Major pertussis-toxin-sensitive GTP-binding protein of bovine lung. Purification, characterization and production of specific antibodies

Two GTP-binding proteins which can be ADP-ribosylated by islet-activating protein, pertussis toxin, were purified from the cholate extract of bovine lung membranes. Both proteins had the same heterotrimeric structure (alpha beta gamma), but the alpha subunits were dissociated from the beta gamma when they were purified in the presence of AlCl3, MgCl2 and NaF. The molecular mass of the alpha subunit of the major protein (designated GLu, with beta gamma) was 40 kDa and that of the minor one was 41 kDa. The results of peptide mapping analysis of alpha subunits with a limited proteolysis indicated that GLu alpha was entirely different from the alpha of brain Gi or Go, while the 41-kDa polypeptide was identical with the alpha of bovine brain Gi. The kinetics of guanosine 5'-[3-O-thio]triphosphate (GTP[gamma S]) binding to GLu was similar to that to lung Gi but quite different from that to brain Go. On the other hand, incubation of GLu alpha at 30 degrees C caused a rapid decrease of GTP[gamma S] binding, the inactivation curve being similar to that of Go alpha but different from that of Gi alpha. The alpha subunits of lung Gi and GLu did not react with the antibodies against the alpha subunit of bovine brain Go. The antibodies were raised in rabbits against GLu alpha and were purified with a GLu alpha-Sepharose column. The purified antibodies reacted not only with GLu alpha but also with the 41-kDa protein and purified brain Gi alpha. However, the antibodies adsorbed with brain Gi alpha reacted only with GLu alpha, indicating antisera raised with GLu alpha contained antibodies that recognize both Gi alpha and GLu alpha, and those specific to GLu alpha. These results further indicate that GLu is different from Gi or Go. Anti-GLu alpha antibodies reacted with the 40-kDa proteins in the membranes of bovine brain and human leukemic (HL-60) cells. The beta gamma subunits were also purified from bovine lung. The beta subunit was the doublet of 36-kDa and 35-kDa polypeptides. The lung beta gamma could elicit the ADP-ribosylation of GLu alpha by islet-activating protein, increase the GTP[gamma S] binding to GLu and protect the thermal denaturation of GLu alpha. The antibodies raised against brain beta gamma cross-reacted with lung beta but not with lung gamma.     

Purification and characterization of two GTP-binding proteins of 22 kDa from human platelet membranes

Two GTP-binding proteins (G-proteins) of 22 kDa were purified to near homogeneity from a sodium cholate extract of human platelet membranes by successive chromatographies on DEAE-Sephacel, Ultrogel AcA-44, phenyl-Sepharose CL-4B, Mono Q HR5/5 and hydroxyapatite columns. They bound maximally 0.89 mol of [35S]guanosine 5'-(3-O-thio)triphosphate per mol of both purified proteins, and this binding was inhibited by GTP and GDP but not by ATP and AppNHp. Their molecular masses were somewhat lower than that of ras p21 and they were not recognized by an anti-v-Ki-ras p21 antibody. These results indicate that human platelet membranes contain at least two low-molecular-mass G-proteins distinct from ras p21, in addition to the heterotrimeric G-proteins, the alpha-subunits of which possess molecular mass values of about 40 kDa.     

Purification of GTP-binding proteins from bovine brain membranes. Identification of heterogeneity of the alpha-subunit of Go proteins

Using high-resolution Mono Q column chromatography, we purified 6 distinct peaks of GTP-binding proteins from bovine brain membranes. Five of them consisted of 3 polypeptides with alpha beta gamma-subunits and served as the substrate of islet-activating protein (IAP), pertussis toxin. The other one was purified as alpha-subunit alone and was also ADP-ribosylated by IAP in the presence of beta gamma-subunits. When each alpha-subunit was characterized by immunoblot analysis using various antibodies with defined specificity, the two of them were identified as Gi-1 and Gi-2, and other 4 appeared to be Go or Go-like G proteins. The alpha-subunits of immunologically Go-like proteins were apparently distinguishable from one another on elution profiles from the Mono Q column. Thus, there was a heterogeneity of the alpha-subunit of Go in the brain membranes.     

Purification of heterotrimeric GTP-binding proteins from brain: identification of a novel form of Go

Using high-resolution Mono-Q anion-exchange chromatography, we purified four distinct GTP-binding proteins from bovine brain. Each consists of alpha and associated beta/gamma subunits, and each is a substrate for pertussis toxin catalyzed ADP-ribosylation. We defined the relationship between the alpha subunits of the purified proteins and cloned cDNAs encoding putative alpha subunits (1) by performing immunoblots with peptide antisera with defined specificity and (2) by comparing the migration on two-dimensional gel electrophoresis of the purified proteins, and of the in vitro translated products of cDNAs encoding alpha subunits. Purified G proteins with alpha subunits of 39, 41, and 40 kDa (G39, G41, and G40 in order of abundance) correspond to the products of Go, Gi1, and Gi2 cDNAs. We purified a novel G protein with an alpha subunit slightly above 39 kDa (G39*). G39* is less abundant than G39, elutes earlier than G39 on Mono-Q chromatography, and has a more basic pI (6.0 vs 5.6) than G39. G39 and G39*, however, are indistinguishable on immunoblots with a large number of specific antisera. The data suggest that G39* may represent a novel form of Go, differing in posttranslational modification rather than primary sequence.     

GTP-binding proteins in bovine brain nuclear membranes.

Nuclear membranes and other subcellular fractions derived from bovine brain cortex were investigated for the existence of GTP-binding proteins. By using photolytic labeling with [alpha-32P]GTP a 29 kDa GTP-binding protein was shown to be present in nuclear membranes which was not present in the plasma membranes nor in microsomal or cytosolic fractions. Two-dimensional gel electrophoresis revealed that this protein is rather acidic with a pI lower than 4.5. Members of the heterotrimeric Gi/o family are not present in the nuclear envelope: a 39 kDa protein, ADP ribosylated by pertussis toxin, was shown to originate from plasma membrane contamination.     

Purification and characterisation of bovine spleen ADPase.

ADPase has been purified from bovine spleen. Electrophoresis revealed a minor contaminent in the preparation that may represent ADPase that has been decreased in size by limited proteolysis during extraction and purification. The native enzyme behaves on SDS/PAGE as a 100-kDa protein but ADPase is a glycoprotein and so its electrophoretic behaviour may be anomalous. The apparent molecular mass is decreased to 70 kDa after removal of carbohydrate by treatment with a glycosidase. The use of a cross-linking reagent followed by electrophoresis suggests that the enzyme is composed of a single subunit. The specific activity of the purified material was 115 U/mg protein. The enzyme catalyses the hydrolysis of nucleoside di- and tri-phosphates but nucleoside monophosphates are not acted upon. The Km for ADP (approx. 10-15 microM) is unaffected by the state of purification of the enzyme, but catalytic activity of the purified material is stimulated by inclusion of detergent in the assay system and by calcium ions. Maximum activity is seen at pH 8.0-8.5 with ADP as substrate but the optimum shifts to 7.5-8.0 for ATP hydrolysis.     

Purification and characterization of two phospholipid exchange proteins from bovine heart.

Two phospholipid exchange proteins from bovine heart have been purified approximately 2000-fold and judged greater than 90% pure. The proteins are similar in molecular weight (both 33,400 by polyacrylamide gel electrophoresis and 23,500 by gel filtration), in amino acid composition, and in specificity, although they differ in isoelectric points, 5.3 and 5.6. The transfer of phospholipids between artificial membranes is catalyzed by these proteins at the following relative rates: 100 for phosphatidylinositol, 35 for phosphatidylcholine, 5 for sphingomyelin, and 0.1 for phosphatidylethanolamine. The use of these exchange proteins in the study of mixed phospholipid vesicle structure is demonstrated. The purified proteins catalyze the substitution of one membrane phospholipid species for another at a rate comparable to true exchange. The phospholipid exchange activity is inhibited by the presence of sphingomyelin, and also by reagents which react with sulfhydryl groups. Evidence is presented for two sites of N-ethylmaleimide binding on these exchange proteins. Reaction with one site has little effect on activity and occurs in the absence of membranes. Reaction with the second site occurs in the presence of phospholipid vesicles and leads to complete, irreversible inhibition of exchange activity.     

Purification of acid ribonucleases from bovine spleen

Two acid RNases were purified from bovine spleen by means of ammonium sulfate fractionation, chromatographies on-phospho-cellulose, heparin-Sepharose CL-6B, poly G-Sepharose, and 2', 5'-ADP-Sepharose, and gel filtration on Toyopearl HW 55F. Both purified preparations were homogeneous as judged by disc electrophoresis at pH 4.3. They were designated as RNase BSP1 and RNase BSP2 in the order of elution from a phospho-cellulose column. RNase BSP2 was immunologically indistinguishable from RNase K2 from bovine kidney. RNase BSP1 was a typical pyrimidine base-specific, uridylic acid-preferential RNase and had very sharp pH optimum at 6.5. RNase BSP1 thus obtained was a glycoprotein giving two major bands on SDS-slap electrophoresis. Although the apparent molecular weight of RNase BSP1 was distributed in the range of 27,000-20,000, it decreased to about 17,000-18,000 after endoglycosidase F digestion. The N-terminal amino acid sequence up to the 20th amino acid had no homology to those of RNase K2 and RNase A.     

Multiple small molecular weight GTP-binding proteins in bovine brain cytosol. Purification and characterization of a 24KDa protein

We have separated multiple small Mr GTP-binding proteins (G-proteins) from bovine brain crude membranes, purified a novel 24KDa G protein (smg p25A) to near homogeneity and characterized it. In this paper, we have studied these small Mr G proteins in the cytosol fraction of bovine brain. [35S]GTP gamma S-binding activity is detected in the cytosol fraction but this activity is one-sixth to one-eighth of that of the crude membrane fraction. When G proteins in the cytosol fraction are purified by successive chromatographies on DEAE-cellulose, Ultrogel AcA-44, hydroxyapatite and Mono Q HR5/5 columns, multiple small Mr G proteins are separated. One of these G proteins shows a Mr of about 24KDa. Its physical, immunological and kinetic properties are indistinguishable from smg p25A. These results indicate that there are also multiple small Mr G proteins in the cytosol fraction of bovine brain, and suggest that one of the cytosol G proteins is the soluble form of smg p25A.     

Purification of the major GTP-binding proteins from human placental membranes

Using minor modifications of procedures developed to purify GTP-binding proteins (G-proteins) from rabbit liver, we have purified the major G-proteins present in human placental membranes. One, referred to as Gi, is the major substrate for pertussis toxin-catalyzed ADP-ribosylation and has an alpha-subunit of 41,000 daltons, and beta-subunit of 36,000 and 35,000 daltons, and a gamma-subunit of 10,000 daltons. The other protein, referred to as Gp, was identified by its ability to bind guanine nucleotides specifically with high affinity. This activity was resolved from Gs and Gi by the second step of purification (AcA-34 chromatography) and was further purified through heptylamine-Sepharose and hydroxylapatite. The guanine nucleotide-binding site, which can be resolved by high performance liquid chromatography procedures and identified by a photolyzable GTP analogue, is associated with a 21,000-dalton protein (Gp alpha) that copurifies with beta gamma-subunits indistinguishable from the beta gamma-subunits associated with Gs and Gi. This protein represents a potentially novel member of the structurally and functionally homologous family of G-proteins that are transducing elements in the receptor-mediated regulation of a variety of cellular processes.     

Purification and characterization of a 2'-phosphodiesterase from bovine spleen

Interferon-induced 2',5'-oligoadenylates are transiently produced during viral infection and are believed to play a role in the interferon-mediated inhibition of replication of at least some viruses. 2',5'-Oligoadenylates must be catabolized but are resistant to degradation by most known ribonucleases. A 2'-phosphodiesterase that degrades 2',5'-oligoadenylates was purified 1500-fold from a low speed homogenate of bovine spleen by precipitation at pH 5.2, ammonium sulfate fractionation, differential ultrafiltration, and successive chromatography on DEAE-Sephacel, hydroxylapatite, and a fast protein liquid chromatography Mono P column. No other 2-5A-degrading activity was observed during the purification procedure. The molecular mass of the enzyme estimated by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 65,000. The enzyme is distinct from bovine spleen phosphodiesterase II. The 2'-phosphodiesterase cleaves 2',5'- and 3',5'-linked oligonucleotides, as well as branched oligoadenylate, A(2'pA)(3'pA), but appears to be most active on 3',5'-oligoribonucleotides. The enzyme cleaves 5'-AMP from the 2' terminus of 2',5'-oligoadenylates and appears to require a free 2' terminus and a 3'-oxygen on the penultimate nucleotide. Substrate length, 5'-phosphorylation, and base composition do not appear to be critical factors in determining enzyme activity. The effects of pH, Mg2+, Mn2+, EDTA, phosphate, 2-mercaptoethanol, and N-ethylmaleimide are also described. This enzyme may be involved in the catabolism of the interferon-induced 2',5'-oligoadenylates and other 2',5'-linked RNAs in the cell.     

Cathepsin S from bovine spleen. Purification, distribution, intracellular localization and action on proteins.

Cathepsin S was detected in bovine kidney, spleen, lymph nodes and lung by immunochemical methods. The immunostaining of cathepsin S in kidney was concentrated to the cells of the proximal tubule, where the enzyme was present in cytoplasmic granules. The purification method for cathepsin S from bovine spleen involved (NH4)2SO4 fractionation, chromatography on CM-Sephadex C-50, gel filtration on Sephacryl S-200 and chromatofocusing (pH 8.0-6.0). The enzyme was partially destroyed by autolysis of the homogenate at pH 4.2. The isoelectric point of cathepsin S was 7.0. Cathepsin S was found to hydrolyse proteins at a similar rate to cathepsin L below pH 7.0. At pH values of 7.0-7.5 cathepsin S retained most of its activity, whereas cathepsin L was completely inactive.     

Molecular characterization of tobacco cDNAs encoding two small GTP-binding proteins

We have isolated two cDNAs encoding small GTP-binding proteins from leaf cDNA libraries. These cDNAs encode distinct proteins which show considerable homology to members of the ras superfamily. Np-ypt3, a 1044 bp long Nicotiana plumbaginifolia cDNA, encodes a 24.4 kDa protein which shows 65% amino acid sequence similarity to the Schizosaccharomyces pombe ypt3 protein. The N-ypt3 gene is differentially expressed in mature flowering plants. Expression of this gene is weak in leaves, higher in stems and roots, but highest in petals, stigmas and stamens. Nt-rab5, a 712 bp long Nicotiana tabacum SR1 cDNA, encodes a 21.9 kDa protein which displays 65% amino acid sequence similarity to mammalian rab5 proteins. The expression pattern of the Nt-rab5 gene is very similar to that of the Np-ypt3 gene. The Nt-rab5 gene is virtually not expressed in leaves, higher in stems and roots, and highest in flowers. Both the Nt-rab5 and Np-ypt3 proteins were expressed in Escherichia coli and shown to bind GTP.     

DRG represents a family of two closely related GTP-binding proteins

In a previous publication we identified a novel human GTP-binding protein that was related to DRG, a developmentally regulated GTP-binding protein from the central nervous system of mouse. Here we demonstrate that both the human and the mouse genome possess two closely related drg genes, termed drg1 and drg2. The two genes share 62% sequence identity at the nucleotide and 58% identity at the protein level. The corresponding proteins appear to constitute a separate family within the superfamily of the GTP-binding proteins. The DRG1 and the DRG2 mRNA are widely expressed in human and mouse tissues and show a very similar distribution pattern. The human drg1 gene is located on chromosome 22q12, the human drg2 gene on chromosome 17p12. Distantly related species including Caenorhabditis elegans, Schizosaccharomyces pombe and Saccharomyces cerevisiae also possess two drg genes. In contrast, the genomes of archaebacteria (Halobium, Methanococcus, Thermoplasma) harbor only one drg gene, while eubacteria do not seem to contain any. The high conservation of the polypeptide sequences between distantly related organisms indicates an important role for DRG1 and DRG2 in a fundamental pathway.     

Purification and characterization of a protein tyrosine kinase from bovine spleen

Protein tyrosine kinase was purified extensively from a 30,000 X g particulate fraction of bovine spleen by a procedure involving four column chromatographies: DEAE-Sepharose, polyamino acids affinity, hydroxylapatite, and Sephacryl S-200 molecular sieving. The purification resulted in more than 3,000-fold enrichment in [Val5]angiotensin II phosphorylation activity (specific activity 202 nmol/min/mg). All column chromatography profiles showed single protein tyrosine kinase activity peaks with the exception of that of affinity chromatography, where about 50% of the enzyme activity appeared with the breakthrough fraction; only the bound enzyme was further purified. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of a purified sample phosphorylated in the presence of [gamma-32P]ATP revealed the presence of a single phosphorylated polypeptide of molecular weight 50,000 which represents about 40% of total protein. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions showed that protein tyrosine kinase activity co-migrated with the phosphoprotein. Stoichiometry of the phosphorylation of the 50-kDa polypeptide was found to be 1.0 mol/mol. The purified sample did not appear to contain phosphotyrosine protein phosphatase activity. Both casein and histone could be phosphorylated by the purified sample, and the phosphorylation occurred only at tyrosine residue, suggesting that there was no protein serine and threonine kinase contamination.     

High molecular weight forms of GTP-binding regulatory proteins from the bovine cerebellum

A new form of a low Km GTPase belonging to the family of regulatory GTP-binding G-proteins has been identified in bovine cerebellum. The molecular weight of this G-protein is several times as high as that of other G-proteins known to be alpha beta gamma heterotrimers: i. e., Gs, Gi, Go, transducin and a new G-protein which had recently been isolated in our laboratory from bovine cerebellum. The high molecular weight G-protein is stable against dissociation; its molecular mass does not change after treatment with DTT, colchicine and NaF. Using antibodies against the alpha-subunit of the formerly isolated cerebellar G-protein and the transducin beta-subunit, it was demonstrated that the both immunoreactive subunits are present in the high molecular weight G-protein. The two forms of the cerebellar G-proteins, i. e., "high" and "low molecular weight" ones, differ drastically in terms of the Mg2+ effect on their GTPase activity. Whereas at submicromolar concentrations of Mg2+ the GTPase activity of the former is virtually absent, the GTPase activity of the latter is more elevated in the presence of EDTA than in the presence of Mg2+.     

Purification of beta-glucosidase activities from bovine spleen by affinity chromatography

Bovine spleen β-d-glucosidase, glucosylceramide: β-d-glucosidase and glucosylsphingosine: β-d-glucosidase were purified by chromatography on a “gluconate” Sepharose column. Ten other lysosomal acid hydrolases, present in the preparation applied to the column, were absent from the glucosidase fraction.     

Purification and characterization of ten new rice NaCl-soluble proteins: identification of four protein-synthesis inhibitors and two immunoglobulin-binding proteins

Ten new proteins from rice (Oryza saliva L. cv. Bahia) including four protein-synthesis inhibitors and two immunoglobulin E (IgE)-binding proteins have been isolated and characterized. These proteins as well as one previously known component, -globulin, were purified from a 0.5 M NaCl extract of rice endosperm by a new, apparently non-denaturing, isolation procedure developed for rice proteins. The method is based on extractions of this complex protein mixture with a diluted volatile salt solution and an aqueous solution of ethanol. This preliminary step results in an improvement in the separation of these proteins, thus facilitating their subsequent purification by reversed-phased high-performance liquid chromatography. These new proteins have similar relative molecular masses (M_rs) from 11000 to 17000. The purity of the proteins was analyzed by micro two-dimensional gel electrophoresis. Four of these components were found to be in-vitro protein-synthesis inhibitors in a cell-free system from rat brain. The NH₂-terminal amino-acid sequences of these four inhibitors were determined from 12 to 26 cycles after direct blotting of the separated proteins from electrophoresis gels. Three of these proteins with M_rs between 16000 and 17000 showed a high degree of homology ranging from 57% to 75% but seem to be unrelated to the fourth inhibitor. In addition, the -globulin and one of the new low-molecular-weight proteins of M_r 12500 seemed to show allergenic properties since they bound IgE antibodies from the sera of hypersensitive patients. Boths proteins have blocked NH₂-terminal amino acids.Abbreviations HMW high molecular weight - IgE immunoglobulin E - LMW low molecular weight - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - RP-HPLC reversed-phase high-performance liquid chromatography - SDS sodium dodecyl sulphate We thank F. Soriano and F. Colillia for technical assistance, and Shirley McGrath for secretarial work. We also appreciate the cheerful assistance of the members of Instituto Nacional de Semillas, specially Mr. L. Solaices, who provided samples of rice. This work was supported by a grant from Comision Asesora de Investigación Ciéntifica y Técnica.