial natriuretic factor R1 receptor from bovine adrenal zona glomerulosa: purification, characterization, and modulation by amiloride

The atrial natriuretic factor (ANF) R1 receptor from bovine adrenal zona glomerulosa was solubilized with Triton X-100 and purified 13,000-fold, to apparent homogeneity, by sequential affinity chromatography on ANF-agarose and steric exclusion high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the purified receptor preparation in the absence or presence of dithiothreitol revealed a single protein band of Mr 130,000. Affinity cross-linking of 125I-ANF to the purified receptor resulted in the labeling of the Mr 130,000 band. The purified receptor bound ANF with a specific activity of 6.8 nmol/mg of protein, corresponding to a stoichiometry of 0.9 mol of ANF bound/mol of Mr 130,000 polypeptide. Starting with 500 g of adrenal zona glomerulosa tissue, we obtained more than 500 pmol of purified receptor with an overall yield of 9%. The purified receptor showed a typical ANF-R1 pharmacological specificity similar to that of the membrane-bound receptor. The homogeneous Mr 130,000 receptor protein displayed high guanylate cyclase activity [1.4 mumol of cyclic GMP formed min-1 (mg of protein)-1] which was not stimulated by ANF. This finding supports the notion that the ANF binding and the guanylate cyclase activities are intrinsic components of the same polypeptide. Finally, the purified ANF-R1 receptor retained its sensitivity to modulation by amiloride, suggesting the presence of an allosteric binding site for amiloride on the receptor protein.     

Purification of the 5-hydroxytryptamine 5-HT3 receptor from NCB20 cells

A 5-hydroxytryptamine 5-HT3 receptor binding site has been purified from deoxycholate-solubilized NCB20 cell membranes. Purification (1,700-fold) was achieved in one step by affinity chromatography with L-685,603 immobilized on agarose. The 5-HT3 selective antagonist [3H]Q ICS 205-930 labeled a single population of receptors in the affinity-purified preparation with a Bmax of 3.1 +/- 0.9 nmol/mg protein and Kd of 0.40 +/- 0.05 nM (mean +/- S.E., n = 3). The rank order of potency for a series of competing compounds confirmed that [3H]Q ICS 205,930 was labeling a 5-HT3 receptor in the purified preparation, and the inhibition constants for all antagonists were unchanged after purification. The purified 5-HT3 binding site eluted from a Sepharose 6B gel filtration column in a similar manner to the crude solubilized preparation (Stokes radius of 4.9 nm, apparent molecular size 250,000). Polyacrylamide gel electrophoresis of the affinity-purified receptor showed two broad bands by silver staining, migrating with apparent molecular masses of 54,000 and 38,000. Gel filtration of the affinity purified material yielded a single peak labeled by [3H]Q ICS 205-930 with an apparent molecular size of 250,000, which was also composed of two bands of 54,000 and 38,000, consistent with these being the constituents of the 5-HT3 receptor.     

Effect of histone H2A on progesterone production by bovine luteal cells

Histone H2A competitively inhibits binding of GnRH to high affinity rat ovarian receptor sites and blocks gonadotropin-stimulated steroid and cAMP accumulation during culture of rat granulosal or luteal cells. The objective of our study was to examine the progesterone suppressive effects of histone H2A on bovine luteal cells. In the first study, luteal cells were treated at Time = 0 h with a partially purified preparation of bovine ovarian histone H2A (3 ng GnRH equivalents, 800 micrograms protein), equivalent amounts of GnRH (3 ng), or BSA (800 micrograms) and incubated for a total of 4 h. At Time = 2 h, cells were treated with 5 ng bovine LH (bLH) or with medium. Histone H2A completely blocked both basal and LH-induced accumulation of progesterone compared with untreated cultures or cultures treated with bLH. Neither BSA nor GnRH suppressed LH-induced progesterone accumulation. In the second study, histone H2A was added to cultures at Time = 0 h and bovine luteal cells were cultured for 8 h. After 2 h of treatment, histone H2A (3 ng GnRH equivalents) was removed from selected cultures and replaced with fresh medium. Four hours later cultures were treated with 5 ng bLH or medium. LH treatment of cultures from which histone H2A had been removed resulted in an increase in accumulation of progesterone compared with control cultures treated throughout the treatment period with histone H2A. The third study examined the effect of 9-181 pg GnRH equivalents (1.7-34 micrograms protein) of a highly purified preparation of bovine ovarian histone H2A on basal and LH-induced progesterone production during 2 or 3 h of culture.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of opioid receptor in the presence of sodium ions.

Opioid receptor was solubilized from rat brain membranes with a mixture of the detergents CHAPS and digitonin in the presence of protease inhibitors and 1 M NaCl. The solubilized receptor bound mu-opioid agonists and antagonists with affinities similar to those of native membrane receptor. The affinity of solubilized receptor for the agonist PL017 was greatly reduced by GTP gamma S, suggesting the receptor is still associated with G-protein. The solubilized material was passed through an opioid antagonist (10cd) affinity column and a wheat germ agglutinin column, set up in series, to obtain a partially purified receptor preparation. This partially purified material bound mu-agonist with low affinity and the binding affinity was no longer affected by GTP gamma S. The partially purified receptor was further purified by repeating the affinity and lectin chromatography with smaller size column. Binding of opioid antagonist [3H]diprenorphine to the partially or purified receptors was dependent upon the presence of sodium ions. The purified receptor showed saturable and stereospecific binding for opioid ligands, was predominantly of the mu-type, and exhibited as a diffuse band with a medium molecular mass of 62 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The average specific binding activity of the purified receptor was 18.8 +/- 2.3 pmol/micrograms protein, a value close to the theoretical estimation.     

A kainic acid receptor from frog brain purified using domoic acid affinity chromatography

A kainic acid receptor was purified from Triton X-100/digitonin-solubilized frog brain membranes. The purification was carried out in two steps: ion exchange chromatography using DEAE-Sepharose CL-6B and affinity chromatography with domoic acid immobilized on Sepharose 4B. The specific binding activity of the affinity-purified receptor is 481-fold higher than that of the crude solubilized preparation and 1617-fold higher than that of the whole membrane fraction. Scatchard analyses of the affinity-purified receptor showed a curvilinear plot which fit a two-site model with dissociation constants of 5.5 and 34 nM and Bmax values of 1700 pmol/mg protein and 4400 pmol/mg protein for the high and low affinity components, respectively. The dissociation constants of the purified receptor are similar to those of the crude soluble preparation (4.8 and 39 nM). Inhibition constants for several kainic acid analogs were also similar for the purified and crude preparations. The active purified receptor migrated with a Mr = 570,000 on gel filtration analysis using Sepharose 6B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the affinity-purified receptor showed a single broad band with silver stain, migrating with a Mr = 48,000.     

Recovery of thrombopoietin during purification

A thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) was previously purified by a six-step purification procedure. However, the exact quantity of TSF that was recovered, through the various purification procedures, was unknown because of the absence of a method for establishing a unit of measure of TSF. In the present work dose-response relationships on both the crude TSF preparations and on the more highly purified TSF were determined. TSF units were calculated from the dose-response curves. A unit of TSF is defined as the amount of material (mg) that is required to increase the percentages 35S incorporation into platelets of immunothrombocythemic mice by 50% above the baseline. The results of determining the TSF units on the crude TSF preparation indicated that 0.11 unit (U) of TSF/mg protein was present. Results showed that the specific activity of TSF can be increased to about 3.6 U/mg by a single purification procedure using Sephadex G-75 column chromatography. Increased specific activities were obtained by additional purification steps, i.e., DEAE-cellulose column chromatography, SE-HPLC, DEAE-HPLC, and SDS-PAGE. The purified product appears to have a specific activity of about 11,000 U/mg of protein with 0.00003% of the protein and 1.1% of the TSF recovered from the starting material. Establishing a unit of measure for TSF will allow calculations of its degree of purity, provide a method for quantitation of recoveries of activities after various purification procedures, and allow comparisons of results from different experiments and different laboratories.     

Purification of the rat hepatic alpha 2-macroglobulin receptor as an approximately 440-kDa single chain protein

alpha 2-Macroglobulin-trypsin complex (alpha 2M.T) and alpha 2M-methylamine bind in a Ca2+-dependent way to a 400- to 500-kDa receptor in rat and human liver membranes (Gliemann, J., Davidsen, O., and Moestrup, S. K. (1989) Biochim. Biophys. Acta 980, 326-332). Here we report the preparation of alpha 2M receptors from rat liver membranes solubilized in 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonic acid (CHAPS) dihydrate and incubated with Sepharose-immobilized alpha 2M-methylamine. The receptor preparation eluted with EDTA (pH 6.0) contained a protein larger than the 360-kDa alpha 2M (nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and some minor contaminants. The reduced large protein was about 440 kDa using reduced laminin (heavy chain: 400 kDa) as a standard. About 10 micrograms of receptor protein was obtained from 100 mg of liver membranes. The receptor preparation immobilized on nitrocellulose sheets bound 125I-alpha 2M.T, and the binding activity co-eluted with the 440-kDa protein. 125I-Labeled rat alpha 1-inhibitor-3 (alpha 1I3), a 200-kDa analogue of the alpha 2M subunit which binds to the alpha 2M receptors, was cross-linked to the 440-kDa protein. The receptor preparation was iodinated, and the 125I-labeled 440-kDa protein was isolated. It showed Ca2+-dependent saturable binding to alpha 2M-methylamine. In conclusion, we have purified the major hepatic alpha 2M receptor as an approximately 440-kDa single chain protein.