Purification and characterization from bovine brain cytosol of a novel regulatory protein inhibiting the dissociation of GDP from and the subsequent binding of GTP to rhoB p20, a ras p21-like GTP-binding protein

A novel regulatory protein for the rho proteins (rhoA p21 and rhoB p20), belonging to a ras p21/ras p21-like small molecular weight (Mr) GTP-binding protein (G protein) superfamily, was purified to near homogeneity from bovine brain cytosol and characterized. This regulatory protein, designated here as GDP dissociation inhibitor (GDI) for the rho proteins (rho GDI), inhibited the dissociation of GDP from rhoB p20 and the binding of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to the GDP-bound form of rhoB p20 but not of that to the guanine nucleotide-free form. The Mr value of rho GDI was estimated to be about 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and from the S value, indicating that rho GDI is composed of a single polypeptide without a subunit structure. The isoelectric point was about pH 5.7. rho GDI made a complex with the GDP-bound form of rhoB p20 with a molar ratio of 1:1 but not with the GTP gamma S-bound or guanine nucleotide-free form. rho GDI did not stimulate the GTPase activity of rhoB p20 and by itself showed neither GTP gamma S-binding nor GTPase activity. rho GDI was equally active for rhoA p21 and rhoB p20 but was inactive for other ras p21/ras p21-like G proteins including c-Ha-ras p21, smg p25A, and smg p21. rho GDI activity was detected in the cytosol fraction of various rat tissues. These results indicate that, in mammalian tissues, there is a novel type of regulatory protein specific for the rho proteins that interacts with the GDP-bound form of the rho proteins and thereby regulates the GDP/GTP exchange reaction of the rho proteins by inhibiting the dissociation of GDP from and the subsequent binding of GTP to them. Since there is a GTPase-activating protein for the rho proteins stimulating the GTPase activity of the rho proteins in mammalian tissues, the rho proteins appear to be regulated at least by GTPase-activating protein and GDI in a dual manner.     

Purification and properties of the cytosolic substrate for botulinum ADP-ribosyltransferase. Identification as an Mr 22,000 guanine nucleotide-binding protein

The substrate for ADP-ribosyltransferase from Clostridium botulinum was purified from the cytosol of bovine adrenal gland. Purification procedures consisted of ammonium sulfate fractionation, chromatographies on columns of DEAE-Sepharose and phenyl-Sepharose, gel filtration on a TSK-gel G3000SW column, and Mono Q fast protein liquid chromatography. On DEAE-Sepharose chromatography, the substrate activity was eluted in two separate peaks, and electrophoretic analyses revealed that the substrates in the two peaks are of similar molecular weight but different isoelectric points. The major peak of the substrate was further purified. It was purified about 1,800-fold with a recovery of 2.2% by the above procedures. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the final preparation showed a single protein band at Mr 22,000. The purified protein served as a substrate for botulinum ADP-ribosyltransferase and was maximally ADP-ribosylated to the extent of about 0.7 mol of ADP-ribose/mol of protein. A guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was co-purified with the ADP-ribosylation substrate, and the purified protein maximally bound about 0.5 mol of GTP gamma S/mol. GTP gamma S binding was effectively competed by GTP and GDP but not by GMP, ATP, and ADP. Thus, the ADP-ribosylation substrate is a GTP-binding protein. This protein, designated Gb (b for botulinum), is widely distributed in various tissues. It was rich in brain, pituitary, and adrenal glands, and poor in heart, smooth, and skeletal muscles.     

Purification of the thromboxane A2/prostaglandin H2 receptor from human blood platelets

S-145 (5Z-7-(3-endo-phenylsulfonylamino-(2.2.1.)-bicyclohept -2-exo-yl) heptenoic acid) is a potent and selective antagonist for thromboxane A2/prostaglandin H2 receptor. Using this compound as an immobilized ligand for affinity chromatography and [3H]S-145 as a radioligand, we have purified the thromboxane A2/prostaglandin H2 receptor from the membranes of human blood platelets. The purification procedures consisted of solubilization of the receptor with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), affinity chromatographies on columns of S-145 affinity gel, wheat germ agglutinin agarose and red agarose, and repeated gel filtration high performance liquid chromatography on a TSK gel G-3000SW column. On the second gel filtration high performance liquid chromatography, the [3H]S-145 binding activity was eluted as a symmetrical peak which overlapped exactly with a peak of ultraviolet absorption at 280 nm. By these procedures, the receptor was purified about 8700-fold from the solubilized extract with a recovery of 6%. The final preparation showed a broad protein band at Mr 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and maximally bound 19.2 nmol of [3H]S-145/mg protein with a Kd of 29.8 nM. The [3H]S-145 binding to the purified receptor was specifically displaced by several thromboxane A2/prostaglandin H2 analogues.     

Cellular retinoic acid-binding protein from rat testis. Purification and characterization

Cellular retinoic acid-binding protein has been purified to homogeneity from rat testes. The procedures utilized in the purification included acid precipitation, gel filtration, and chromatography on DEAE-cellulose. The binding protein was purified approximately 12,000-fold, based on total soluble testicular protein. The protein is a single polypeptide chain with a molecular weight of 14,600, determined by information from gel filtration and sodium dodecyl sulfate-polyacrylamide electrophoresis. The protein binds retinoic acid with high affinity; the apparent dissociation constant was determined by fluorometric titration to be 4.2 X 10(-9) M.     

Bioluminescent immunoassay using a fusion protein of protein A and the photoprotein aequorin

Aequorin is a photoprotein that emits light in the presence of Ca2+ ions. To develop a bioluminescent immunoassay based on the light emission property of aequorin, we have expressed the apoaequorin fusion protein with S. aureus protein A in E. coli by recombinant DNA techniques. The fusion protein expressed was purified by IgG-Sepharose affinity chromatography, gel filtration and HPLC procedures. The purified protein A-apoaequorin fusion protein has both the luminescent activity of aequorin and the IgG-binding ability of protein A. We compared results obtained using the protein A-aequorin fusion protein with those obtained using a protein A conjugated horseradish peroxidase based immunoassay, and found them to yield similar results.     

Purification of the 22 kDa protein substrate of botulinum ADP-ribosyltransferase C3 from porcine brain cytosol and its characterization as a GTP-binding protein highly homologous to the rho gene product

The 22 kDa protein substrate of botulinum ADP-ribosyltransferase C3 was purified from porcine brain cytosol by acetone precipitation, CM-Sephadex, octyl-Sepharose and TSK phenyl-5PW HPLC chromatography to apparent homogeneity. ADP-ribosylation of the protein was increased by guanine nucleotides (GTP, GDP, GTP gamma S, each 100 microM) but not by GMP, ATP or ATP gamma S. The purified 22 kDa protein bound maximally 0.9 mol [35S]GTP gamma S and hydrolyzed GTP with the rate 0.007 mol per mol protein. Amino acid sequences were obtained from two tryptic peptides, selected from an in situ digestion of Immobilon electrotransferred, gel purified ADP-ribosylated substrate. The two sequences obtained, cover 23 residues from the corresponding sequences in human rho.     

The rho gene product expressed in E. coli is a substrate of botulinum ADP-ribosyltransferase C3

The ras-related rho A protein expressed in E. coli, was ADP-ribosylated by botulinum ADP-ribosyltransferase C3. C3 also modified the valine-14 mutant rho protein but not the products of H-ras, R-ras, ral, ypt, and rap 1 genes. A ras-rho chimaera consisting of 60 amino acids from the amino terminus of ras fused to 133 amino acids from the carboxy terminus of rho was not modified by C3. Antibodies raised against the porcine brain cytosolic substrate of C3 cross reacted with the rho, valine-14 rho and ras-rho proteins, but not with the gene products of H-ras, R-ras, ral or rap 1. Polyclonal anti-H-ras antibodies cross reacted with H-ras but not with ral, rho, or the C3 substrate purified from porcine brain.     

Purification and characterization of a high-affinity binding protein for pancreatic-type phospholipase A2.

A high-affinity and specific binding site for mammalian group I phospholipase A2 (PLA2-I) was found on the membranes of bovine corpus luteum. Affinity labeling experiments revealed that PLA2-I binds to a single polypeptide with a mass of 190-200 kDa. The PLA2-I binding protein in the membranes was solubilized in an active form with n-octyl beta-D-thioglucoside, and then purified approx. 16,000-fold. The purification procedures consisted of diethylaminoethyl-Sephacel chromatography, PLA2-I-affinity gel chromatography and gel-filtration high-performance liquid chromatography on a TSKgel G3,000SWXL column. The final preparation migrated as a single molecular species of 190 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and identification of the 190 kDa protein as the PLA2-I binding protein was demonstrated by ligand blotting analysis. The purified protein possessed a binding capacity with high affinity and specificity for a mammalian mature type of PLA2-I. Treatment of the purified material with N-glycosidase F resulted in increased mobility of the protein on SDS-PAGE as well as considerable abolition of the PLA2-I binding activity, thus suggesting the requirement of the carbohydrate moiety of the PLA2-I binding protein for receptor-ligand interactions.     

Purification and subunit structure of mouse liver cystathionase

Cystathionase has been purified from mouse liver by ammonium sulfate precipitation, ethanol precipitation, column chromatography on DEAE-cellulose and on hydrox-ylapatite, as well as Sephadex G-200 gel filtration. These procedures yielded a chromatographically homogeneous enzyme which was purified more than 1000-fold relative to whole liver extract. Overall recovery was approximately 4%. The purified enzyme does not contain detectable carbohydrate and migrates as a single protein component on analytical disc gel electrophoresis. A sedimentation coefficient of 8.3 S has been determined for the active enzyme by rate zonal centrifugation in glycerol gradients. This value suggests a molecular weight for the native enzyme of approximately 160,000 g/mol, a value similar to that estimated by gel filtration. Following sodium dodecyl sulfate gel electrophoresis in the presence of reducing agent and at different gel concentrations, a single protein component with a molecular weight of 40,000 g/mol was obtained. Thus, the enzyme appears to consist of four subunits of equal size. The K_m value for cystathionine at pH 8.1, 37 °C, and in the presence of 1 mm dithioerythritol is approximately 1 mm.     

人胃腺苷脱氨酶的分离纯化及性质研究

用硫酸铵分级沉淀、ＤＥＡＥ－纤维素离子交换层析、免疫亲和层析、ＳｅｐｈａｄｅｘＧ１００凝胶柱层析从人胃组织中提取出腺苷脱氨酶,酶纯化１９３２４倍,比活力为５７９７Ｕ／ｍｇ蛋白．提取酶液经ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和等电聚焦只呈现一条区带。测得该酶的分子量为４１．２ｋＤ,等电点为ｐＨ４．８．氨基酸组成分析表明该酶由３８８个氨基酸残基组成,Ｎ端氨基酸为精氨酸。酶的最适ｐＨ为６．５,ｐＨ小于５．０或大于９．０时不稳定;最适温度为３７℃,对热不太稳定,以腺苷及２－脱氧腺苷作为底物,其Ｋｍ分别为８７μｍｏｌ／Ｌ和４１μｍｏｌ／Ｌ。     

Brain neurite-stimulating protein

A protein possessing the neurite-stimulating activity in organotypic cultures of chicken embryo spinal ganglia was isolated and purified from bovine brain tissue. The isolation and purification procedures included acid extraction, ultrafiltration, preparative polyacrylamide gel electrophoresis and chromatography on heparin-Sepharose. The molecular weight of the protein is about 15000 Da. The neurite-stimulating activity of the purified protein manifests itself at a protein concentration of about 10(-9) M.     

Purification and characterization of an extracellular beta-1,4-mannanase from a marine bacterium, Vibrio sp. strain MA-138.

A beta-mannanase (EC 3.2.1.78) from Vibrio sp. strain MA-138 was purified by ammonium sulfate precipitation and several chromatographic procedures including gel filtration, adsorption, and ion-exchange chromatographies. The final ion-exchange chromatography Mono Q yielded one major active fraction and three minor active fractions. The major active fraction was purified to homogeneity on the basis of native polyacrylamide gel electrophoresis (PAGE). This purified enzyme was identified as a glycoprotein by periodic acid-Schiff staining and a monomeric protein with a molecular mass of 49 kDa by sodium dodecyl sulfate-PAGE. The pI of the enzyme was 3.8. The purified enzyme exhibited maximal activity at pH 6.5 and 40 degrees C and hydrolyzed at random the internal beta-1,4-mannosidic linkages in beta-mannan to give various sizes of oligosaccharides. The first 20 N-terminal amino acid sequence of the purified enzyme showed high homology with the N-terminal region of beta-mannanase from Streptomyces lividans 66.     

Copurification of rho protein and the rho-GDP dissociation inhibitor from bovine neutrophil cytosol. Effect of phosphoinositides on rho ADP-ribosylation by the C3 exoenzyme of Clostridium botulinum.

The substrate of the C3 exoenzyme from botulinum toxin is a protein which is particularly abundant in the cytosol of neutrophils [Stasia, M. J., Jouan, A., Bourmeyster, N., Boquet, P., & Vignais, P. V. (1991) Biochem. Biophys. Res. Commun. 180, 615-622]. Optimal conditions for the ADP-ribosylation of the C3 substrate have been established in order to follow the course of its purification from bovine neutrophil cytosol. In particular, phosphoinositides at micromolar concentrations were found to enhance the ADP-ribosylation capacity of the C3 substrate in crude neutrophil cytosol and partially purified fractions. A [32P]ADP-ribosylatable protein, migrating on SDS-PAGE with a mass of 24 kDa, was copurified with a 29-kDa protein by a series of chromatographic steps on DEAE-Sephacel, Biogel P60, and Mono Q. In the case of the C3 substrate, isoelectric focusing revealed two major labeled bands with pI values of 6.2 and 5.6; the pI of the 29-kDa protein was 4.8-5.0. On the basis of the amino acid sequence of peptides resolved after proteolytic digestion, the 24-kDa protein and the 29-kDa protein were identified respectively as rho and the GDP dissociation inhibitor (GDI), suggesting that rho and GDI copurify from bovine neutrophil cytosol in the form of a complex. The presence of a number of amino acid residues specific of rho A in the enzymatic digest originating from rho indicates that, among the rho proteins, at least rho A belongs to the GDI-rho complex.     

A virus-inactivating protein isolated from the digestive juice of the silkworm, Bombyx mori

A protein that can precipitate nuclear polyhedrosis virus (NPV) in vitro was isolated from the digestive juice of silkworm larvae (Bombyx mori) by the procedures of gel filtration and ion-exchange and hydroxylapatite column chromatography. The SDS-polyacrylamide gel electrophoretic and the ultracentrifugal analyses showed that the purified substance was a homogenous simple protein. The molecular weight of the purified protein was 27,000–28,000 and the sedimentation coefficient was 2.61 S. This protein had an additional activity to inactivate NPV of B. mori in vitro, somewhat analogous to serological neutralization by serum proteins. Electron microscope observations showed that amorphous materials could be found on the surface of envelopes and that the nucleocapsids disappeared.     

Purification and partial amino acid sequences of a phospholipase C-associated GTP-binding protein from calf thymocytes

We recently identified a phosphoinositide-specific phospholipase C (PI-PLC)-stimulating GTP-binding protein (G protein) in calf thymocyte cytosol (Wang, P., Toyoshima, S., & Osawa, T. (1987) J. Biochem. 102, 1275-1287; and (1988) 103, 137-142). In this study we completely purified a G protein whose properties are quite similar to the G protein mentioned above from the calf thymocyte membrane and determined partial amino acid sequences of it. The purification was achieved by first treating the membrane with GTP gamma S, followed by sequential column chromatographies on DEAE-Sepharose CL-6B, Sephacryl S-200, Mono Q, and Mono S. The G protein was purified in a GTP gamma S-binding form and assayed as to the radioactivity of the [35S]GTP gamma S-bound PI-PLC-associated G protein standard obtained from calf thymocyte cytosol. The purified G protein could stimulate the activity of a partially purified PI-PLC for phosphatidylinositol 4,5-bisphosphate hydrolysis. From approximately 5.6 g of membrane protein we obtained about 5 micrograms of a purified sample. The purified G protein showed a molecular weight of 21 kDa on SDS-PAGE and one of 25 kDa on gel filtration. The partial amino acid sequences were determined by treating the purified sample with lysylendopeptidase, purifying the resultant peptide fragments on a HPLC-reverse phase column and then sequencing the peptide fragments with a sequencer. Comparison of the obtained sequences with those of known lower molecular weight GTP-binding proteins suggested that, although structurally similar to rho gene products, this is a novel G protein.     

Ca2+-binding protein from human kidney. Purification and properties.

A Ca2+-binding protein (CaBP) from human kidney was purified by two different procedures. The first involved heat-precipitation of a kidney cytosol fraction followed by gel filtration and chromatofocusing. This resulted in a 200-fold increase in the specific Ca2+-binding activity with a yield of 10%. A specific antibody was raised against the purified CaBP, as demonstrated by one precipitate in crossed immunoelectrophoresis of a kidney cytosol fraction. The antibody was coupled to Sepharose 4B and CaBP was then purified by immunoadsorbent chromatography. Applying this technique, a 500-fold purification of CaBP with a yield of 50% was obtained. Both preparations appeared homogeneous in crossed immunoelectrophoresis against a polyvalent antiserum and migrated as a single band corresponding to a mol.wt. of 26000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In gel filtration under non-denaturing conditions CaBP was eluted corresponding to a mol.wt. of 28000. The association constant for the high-affinity Ca2+-binding sites of CaBP was estimated by gel filtration to be 0.1 X 10(6)M-1, and the protein displayed Ca2+-dependent electrophoretic mobility, with more rapid anodic migration in the presence of EDTA. The protein eluted at a position corresponding to a pI of 4.5 in chromatofocusing. Immunochemical experiments with the specific antibody showed no cross-reaction between renal and intestinal CaBP.     

Purification and partial characterization of cellular retinoic acid-binding protein from human placenta

Cellular retinoic acid-binding protein (CRABP) has been purified to homogeneity from human placenta by a series of procedures, including acetone powder extraction, gel filtration on Sephadex G-50, and ion-exchange chromatography on DEAE-cellulose and on SP-Sephadex. Cellular retinol-binding protein (CRBP) was isolated concurrently. CRABP was purified 75,400-fold, based on total soluble acetone powder extract of placenta. The protein is a single polypeptide chain with a molecular mass of 14,600 Da, estimated by sodium dodecyl sulfate (SDS) gel electrophoresis or gel filtration, and has an isoelectric point of 4.78 (apo-CRABP, 4.82). On analysis of absorption and fluorescence spectra, the protein was seen to exhibit an absorption peak at 350 nm, fluorescence excitation maxima at 350 and 370 nm, and a fluorescence emission maximum at 475 nm. Human CRABP was immunologically distinct from human CRBP and serum retinol-binding protein.     

Studies on phospholipase A in Trimeresurus flaoviridis venom. III. Purification and some properties of phospholipase A inhibitor in Habu serum.

Phospholipase A [EC 3.1.1.4] inhibitor was purified from Habu (Trimeresurus flavivurudls) serum by gel filtration on Sephadex G-200, chromatography on DE-23 cellulose and affinity chromatography on a Sepharose 4B-phospholipase A column. By these procedures, a 31-fold increase in specific activity was attained with a yield of 15%. The purified material was homogeneous as judged by cellulose acetate and polyacrylamide gel electrophoresis. It had an apparent molecular weight of 100,000 as measured by gel filtration on Sephadex G-200. The purified inhibitor was stable for 20 min at 80 degrees and was unstable below pH 6. It migrated before albumin in cellulose acetate electrophoresis and did not form any precipitin line with the crude venom or with purified phospholipase A in immunodiffusin tests. An 8-fold excess of the purified inhibitor by weight was required to inhibit completely both the egg yolk clearing action and the hemolytic action of phospholipase A.     

Expression and purification of recombinant and native calreticulin.

Calreticulin is a 60-kDa Ca(2+)-binding protein of the endo(sarco)plasmic reticulum membranes of a variety of cellular systems. The protein binds approximately 25 mol of Ca2+ with low affinity and approximately 1 mol of Ca2+ with high affinity and is believed to be a site for Ca2+ binding/storage in the lumen of the endo(sarco)plasmic reticulum. In the present study, we describe purification procedures for the isolation of recombinant and native calreticulin. Recombinant calreticulin was expressed in Escherichia coli, using the glutathione S-transferase fusion protein system, and was purified to homogeneity on glutathione-Sepharose followed by Mono Q FPLC chromatography. A selective ammonium sulfate precipitation method was developed for the purification of native calreticulin. The protein was purified from ammonium sulfate precipitates by diethylaminoethyl-Sephadex and hydroxylapatite chromatography procedures, which eliminates the need to prepare membrane fractions. The purification procedures reported here for recombinant and native calreticulin yield homogeneous preparations of the proteins, as judged by the HPLC reverse-phase chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified native and recombinant calreticulin were identified by their NH2-terminal amino acid sequences, by their Ca2+ binding properties, and by their reactivity with anticalreticulin antibodies.