Purification of phosphatidylinositol kinase from bovine brain myelin.

A membrane-bound phosphatidylinositol (PI) kinase (EC 2.7.1.67) was purified by affinity chromatography from bovine brain myelin. This enzyme activity was solubilized with non-ionic detergent and chromatographed on an anion-exchange column. Further purification was achieved by affinity chromatography on PI covalently coupled to epoxy-activated Sepharose, which was eluted with a combination of PI and detergent. The final step in the purification was by gel filtration on an Ultrogel AcA44 column. This procedure afforded greater than 5500-fold purification of the enzyme from whole brain myelin. The resulting activity exhibited a major silver-stained band on SDS/polyacrylamide-gel electrophoresis with an apparent Mr 45,000. The identity of this band as PI kinase was corroborated by demonstration of enzyme activity in the gel region corresponding to that of the stained protein. The purified enzyme exhibited a non-linear dependence on PI as substrate, with two apparent kinetic components. The lower-affinity component exhibited a Km similar to that observed for the phosphorylation of phosphatidylinositol 4-phosphate by the enzyme.     

Glycoproteins containing peanut-agglutinin receptors from human-peripheral-blood T-lymphocyte plasma membranes

A glycoprotein fraction possessing peanut agglutinin receptors has been isolated from detergent extracts of neuraminidase-treated human peripheral blood T-lymphocyte plasma membranes with affinity matrices comprising the peanut agglutinin co-valently immobilised on Sepharose 4B. This fraction could be specifically eluted from affinity columns using buffer solutions supplemented with 0.2 M D-galactose and was shown, by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (0.1%), to contain four major glycoprotein components with apparent molecular weights of 200000, 190000, 110000 and 95000. It is suggested, from the observed reactivity of these glycoproteins with various lectins in double-diffusion experiments, that they possess both O-glycosidically and N-glycosidically linked carbohydrates.     

Solubilization of alkyldihydroxyacetone-P synthase from Ehrlich ascites cell microsomal membranes.

The enzyme, alkyldihydroxyacetone-P synthase, has been solubilized and partially purified from microsomal preparations of Ehrlich ascites cells after treatment with Triton X-100 and phospholipase C, followed by chromatography on Sepharose 4B. When the Triton X-100 was removed after solubilization the enzyme was still active but eluted in the void volume of the Sepharose 4B column, whereas in the presence of detergent it eluted much later as a single peak of activity, indicating that the solubilized enzyme tends to aggregate unless detergent is present. The lower molecular weight form of alkyldihydroxyacetone-P synthase (in detergent) had an estimated molecular mass of 250,000–300,000 daltons.     

Physical properties of collagen--sodium dodecyl sulfate complexes.

Sodium dodecyl sulfate (NaDodSO4)--polyacrylamide gel electrophoresis and gel filtration chromatography of protein--NaDodSO4 complexes are frequently used to characterize collagen-like polypeptide components in mixtures obtained from extracts of basement membranes. However, electrophoresis yields anomalously high apparent molecular weights for collagenous polypeptides when typical globular proteins are used as molecular weight standards, and the use of gel filtration chromatography for this purpose was suspect because Nozaki et al. [Nozaki, Y., Schechter, N. M., Reynolds, J. A., & Tanford, C. (1976) Biochemistry 15, 3884--3890] found that asymmetric particles, including NaDodSO4--protein complexes, coeluted with native globular proteins of lower Stokes radius, when Sepharose 4B was used. To understand these effects and to improve the characterization of collagenous polypeptides, we investigated the secondary structure of NaDodSO4--collagen complexes with the use of circular dichroism, measured the NaDodSO4 content, studied the dependence of electrophoretic mobility on gel concentration, and extended work on gel filtration by use of a more porous gel, Sepharose CL-4B. We found that the anomalous behavior of collagen chains on NaDodSO4--polyacrylamide gel electrophoresis is due in large part to treatment of data and that the method can be used to determine rather accurate values for the number of residues per polypeptide chain. Our gel filtration results indicated that reliable molecular weights can be obtained when Sepharose CL-4B is used. These methods can be applied equally well to collagenous and noncollagenous polypeptides.     

Heparan sulfate proteoglycans of human lung fibroblasts. Occurrence of distinct membrane, matrix and secreted forms

Human lung fibroblasts (HLF) were labeled with 35SO2-4 for 48 h and extracted with a guanidinium chloride buffer. A fraction of the extracted heparan sulfate proteoglycan (HSPG) appeared micelle-associated. In the absence of detergent these HSPG eluted in the void volume of Sepharose CL2B columns. In the presence of detergent these HSPG were included in Sepharose CL2B (Kav = 0.55) and 4B (Kav = 0.3) columns. This type of HSPG was specifically associated with isolated HLF cell membranes, suggesting that it may represent a fraction of integral membrane proteoglycans. Most of the HSPG in the HLF monolayers, however, eluted in the included volume of Sepharose CL2B (Kav = 0.4) and CL4B (Kav = 0.1) columns in the absence of detergent. This type of HSPG was not affected by detergent and was specifically retained in 'extracellular matrix' preparations. The medium of HLF monolayers contained HSPG of similar Mr as the membrane-associated HSPG. Of these three distinct HSPG fractions only the membrane-associated form could be incorporated in liposomes, confirming that the HSPG in this fraction may be integral membrane components.     

Isolation and characteristics of dihydropyridine calcium antagonist receptor from rabbit skeletal muscles

Up to 80% of the dihydropyridine receptor is solubilized from transverse tubules of rabbit skeletal muscle by 3-[(3-cholamidopropyl)-dimethylammonium]-2-oxy-1-propane sulfonate (CHAPSO). The DHP receptor is an oligomeric complex made up of two subunits with molecular masses of 160 and 53 kD as shown by DHP-Sepharose affinity chromatography and SDS gel electrophoresis of specifically eluted proteins. The reduction of disulfide bridges of the 160 kD subunit is accompanied by a decrease in its apparent molecular mass up to 125 kD. A method is proposed for preparative isolation of the DHP receptor which is based on ion-exchange chromatography and WGA-Sepharose chromatography. Individual subunits of DHP receptor were isolated by Sepharose 4B gel filtration in SDS; their amino acid composition was determined. Both the 160 and 53 kD subunits are N-glycosylated, and the oligosaccharide portions make up to 7.5% and 6.6%, respectively.     

Investigation into the use of graft copolymer-based cibacron blue F3GA columns for the purification of proteins

The graft copolymers Nylon-co-hydroxyethylmethacrylate and poly(ethylene)-co-hydroxyethylmethacrylate coupled to Cibacron blue F3GA at wet volume levels similar to those obtained with Sepharose 4B. However, the graft copolymers removed protein from human serum to a far lesser extent than did Sepharose 4B. Further investigations involved the preparation of hydrolyzed poly(vinyl acetate) copolymers of nylon and polyethylene and of cellulose-co-hydroxyethylmethacrylate and study of the ability of the copolymers to remove human serum albumin and lactic dehydrogenase. Comparisons were made with Sepharose 4B-, Sephadex G15-, and G25-based Cibacron blue F3GA systems. The effectiveness of Sepharose 4B-Cibacron blue F3GA is thought to be due to the manner in which the dye is located within the pores of the gel.     

Purification of detergent-solubilized form and membrane-binding domain of rat γ-glutamyltransferase by immuno-affinity and hydrophobic chromatography

A new method to purify papain- or detergent-solubilized form (papain or detergent form) of γ-glutamyltransferase from rat hepatomas as well as from rat kidney by immuno-affinity column chromatography is presented. The antibody-column was prepared by coupling the anti-kidney papain form antibody, which had been purified by using a kidney papain form-Sepharose column, to CNBr-activated Sepharose 4B. The enzyme bound to the antibody-column was eluted with 0.04 M NH₄OH. By this method, detergent forms were purified 300 and 1600-fold in approx. 50% yields from rat kidney and rat ascites hepatoma AH 13, respectively, and the papain form was also purified 16 000-fold in a similar yield from primary hepatoma which has a very low activity of this enzyme. Preparations thus obtained apparently did not contain any peptide other than heavy and light subunit peptides of this enzyme on SDS-polyacrylamide gel electrophoresis. The detergent form of kidney enzyme was preferentially adsorbed to a hydrophobic column of aminooctyl-Sepharose, while the papain form was not, suggesting that the detergent form might be adsorbed to the column through hydrophobic interaction of the membrane-binding domain. The domain peptide was also purified by the hydropholic column after release from the detergent form by papain treatment. The molecular weight of the peptide was estimated to be about 16 000 on SDS-polyacrylamide gel electrophoresis. On double immunodiffusion, the domain peptide reacted with anti-detergent form antibody but not with anti-papain form antibody. The domain-specific antibody was also purified from the anti-detergent form antibody.     

Purification of detergent-solubilized form and membrane-binding domain of rat gamma-glutamyltransferase by immuno-affinity and hydrophobic chromatography

A new method to purify papain- or detergent-solubilized form (papain or detergent form) of gamma-glutamyltransferase from rat hepatomas as well as from rat kidney by immuno-affinity column chromatography is presented. The antibody-column was prepared by coupling the anti-kidney papain form antibody, which had been purified by using a kidney papain form-Sepharose column, to CNBr-activated Sepharose 4B. The enzyme bound to the antibody-column was eluted with 0.04 M NH4OH. By this method, detergent forms were purified 300 and 1600-fold in approx. 50% yields from rat kidney and rat ascites hepatoma AH 13, respectively, and the papain form was also purified 16 000-fold in a similar yield from primary hepatoma which has a very low activity of this enzyme. Preparations thus obtained apparently did not contain any peptide other than heavy and light subunit peptides of this enzyme on SDS-polyacrylamide gel electrophoresis. The detergent form of kidney enzyme was preferentially absorbed to a hydrophobic column of aminooctyl-Sepharose, while the papain form was not, suggesting that the detergent form might be adsorbed to the column through hydrophobic interaction of the membrane-binding domain. The domain peptide was also purified by the hydrophobic column after release from the detergent form by papain treatment. The molecular weight of the peptide was estimated to be about 16 000 on SDS-polyacrylamide gel electrophoresis. On double immunodiffusion, the domain peptide reacted with anti-detergent form antibody but not with anti-papain form antibody. The domain-specific antibody was also purified from the anti-detergent form antibody.     

Development of radioimmunoassay for thromboxane B2

A simple method for the preparation of rat liver urate oxidase is described. The enzyme was purified from rat liver homogenate by cell fractionation, detergent treatment, alkali treatment, and affinity chromatography on 8-aminoxanthine-bound Sepharose 4B. This enzyme preparation had a specific activity of 9.1 U/mg of protein and was purified about 1000-fold from the liver homogenate. After sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue, this preparation yielded one protein band at a position corresponding to a molecular weight of 33,000.     

膜上tRNA结合蛋白的分离与初步鉴定

用ＴｒｉｔｏｎＸ－１１４分相法分离啤酒酵母的膜总蛋白,经过酵母ｔＲＮＡ分子交联的Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析,用０－０．８ｍｏｌ／Ｌ（ＮＨ４０２ＳＯ４梯度缓冲液洗脱ｔＲＮＡ结合的蛋白质。凝胶阻滞电泳实验室鉴定出两种主要的与ｔＲＮＡ分子特异性结合的蛋白质。     

Characterization of solubilized insulin receptors from rat liver microsomes. Existence of two receptor species with different binding properties

Insulin receptors were solubilized from rat liver microsomes by the nonionic detergent Triton X-100. After gel filtration of the extract on Sepharose CL-6B, two insulin-binding species (peak I and peak II) were obtained. The structure and binding properties of both peaks were characterized. Gel filtration yielded Stokes radii of 9.2 nm (peak I) and 8.0 nm (peak II). Both peaks were glycoproteins. At 4 degrees C peak I showed optimal insulin binding at pH 8.0 and high ionic strength. In contrast, peak II had its binding optimum at pH 7.0 and low ionic strength, where peak I binding was minimal. For peak I the change in insulin binding under different conditions of pH and ionic strength was due to a change in receptor affinity only. For peak II an additional change in receptor number was found. Both peaks yielded non-linear Scatchard plots under most of the buffer conditions examined. At their binding optima at 4 degrees C the high affinity dissociation constants were 0.50 nM (peak I) and 0.55 nM (peak II). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of peak I revealed five receptor bands with Mr 400 000, 365 000, 320 000, 290 000, and 245 000 under non-reducing conditions. For peak II two major receptor bands with Mr 210 000 and 115 000 were found. The peak II receptor bands were also obtained after mild reduction of peak I. After complete reduction both peaks showed one major receptor band with Mr 130 000. The reductive generation of the peak II receptor together with molecular mass estimations suggest that the peak I receptor is the disulfide-linked dimer of the peak II receptor. Thus, Triton extracts from rat liver microsomes contain two receptor species, which are related, but differ considerably in their size and insulin-binding properties.     

Characterization of the nontransformed and transformed androgen receptor and heat shock protein 90 with high-performance hydrophobic-interaction chromatography

The hydrophobicity of the nontransformed and transformed androgen receptor from rat submandibular gland and heat shock protein 90 (hsp90) from rat submandibular gland and liver was characterized by using high-performance hydrophobic-interaction chromatography on TSK gel Ether-5PW. In the absence of molybdate, cytosol [3H]R1881-androgen receptor complexes were mainly eluted in the 1.3 M region (Peak 1) with a small peak in the 0.8 M region (Peak 2) of a descending salt gradient (2 to 0 M) of ammonium sulfate. In the presence of molybdate, Peak 2 was predominant. When labeled-cytosol was applied after being heated at 25 degrees C for 30 min, a third peak (Peak 3) at around 0.64 M ammonium sulfate was newly observed. Peaks 2 and 3 were observed, while Peak 1 completely disappeared with the labeled-cytosol precipitated at 40% saturated ammonium sulfate. The Stokes radius of Peak 1 was 7 nm, and of Peak 2 was 8 nm. Both peaks were retained poorly by DNA-cellulose but bound rather well to DEAE-cellulose. These results suggest that these two peaks represent the nontransformed receptor, indicating that there are isoforms of the nontransformed androgen receptor which are distinguished by their hydrophobic properties and Stokes radii. Peak 3 had a Stokes radius of 5 nm and preferentially bound to DNA-cellulose, suggesting that this peak corresponds to the transformed receptor. These results indicated that the transformation of the androgen receptor accompanies the enrichment of the hydrophobicity of the receptor molecule. Hsp90 purified from rat livers and hsp90 in the cytosol both from livers and submandibular glands were eluted from Ether-5PW at 0.8 M ammonium sulfate, at almost the same position as Peak 2. This finding suggests that the enrichment of hydrophobicity on transformation is due to dissociation of hsp90 from the nontransformed androgen receptor.     

Proteoglycans of human umbilical cord arteries

Proteoglycans (PGs) were dissociatively extracted from human umbilical cord arteries (UCAs) with 4 M guanidine hydrochloride containing Triton X-100 and protease inhibitors, purified by Q-Sepharose anion exchange chromatography and lyophilized. They were analysed by gel filtration, SDS/PAGE and agarose gel electrophoresis before and after treatment with chondroitinase ABC. It was found that the PG preparation was especially enriched in chondroitin/dermatan sulphate PGs. The predominant PG fraction included small PGs that emerged from Sepharose CL-2B with Kav = 0.74. Their molecular mass, estimated by SDS/PAGE, was 160-200 kDa and 90-150 kDa, i.e. it was typical for biglycan and decorin, respectively. Treatment with chondroitinase ABC yielded the core proteins of 45 and 47 kDa, characteristic for both small PGs. Remarkable amounts of the 45 kDa protein were detected in non-treated PG samples, suggesting the presence of free core proteins of biglycan and decorin. Large PGs were present in lower amounts. In intact form they were eluted from Sepharose CL-2B with Kav = 0.17 and 0.43. Digestion with chondroitinase ABC yielded the core proteins with a molecular mass within the range of 180-360 kDa but predominant were the bands of 200, 250 and 360 kDa. The large PGs probably represent various forms of versican or perlecan bearing chondroitin sulphate chains.     

Isolation and characterization of root nodule proteins from lupin

A group of root nodule-specific plant proteins (nodulins) has been isolated from yellow lupin (Lupinus luteus) by immunoaffinity chromatography. The cytoplasmic nodule protein extract was initially enriched in nodulins on a column with immobilized IgG fraction. It was then purified by chromatography on Sepharose 4B - bound IgG against uninfected root proteins and finally on Sepharose 4B - bound IgG against Rhizobium lupini proteins. Rocket immunoelectrophoresis showed that the nodulin preparation did not react with antibodies against root or bacterial proteins. SDS gel electrophoresis of lupin nodulins revealed at least 23 polypeptides ranging in Mr, from 7,000 to 70,000, probably representing protein subunits.     

Identification and purification of a renal amiloride-binding protein with properties of the Na+-H+ exchanger

The aim of this study was to identify and purify the Na+-H+ exchanger from rabbit renal brush border membranes by use of affinity chromatography. Triton-solubilized membranes were equilibrated with an affinity matrix consisting of the amiloride analogue A35 (5-N-(3-aminophenyl)amiloride) covalently coupled to Sepharose CL-4B beads through a triglycine spacer arm. The matrix was then washed extensively with buffer and sequentially eluted with buffer, buffer containing 5 mM amiloride, and 1% sodium dodecyl sulfate (SDS). Eluates were concentrated and subjected to SDS-polyacrylamide gel electrophoresis. The silver-stained gel revealed a 25-kDa protein that was not visible in the initial solubilized brush border membrane extract, was not eluted from the affinity matrix by buffer alone, but was eluted with 5 mM amiloride. A subsequent elution with 1% SDS did not release any more of the 25-kDa protein, indicating that it had been completely eluted from the affinity matrix by amiloride. The presence of 5 mM amiloride during equilibration of the solubilized brush border extract with the affinity matrix completely blocked adsorption of the 25-kDa protein. The relative abundance of this protein correlated closely with Na+-H+ exchange activity when preparations of cortical brush border membrane vesicles, outer medullary brush border membrane vesicles, and cortical basolateral membrane vesicles were compared. Moreover, binding of the protein to the affinity matrix was inhibited by amiloride and amiloride analogues with a rank order identical to that for inhibition of Na+-H+ exchange activity. These findings strongly suggest that the 25-kDa protein is a structural component of the Na+-H+ exchanger.     

Use of nile red as a fluorescent probe for the study of the hydrophobic properties of protein-sodium dodecyl sulfate complexes in solution.

Our results show that the noncovalent dye 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one (Nile red) can be used as a fluorescent probe to study the hydrophobic properties of proteins associated with the anionic detergent sodium dodecyl sulfate (SDS). Nile red can interact with both SDS micelles and protein-SDS complexes. The enhancement of Nile red fluorescence observed with diverse types of proteins occurs at SDS concentrations lower than the critical micelle concentration of this detergent. This is also observed using the covalent fluorophore rhodamine B isothiocyanate. Additional results obtained in studies in solution show that the fluorescence intensity and the spectral characteristics of Nile red associated with different proteins complexed with SDS are very similar. These spectroscopic similarities are probably related to the equivalent synchrotron X-ray scattering results found for various protein-SDS complexes in solution. The scattering results suggest that SDS induces the formation of complexes in which the basic structural properties are independent of the different initial structures of native proteins. We speculate that Nile red is bound to regions with equivalent hydrophobic characteristics located in the uniform structures produced by the association of SDS with proteins.     

Effect of sodium dodecyl fulfate on the dissociation of bovine liver catalase.

Native bovine liver catalase [EC 1.11.1.6] and catalase acetylated with N-acetylimidazole (AI) both combined with sodium dodecyl sulfate (SDS) to form catalase-SDS complexes. The differences between native and acetylated catalase bound to SDS were investigated as regards enzymatic activity, absorption spectra, ORD and CD, sedimentation velocity and fluorescence spectra. It was found that the binding of SDS with both catalases depended on incubation time and SDS concentration, and that the acetylation of catalase had some protective effect on the denaturation of the molecule by SDS, which may be ascribed to a reduction of ionic interaction between SDS and the protein on acetylation. The native catalase was found to split into three smaller components on incubation with 1% SDS for 96 hr, whereas the acetylated catalase split into two smaller components. These smaller components were isolated by gel filtration through Sephadex G-100. The isolated components has estimated molecular weights of 60,000, 30,000, aide. It seemed likely that the modification occurred stepwise. Approximately 26% of the carboxyl groups of fibrinogen was modified finally. The modified fibrinogen had no interaction with cationic detergent, and did not form any complex with the detergent. In dilute acid, fibrinogen was observed to show only a slight interaction with cationic detergent. It is probable that the exposed and ionized carboxyl groups are essential for the formation of a complex between fibrinogen and cationic detergent.     

Use of the hydrophobic probe Nile red for the fluorescent staining of protein bands in sodium dodecyl sulfate-polyacrylamide gels.

In a previous work (J.-R. Daban, M. Samsó, and S. Bartolomé, Anal. Biochem. 199, 162-168, 1991) we observed that, in the presence of the detergent sodium dodecyl sulfate (SDS), diverse types of proteins produced a high increase in the fluorescence intensity of the hydrophobic probe 9-diethylamino-5H-benzo[alpha]-phenoxazine-5-one (Nile red). This enhancement of Nile red fluorescence was observed at SDS concentrations lower than the critical micelle concentration (CMC) of this detergent in the buffer (0.025 M Tris and 0.192 M glycine, pH 8.3) currently used in SDS-polyacrylamide gel electrophoresis. This observation led us to introduce a modification in the typical (U. K. Laemmli, Nature 227, 680-685, 1970) SDS-polyacrylamide gels, in which the SDS concentration in the gel after electrophoresis is lower than the CMC of this detergent but high enough to maintain the stability of the protein-SDS complexes in the bands. The staining of these modified gels with Nile red produces very high fluorescence in the protein-SDS bands and low background fluorescence. The Nile red staining method described in this paper is very rapid (i.e., the bands can be visualized and photographed within 6 min after the electrophoretic separation) and has a high sensitivity, similar to that obtained with the covalent fluorophores rhodamine B isothiocyanate and carboxytetramethyl-rhodamine succinimidyl ester also investigated in this work. Furthermore, our quantitative estimates indicate that most of the protein bands stained with Nile red show similar values of the fluorescence intensity per unit mass.     

Type B nucleoside-diphosphatase of rat brain. Purification and properties of an enzyme with high thiamin pyrophosphatase activity

Type B nucleoside-diphosphatase was purified from membranes of rat brain by solubilization with a non-ionic detergent and successive column chromatographies on DEAE-cellulose DE-52, concanavalin-A-Sepharose, Bio-Gel HT, blue-Sepharose CL-6B, chelating Sepharose 6B, Ultrogel AcA44 and TSK gel G3000 SW. The purified enzyme gave a single protein band on SDS/polyacrylamide gel electrophoresis and its molecular mass was estimated to be 75 kDa. It hydrolyzed thiamin diphosphate as well as GDP, IDP and UDP. Thiamin diphosphate (TPP) was hydrolyzed twice as efficiently as nucleoside diphosphates in the presence of Mn2+ at pH 7.4. The Km values for TPP, GDP, IDP and UDP were 0.66, 0.40, 0.54 and 1.06 mM respectively. ATP, ADP and pyridoxal 5'-phosphate inhibited thiamin-pyrophosphatase activity competitively and their Ki values were 2.3 mM, 1.0 mM and 0.59 mM respectively. The optimum pH of thiamin-pyrophosphatase activity was 7.4 in the presence of Mn2+ and that of GDP-hydrolytic activity was 6.5 in the presence of Mg2+.